The interaction of viruses with fetal and adult ovine cells in vitro

Studies were undertaken to investigate the role of peripheral blood leukocytes and spleen cells in age-related host resistance to viral infection. Initially, peripheral blood leukocyte and spleen cell cultures derived from adult ewes and 70 to 145 day gestational age fetal lambs were examined for their ability to produce interferon and support viral replication (the complete ovine gestational period is normally 150 days). Bleutongue (BTV), Chikungunya (CV), Semliki Forest (SFV), Newcastle disease (NDV), vesicular stomatitis (VSV), and vaccinia viruses, and herpesvirus hominis type II (HVH-II), failed to replicate to detectable levels in either fetal or adult cell cultures. No difference in either the levels or the kinetics of interferon production between virus-infected adult and fetal cell cultures was observed. Mean levels of peak interferon production induced by BTV, CV, SFV, NDV, and HVH-II or approximately 1000-3000 units/ml were demonstrated by 24 hrs post-virus inoculation. In contrast, mean interferon titers induced by VSV and vaccinia virus were significantly lower ( < 40-550 units/ml) and did not reach peak levels until 48 hrs post-inoculation. Variations in interferon levels induced on separate occasion using cells from the same adult donor and from the same donor age group were also observed. The interferon induced in both the fetal and adult cell cultures fulfilled the usual criteria for characterization. Blood leukocytes and spleen cells from adult sheep and 70 to 145 day gestation fetal lambs were found equally capable of inhibiting HCH-II and BTV infection in fetal lamb kidney (FLK) cell monolayers. Decreased cytopathic effect as well as diminished viral replication was demonstrated in the mixed cell cultures as compared with control FLK cultures. Increasing the total number of leukocytes or spleen cells from 0.5 x 10[6] to 2.0 x 10[6] resulted in enhanced antiviral protection and was accompanied by a concurrent increase in interferon production in mixed leukocyte-FLK cell cultures but not in mixed cultures of spleen and FLK cells. Ovine leukocytes did not inhibit the progression of HVH-II infection in mouse embryo fibroblast cultures, indicating that the antiviral effect was species specific. AN interferon-like substance was also detectable in mixed cultures containing leukocytes or spleen cells and uninfected FLK cell monolayers. Similar levels of the interferon-like substance were found when cultures of ovine leukocytes were added to either mycoplasma-contaminated FLK cell or antibiotic-treated FLK cells free of detectable mycoplasma. Uninfected ovine leukocyte, spleen cell, or FLK cell cultures did not produce interferon. A mycoplasmal species, Acholeplasma laidlawii, was subsequently isolated as a contaminant from the FLK cell line, and was shown to be associated with the induction of interferon in culture of ovine peripheral blood leukocytes. Broth cultures of the mycoplasma induced between 20 and 230 units/ml of interferon in leukocytes from two adult ewes. The amount of interferon produced correlated with the inoculum size of mycoplasma. Interferon production was associated with replication of the mycoplasma in the leukocyte cultures. Interferon was not induced by sterile mycoplasmal broth, a cell-free filtrate of the mycoplasmal cultures, or heat-inactivatied mycoplasma. The antiviral substance was characterized as interferon by the usual criteria.

THE INTERACTION OF VIRUSES vlITH FETAL AND ADUL'r OVI~"'E CELLS IN VITRO by Charles Rienzi Rinaldo, Jr • .:. ~ dissertation submi tted to the faculty of the University of utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology university of Utah August 1973 This dissertation for the Doctor of Philosophy Degree by Charles Rienzi Rinaldo, Jr. has been approved July 1973 man, Supervisory Committee Member -, Member Mefuber /� Member {/' Member ��-ZC�-<-�---­ Chairman, Major department ACKNOWLEDGE!1ENTS This thesis is dedicated to my wife, Donna, and my son, Jonathan, for the sacrifices they have made and for the steadfast support and love they have given. I would also like to thank my parents for their support throughout this endeavor. I'·'Iy sincere appreciation is expressed to Dr. James C. Overall, Jr., and Dr. Lowell A. Glasgow for their support and guidance throughout the course of these investigations. I am also grateful for the advice and guidance given by Dr. Douglas ">1. Hill, Dr. Barry alNeill, and Dr. Charles ~l. c. De~"Jitt Cole, Dr. Frank J. during the preparation of this dissertation. I am indebted to Hs. Jennifer Fischbach for laboratory assistance during several of the experiments, and to Dr. Carter Bishop for his guidance and assistance in develo9ing the procedure for extraction of leukocytes from the peripheral blood. This research was partially supported by grant AI-I0217 from the National Institute of Allergy and Infectious Disease, and grant 1~1-02255 from the National Institute of Arthritis and Hetabolic Disease. 'rABLE OF CONTENTS Z>..BSTRACT • • • ...... • • ... ·. ........ ....... RBVIS:;-J OF LITERATURE: The interaction of viruses with cells of the blood, reticuloendothelial, and lymphoid systems • • • • • • • • • • ·........ Text .................. Tables • • · . . . . . .. • • Literature Cited • • . . . . . . . . . . • ·. • • • ·....·..• Table of Contents E.{PERIN~?NTAL I. R::';SULTS • The interaction of viruses with fetal and adult ovine leukocytes and spleen cells in vitro. I. Interferon production and viral replication •••••••••••• ·............... Introduction • • • · . ..·... Haterials and Hethods · . . . . . . . . . Results . . . . . . . ·..... Discussion · . • • · . · . . . . · . Literature Cited • • • · . . . · . . . · . lrables • • · . . • • · . . . · . . . . Figures ..... .......... Abstract • II. The interaction of viruses with fetal and adult ovine leuJ<.ocytes and spleen cells in vitro. II. Viral infection in a mixed cell system •••••••••••••• vi 1 5 6 8 46 64 90 90 91 92 95 100 104 113 121 124 132 Page Abstract • Introduction • · • 133 • • 134 • Nateria1s and Methods Hesu1ts • • Discussion · 133 • • • • • 144 • • • ·• • • • • • • • • • Figures III. • • • Literature Cited Tables 135 148 154 • • 161 induction of interferon in ovine leukocytes • 169 • ~~cop1asma-associated Abstract • • t'·1ateria1s and nethods Results • • • • Literature Cited • • • • . • v 177 183 • • • • • 190 196 • • • • 171 172 • • • • • • • • • • • Discussion • • • • Tables 170 • • • • Introduction • vrr:::. • • 200 ABSTRACT Studies were undertaken to investigate the role of peripheral blood leukocytes and spleen cells in age-related host resistance to viral infection. Initially, peripheral blood leukocyte and spleen cell cultures derived from adult ewes and 70 to 145 day gestational age fetal lambs were examined for their ability to produce interferon and support viral replication (the complete ovine gestational period is normally 150 days). Bleutongue (BTV), Chikungunya (Cv), Semliki Forest (SFV), Newcastle disease (NDV), vesicular stomatitis (VSV), and vaccinia viruses, and Herpesvirus hominis type II (HVH-II), failed to replicate to detectable levels in either fetal or adult cell cultures. No difference in either the levels or the kinetics of interferon production between virus-infected adult and fetal cell cultures was observed. Mean levels of peak interferon production induced by BTV, CV, SFV, NOV, and HVH-II of approximately 1000-3000 units/ml were demonstrated by 24 hrs post-virus inoculation. In contrast, mean interferon titers induced by VSV and vaccinia virus were significantly lower «40-550 units/ml) and did not reach peak levels until 48 hrs postinoculation. Variations in interferon levels induced on separate occasions using cells from the same adult donor and from the same donor age group were also observed. The interferon induced in both the fetal and adult cell cultures fulfilled the usual criteria for characterization. Blood leukocytes and spleen cells from adult sheep and 70 to 145 day gestation fetal lambs were found equally capable of inhibiting HVH-II and BTV infection in fetal lamb kidney (FLK) cell monolayers. Decreased cytopathic effect as well as diminished viral replication was demonstrated in the mixed cell cultures as compared with control FLK cultures. Increasing the total number of leukocytes or spleen cells from 0.5 X 10 6 to 2.0 X 10 6 resulted in enhanced antiviral protection and was accompanied by a concurrent increase in interferon production in mixed leukocyte-FLK cell cultures but not in mixed cultures of spleen and FLK cells. Ovine leukocytes did not inhibit the progression of HVH-II infection in mouse embryo fibroblast cultures, indicating that the antiviral effect was species specific. An interferon-like substance was also detectable in mixed cultures containing leukocytes or spleen cells and uninfected FLK cell monolayers. Similar levels of the interferon-like substance were found when cultures of ovine leukocytes were added to either mycoplasma-contaminated FLK cells or antibiotic-treated FLK cells free of detectable mycoplasma. Uninfected ovine leukocyte, spleen cell, or FLK cell cultures did not produce interferon. A mycoplasmal species, Acholeplasma laidlawii, was subsequently isolated as a contaminant from the FLK cell vii line, and was shown to be associated with the induction of interferon in cultures of ovine peripheral blood leukocytes. Broth cultures of the mycoplasma induced between 20 and 230 units/ml of interferon in leukocytes from two adult ewes. The amount of interferon produced correlated with the inoculum size of mycoplasma. Interferon production was associated with replication of the mycoplasma in the leukocyte cultures. Interferon was not induced by sterile mycoplasmal broth, a cell-free filtrate of the mycoplasmal cultures, or heat-inactivated mycoplasma. The antiviral substance was characterized as interferon by the usual criteria. viii INTRODUCTION Abortion, stillbirth, intrauterine infection, and congenital malformation may be associated with various systemic viral diseases of the pregnant animal. The most per- plexing aspect of this situation is that in the majority of cases of fetal and neonatal infection, the viral disease is quite innocuous to the mother and may even remain subclinical and asymptomatic (e.g., rubella virus and cytomegalovirus infection of humans, bluetongue virus infection of sheep). It is apparent, therefore, that the mammalian fetus reacts in an immature manner to viral infections frequently resulting in severe disease. In an attempt to explain the basis for enhanced fetal susceptibility to viral infection, conflicting data have been presented. Inability to produce interferon and changes in viral interaction with cells of the blood, lymphoid, and reticuloendothelial systems have been proposed as two possible mechanisms for this immaturity. The prominent role of these cells in antiviral host resistance of adults has already been substantially documented. In our research of age-dependent resistance, we consequently chose to utilize cells of the spleen and blood derived from fetal and adult sheep. One of the principle advantages of the sheep is the large animal size. Ample tissue and blood samples can be 2 obtained from adults and fetuses as young as mid-second trimester of gestation. This has permitted a comprehensive examination of both the in vivo and tissue culture responses of the fetal host to viral infection. ponse The interferon res- of the intact fetal lamb has been determined over the last four years in our laboratory. The validity of the results obtained in tissue culture could therefore be confirmed or contrasted with these in vivo data. The major questions to be answered by this research included first whether the inability to produce interferon was a basis for fetal immaturity to viral infections. The in vivo data obtained in our laboratory suggested that the fetal lamb was quite competent to produce interferon as early as first trimester gestation. Levels of serum inter- feron following viral infection, moreover, were equal to or many-fold higher than those in adult sheep following inoculation with Chikungunya virus. We proposed to examine virus-infected cultures of leukocytes and spleen cells from donors of various ages for evidence of interferon production. Would infected cultures from fetal animals produce strikingly higher interferon levels than those from adults, paralleling the in vivo situation? Or could we detect an ontogenic development in the ability to produce interferon in vitro which is apparently not found in the intact animal? Answers to these questions could also enable further evaluation of the relevancy of studies of age-related interferon production in humans. The majority of these investigations 3 have been based on the in vitro production of interferon by blood leukocytes. It is possible that this model system does not accurately reflect the ability of the intact human fetus to produce interferon. A further question to be answered was whether enhanced replication of viruses occurred in fetal cells, which could then lead to an inability of the intact host to cope with viral infections. To study this problem, viral replication was investigated in leukocyte and spleen cell cultures. Was it possible that fetal cells would produce significantly higher titers of virus than did adult cells? Would cells from earlier gestational ages produce progressively more virus? If we found differences in the way fetal and adult cells react to viral infection, many avenues of approach would be available for examining the reasons for such a phenomenon. For example, future research could investigate the complete viral replication cycle in cells from donors of various ages. If no differences were found, further research could concentrate on other aspects of the host response. A mixed cell system was devised to provide another parameter of host antiviral resistance in vitro. Cells from fetal and adult sheep were added to virus-infected and control cell monolayers. The mixed cell cultures were examined for levels of viral replication, interferon production, and cytopathic effect. The question posed was whether cells from fetal lambs were as competent as those from adult ewes 4 to inhibit viral infection in the cell monolayers. During the course of these studies, a mycoplasmal contaminant was isolated from the semi-continuous line of ovine cells being used. This led to the unexpected dis- covery that mycoplasmas could induce interferon in sheep leukocyte cultures. These investigations represented the final portion of the thesis research project. REVIEW OF LITERATURE THE INTERACTION OF VIRUSES WITH CELLS OF THE BLOOD, RETICULOENDOTHELIAL, AND LYMPHOID SYSTEMS In preparation for submission to Bacteriological Reviews rrABLi:: OF' CONTENTS .............. 8 CONSID.t;RATIONS INVOLVED IN EXPERIMENTATION • • 9 Cn~c) 9 Definition of host resistance cells ·....... Metabolic alterations of HRC Contaminating cells in HRC cultures :\SSOCI;'-~TION OF R~PLICATION OF VIRUSES IN HRC VIRUS~S 'dITH ERC IN VIRAL IHT3RFERON RSSPONSS OF HRC • • • 13 DISE~Sg:3 ·..... ...·... Interferon response of HRC to RNA viruses Picornaviruses ..... ·... 1,1yxoviruses 14 16 22 25 25 25 paramyxoviruses ..... Arboviruses, group A • ~irboviruses, rrogaviruses Coronaviruses ... l!.-denoviruses • ... .. ·.. ·........ ·....... ... ·..... Interferon response of HRC to Papovaviruses .. group B • Rhabdoviruses :,?oxviruses 11 DN~ viruses ... ·..... ·............ ...... ..... .... Immune soecific induction of interferon and other soluble factors in HRC cultures • • • • 26 31 32 33 33 33 33 33 34 34 34 36 39 7 ;:\ssociation of viruses \vith HRC in congenital viral diseases of humans • 39 .:':.ge-dependent interaction of HRC with viruses in eX9crimental infections • • 42 conCLUSION • 44 46 64 INTRODUCTION The role of cells of the blood, reticuloendothelial (RE), and lymphoid systems (which have been termed host resistance cells - HRC - for purposes of this review) in viral infections remains ill defined after years of research. Early studies (for reviews, see 76, 116) indicated that the interaction of HRC with viruses may differ sharply from that with bacteria. More recently, well-defined tissue culture techniques have shown that a number of viruses are intimately associated with HRC at various stages of host infection. This interrelationship between the parasite and the host cells could augment the pathogenic process by protecting the virus from antibodies, transporting it throughout the host, or by acting as a major site of replication. The interaction could contribute to host resistance by directly inactivating the virus, by production of the antiviral protein interferon, or by initiating the host's immune response. Recent studies have shown, furthermore, that age-related resistance to viral infection may be at least partially based on several unique variations in these virus - HRC interactions. The purpose of this paper is to present a comprehensive review of the various aspects of viral infections of HRC, with emphasis on the two major areas which experimental research has concentrated on, i.e., viral replication and interferon 9 production. A third area which has generated considerable interest is age-related interactions of viruses with HRC and is also included as the final portion of this review. CONSIDERATIONS INVOLVED IN EXPERIMENTATION Definition of HRC. The term "host resistance cells," or HRC, as used in this review refers to the gamut of lymphoid (114), RES (58), and blood cells (190) intimately involved in resistance to viral infections, in contrast to, for example, liver parenchymal or tissue fibroblast cells. Although these systems can be examined as three distinct entities, they share several common cell types and sites of origin in the individual. It is now generally accepted that HRC are derived from common multipotential stem cells (58, 114,190), which evolve into the various unipotential HRC types found throughout the animal. In relation to host resistance, two distinct classes of lymphoid cells have been described (32, 114): thymus- dependent, long-lived small lymphocytes (T cells) involved in cellular immune reactions and the development of humoral immunity, and bursal-influenced, short-lived small lymphocytes (B cells) which serve as precursors of, or directly as, antibody-forming cells (i.e., plasma cells). These may be found in the various lymphoid tissues, and in the blood, spleen, and tissue inflammatory areas, and initially evolve from precursor cells of the bone marrow. The cells of the RES were originally defined strictly 10 as phagocytes (58), although poorly phagocytic cells such as endothelial cells, fibrocytes, and reticular cells were included. A newly proposed classification (58) excludes these latter types by placing all highly phagocytic mononuclear cells and their precursors in what is termed the "mononuclear phagocyte system. u Free and fixed macro- phages, the major cells of this system, are known to evolve principally from circulating blood monocytes which in turn have originated from bone marrow precursor cells. Hacro- phages are localized in the connective tissue (histiocytes), liver (Kupffer cells), lung (alveolar macrophages), spleen and lymph node (free and fixed macrophages), bone marrow, and serous cavity (peritoneal macrophages). Evidence has been presented which suggests that certain populations of macrophages (e.g., inflammatory and peritoneal exudate) arise from small lymphocytes (47, 179). If this is true, an obvious dilemma confronts the experimenter who attempts to utilize lymphocytes and/or macrophages as "pure ll cultures. Blood leukocytes (190) consist of five types. The monocytes are relatively active mononuclear phagocytes of the RES which evolve into tissue rnacrophages. As mentioned, the principal classes of lymphocytes pertinent to host resistance consist of the Band T cells. Finally, the granulocytes (basophils, eosinophils, and neutrophils) are highly phagocytic cells with a polymorphonucleus (PMN) incapable of division under normal conditions, and which 11 originate from bone marrow presursor cells • .f'.1etabolic alterations of HRC. studies of virus - HRC interactions have mainly employed well-defined in vitro techniques. These artificial conditions are far from ideal, hOvlever I as they generate several potential problems for the investigator. Cells conserved under these conditions do not maintain the same structure and function as they possessed in vivo. Furthermore, the use of anticoagulants in studies of blood cells and sterile inflammatory stimulants in the examination of peritoneal exudate cells tends to further alter the cells. Granulocytes in culture display serious effects of aging by 24 hours as evidenced by a decrease in phagocytic properties and adherence to surfaces, leading to cell death (5). The use of heparin, a polyanionic polysaccharide, as an anticoagulant has been shown to increase phagocytic ability (95), inhibit viral replication by complexing viral particles (176), and alter interferon production (94). Ethylenediaminetetraacetic acid (EDTA), another frequently used anticoagulant, has been observed to grossly inhibit phagocytosis (T. F. Dougherty, personal communication). Lymphocytes placed in culture exhibit variable responses (5, 108, 184). In general, the majority of lympho- cytes are in the resting metabolic phase and undergo progressive degeneration over a several day period leading to cell death. A small percentage have the potential to trans- form without the stimulus of mitogens into large mononuclear 12 cells. These alterations are quite important to virus - HRC interactions, as such lymphocytes undergoing blastogenesis support the replication of virus and "spontaneously" produce interferon, whereas resting lymphocytes usually do neither of these (184). In vitro, macrophages have been shown to evolve from monocytes shortly after attachment (30). In studies of virus reactions with cells of the peritoneal cavity, the majority of studies employ sterile inflammatory exudates elicited by a variety of agents. Normally, the peritoneal cavity of the mouse, for example, contains an almost equal admixture of macrophages and lymphocytes (29). The pro- portion of macrophages to lymphocytes changes sharply after injection of the stimulators, which is the reason such treatment is of value. However, several metabolic and morphologic alterations also occur as a consequence of such stimulation. Injection of rodents intraperitoneally (i.p.) with thioglycollate broth, for example, leads to so-called "stimulated" macrophages with increased numbers of vacuoles and bundles of filaments in the cytoplasm (84). Peptone stimulation also induces vacuolization in peritoneal macrophages (137), and glycogen elicits an enhancement of size, morphology, "stickiness," content of acid phosphatase, cytotoxicity for fibroblasts, and phagocytosis above that found in normal cells (Ill, 137). All three of these irri- tants evoke mesothelial cells into the peritoneal exudate, which are capable of adhering to surfaces and can replicate 13 slowly in vitro (29). Stimulated macrophages can undergo division (147) whereas unstimulated macrophages do not divide under normal culture conditions (30). Brain heart infusion broth has also been reported to increase the cytotoxicity capabilities and acid phosphatase levels of macrophages above normal (111). One of the few studies on the effects of stimulated macrophages on viral infection has shown that proteosepeptone stimulated adult peritoneal macrophages when transferred i.p. to suckling mice protected the immature animals against Herpesvirus hominis type 1 (HVH - I) infection (85). Stimulated macrophages from suckling mice had no such protective ability. Possible mechanisms behind the transfer of resistance were enhanced interferon production, inhibited intracellular viral replication, and increased viral ingestion by the peptone-elicited macrophages. Contaminating cells in HRC cultures. v~ny investi- gations of viral interactions with HRC fail to fully describe the cell types used in their cultures. Peritoneal exudates, for example, contain not only macrophages and lymphocytes, but also a small amount of granulocytes and erythrocytes. Granulocytes have been reported to influence the formation of macrophages from lymphocytes in vitro (47). During the separation of leukocytes from whole blood, a large number of platelets (i.e., thrombocytes) are usually carried over into the final cultures (76). In a recent study (104), Chikungunya virus (CV) was markedly stabilized against 14 thermal inactivation of infectivity at 37C by incubation with washed human thrombocytes. High titers of infectious virus were recoverable when the virus-platelet material was inoculated into susceptible cell cultures, indicating the complex was reversible. Numerous investigators have attempted to utilize "pure" cultures of specific cell types to study virus - HRC interactions. However, most of these systems have at least 1% contamination with other cells, which could significantly alter the experimental results. Sawyer (149) has demonstrat- ed that a small number of guinea pig macrophages mixed with pure PMN leukocytes could directly influence the elimination of influenza A (WS strain) virus from the supernatant. Interferon production by pure cultures of human lymphocytes was also greatly enhanced by the addition of a small quantity of macrophages (48). Blastogenic transformation of lymphocytes, furthermore, may be significantly enhanced by the presence of macrophages (184). ASSOCIATION OF VIRUSES WITH HRC IN VIRAL DISEASES During viral infections of the adult animal, various associations with HRC can occur. In the blood, leukocytosis or leukopenia result during most viral diseases (76, 184). The fact that such gross changes in the quantities of blood leukocytes occur is the first indication that there is an intimate virus-leukocyte relationship during many host infections. An association with the appearance of atypical 15 lymphocytes has also been established in several diseases of suspected viral etiology, such as infectious mononucleosis and Burkitt's lymphoma (184). Lymphocytes from patients with several types of viral diseases (e.g., measles and rubella) have been demonstrated to have a high frequency of chromosomal aberrations (162). Similar chromosomal defects have been observed in lymphocytes infected with viruses in vitro (130). Numerous associations between viruses and blood HRC have been established using electron microscopy, direct isolation of virus, and immunofluorescence (76, 159, 184). Viruses such as measles, rubella, mumps, herpes, and cytomegalovirus (CMV) in humans, and canine distemper virus, several poxviruses, poliovirus, and bluetongue virus in animals are known to be aSSOCiated with blood HRC. The actual role of these interactions to overall host resistance has not been elucidated, however. Free and fixed macrophages may significantly alter the outcome of viral disease in experimental situations. Mims (116) has reviewed evidence that most nonpathogenic viruses were largely taken up and destroyed by Kupffer cells of the liver after intravenous (i.v.) injection. Other, more pathogenic agents were either passively transferred through or replicated within the macrophages, and subsequently infected the parenchymal cells of the liver. Macrophages also exhibit genetically-determined defects which directly regulate host susceptibility to mouse hepatitis virus (9) and West Nile virus (70) infections of mice. 16 In summary, ample evidence exists demonstrating the frequency of viral interaction with HRC during several host infectious processes. The exact role of blood and lymphoid cells in this response remains to be fully defined, while macrophages appear to be of paramount value in host resistance to many viral infections. REPLICATION OF VIRUSES IN HRC Numerous studies over the last 50 years have examined the replication of viruses in animal HRC. Table 1 lists the viruses which either have been proven to multiply or have failed to replicate in HRC from different animal species and donor sites. There is evidence of viral growth by mem- bers of all the major DNA and RNA viral groups researched to date. Most of the data listed in Table 1 has been gleaned from in vitro HRC culture systems wherein the experimenter could easily quantitate viral replication. Usually, timed- interval samples taken during the course of the virus - HRC infection were assayed for virus by the plaque technique on suitable cell monolayers. Where the initial diminution in titer due to the virus-cell eclipse phase has been followed by a significant rise in titer, one has strong positive evidence for viral replication. Other methods widely used for documenting viral multiplication in HRC include visual evidence of gross cytopathic effect (ePE) and fluorescent antibody detection. The former technique involves a direct 17 effect of viral replication on the HRC resulting in giant cell formation, cytoplasmic granulation and vacuolation or actual disintegration of infected cells as compared to normal, uninfected control cultures. With some viruses, these effects may be serially transmitted to fresh HRC suspensions as further proof of viral growth. Indirect or direct fluorescent labeling of infected cultures gives evidence for the presence of viral proteins within the cells and can qualitatively detect levels of multiplication which are too low for examination by more quantitative techniques. An increase in fluorescence with time is taken as proof of viral replication. The serious drawback in these latter two methods is that they fail to give precise enumerations of viral titers. Either of these effects (increase in CPE or intracellular viral proteins) cannot be taken as indisputable evidence of complete infectious virus formation. Danes and associates (33), for example, have reported that rabbit alveolar macrophage cultures infected with a dermatotropic strain of vaccinia virus displayed positive fluorescence even when no infectious virus was recoverable. Certain viral infections at high multiplicities have also been shown to cause host cell toxicity in the absence of viral growth (76, 155). Several of the virus - HRC systems listed in Table 1 exhibited no apparent cytopathic effect while a rise in viral titer was recorded. Obviously, if these experimenters had only assayed for an increase in CPE, they would have found no 18 evidence of viral replication. It is therefore wise to remain somewhat skeptical about data which purports to show positive viral multiplication through increases in CPE or immunofluorescence alone. The specific role of each leukocyte type in viral infections has only been examined during recent years. On the basis of differential adherence to and elution from siliconized glass bead columns (142), vfueelock (182) separated human lymphocytes and polymorphonuclear leukocytes into 95-99% homogeneous suspensions. No significant degree of Newcastle disease virus (NDV) replication could be demonstrated in either pure cell culture, although substantial viral growth occurred in mixed leukocyte cultures. Further studies (44, 185) showed that peripheral blood monocytes were able to support the replication of vesicular stomatitis (VSV) and 17 D yellow fever viruses. There is no conclusive evidence that viruses multiply in polymorphonuclear leukocytes, although numerous reports have cited the presence of viral antigens and possibly whole viral particles in blood neutrophils (reviewed in 159). Since these examinations employed immunofluorescent labelling, one must remain skeptical as to the relevance of these findings to viral replication. Many of the virus - HRC systems described in Table 1 utilized cultures pre-treated with mitogens such as phytohemagglutinin (PHA), an extract of the kidney bean Phaseolus vulgaris. PHA was originally used to separate 19 erythrocytes from leukocytes of the blood (129). In 1960 this substance was discovered by Nowell (133) to be a nonspecific mitogenic stimulant which could induce many diverse responses in small lymphocytes in vitro (129). PHA-stimu- lated lymphocytes form a new basophilic, pyroninophilic cytoplasm along with nuclear changes from strongly heterochromatic to euchromatic type. Uptake of tritiated thymidine into DNA is measurable after several days in culture, whereas tritiated uridine incorporation into cellular RNA can be followed at an earlier time. During this period PHA converts the small lymphocytes into large blastoid cells which may divide beginning two days after the start of culturing. Similar in vitro responses have been elicited by specific stimulants consisting of antigens of various kinds, cultivated with cells derived from individuals known to be sensitized to these antigens (21). In addition, many lymphoid cell lines derived from diseased and normal patients' peripheral blood have been established in permanent suspension cultures (121, 122). They grow as free-floating pleomorphic forms which resemble the immature Ublast-like tl transformed cells seen after PHA or specific antigen stimulation. Nucleic acid hybridization studies have revealed that all such lymphoid cell lines, irrespective of origin, contain Epstein-Barr virus (132, 192). The mechanisms of enhanced viral replication in PHAstimulated human leukocytes have been thoroughly examined by Willems and colleagues (186-188). These authors found 20 that viral replication in PHA-treated lymphocytes was limited primarily to viruses which replicated in the cell cytoplasm (186). Poliovirus (187) and vaccinia virus (113) replication in peripheral blood leukocyte cultures was enhanced by PHAstimulation through an increase in the number of cells producing virus. Viral yield was approximately the same per producing cell, both in treated and nontreated cultures. By examining each step of viral infection separately, Willems and associates (187) discovered that stimulated lymphocytes adsorbed poliovirus more efficiently. Over 50% of the virus penetrating unstimulated lymphocytes resulted in abortive infections, whereas almost 100% correlation between penetration and replication took place in stimulated cells. It was suggested that the formation of new ribosomal RNA, known to follow PHA stimulation (31), allowed more efficient viral replication. These data suggest interesting possibilities as to the role of lymphoblastic cells during in vivo viral infection. Numerous studies have shown that initially many lymphoid cells are in immature forms during embryogenesis (158). Although little experimental evidence is available, it is not unreasonable to assume that lymphocytopoiesis could contribute to enhancement of viral infections of the fetal host. It has also been noted that due either to antigenic stimuli or during an essentially reparative, restocking process, small lymphocytes in spleen and mesenteric lymph nodes of adults yield blastoid cells prior to the cell 21 division (71). In the adult animal, viral infections of the lymphoid tissues could lead to enhanced viral replication by growth of the virus in the highly susceptible lymphoblastoid cells. An interesting finding by Wallace (180) is that during seven months of continuous culture of human lymphoblasts, concomitant poliovirus type 1 replication and cell multiplication occurred without recognizable cell destruction by virus. Human adenovirus type 5, rhinovirus type 2, reo- virus type 1, and mumps virus also were able to multiply in carrier cultures for long periods of time. It is con- ceivable that factors maintaining and controlling persistent viral infections in these lymphoblastic cells could be of importance in latent viral infections of the intact host. In this regard, Wheelock and Edelman (183) have suggested that 17 D yellow fever virus induces a state of longlasting immunity in man by remaining in a slowly replicating or non-replicating latent state in the lymphocytes. Once these infected cells are stimulated by any of a variety of factors, including viral antigens, the virus may then propagate to high titers within transformed lymphocytes but be confined to the lymphatics by Circulating antibody. Repeated cycles of viral replication could then serve as antigenic boosters for long term virus-specific antibody production. In summary, viral agents from all of the major DNA and 22 RNA virus groups have been proven to replicate in HRC in vitro. Some viruses multiply well in unstimulated HRC, others display enhanced growth in stimulated cells, while many viruses require stimulated, immature lymphocytes in order to multiply at all. It is still a matter of specu- lation, however, whether any of these concepts are applicable to in vivo situation. INTERFERON RESPONSE OF HRC In 1957 the antiviral substance, interferon, was discovered by Isaacs and Lindenmann (87). The interferon system has since been divided into several components (77) which will briefly be reviewed here. Interferon is a pro- tein or proteins which are produced or released by cells following viral infection and certain nonviral stimuli. It is not in itself antiviral, but rather reacts with cells to induce the formation of a new intracellular polypeptide or protein which mediates the antiviral activity. Included among interferon's more important properties are a relative inactivity in cells of heterologous species and a strikingly poor antigenicity. Evidence suggests that the biological significance of interferon lies in its importance to the body's nonimmune defenses, which are probably the major causes of recovery from already established viral infections (10). In contrast, the immune defenses may not be essen- tial for recovery but they function to limit viremic spread during primary infection and to prevent reinfection. The 23 interferon system can also serve to control viral spread through the bloodstream by acting either at the local site of infection or in target organs, and has been shown to be operative in mammals, birds, reptiles, fish, and possibly in plants (10). In reviewing the relatively massive amount of data on in vitro interferon production by leukocytes, one must first consider whether it has importance to the in vivo situation. Certainly the RES is the most prominent source of interferon for many inducing substances in the whole animal. Studies of explanted or homogenized spleen and liver tissue have revealed these organs to be primary factories for interferon after in~ and in vitro viral infection (57, 99, 168, 178). Splenectomized mice showed significantly depressed serum interferon levels after injection of virus (57, 168), as did mice whose RES had been blockaded with various agents (22, 99). Antilymphocyte serum (ALS) pretreatment reduced serum interferon titers after virus infection (12, 78), depending upon the specific inducer used (36). Others have shown, however, that ALS treatment had no effect on interferon production (67). The strongest evidence that peripheral blood leukocytes are primary contributors to interferon synthesis been presented by the De Maeyer and associates. n vivo has Production of circulating interferon upon stimulation with Sindbis virus or NDV was markedly depressed in C3H mice following total-body X-irradiation (93). This observation has been 24 confirmed by Glasgow (67). Inhibition of interferon synthe- sis was ascribed to an impairment of hematopoietic function and was reversed by grafting syngeneic or even xenogeneic bone marrow cells into lethally irradiated recipients (35, 37). They found, furthermore, that the circulating inter- feron response to myxoviruses and paramyxoviruses was strongly reduced by X-ray or ALS treatment, while under similar conditions the interferon response remained unaltered to encephalomyocarditis or vaccinia virus and was of intermediate radio-sensitivity to VSV and Semliki Forest (SFV) virus (36, 37). Finally, the only in vitro system which consistently reflected the in vivo situation was NDV infection of whole blood suspensions (34). These results clearly indicate that induction of interferon after i.v. myxovirus or paramyxovirus injection of mice is principally occurring in the radiosensitive, circulating lymphocytes. Other authors have argued that blood leukocytes of the mouse are not significant participants in interferon production during myxovirus and paramyxovirus infection (67). Subrahrnanyan and Xtims (169) failed to detect interferon in whole heparinized blood extracted at timed intervals from i.v. influenza virus-infected mice. Although no supporting evidence was presented, Hellman and associates (82) have stated that combined X-irradiation, splenectomy, and RES blockade did not influence the LDSO or circulating interferon response in mice induced by intranasal influenza virus inoculation. These authors did not consider that 25 pulmonary phagocytes resistant to these treatments may have been the key host factors during the infection. Nagano and colleagues (127) examined X-irradiated mice and reported that a greater than 90% decrease in peripheral blood leukocyte count was accompanied by 40-80% lower level of NDVinduced, and a 1-75% drop in endotoxin-induced, serum interferon response. They concluded that a lack of parallelism existed between the fall in leukocyte count and production of circulating interferon, and therefore blood leukocytes did not synthesize interferon. From their data it would appear an opposite interpretation could be made. Interferon Response Of HRC To RNA Viruses Picornaviruses. Gresser and Chany (75) incubated dilu- tions of interferon induced by Sendai virus in human leulcocytes wi th peripheral blood leukocyte preparations derived from healthy donors. The results suggested that human white cells were markedly sensitive to the antiviral action of interferon since a SOO-fold dilution of interferon significantly reduced poliovirus replication in these cells. Poliovirus-induced interferon production was not determined in this study. ;,lyxoviruses. ~:i2' Various strains of influenza A, influenza s.nd influenza B viruses failed to induce more than minimal amounts of interferon in human peripheral blood suspensions by 24 hr post-infection (164). Subrahmanyan and r':ims (164) supported these findings by failing to 26 demonstrate detectable interferon production in influenza A (PR 8 strain)-infected heparinized mouse blood after eight hr incubation. These latter authors may not have observed long enough for interferon production, as optimal interferon yields appear to occur by 24 hr post-infection in adult leukocyte cultures following other types of viral infection (164) • In contrast to the results using blood leukocytes, influenza A (Moscow strain) induced significant titers of interferon in mouse peritoneal cells in vitro (100). These data were questioned by Subrahmanyan and Biros (170), who could not detect any interferon in peritoneal cell cultures from nonstimulated mice and mice injected with nutrient broth after 48 hr infection with influenza A (PR-8 strain) virus. The same study failed to show interferon production by the virus in unstimulated macrophages which had been separated by adherency to glass. paramyxoviruses. Viruses of the paramyxovirus group have been extensively scrutinized for their ability to induce interferon in peripheral blood leUkocytes. Parain- fluenza 1 (i.e., Sendai) virus was used by Gresser (74) in the initial report on the induction of interferon in blood leukocyte cultures. Substantial levels of interferon pro- duction ",'ere recorded 72 hr after Sendai infection of leukocyte suspensions derived from normal and diseased patients. The amount of interferon released appeared roughly proportional to the number of white cells in the cultures. 27 Lee and Ozere (106) verified Gresser's (74) observation that the level of interferon induced by Sendai virus was somewhat proportional to the number of blood leukocytes present. purified mononuclear cell cultures were also established by magnetic removal of phagocytic cells (i.e., mostly PMN cells) which had ingested iron powder. Interferon production in mixed cultures of PMN and monocytic cells was ShO\'ln to be three-fold or more enhanced than in the "pure II cultures of mononuclear cells. This represents the only documented evidence where neutrophils have been responsible for in vitro interferon production. UV-irradiated and non-irradiated Sendai virus were equally potent interferon inducers in human peripheral leukocyte suspensions (26, 164). Levels of interferon were first detectable two hr post-infection, with peak titers being reached by seven hr. Interferon production apparently ceased within 24 hr after the cultures were inoculated with the virus, and the cells became refractory to subsequent inductions as witnessed by significantly reduced yields of interferon (165). preincubating the white blood cells for 24 hr before use in these studies only slightly lowered the interferon producing cell capabilities. The Sendai virus-human leukocyte culture system has been utilized to examine the effects of serum and actinomycin D on interferon production. Hadhazy and coworkers (79) reported suspensions of human blood leukocytes began producing interferon by the same time after induction by 28 Sendai virus t-lhether the medium contained serum (inactivated calf serum) or not. In the absence of serum, the interferon titers leveled off after five hr and only reached 10% of that obtained \vhen serum was present. strander (163) found that this phenomenon was due to a critical macromolecular, age-independent, and species-nonspecific principle in serum. Leukocytes synthesized interferon de ~, since they be- came insensitive to the effects of actinomycin D \vhen it was added within a few hours after Sendai virus inoculation (79, 105). Additional studies with parainfluenza I virus have sho,\,ln that human leuKocytes derived from bone marrow and blood had approximately equivalent interferon producing ca9abilities (105). Finally, whole, heparinized mouse blood samples infected ""ith Sendai virus in vitro displayed only very 10\'1 interferon titers by four to eight hr post- inoculation (164). Parinfluenza 3 virus has elicited substantial amounts of interferon in rabbit alveolar macrophage cultures by 24 hr after in vitro infection (1). Pretreatment of rabbit lung macrophages with this interferon protected the cells against virulent rabbitpox challenge. In further studies, alveolar macrophages from rabbits inoculated in vivo with parainfluenza 3 virus were resistant to in vitro infection vlith rabbitpox virus (2), unless the animals had been exposed to air pollutants such as N0 2 before being injected with the parinfluenza 3 agent (177). 29 NDV has been employed in numerous virus-leukocyte studies during the last decade. Strander and Cantell (164, 165) found it to be the most potent inducer of interferon in human peripheral blood white cell suspensions, whether the virus had been UV-inactivated or not. NDV-induced interferon production begins between two to four hr after viral inoculation of human leukocyte cultures, and reaches maximal levels by 14 hr (182). contrary to the results of Strander and Cantell (164), vlheelock (182) demonstrated that incubating the cells for 24-48 hr totally eliminated their induced interferon producing capabilities. ~DV­ It has also been reported that leukocyte cultures from 52 human patients varied significantly in the amount of interferon produced in response to ~IDV (138). Of greater significance is that actinomycin D treatment of NDV-infected bovine leukocytes suppressed interferon production but did not enhance the growth of the virus within the same cells (98). It was speculated, therefore, that interferon produced by peripheral leukocytes could not inhibit the multiplication of the virus in white blood cells but could act against NDV in other target cells. The lymphocytes of the blood appear to be the main synthesizers of NDV-induced interferon in vitro. cultures of lymphocytes infected with ~~V Purified made significant quantities of interferon whereas "pure" polymorphonuclear cell cultures did not produce any interferon after exposure to the virus (182). De ~~eyer and his colleagues (36) 30 have shown that NOV-induced circulating mouse interferon was primarily made by very radio-sensitive, bone-marrow derived cells, most likely lymphocytes. other in vivo sources are quite possible, however, as for example peritoneal exudate cells produced substantial amounts of interferon in vitro after extraction from mice previously infected i.p. with W~V (100). Smith and Wagner (159) indirectly demonstrated that the monocytic (i.e., macrophage) portion of the peritoneal exudate from rabbits injected with glycogen was the principle manufacturer of NDVinduced interferon. Interferon yields in mixed PIvlN-mono- nuclear cell cultures seemed to be a function of the number of macrophages present. They also reported that normal, non-stimulated rabbit macrophages had a reduced capacity to synthesize interferon than did glycogen-stimulated macrophages, quite analogous to HVH-l infection of normal and proteosepeptone stimulated mouse macrophages (85). Enhanced yields of NOV-induced interferon have been achieved by incubating the virus-macrophage cultures at 39 C as compared to 37 C (102). Mouse peritoneal macrophages have also been reported to become hyporeactive to induction of interferon by NDV by 48 hr after the initial viral infection (159). Humps virus has been shown to induce appreciable amounts of interferon in PHA and non-FHA stimulated human peripheral leukocyte cultures (41, 164). Measles virus has also been described as an inducer of interferon in human 31 peripheral leukocyte cultures, although interferon levels \-lere some 3D-fold lower than those produced by Sendai virus infection (74). l~boviruses, group A. Glasgow (64) investigated the interaction of CV with peritoneal exudate cells from immune and nonimmune mice. cv- In most instances, cultures of peritoneal cells from immune animals produced two to ten times greater interferon quantities than did control cells after CiJ-infection. specific for cv, This enhanced interferon response was and humoral or cell-bound antibody played no apparent role in the reaction. rrransfer of peritoneal macrophages previously exposed to CV in vitro to mice infected with SFV or encephalomyocarditis virus protected the animals against these normally lethal challenges (65). It was postulated that interferon production initiated in vitro in the macrophages was primarily responsible for the antiviral resistance transferred in yjvo. In companion studies, interferon titers in cv- inoculated macrophage cultures were of lesser magnitude than serum interferon levels after in vivo CV infection, indicating that the situation in the animal may not be equated with the in vitro system (66). SFV induced only barely detectable levels of interferon after 48 hr exposure to unstimulated, nutrient broth stimulated, and semipurified mouse peritoneal macrophage cultures (170). Sindbis virus has also been shown to produce 10\.., levels of interferon in heparinized rabbit blood samples by 32 24 hr post-infection (99). Arboviruses, group B. The 17 D vaccine strain of yellow fever virus induced interferon in mixed cultures of human leukocytes from peripheral blood (183). studies with nearly homogeneous cultures of each leukocyte type revealed that interferon was induced mainly in monocytes, slightly in lymphocytes, and not at all in neutrophils. PHA-stimulation of lymphocytes enhanced 17 D virus production in these cells even in the presence of interferon. Treatment of leukocytes with interferon prior to inoculating them with the virus markedly inhibited viral replication. It is possible that the interferon produced by the PHA-treated leukocytes did not have enough time to act upon these cells to prevent viral multiplication. Peritoneal macrophages from mouse strains naturally resistant and susceptible to West Nile virus have been compared for their reactions with the virus in vitro (81). Although no difference in interferon production existed between macrophages of the murine strains, cells from resistant mice were three times more sensitive to the effects of interferon derived from either mouse strain. Oddly enough, this increased sensitivity to interferon was specific for group B arboviruses. It is also pertinent to note that the authors did not state which type of interferon (i.e., macrophage, serum, or brain) was utilized in their studies, since mouse macrophage interferon appears ineffective against Sendai virus infection in other 33 macrophages (85). Rhabdoviruses. VSV did not induce detectable levels of interferon in cultures of mixed human leukocytes (26, 44, 164). NDV-induced leukocytic interferon added to leukocyte cultures prior to VSV inoculation reduced viral yields, indicating the cells were sensitive to the effects interferon. VSV normally multiplies in the monocyte fraction (44) but PHA (45, 182) and ALS (46) treated lymphocytes also serve as host cells for viral replication, even in the presence of interferon induced by this agent. Togaviruses. In two separate studies it has been re- vealed that lactic dehydrogenase (LDH) virus did not induce interferon in mouse peritoneal macrophage cultures (40, 52). ~·\.s displayed in other virus-HRC systems, there ,,'las a significant inhibition of LDH virus replication in macrophage cultures treated with interferon prepared from LDH virus-infected mouse serum (52). Coronaviruses. Shif and Bang (153) investigated the relationships of mouse hepatitis virus with peritoneal macrophages retrieved from mice genetically-resistant to the virus. L~'o interferon activity was detected in either normal or virus-infected cells by 20 hr after infection. Interferon Response Of HRC To DNA Viruses Adenoviruses. Chicken blood leukocytes were good sources for interferon synthes after in vitro exposure to human adenoviruses 5, 6, 8, 12, 14, or 16 (126). Increasing the 34 cell concentration concomitantly enhanced interferon production, but preincubating the leukocytes for up to 48 hr did not alter the interferon producing capabilities, in contrast to the data of ~Vheelock (182). By eliminating serum from the growth media, it has also been shown that leukocytes could still produce substantial amounts of interferon after adenovirus 12 infection (126). Papovaviruses. A highly oncogenic strain of polyoma virus induced the production of interferon in hamster peritoneal exudate cells (171). Herpesviruses. Hirsch and colleagues (85) reported that proteosepeptone stimulated mouse peritoneal macrophages manufactured two times more interferon 24 hr after HVrI-I infection than did unstimulated cells from adult animals. No interferon \~as inducible by HVH-I in unstimulated or stimulated suckling mouse macrophage cultures. As noted previously, adult macrophage interferon proved ineffective against Sendai virus infection of other adult peritoneal macrophages in vitro. Poxviruses. Vaccinia virus initiated interferon pro- duction in mouse peritoneal exudate cells by 48-72 hr post infection (68), although negative results have been reported (170). Peritoneal exudate cells transferred intraperito- neally to mice after three hr in vitro exposure to uv- inactivated vaccinia virus enhanced the animal's resistance to intracerebral VSV challenge (68). It was speculated that vaccinia-induced interferon production by the transferred 35 cells resulted in the protection. Glasgow (63) developed another unique model to examine the role of HRC in host response to viral infections. Pri- mary mouse embryo cell monolayers were treated with either a low inoculum of vaccinia virus or peritoneal exudate cells alone. plus mouse peritoneal cells, The peritoneal exudate cells suppressed viral proliferation in the mixed cell system as witnessed by decreased titers in the supernatant fluid and by the inhibition of spread of viral ePEe Evidence that interferon \'I7as the mediating factor of this in vitro protection model included: a) a four-fold increase in inter- feron titers in the mixed cell cultures as compared with monolayer cultures with virus alone, and b) no protection in heterologous cell monolayers (i.e., mouse peritoneal cells did not inhibit vaccinia in chick embryo cells). No evidence of interferon synthesis was detected in human peripheral blood leukocytes after vaccinia virus infection (26, 164). Since these authors only observed for inter- feron during the first 24 hr post-infection, the bulk of the interferon production may not yet have taken place (145). Subrahmanyan and Mims (170) published data that described a total lack of interferon production in unstimulated and nutrient broth stimulated peritoneal macrophages, s.nd semi-purified peritoneal macrophages, following ectromelia or cowpox virus infection. Macrophages were found highly sensitive to mouse brain interferon, as demonstrated by a sharp decrease in ectromelia virus infection of cells 36 pretreated with the interferon. Immune Specific Induction of Interferon and Other Soluble Factors in HRC Cultures Several recent reports have demonstrated that interferon is directly involved in the cell-mediated immune response. Convincing evidence for this phenomenon is the observation that mixed lymphocyte cultures from mice with marked differences in histo-compatibility antigens were found to produce interferon (61). Lackovic and Borecky (101) have des- cribed a "spontaneous ll release of interferon in L-cell cultures mixed with mouse peritoneal or spleen cells from Lcell i~nunized donors. Immune specific induction of inter- feron has also been witnessed in human blood lymphocyte cultures stimulated with purified protein derivative of tuberculin (PPD), tetanus toxoid, or diphtheria toxoid (72). The induced response was specific for cells obtained from immune donors, and it was further suggested that interferon production correlated with the degree of blastogenesis in the cultures (73). Mice sensitized to Mycobacterium tuber- culosum, BeG strain, produced strikingly greater levels of serum interferon following intravenous PPD inoculation than did normal animals (161). Mouse peritoneal exudate cells (115) and human blood leukocytes (49) from tuberculinsensitized donors have also been observed to produce interferon following in vitro exposure to PPD. This process was shown to require the interaction of macrophages with lymphocytes in the cul tures. 37 It is also pertinent that continuous lymphoblastoid cell lines and mitogen-stimulated lymphocytes produce interferon (184). It is now fairly well established that such lymphoblastoid cells bear a close relationship to the large pyroninophilic "activated" lymphocytes found during the in vivo cellular immune response (21). paradoxically, lymphocyte transformation is inhibited during the course of several different viral infections (184), which conceivably could augment the particular viral disease by reducing the cell-mediated immune reaction. The value of the interferon induced during the cellular immune response may lie in its antiviral effect. Glasgow (64) originally reported that mouse peritoneal exudate cells from donors immunized to of interferon on reexposure to ev produced elevated levels ev. This response could not be correlated with enhanced ingestion of Virus, cell-bound antibody, or the presence of neutralizing antibodies. Yamada and co-workers (191) have confirmed and extended these results by showing that mouse peritoneal exudate cells derived from NOV-immune animals produced enhanced interferon titers in response to in vitro NDV infection. It was suggested that interferons induced in "immune" macrophages may have differed in molecular weight from those synthesized by "non-immune" cells (6). These authors also showed that NDV induced enhanced interferon yields in "non-immune" macrophages pretreated with anti-NDV antiserum, which may have been linked to an increased virus-to-cell adsorption 38 rate (7). Mention should be made that other investigators (170) have reportedly been unable to repeat the results of Glasgow or the latter group of authors. Epstein and associ- ates (50), however, have demonstrated a selective increase in the interferon response of lymphocytes obtained from reimmunized human donors to vaccinia virus antigen. This response was greatly augmented by the presence of macrophages in the cultures. Recent evidence suggests that interferon induced during the cellular immune response may play a more broad role in host resistance. For example, interferon has been shown to enhance the specific cytotoxicity of sensitized mouse lymphocytes for allogeneic target tumor cells (107). In- terferon may also have the ability to non-specifically enhance phagocytosis, as has been observed in mouse peritoneal macrophage cultures (86). Salvin and co-workers (148) have provided intriguing evidence that migration inhibitory factor (MIF) and interferon have very similar properties, which suggest that they may be the same molecules. Fol- lowing i. v. injection of mice ,,,ith M. tuberculosis strain BCG or old tuberculin cells, maximal serum levels of roth MIF and interferon occurred at the same time. Furthermore, the two mediators were of similar molecular weight and were both unstable at pH 2. Antigen-specific inhibition of macrophage migration has been described for vaccinia (174), fibroma (173), influ.enza (53), and LDH, lymphocytic choriomeningitis (LCN), 39 and mumps viruses (175). Oldstone and Dixon (134) have observed the immune-specific release of a cytotoxic factor similar to lymphotoxin following exposure of lymphoid cells to LCr~ virus. Evidence has recently been provided that suggests the susceptibility to recurrent HVH-I infection in humans may be due to an impaired production of MIF and lymphotoxin (189). other factors produced by lymphocytes following antigenic exposure (121), such as transfer factor, blastogenic factor, immunoglobulins, and leukotactic factors, have not been investigated for their association with viral disease. AGE-RELATED INTERACTION OF HRC WITH VIRUSES Association of viruses with HRC in congenital viral diseases of humans. Several excellent reviews have detailed the complex age-related interrelationships between viruses and animals (117, 136, 151, 154). For purposes of this review, only a brief synopsis of the literature will be presented. Age-dependent resistance to viral infections may vary according to the pathogen, pathway of infection, and animal involved (117, 154). In general, resistance is associated with limited dissemination of virus and decreased intensity of cellular damage, which may relate to decreased adrenocortical activity or inherent susceptibility of different tissues, increased fetal immunocompetence and/or interferon production, and a decline in the ability of virus to persist 40 within cells. The mammalian fetus is commonly neither in- fected nor affected following maternal viral infection, but at least ten viruses are known to produce embryonic malformations in animals (151). Rubella virus represents the hallmark infective agent of congenital disease in humans and has been extensively reviewed elsewhere (42, 150). The key to rubella virus in- fection may be at the level of virus-HRC interaction and cell-mediated immunity. Thymus-derived, long-lived small lymphocytes (i.e., T cells) are directly involved in cellmediated immunity and in the PHA-induced blastogenic response, as mentioned. As rubella virus is of low cyto- pathogenicity (42, 150) and has been recovered from the thymus (119) and blood leukocytes (110, 156) of congenitally infected infants, it is quite possible that the virus could infect and survive within these lymphocytes for long time periods (143, 156). The persistent humoral antibody res- ponse observed during infection could result from the normal death of infected cells with concomitant release of virus. This virus-cell relationship could also account for the PHAunresponsiveness of lymphocytes from congenital rubella patients (135), since rubella virus added to normal lymphocytes in vitro produces the same inhibitory effect (120). The pathogenesis of rubella disease could directly benefit in at least two ways from the virus' ability to prevent cell blastogenesis: a) rubella virus cannot re- plicate well in PHA-stimulated lymphocytes (135) or in 41 lymphoblastic cell lines (180), but can multiply in unstimulated, non-blastogenic lymphocytes (135)1 b) cell-mediated immunity against rubella antigen-containing somatic cells could be blocked by viral infection of thymus-dependent lymphocytes. Relatively small amounts of virus would be needed to impede the response of primordial lymphocytes in the first trimester fetus, whereas large quantities would be necessary to create persistent infection in adults (143). Cytomegalic inclusion disease of the newborn resembles certain features of congenital rubella (181), the pathology of which will not be belabored here. In vitro, CMV produces CPE leading to cell death, and it therefore seems unlikely to react with leukocytes in the same manner as rubella virus. CMV has been obtained from buffy coat portions of blood taken from newborn (166), adult leukemic (89), and blood donor patients (39), and from congenitally infected babies· lymphocytes (103, 110). In each study, isolations could be achieved in the presence of specific circulating antibody, suggesting that CMV was within the leukocytes. Although these results indicate the prominence of CMV-leukocyte interaction during the disease, the role of cellular immunity remains to be accurately assessed. Lymphocytes derived from infected infants (110) and actively infected adults (139) have responded normally to phytohemagglutinin. Active CI~V infections are now recognized as cornmon in immunosuppressed allograft recipients (80), however, which implies that a depression in cell immunity may lead to the enhanced disease. 42 Age-dependent interaction of HRC with viruses in experimental infections. Johnson investigated the n vivo and in vitro role of peritoneal macrophages in the spread of H~i-I encephalitis in mice (92). He discovered that peritoneal macrophage cultures from suckling mice mirrored the immature host's enhanced susceptibility to HVH-I i.p. infection. Although both adult and suckling mouse macro- phages were as easily infected with virus, adult cells did not display cytopathic effect or allow HWI-I to spread through neighboring macrophages. infection, Hirsch et ale With the same model (85) found that adult peritoneal cells in vitro produced greater amounts of interferon and nlore efficiently inhibited release of complete infectious virus than did suckling mouse macrophages. Viral compo- nents were inefficiently assembled within adult macrophages due probably to an error in viral DNA metabolism (160). Albino rats exhibit an age-related resistance to intraperitoneal vaccinia virus infection (90), which is quite Similar to the herpesvirus model. Fluorescent antibody and ALS studies (90) showed that peritoneal cells in older animals restricted the virus more effectively than did cells in young rats after intraperitoneal infection. However, peritoneal macrophages derived from all donor age groups failed to allow viral replication in vitro (91). Donor cells did show a progressive increase in migration with age in response to in vitro exposure to vaccinia Virus, but results were somewhat inconclusive (96). 43 Liver macrophage cultures developed from mouse tissue explants displayed increased resistance with age to mouse hepatitis virus (59). This system paralleled the ontogeny or resistance of C3H mice to i.p. viral infection. Specific mechanisms were not elucidated. subrahmanyan (167) has reported an age-dependent susceptibility to cowpox virus in mice. He found no age- related difference in the ease of peritoneal macrophage infection or in ability of virus to infect surrounding cells. This represents the only model system of those re- ported where in vitro macrophage susceptibility to viral infection does not mimic in vivo resistance with age. Human blood leukocytes from differing aged donors have been examined for their competence to produce interferon after viral infection. Cantell and co-workers (27) chal- lenged whole, heparinized blood samples from aborted and premature fetuses, newborns, children, and adult volunteers with Sendai virus. Supernatant fluids taken at 24 hr post- infection exhibited slightly increasing mean interferon titers with rise in donor age. When expressed as units of interferon per lymphocyte rather than units/ml, however, interferon yields appeared the same for each donor age. 'rhis may be significant if one considers the lymphocyte to be the major interferon-producing cell of the blood (182). Irhese results were confirmed by Ray (144) using Sendai virus as the inducer, but employing peripheral blood lymphocyte cultures from differing aged humans rather than whole blood. 44 Banatvala and colleagues (8) reported that interferon induction by Sendai and rubella viruses in whole, heparinized blood suspensions, and cell tissue cultures derived from 10 to 23 \"eek gestation fetuses was unrelated to fetal age. CONCLUSION In preparing a comprehensive review on virus-HRC literature, one is amazed at the enormity of publications which have emerged since the mid-60's. The persistent dilemma of the validity of contrasting and comparing in vivo with in vitro studies arises also, and one is forced to accept the inequities of the situation since no suitable alternative exists. The only experimental models of sig- nificance have utilized human blood leukocytes and mouse peritoneal exudate cells, which present obvious limitations in comparing responses of different animals species and cell types to viral infection. A few conspicuous patterns do ascend from the massive amount of literature reviewed herein. The first is that virus-leukocyte interactions may differ as to the species of virus and animal, cell type, and conditions employed. It is also apparent that mobile and fixed macrophages may hold the key to in vivo spread of viral infection, whereas blood leukocytes are generally concluded to be of lesser importance in antiviral resistance. The possible excep- tion to this may be in the age-related response of the host to certain viral infections. PHA has provided a valuable 45 integration of cell immunity, interferon response, and viral replication in lymphocytes, and possibly in deciphering the response of the various lymphocyte populations to viral infection. Finally, it appears that the importance of in- terferon in virus-ERC interactions may lie more in its antiviral capabilities in HRC and its role in cellular immunity than in the ability of viruses to induce its production. TABLS 1: VIRUS CELL SPECIES Replication of RNA-Viruses in HRC CELL TYPE VIRUS REPLICATION REFERENCES COi"'!!',1ENT Picornaviruses Poliovirus type I Human peripheral blood leukocytes 17 PHA used during cell extraction 20 Poliovirus type II & III + 75 PHA used during cell extraction + 186 Cells PHA-stimulated + 187 Cells PHA or non-PHA stimulated Peritoneal macrophages + 187 Cells PHA-stimulated Lymphoblasts + 180 Virus persisted in long-term culturesr no CPE Monkey Peritoneal exudate cells + 11 No apparent CPE Human Lymphoblasts + 180 No apparent CPE ,r::,. (J'\ TABLE 1: VIRUS CELL SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCE COlv'fi'/i.E NT Coxsackie A virus Human Lymphoblasts + 180 Echo 9 virus Human peripheral blood leukocytes + 17 PHA used during cell extraction peripheral blood lymphocytes + 19 Cells PHA-stimulated had lOX greater virus replication than nonPHA treated cells peripheral blood leukocytes + 186 Cells PHA-stimulated Lymphoblasts + 180 Virus persisted without apparent CPE Echo 11 virus Human Encephalomyocarditis virus Nouse Peritoneal exudate cells Mengovirus House Lymph node lymphocytes Extensive CPE developed 38 + 51 Cells PHA-stimulated~ no replication in non-PHA stimulated cultures ~ .....,J TABLE 1: VIRUS Rhinovirus type 2 CELL SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCES CO~~E~"T 180 High viral titers without apparent CPE 186 Cells PHA-stimulated~ neither A-PRS nor A2..."'" .Af:.. strain replicated Human Lymphoblasts Human Peripheral blood leukocytes Mouse Peritoneal exudate cells Guinea pig Peritoneal macro phages 149 Influenza A-WS strain Guinea pig Mouse Rat Peritoneal exudate cells 149 Influenza A-NWS strain Human peripheral blood leukocytes 186 Cells PHA-stimulated~ Influenza B-Lee strain Guinea pig Mouse Rat Peritoneal exudate cells 149 Influenza B-Lee strain + Myxoviruses Influenza A virus Influenza B virus 23 Influenza Al-IvlOSCOW strain ~ CD TABtZ 1: VIRUS Fowl plague virus CELL SPECIES CELL TYPE Chicken Peripheral blood monocytes Human peripheral blood leukocytes Continued VIRUS REPLICATION REFERENCE + 56 Immunofluorescent detection 74 PHA used during cell extraction COMMENT param~xoviruses parainfluenza 1 (Sendai) virus 165 Virus failed to replicate at various MOl 105 186 parainfluenza 3 virus Nouse Peritoneal macrophages Mouse Lymph node lymphocytes Human Lymphoblasts Rabbit Alveolar macrophages + 3 51 Cells PHA-stimulated No apparent CPE Cells PHA-stimulated 180 1 Only observed for repli. during 1st 9hrs post-infection~exten- sive CPE developed ~ \0 TABLE 1: VIRUS Newcastle disease virus CELL SPECIES HlL"1lan CELL TYPE Peripheral blood leukocytes Continued VIRUS REPLICATION Peripheral blood leukocytes Bovine r"1wnps virus House Peritoneal exudate cells Human Peripheral blood leukocytes Lymphoblasts COViMENT + 182 + 165 Virus only replicated when inoculated at low multiplicity 186 Cells FHA-stimulated Lymphoblasts Chicken REFERENCES 180 + 43 + 98 23 Extensive CPE noted + 41 Cells PHA-stimulated1 no replication in nonPHA treated cultures + 186 Cells PHA-stimulated + 180 No replication observed until 25 days postinfection7virus then persisted in long-term cultures U1 o _~.. 3L3 1: CELL .. \, .... ,::) V .L'-nT"""' ~ 1eas s virus Rinderpest virus SPEcr::;.:; Continuec: VIRUS CSLL irYI)~ !-Iuman ::onkey peripheral blood leukocytes Canine Rabbit Guinea pig Mouse Chicken Peripheral blood leukocytes Bovine peripheral blood leukocytes 2:P LICATI03 + RZ:;F=:RL;~'JC.sS 18 C Col'. :I'~'8 :·JT F3A used during cell virus icated in mononuclear cells extraction~ 18 + 139 PH~~ used during cell extraction Extensive CPE developed Ul I--' TABLE 1: VIRUS Canine distemper virus CELL SPECIES CELL TYPE Continued VIRUS REPLICP}.TION Peripheral blood lymphocytes + 140 Alveolar macrophages + 4 Canine Ferret Alveolar,peritoneal, spleen, and liver macrophages + 141 Human Peripheral blood leukocytes and lymphocytes 104 Ovine Peripheral blood leukocytes 145 Mouse Peritoneal exudate cells Canine COHMENT REFERENCES Virus replicated up to BOX higher in ALStreated cultures than in non-treated cells Giant cell formation occurred Used neonatal donor cells Arboviruses, 9J:'ouP A Chikungunya virus No replication in fetal or adult donor cells 66 No replication in fe145 Sernliki Peripheral blood Ovine tal, or adult donor Forest leukocytes __________________________ --________________________________________________ ________________ virus ~c~e~l~l~s~ ~ ~ TABLE 1: VIRUS Sindbis virus CELL SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCE CO~.u."1E ~""T Human peripheral blood leukocytes + 186 Cells PHA-stimulated Human Peripheral blood leukocytes + 183 Virus replicates best in monocytes1small amount of replication in lymphocytes + 185 Small amount of replication1 oc curred irregularly Arboviruses, group B Yellow fever 17 D virus ~.yest virus Nile Mouse Peripheral blood lymphocytes + 183, 185 Cells PHA-stimulated peritoneal macrophages + 70 Virus replication occurred only in cells from genetically susceptible PRI mice + 81 Virus replicated in cells from genetically resistant and susceptible C H mice 3 U1 W TABLE 1: VIRUS Vescular stomatitis virus CELL SPECI3S Human CELL TYPE Continued VIRU.3 REPLICATION REFERENCES COMMEN""T + 44 + 186 Cells PHA-stimulated + 45 Cells PHA-stimulated + 46 Cells pretreated with ALS were 100X more susceptible than controls peripheral blood myeloblasts + 13 Cells derived from patients with acute myeloid leukemia Lymphoblasts + 25, 180 Extensive CPE developed 145 Fetal and adult donor cells examined Peripheral blood leukocytes Peripheral blood lymphocytes Ovine Peripheral blood leukocytes House Peritoneal macrophages + 3 Lymph node lymphocytes + 51 Virus replicated in monocytes only No CPE observed Cells PHA-stimulated~ no replication in nonPHA stimulated cultures U1 ~ TABLE 1: VIRUS CELL SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCES CO~J.1ENT Reoviruses Reovirus type 1 Human Lymphoblasts Reovirus type 2 Mouse Lymph node lymphocytes Reovirus type 3 Human peripheral blood leukocytes Mouse Lymph node lymphocytes OVine peripheral blood leukocytes Chicken peripheral blood leukocytes + 180 No apparent CPE 51 Cells PHA-stimulated 182 Cells PHA-stimulated 51 Cells PHA-stimulated 145 Fetal and adult cells examined DiElornaviruses Bluetongue virus Leukoviruses Rous sarcoma virus + 28 U1 U1 TABLE 1: CELL VI~US SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCES CO}fJ.f~NT Arenaviruses Lymphocytic choriomeningitis virus Nouse Peritoneal macro phages + 118 Mouse Peritoneal macrophages + 52 Immunofluorescent detection7no visible CPE Togaviruses Lactic dehydrogenase virus 52 Peritoneal lymphocytes Rubella virus Human peripheral blood leukocytes + Lymphoblasts No visible CPE 112 Cells were obtained from normal adult and congenital rubella syndrome babies7virus may persist in cells, suggesting low-level replication 135 Used normal adult donor cells7no replication in PHA-stimulated cells 180 Ul 0'\ TABLE I: VIRUS CELL SPECIES Continued VIRUS REPLICATION REFERENCES CO!v1MENT Liver macrophages + 9 Peritoneal macrophages + 97 Virus replicated and induced extensive CPE in cells from genetically susceptible PRI mice but not in macrophages from genetically resistant C3H mice CE LL 'lOY P .t; Coronaviruses I~use IJIouse hepatitis virus Unclassified RNA viruses Hog cholera virus porcine Peripheral blood leukocytes + 43 Equine infectious anemia virus Equine peripheral blood leukocytes + 97 Continuous-passage leukocytes + 123 Monocytes were the probable sites of virus replication Extensive CPE noted 01 '" TABLE 1: VIRUS CELL SPECIES Replication of DNA-Viruses in HRC CELL TYPE VIRUS REPLICATION REFERENCES COl'JfJ'1ENT Adenoviruses Adenovirus type 2, type 7, and type 12 Human peripheral blood leukocytes Adenovirus type 5 Human Lymphoblasts IJIouse adenovirus f,10use Lymph node lymphocytes Human peripheral blood leukocytes Human peripheral blood leukocytes + 186 Cells PHA-stimulated 180 No apparent CPE 51 Cells PHA-stimulated 186 Cells PHA-stimulated Papovaviruses SV 40 Herpesviruses Hereesvirus hom~nis + 69, 128 type 1 186 Cells PHA-stimulated: no replication in non-PHA treated cultures Cells PHA-stimulated U'I (l) 'rABLE 1: VIRUS C.t;LL SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCES C OlViY'lE NT Herpesviruses Continued !-louse Peripheral blood lymphocytes + 24 Cells PHA-stimulated supported significantly greater virus replication than nontreated cells Lymphoblasts + 15 Nuclear irregularities prominent + 180 + 83 Virus multiplied in both EB3 and P3J cell lines + 55 Virus replication was much less in EB3 cell lines than in P3J 51 Cells PHA-stimulated 85, Virus replicated in cells from suckling but not adult mice Lymph node lymphocytes Peritoneal macro phages + 92 + 3 Extensive CPE noted No apparent CPE U1 \.0 TABLE 1: VIRUS CBLL SPECIES CELL TYPE Continued VIRUS REPLICATION REFERENCES COVl.HENT Heroesviruses Continued Herpesvirus hominis type II Varicella zoster virus 60 Virus taken up with extracellular "fragments" survived longer inside cells 160 Viral components were inefficiently assembled inside host cells 55 Cell strains differed as to ability to support viral replication Mouse peritoneal macrophages Human Lymphoblastoid Ovine Peripheral blood leukocytes 145 Human peripheral blood leukocytes 69 + No replication in fetal, newborn, or adult donor cells No replication in PHA or non-PHA treated cells 0'1 o TABLE 1: VIRUS Cytomegalovirus Epstein-Barr virus CELL SPECIES C~LL TYPE Continued VIRUS REPLICATION REFERENCES C Oi1,.1V!E.NT Human Peripheral blood leukocytes Mouse Peritoneal macrophages + 172 Human Lyrnphoblasts + 124 Quantitated by microtiter assay of leukocyte transformation Human peripheral blood leukocytes + 113 Cells PHA-stimulated~ no replication in non-PHA treated cultures 186 Cells PHA-stimulated Virus perSisted in cultures without apparent CPE 69 Virus failed to replicate in either PHA or non-PHA stimulated cells Poxviruses Vaccinia Rabbit Lymphoblasts + 180 peripheral blood leukocytes + 54 Virus persisted1replication occurred in mononuclear cells (j\ ~ TABLE 1: VIRUS CELL SPECIES poxviruses CELL TYPE Alveolar macrophages Continued VIRUS REPLICATION REFERENCE + 16 Neurotropic virus strain 33 Dermatotropic virus COII"uvJE NT Continued strain~immunofluores- cent studies were positive even when no infectious virus was recoverable + 174 Virus replicated in cells from either immune or non-immune donors Peritoneal macrophages + 174 Virus did not replicate in cells from immune donors Rabbit Liver macrophage + 14 Mouse Peritoneal exudate cells Rat 68 Peritoneal macrophages 131 Peritoneal macrophages 91 Cells taken from newborn, young, and adult donors O"t N T..~BLE 1: VIRUS Poxviruses CELL SPECIES CELL TYPE Continued VIRUS REPLICA'rION Ovine Peripheral blood leukocytes 1'lOuse Peritoneal exudate cells + REP£RENCE 145 No replication in fetal or adult donor cells 146 Immunofluorescent replicated in cells from either immune or non - immune donors Continued Ectromelia COIv!i..:E ~7 detection~virus Rabbitpox Rabbit Alveolar macrophages + 1 IvIyxorna Rabbit Peritoneal exudate cells + 109 Fibroma Rabbit Peripheral blood monocytes + 62 peritoneal macrophages + 62 Peripheral blood leukocytes + 125 Unclassified DNA viruses African swine fever virus Porcine (j\ w LITERATURE CITED 1. Acton, J.D., and Q.N. Myrvik. Production of in- 1966. terferon by alveolar macrophages. J. Bacteriol. 91: 2300-2304. 2. Acton, J.D., and Q.N. Myrvik. Resistance of 1968. alveolar monocytes to rabbitpox virus following intratracheal injection of parainfluenza 3 virus. J. Reti- culoendothel. Soc. 5:68-78. 3. Allison, A.C. 1970. On the role of macrophages in some pathological processes, p. 422-440. In R. van Furth (ed.), l'lononuclear phagocytes. Davis, Philadel- F.l\. phia. 4. Appel, M.J.G., and O.R. Jones. 1967. Use of alveolar macrophages for cultivation of canine distemper virus. 5. Proc. Soc. Exp. BioI. Med. 126:571-574. :l.une, J.l-1., and N. J. 'vvade. leukocytes. I. 1970. Physiology of aging Phagocytosis and transformation. proc. Soc. Exp. BioI. Med. 134:421-426. 6. .:\zuma, M., £-1. Yamada, and R. Nishioka. 1970. Relation- ship between immunity and interferon production in macrophages. II. Nolecular weights of interferon produced in macrophage cultures. 14:319-323. Japa~J. Microbiol. 65 7. Azuma, M., M. Yamada, and R. Nishioka. 1970. Relation- ship between immunity and interferon production in macrophages. III. Enhancement of interferon produc- tion by antiserum. 8. Japan. J. Microbiol. 14:325-331. Banatvala, J.E., J.E. potter, and J.M. Best. 1971. In- terferon response to Sendai and rubella viruses in human foetal cultures, leucocytes and placental cultures. 9. J. Gen. Virol. 13:193-201. Bang, PO. B., and A. '/larwick. 1960. Mouse macrophages as host cells for the mouse hepatitis virus and the genetiC basis of their susceptibility. Proc. Nat. Acad. Sci. USA 46:1065-1075. 10. Baron, S. 1970. The biological significance of the interferon system. 11. Barski, G. 1957. Arch. Intern. r~ed. 126 :84-93. Multiplication of poliovirus in reticuloendothelial cells without generalized cytopathogeniC effect. 12. Science 125:448. Barth, R.F., R_M. Friedman, and R.A. Malmgren. 1969. Depression of interferon production in mice after treatment with antilymphocyte serum. 13. Baumberger, U. 1970. Lancet 2:723-724. Growth of vesicular stomatitis virus in human myeloblasts. Pathol. Microbiol. 36: 161-172. 14. Beard, J.W., and P. Rous. 1938. The fate of vaccinia virus on cultivation in vitro with Kupffer cells (reticuloendothelial cells). J. Exp. riled. 67 :883-910. 66 15. Bedoya, V., A.S. Rabson, and P.M. Grimley. 1968. Growth in vitro of herpes simplex virus in human lymphoma cell lines. 16. J. Nat. Cancer Inst. 41:635-652. Benda, R., L. Danes, and J. Cinat1. 1970. Infection of rabbit alveolar macrophages with a neurotropic vaccinia virus strain in vitro. Acta Virol. 14:353- 361. 17. Berg, R. B. 1961. r1ul tiplication of ECHO 9 virus in suspensions of human leucocytes. Proc. Soc. Exp. BioI. Med. 108:742-744. 18. Berg, R.B., and M.S. Rosenthal. m~asles Propagation of virus in suspensions of human and monkey leu- cocytes. 19. 1961. proc. Soc. Exp. BioI. !v1ed. 106:581-585. Berkovich, S., E.M. Smithwick, and M. Steiner. 1969. Effects of echo 9 virus infection on tuberculin sensitivity. 20. AIDer. Rev. Resp. Dis. 100:640-644. Berman, L., and C.S. Stulberg. 1962. primary cul- tures of macrophages from normal human peripheral blood. 21. Lab. Invest. 11:1322-1331. Bloom, B.R. 1971. In vitro approaches to the mechan- ism of cell-mediated immune reactions. Advan. Immunol. 13:101-208. 22. Borden, E.C., F .A. Murphy, and G.~v. Gary. 1970. At- tempted suppression of the interferon response in the mouse. Proc. Soc. Exp. BioI. Med. 134:865-869. 67 23. Borecky, L., and V. Lackovic. 1964. The reticulo- endothelial system and virus infection. I. The re- action of mouse peritoneal mononucleate cells to infection with the mouse and egg line of influenza virus. Persistence and dissemination of the virus in the mouse organism. 24. Acta Virol. 8:208-216. Bouroncle, B.A., K.P. Clausen, and E.M. Darner. 1970. Replication of herpes simplex virus in cultures of phytohemagglutinin-stimulated human lymphocytes. J. Nat. Cancer Inst. 44:1065-1078. 25. Cantell, K. 1970. Attempts to prepare interferon in continuous cultures of human leukocytes. Ann. N. Y. Acad. Sci. 173:160-168. 26. Cantell, K., H. Strander, G.Y. Hadhazy, and H.R. Nevanlinna. 1968. How much interferon can be pre- pared in human leukocyte suspensions, p. 223-232. G. Rita (ed.), The interferons. In Academic Press, New York. 27. Cantell, K., H. Strander, L. Saxen, and B. Meyer. 1968. Interferon response of human leukocytes during intrauterine and postnatal life. 28. Carrel, A. 1925. ignant cell. 29. Cohn, A. Z. p. 50-58. cytes. J. Immunol. 100:1304-1309. Essential characteristics of a mal- J. Amer. Med. Ass. 84:157-158. 1970. Lysosomes in mononuclear phagocytes, In R. van Furth (ed.), Mononuclear phago- F. A. Davis, Philadelphia. 68 30. Cohn, Z.A., and B. Benson. 1965. of mononuclear phagocytes. 31. Cooper, R.L., and A.D. Rubin. The differentiation J. Exp. Ned. 121:153-170. 1965. RNA metabolism in lymphocytes stimulated by phytohemagglutinin: Blood 25:1014-1027. responses to phytohemagglutinin. 32. initial 1971. Craddock, C.G., R. Longmire, and R. !·1cHillan. N. Engl. J. Lymphocytes and the irrunune response. Med. 285:324-331, 378-384. 33. Danes, L., R. Benda, and V. Kliment. 1970. Infection of rabbit alveolar macrophages with a dermatotropic vaccinia virus strain in vitro. Acta Virol. 14:363- 368. 34. DeHaeyer, E., and J. DeMaeyer-Guignard. 1970. A gene with quantitative effect on circulating interferon induction--further studies. l~nn. N. Y. Acad. Sci. 173:228-238. 35. De!"1aeyer, E., P. Jullien, and J. Delvlaeyer-Guignard. 1967. II. Interferon synthesis in x-irradiated animals. Restoration by bone marrow transplantation of circulating interferon production in lethally irradiated mice. 36. Int. J. Radiat. BioI. 13:417-431. Delvlaeyer-Guignard, J., and E. DeHaeyer. 1971. Effect of antilymphocytic serum on circulating interferon in mice as a function of the inducer. 229:212-214. Nature, New BioI. 69 37. DeHaeyer-Guignard, J., E. DeMaeyer, and P. Jullien. 1969. IV. Interferon synthesis in x-irradiated animals. Donor-type serum interferons in rat-to-rnouse radiation chimeras injected with Newcastle disease virus. 38. Proc. Nat. Acad. Sci. USA 63:732-739. Dickinson, L., and A.J. Griffiths. 1966. The patho- genesis of experimental infections with encephalomyocarditis (EMC) virus. Brit. J. Exp. Pathol. 47:35- 44. 39. Diosi, P., E. Noldovan, and N. Tomescu. 1969. cytomegalovirus infection in blood donors. Latent Brit. IV:ed. J. 4:660-662. 40. DuBuy, H.G., and M.L. Johnson. 1966. Studies on the in vitro and in vivo multiplication of the LDH virus of mice. 41. J. Exp. Med. 123:985-998. Due-Nguyen, H., and V~. Henle. 1966. Replication of mumps virus in human leukocyte cultures. J. Bacteriol. 92:258-265. 42. Dudgeon, J.A. 1969. sis and immunology. 43. Congenital rubella. pathogene- Amer. J. Dis. Child. 118: 35-t~. 4. Dunne, H.:,{j., A.J. Luedke, and J.F. Hokanson. 1958. The gro'idth of animal leukocytes and their use in the cultivation of animal viruses. 19:707-711. Aroer. J. Vet. Res. 70 44. ::;delman, R., and E.F. \l'neelock. 1967. Specific role of each human leukocyte type in viral infections. I. Monocyte as host cell for vesicular stomatitis virus replication in vitro. 45. Edelman, R. t and E.F. J. Virol. 1:1139-1149. ~'fueelock. 1968. Specific role of each human leukocyte type in viral infections. II. Phytohemagglutinin-treated lymphocytes as host cells for vesicular stomatitis virus replication in vitro. 46. J. Virol. 2:440-448. Edelman, R., and E.F. ~vl1eelock. 1968. Enhancement of replication of vesicular stomatitis virus in human lymphocyte cultures treated with heterologous antilymphocyte serum. 47. Elves, N. ~'J. 1966. Lancet 1:771-775. The lymphocytes. Lloyd-Luke, London. 48. Epstein, L.B., M.J. Cline, and T.C. Merigan. 1971. The interaction of human macrophages and lymphocytes in the phytohemagglutinin-stimulated production of interferon. 49. J. Clin. Invest. 50:744-753. Epstein, L.B., M.J. Cline, and T.e. Merigan. PPD-stimulated interferon: 1971. in vitro macrophage- lymphocyte interaction in the production of a mediator of cellular immunity. 50. Cell. Immunol. 2:602-613. Epstein, L.B., D.A. Stevens, and T.C. Merigan. 1972. Selective increase in lymphocyte interferon response to vaccinia antigen after revaccination. Acad. Sci. USA 69:2632-2636. Proc. Nat. 71 51. Eustatia, J .X·1. I and J. Van Der Veen. 1971. Viral re- plication in cultures of phytohemagglutinin-treated mouse lymphocytes. Proc. Soc. Exp. BioI. Hed. 137:424- 428. 52. Evans, R. 1970. Further studies on the replication of the lactate dehydrogenase-elevating virus (LDB virus) in mouse peritoneal macrophage cultures. Froc. Soc. Exp. BioI. Med. 133:831-836. 53. Feinstone , S.M., E.H. Beachey, and M.vl. Rytel. 1969. Induction of delayed hypersensitivity to influenza and mumps viruses in mice. 54. J. Immunol. 103:844-849. Florman, A.J. t and J.F. Enders. 1942. The effect of homologous antiserum and complement on the multiplication of vaccinia virus in roller-tube cultures of blood mononuclear cells. 55. Floyd, R. , R. Glasser, V. Vonka, and M. Benyesh-Melnick. 1971. Studies on the growth of herpes simplex virus in lymphoblastoid cells. 56. J. Immunol. 43:159-174. E'ranklin, R.N. 1958. Acta Virol. 15:133-142. The growth of fowl plague virus in tissue cultures of chicken macrophages and giant cells. 57. Virology 6:81-95. Fruitstone, 11.J., B.S. Michaels, D.A.C. Rudloff, and M.M. Sigel. 1966. production in mice. 1008-1011. Role of the spleen in interferon Proc. Soc. Exp. BioI. Hed. 122: 72 58. van Furth, R., Z.A. Cohn, J.G. Hirsch, J.H. Humphrey, ~v.G. Spector, and H.L. Langevoort. nuclear phagocyte system: 1972. The mono- a new classification of macrophages, monocytes, and their precursor cells. Bull. W.H.O. 46:845-852. 59. Gallily, R., A. Warwick, and F.B. Bang. 1967. onto- geny of macrophage resistance to mouse hepatitis in vivo and in vitro. 60. J. Exp. Med. 125:537-548. Garabedian, G.A., and L.V. scott. 1970. Quantitative estimation of phagocytosed herpes simplex virus in mouse peritoneal cells. Arch. Gesamte Virusforsch. 32:59-65. 61. Gifford, G.E., A. Tibor, and D.L. Peavy. 1971. Inter- feron production in mixed lymphocyte cultures. Infec. Immun. 3:164-166. 62. Ginder, D.R. tion. Resistance to fibroma virus infec- The role of immune leukocytes and immune mac- rophages. 63. 1955. J. Exp. Med. 101:43-58. Glasgow, L.A. 1965. Leukocytes and interferon in the host response to viral infections. I. House leuko- cytes and leukocyte-produced interferon in vaccinia virus infection in vitro. 64. Glasgow, L.A. 1966. J. Exp. 1'1ed. 121 :1001-1018. Leukocytes and interferon in the host response to viral infections. II. Enhanced interferon response of leukocytes from immune animals. J. Bacterial. 91:2185-2191. 73 65. Glasgow, L.A. macrophages: 1970. Transfer of interferon-producing new approach to viral chemotherapy. Science 170:854-856. 66. Glasgow, L.A. 1970. Interferon production by macro- phages in vivo, p. 315-324. In C. Chany (ed.), Pro- ceedings of the international symposium on interferon, 2nd. Inst. de 1a Sante de la Recherche Medica1e, France. 67. Glasgow, L.A. 1971. Immunosuppression, interferon, and viral infections. 68. Fed. Proc. 30:1846-1951. Glasgow, L.A., and K. Habel. 1963. Interferon pro- duction by mouse leukocytes in vitro and in vivo. J. Exp. Med. 117:149-160. 69. Gonczol, E., E. Jeney, L. Gergely, and L. Vaczi. 1969. Die vermehrung der viren der herpes-gruppe in humaner uber1ebender 1eukozytenku1tur. Zb1. Bakt., I. Abt. Orig. 210:289-297. 70. Goodman, G.T., and H. Koprowski. 1962. Macrophages as a cellular expression of inherited natural resistance. 71. Proc. Nat. Acad. Sci. USA 48:160-165. Gowans, J.L. 1962. The fate of parental strain small lymphocytes in F1 hybrid rats$ Ann. N. Y. Acad. Sci. 99:432-455. 72. Green, J.A., S.R. Cooperband, and S. Kibrick. 1969. Immune specific induction of interferon production in cultures of human blood lymphocytes. 1417. Science 164:1415- 74 73. Green, J.A., S.R. Cooperband, L.F. Kleinman, and S. Kibricl<. 1970. Immune stimulation of interferon in human leukocyte cultures by non-viral antigens. Ann. N.Y. l\cad. Sci. 173:736-741. 74. Gresser, I. 1961. Production of interferon by sus- pensions of human leukocytes. Froc. Soc. Exp. Biol. Med. 108:799-803. 75. Gresser, I., and C. Chany. 1964. Multiplication of poliovirus type I in preparations of human leukocytes and its inhibition by interferon. J. Immunol. 92: 889-895. 76. Gresser, I., and D.J. Lang. 1966. between viruses and leucocytes. Relationships Progr. Ned. Virol. 8:62-130. 77. Grossberg, S.E. ducers: N. 78. 1972. The interferons and their in- molecular and therapeutic considerations. Eng. J. Med. 287:13-19, 79-85, 122-128. Haahr, S. 1969. The effect of antilymphocyte serum on viraemia and serum interferon of mice infected with 'dest Nile virus. Acta. pathol. Microbiol. Scand. 77:167-168. 79. Hadhazy, Gy., H. Strander, and K. Cantell. 1969. Serum requirement for interferon production by suspended human leucocytes: Studies on action of serum. J. Gen. Virol. 5:351-358. 80. Hanshaw, J.B. etion: 1971. Congenital cytomegalovirus in- a fifteen year perspective. 123:555-561. J. Infec. Dis. 75 81. Hanson, B., H. Koprowski, S. Baron, and C.E. Buckler. 1969. Interferon-mediated natural resistance of mice to arbo B virus infection. 82. Hellman, Microbios IB:5l-68. A., D.H. Iv1artin, and L.J. Wopschall. 1968. The influence of x-irradiation on survival and interferon levels in viral infected mice. Froc. Soc. Exp. BioI. Hed. 128 :455-458. 83. Henle, W., G. Henle, and H. ZurHausen. 1969. Effect of herpes simplex virus on cultured BurJ<.itt tumor cells and its failure to influence the Epstein-Barr virus carrier state. 84. Cancer Res. 29:489-494. Hirsch, J.G., and M.E. Fedorko. 1970. Morphology of mouse mononuclear phagocytes, p. 7-28. Furth (ed.), Hononuclear phagocytes. In R. van F.A. Davis, Philadelphia. 85. Hirsch, B.S., B. Zisman, and A.C. Allison. 1970. l'1acrophages and age-dependent resistance to herpes simplex virus in mice. 86. J. Immunol. 104:1160-1165. Huang, K-Y., R.M. Donahoe, F.B. Gordon, and H.R. Dressler. 1971. Enhancement of phagocytosis by interferon-containing preparations. Infec. Immun. 4:581-588. 87. Isaacs, A., and J. Lindenmann. ference. I. 147:258-267. The interferon. 1957. Virus inter- Froc. Roy. Soc. B. 76 88. Jack, I., and J. Grutzner. 1969. Cellular viraemia in babies infected with rubella virus before birth. Brit. Med. J. 1:289-292. 89. Jack, I., H. Todd, and E.K. Turner. 1968. Isolation of human cytomegalovirus from the circulating leukocytes of a leukaemic patient. Med. J. Aust. 1:210- 213. 90. Jandasek, L. 1970. Influence of anti-leucocyte serum on intraperitoneal vaccinia virus infection in rats. Acta Virol. 14:467-473. 91. Jandasek, L., and M. votava. 1970. Sensitivity of rat macrophages to the vaccinia virus. Z. Immun.-Forsch. 139:439-444. 92. Johnson, R.T. encephalitis. 1964. II. The pathogenesis of herpes virus A cellular basis for the deve- lopment of resistance with age. J. Exp. Med. 120:359- 374. 93. Jullien, P., and E. DeNaeyer. 1966. in x-irradiated animals. I. Interferon synthesis Depression of circulat- ing interferon in C3H mice after whole-body irradiation. 94. Int. J. Radiat. Biol. 11:567-576. Kato, N., and H.J. Eggers. terferon synthesis: 1969. Depressors of in- inhibition of interferon syn- thesis by chick allantoic mucopolysaccharide, capsular polysaccharide of Klebsiella pneumoniae and heparin. J. Gen. Viral. 5:369-377. 77 95. Kitchen, A.G., and R. Megirian. 1971. Heparin en- hancement of Kupffer cell phagocytosis in vitro. J". 96. Reticuloendothel. Soc. 9:13-22. Klhufek, J., M. Blatna, and L. Jandasek. 1970. ~f­ fects of vaccinia virus on the migration of rat peritoneal cells. Zbl. Bakt., I. Abt. Orig. 214:9- 12. 97. Kobayashi, K., and Y. Kono. 1967. propagation and titration of equine infectious anemia virus in horse leukocyte culture. Nat. Inst. Anim. Health Quart. (Tokyo) 7:8-20. 98. Kono, Y. 1967. Rapid production of interferon in bovine leucocyte cultures. Proc. Soc. Exp. Biol. Ned. 124:155-160. 99. Kono, Y., and M. Ho. 1965. The role of the reticulo- endothelial system in interferon formation in the rabbit. 100. Virology 25:162-166. Lackovic, V., and L. Borecky. 1965. thelia1 system and virus infection. The reticuloendoII. Production of interferon and antibody-like substances in mouse peritoneal cells infected with myxoviruses in vivo. Arch. Gesamte Virusforsch. 17:619-630. 101. Lackovic, V., and L. Borecky. 1970. Release of an interferon-like virus inhibitor during contact of mouse leukocytes with target cells. 178. Acta Virol. 14: 78 102. Lackovic, V., L. Borecky, D. Sikl, L. Mosler, and S. Bauer. 1967. The temperature requirement for inter- feron production in cells stimulated by Newcastle disease virus or microbial agents in vitro. Acta Virol. 11:500-505. 103. Lang, L.J., and B. Noren. 1968. lowing congenital infection. 104. Cytomegalovirus fol- J. Pediat. 73:812-819. Larke, R.P.B., and E.F. Wheelock. 1970. Stabiliza- tion of Chikungunya virus infectivity by human blood platelets. 105. Lee, S.H.S. J. Iniec. Dis. 122:523-531. 1969. Interferon production by human leuKocytes in vitro: some biological characteristics. Appl. Microbiol. 18:731-735. 106. Lee, S.H.S., R.L. Ozere, and C.E. van Rooyen. 1966. Interferon production by human leucocytes in vitro. Reduced levels in lymphatic leukemia. Proc. Soc. Exp. BioI. Med. 122:32-39. 107. Lindahl, P., P. Leary, and I. Gresser. 1972. En- hancement by interferon of the specific cytotoxicity of sensitized lymphocytes. Proc. Nat. Acad. Sci. USA 69:721-725. 108. Ling, N.R. Holland, 109. Maral, R. 1968. Lymphocyte stimulation. North- ~~sterdam. 1957. Etude de developpement du virus de la myxomatose en cultures de tissus. pasteur 92:742-751. Ann. Inst. 79 110. W.C., W.A. Cope, J.F. Soothill, and J.A. ~~rshall, Dudgeon. 1970. In vitro lymphocyte response in some immunity deficiency diseases and in intrauterine virus infections. Ill. Proc. Roy. Soc. Med. 63:351-354. MCLaughlin, J.F., N.H. Ruddle, and B.H. Waksman. 1972. Relationship between activation of peritoneal cells and their cytopathogenicity. J. Reticuloendothel. Soc. 12.293-304. 112. Mellman, W.!>1., S.A. Plotkin, P.S. Moorhead, and E.H. Hartnett. cytes. 113. 1965. Rubella infection of human leuko- Amer. J. Dis. Child. 110:473-476. Miller, G., and J.F. Enders. Vaccinia virus 1968. replication and cytopathic effect in cultures of phytohemagglutinin-treated human peripheral blood leukocytes. 114. J. Virol. 2:787-792. lvliller, J.F.A.P., and G.F. Mitchell. and antigen reactive cells. 1969. Thymus Transplant. Rev. 1:3- 42. 115. r1ilstone, L.N., and B.H. Vlaksman. 1970. Release of virus inhibitor from tuberculin-sensitized peritoneal cells stimulated by antigen. J. Immunol. 105: 1068-1071. 116. Mirns, C.~. 1964. virus diseases. 117. Mirns, C.A. the fetus. 1968. Aspects of the pathogenesis of Bacteriol. Rev. 28:30-71. pathogenesis of viral infections of Progr. filed. Viro1. 10:194-237. 80 118. Mirns, C.!\., and T.P. Subrahrnanyan. 1966. Immuno- fluorescent study of the mechanism of resistance to J. superinfection in mice carrying the LCM virus. Pathol. Bacterio1. 91:403-415. 119. Monif, G.R.G., G.B. Avery, S.B. Korones, and J.L. 1965. Sever. Postmortem isolation of rubella virus from three children with rubella-syndrome defects. Lancet 1:723-725. 120. i ..1ontgomery, J.R., r·l. A. South, H.E. Rawls, G.B. Olson, P.B. Dent, and R.A. Good. J. L. Melnick, 1967. Viral inhibition of lymphocyte response to phytohemagglutinin. 121. Science 157:1068-1070. i"loore, G.E:., R.E. Gerner, and H.A. Franklin. Culture of normal human leukocytes. 1967. J. Aroer. Med. Ass. 199:519-524. 122. Hoore, G.E., E. Ito, K. Ulrich, and A.A. Sandberg. 1966. Culture of human leukemia cells. Cancer 19: 713-723. 123. i·loore, 1970. R.~V., H.E. Redmond, M. Katada, and M.~'lallace. Growth of the equine infectious anemia virus in a continuous passage horse leukocyte culture. lmer. J. vet. Res. 31:1569-1575. 124. Noss, D.J., and J.H. pope. 1972. Assay of the in- fectivity of Epstein-Barr virus by transforming of human 1eucocytes in vitro. 236. J. Gen. Virol. 17:233- 81 125. Moulton, J., and L. Coggins. 1968. Synthesis and cy- topathogenesis of African swine fever virus in porcine cell cultures. 126. Amer. J. vet. Res. 29:219-232. l"!usci, I., R. pusztai, I. Beladi, and M. Bakay. 1970. Production of high titers of interferon in chicken leukocyte cultures inoculated with human adenovirus type 12. 127. Acta Virol. 14:453-458. Nagano, Y., N. Maehara, and N. Nakamura. 1970. Role of circulating leukocytes in the production of virusinhibiting factor or interferon. Japan. J. Microbiol. 14:209-213. 128. Nahmias, t;.J., S. Kibrick, and R.C. Rosan. Viral leukocyte interrelationships. tion of a cultures. 129. D~~ I. 1964. I',1u1tiplica- virus-herpes simplex in human leukocyte J. Immuno1. 93:69-74. Naspitz, Ch. K., and M. Richter. 1968. The action of phytohemagglutinin in vivo and in vitro, a review. Progr. Allergy 12:1-85. 130. Nichols, ~.w. normalities. 131. 1970. Virus-induced chromosome ab- Annu. Rev. Microbial. 24:479-500. Nishmi, 1'1., and H. Niecikowski. 1963. Interaction of vaccinia virus and cells in primary and continuous culture. 132. Nature 199:1117-1118. Nonoyama, M., and J. pagano. 1971. Detection of Epstein-Barr viral genome in non-productive cells. clature, New BioI. 233:103-106. 82 133. NO~tlell, 1960. P.C. Phytohemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20:462-466. 134. Oldstone, ~B.A., and F.J. Dixon. 1970. Tissue injury in lymphocytic choriomeningitis viral infection: virus-induced immunologically specific release of a cytotoxic factor from immune lymphoid cells. Virol- ogy 42:805-813. 135. Olson, G.B., P.B. Dent, W.E. Rawls, M.A. South, J.R. I'·1ontgomery, J .L. MelnicJ<, and R .A. Good. 1968. Ab- normalities of in vitro lymphocyte responses during rubella infections. 136. J. Exp. Hed. 128:47-68. Overall, J.C., Jr., and L.A. Glasgow. 1970. infections of the fetus and newborn infant. Virus J. Pediat. 77:315-333. 137. Perkins, E.H. 1970. Digestion of antigen by perito- neal macrophages, p. 562-578. I\'!ononuclear phagocytes. 138. Pidot, l~.• L.R., G. O'Keefe, I'ilclntyre. 1972. In R. van Furth (ed.), F.A. Davis, Philadelphia. III, N. HcManus, and O.R. Human leukocyte interferon: the variation in normals and correlation with PHA transformation. 139. Proc. Soc. Exp. BioI. Med. 140:1263-1269. Pigoury, L., B. Vacher, C. Chabassol, and A. Poussot. 1967. Note sur la culture du virus bovipestique lapinise en cultures cellularies de leucocytes de boeuf. Ann. Inst. Pasteur 113:631-634. 83 140. poste, G. 1970. Increased growth of viruses in lym- phocytes treated with heterologous antilymphocyte serum. 141. Transplantation 10:106-115. Poste, G. 1971. The growth and cytopathogenicity of virulent and attenuated strains of canine distemper J. Comp. pathol. virus in dog and ferret macrophages. 81:49-54. 142. Rabinowitz, Y. 1964. Separation of lymphocytes, polymorphonuclear leukocytes and monocytes on glass columns, including tissue culture observations. Blood 73:811-828. 143. Rawls, W.E. 1968. Congenital rubella: cance of virus persistence. the signifi- Progr. Med. Virol. 10: 238-285. 144. Ray, C.G. 1970. The ontogeny of interferon produc- tion by human leukocytes. 145. Rinaldo, C.R., Jr., J.C. Overall, Jr., and L.A. Glasgow. 146. J. Pediat. 76:94-98. 1973. Roberts, J.A. In preparation. 1964. Growth of virulent and atten- uated ectromelia virus in cultured macrophages from normal and ectromelia-immune mice. J. Immunol. 92: 837-842. 147. Roser, B. 1970. The origin, kinetics, and fate of macrophage populations. 8:139-161. J. Reticuloendothel. Soc. 84 148. Salvin, S.B., J.S. Youngner, and W.H. Lederer. l~igration 1973. inhibitory factor and interferon in the cir- culation of mice with delayed hypersensitivity. In- fec. Immun. 7:68-75. 149. sawyer, v·T.D. 1969. Interaction of influenza virus J. with leukocytes and its effect on phagocytosis. Infec. Dis. 119:541-556. 150. schiff, G.M., and J.L. Sever. 1966. laboratory and clinical advances. Rubella: recent Progr. Med. Virol. 8:30-61. 151. Sever, J.L. 1971. Virus infections and malformation. Fed. Proc. 30:114-117. 152. Shif, I., and F.B. Bang. 1970. In vitro interaction of mouse hepatitis virus and macrophages from genetically resistant mice. growth curves. 153. I. Adsorption of virus and J. Exp. Med. 131:843-850. Shif, I., and F.B. Bang. 1970. In vitro interaction of mouse hepatitis virus and macrophages from genetically resistant mice. II. Biological characteri- zation of a variant virus MHV (C3H) isolated from stocks of MHV (PRI). 154. Sigel, l1.M. 1952. J. Exp. Med. 131:851-862. Influence of age on susceptibility to virus infections with particular reference to laboratory animals. 155. Silverstein, s. 1970. Annu. Rev. Microbiol. 6:247-280. Macrophages and viral immunity. Semin. Hemat. 7:185-214. 85 156. Simons, H.J. 1968. logical paradox? 157. Congenital rubella: an immuno- Lancet 2:1275-1278. Smith, T.J., and R.R. phage interferons. ~vagner. I. 1967. Rabbit macro- Conditions for biosynthesis by virus-infected and uninfected cells. J. Exp. Med. 125:559-577. 158. Solomon, J.B. 1971. Foetal and neonatal immunology. American Elsevier, New York. 159. Sommerville, R.G. 1968. Rapid diagnosis of viral in- fections by immunofluorescent staining of viral antigens in leucocytes and macrophages. Progr. Ned. Virol. 10:398-414. 160. stevens, J.G., and M.L. Cook. 1971. Restriction of herpes simplex virus by macrophages. the cell-virus interaction. 161. An analysis of J. Exp. Med. 133:19-38. Stinebring, W.R., and P.M. Absher. 1970. Production of interferon following an immune response. Ann. N. Y. Acad. Sci. 173:714-718. 162. stitch, H.F., and D.S. Yohn. chromosomes. 163. Strander, H. 1970. Viruses and Progr. Med. Virol. 12:78-127. 1969. production of interferon in serum- free human leukocyte suspensions. Appl. Microbiol. 18:810-815. 164. strander, H., and K. Cantell. 1966. Production of interferon by human leukocytes in vitro. Exp. Fenn. 44:265-273. Ann. Med. 86 165. strander, H., and K. Cantell. 1967. Further studies on the production of interferon by human leukocytes in vitro. 166. Ann. Med. Exp. Fenn. 45:20-29. Stulberg, C.S., vv.W. and H.J. Brough. Zuelzer, R.B. page, P.E. Taylor, 1966. Cytomegalovirus infections with reference to isolations from lymph nodes and blood. 167. Prac. Soc. Exp. Biol. r1ed. 123:976-982. Subrahmanyan, T.P. 1968. A study of the possible basis of age-dependent resistance of mice to poxvirus disease. Aust. J. Exp. Bio1. Med. Sci. 46: 251-265. 168. Subrahmanyan, T.P., and C.A. Mims. 1966. Fate of intravenously administered interferon and the distribution of interferon during virus infections in mice. 169. Brit. J. Exp. patho1. 47:168-176. Subrahmanyan, T.P., and C.A. Mirns. 1967. A study of the production, source and action of interferon appearing in mice after the intravenous injection of influenza virus. 170. Subrahmanyan, Brit. J. Exp. Patho1. 48:568-577. rr.p., and C .A. t1ims. 1970. production by mouse peritoneal cells. Interferon J. Reticu- loendothe1. Soc. 7:32-42. 1 71. Ira1as, M. I G. 'Nei sZfei1er, and L. Ba tka1. ter interferons induced by polyoma virus. Viral. 12:378-380. 1968. Acta Hams- 87 172. Tegtmeyer, P.J., and J.E. Craighead. 1968. Infec- tion of adult mouse macrophages in vitro with cytomegalovirus. 173. Proc. Soc. Exp. Bio1. Med. 129:690-694. Tompkins, W.A.F., C. Adams, and W.E. Rawls. 1970. An in vitro measure of cellular immunity to fibroma virus. 174. J. Immuno1. 104:502-510. Tompkins, W.A.F., J.M. Zarling, and W.E. Rawls. 1970. In vitro assessment of cellular immunity to vaccinia virus: contribution of lymphocytes and macrophages. Infec. Immun. 2:783-790. 175. Tubergen, D.G., and M.B.A. Oldstone. 1971. Release of macrophage migration inhibitory factor by virusinfected spleen cells. 176. J. Immunol. 107:1483-1485. Valeri, A., and K. Cantell. 1963. heparin on herpes simplex virus. 177. The effect of Virology 21:661-662. Valand, S.B., J.D. Acton, and Q.N. Myrvik. 1970. Nitrogen dioxide inhibition of viral-induced resistance in alveolar macrophages. Arch. Environ. Health 20:303-309. 178. Van Rossum, W., and P. De Somer. 1966. of the interferon production in vivo. Some aspects Life Sci. 5: 105-113. 179. Vernon-Roberts, B. 1969. Lymphocyte to macrophage transformation in the peritoneal cavity preceding the mobilization of peritoneal macrophages to inflammed areas. Nature (London) 222:1268-1288. 88 180.llallace, R.E. 1969. Susceptibility of human lympho- blasts (RPMI 7466) to viral infections in vitro. Proc. Soc. Exp. Biol. Med. 130:702-710. 181. Weller, T.H. 1971. The cytomegaloviruses: ubiquitous agents with protean clinical manifestations. N. Engl. J. tiled. 285 :203-214, 267-274. 182. ;,\1heelock, E.F. 1966. Virus replication and high- titered interferon production in human leukocyte J. cultures infected with Newcastle disease virus. Bacteriol. 92:1415-1421. 183. 'iV'heelock, E.F., and R. Edelman. 1969. Specific role of each human leukocyte type in viral infections. III. 17 D yellow fever virus replication and inter- feron production in homogeneous leukocyte cultures treated with phytohemagglutinin. J. Immunol. 103: 429-436. 184.'dheeloc1(, E.F., and S.T. Toy. 1973. of lymphocytes in viral infections. participation Advan. Immunol. 16:123-184. 185. Wheelock, E.F., S.T. Toy, and R.L. Stjernholm. Lymphocytes and yellow fever. I. 1970. Transient virus refractory state following vaccination of man with the 17-D strain. 186. J. Immunol. 105:1304-1306. I!'Jillems, F.T.C., J.L. Melnick, and ~v.E. Rawls. 1969. Viral inhibition of the phytohemagglutinin response of human lymphocytes and application to viral hepatitis. Proc. Soc. Exp. BioI. Med. 130:652-661. 89 l87.~villems, F.T.C., J.L. Melnick, and ';"l.E. Rawls. 1969. Replication of poliovirus in phytohemagglutininstimulated human lymphocytes. 188. J. Virol. 3:451-457. Willems, F.T.C., and W.E. Rawls. 1969. Poliovirus inhibition of the phytohemagglutinin response in human lymphocytes. 189. Arch. Gesamte Virusforsch. 27:352-363. Wilton, J.l-1.A., L. Ivanyi, and T. Lehner. 1972. Cell-mediated immunity in Herpesvirus hominis infections. 190. Brit. Med. J. 1:723-726. ~introbe, M.M. 1967. Clinical hematology, 6th ed. Lea and Febiger, Philadelphia. 191. Yamada, M., M. Azuma, R. Nishioka, and T. Togashi. 1970. Relationship between immunity and interferon production in macrophages. on interferon production. I. Effect of immunity Japan. J. Microbiol. 14: 311-318. 192. Zur Hausen, H., and H. Schulte-Holthausen. 1970. Presence of EB virus nucleic acid homology in a "virus-free n line of Burkitt tumour cells. (London) 227:245-248. Nature EXPERIMENTAL RESULTS SECTION I THE INTERACTION OF VIRUSES WITH FETAL AND ADULT OVINE LEUKOCYTES AND SPLEEN CELLS IN VITRO I. INTERFERON PRODUCTION AND VIRAL REPLICATION In preparation for submission to Infection and Immunity ABSTRACT Peripheral blood leukocyte and spleen cell cultures derived from adult ewes and fetal lambs of 70 to 145 day gestational age were examined for their ability to produce interferon and support viral replication. Bluetongue (BTV), Chikungunya (CV), Semliki Forest (SF'V), Newcastle disease (NDV), vesicular stomatitis (VSV), and vaccinia viruses and Herpesvirus hominis type II (HVH-II), failed to replicate to detectable levels in either fetal or adult cell cultures. No difference in either the levels or the kinetics of interferon production between virus-infected adult and fetal cell cultures was observed. Mean levels of peak interferon pro- duction induced by BTV, CV, SFV, NDV, and HVH-II of approximately 1000-3000 units/ml were demonstrated by 24 hr postvirus inoculation. In contrast, mean interferon titers in- duced by VSV and vaccinia virus were significantly lower ( <40-550 units/ml) and did not reach peak levels until 48 hr post-inoculation. Variations in interferon levels induced on separate occasions using cells from the same adult donor and from the same donor age group were also observed. The interferon induced in both the fetal and adult cell cultures fulfilled the usual criteria for characterization. INTRODUCTION The mammalian fetus has been shown to react in an immature manner to several types of viral infections. Abor- tion, stillbirth, intrauterine infection, and congenital malformation may be associated with various systemic diseases of the mother (40). In the natural setting, congenital rubella virus and cytomegalovirus infections represent the prototype diseases in the human fetus. Bluetongue virus (3TV) of sheep, hog cholera virus of sv.Tine, and bovine viral diarrhea-mucosal disease virus of cows are among the several agents associated with disease of the fetus and newborn in animals (6). These agents result in severe generalized symptoms and malformations following invasion of susceptible fetuses. Snhanced susceptibility of fetuses and newborns has been described in many viral infections of experimental animals (11,50). These reports confirmed the fact observed in nature that host resistance to some viral infectionsis age-related. Several investigators have suggested that the inability of fetal and newborn animals to cope with viral infections may be based on an inefficient interferon response (2, 8, 16, 19, 48). Overall and Glasgow (39) have presented evi- dence, however, that third trimester fetal lambs are capable 93 of producing 100 to 1000-fold higher levels of serum interferon than adult sheep following intravenous (i.v.) inoculation with Chikungunya virus (Cv). In a recent extension of these results (41) it was shown that first and second trimester fetal lambs produced levels of interferon equal to or greater than the adult in response to ev. It is apparent from these data that, at least in the ovine species, fetal immaturity to viral infections may not be fully explained by an inability to produce interferon. Several studies have attempted to evaluate the ability of the human fetus to produce interferon by utilizing peripheral blood leukocytes from donors of various ages (1, 5, 46). The use of this model has been based on the suggestion that interferon production by blood leukocytes (13, 57, 59) and cells of the reticuloendothelial system (RES) (25) following exposure to viruses in vitro may reflect the host's ability to produce interferon in vivo. A major in- herent drawback to the human model, is, of course, the extreme difficulty in comparing the in vitro interferon response with that of the intact animal. In this regard, the fetal lamb system has provided us with the unique opportunity of comparing the interferon response of fetal cell cultures with that of the intact host. It has also been reported that fixed and mobile tissue macrophages may have a paramount role in regulating agerelated host resistance to mouse hepatitis virus (12), vaccinia virus (21), and HerEesvirus hominis type I (HVH-I) 94 (17, 23, 54) infection of rodents. The importance of viral interaction with peripheral blood leukocytes has been indicated in studies of congenital rubella (20, 45) and cytomegalovirus (14, 26) infection of humans. 'ile have, therefore I chosen to investigate several para- meters of the interaction of viruses with fetal cells in vitro. In this report, seven viral agents are examined for their capacity to replicate and induce interferon in fetal and adult ovine leukocytes and spleen cells. An accompany- ing paper describes the relative ability of cells from fetal and adult sheep to inhibit viral infection in an ovine kidney cell monolayer system (47). MATERIALS AND METHODS Cells and media. The cells and cultivation procedures have been described (Rinaldo, C.R., Jr., J.C. Overall, Jr., B. Cole, and L.A. Glasgow, submitted for publication Section III, this thesis). All cell cultures were maintained on Eagle's minimum essential media (MEM) supplemented with 10% (vol/vol) fetal calf serum (FCS) and antibiotics. The cell lines used included semi-continuous lines of fetal lamb kidney (FLK) and human embryonic lung WI-38 cells, as well as cloned, continuous lines of mouse L 929 and baby hamster kidney-2l/Cl3 (BHK-2l/CI3) cells, as described (Rinaldo, et al., submitted for publication). In addition, a continuous line of CV-I monkey kidney cells obtained originally from the American Type Culture Collection Repository (Rockville, Md.) was also used. primary cultures of chick embryo cells, mouse embryo fibroblasts (MEF) derived from CD-l albino Swiss-Webster mouse embryos (Charles Rivers Breeding Laboratories, Brookline, Mass.), and rabbit kidney cells derived from a four to six-week old rabbit were prepared in a similar manner as were the FLK cells. Viruses. All of the viruses used in these experiments were assayed as the number of plaque forming units (PFU) per ml under 0.5% (wt/vol) agarose in concentrated amino acids and vitamins. ~~M containing double 96 Vesicular stomatitis virus (VSV), titering at 1.5 x 10 7 PFU/ml on CV-l cells, and the vaccine strain BTV, which titered at 4 x 10 6 PFU/ml on L cells, have been described (Rinaldo, et al., submitted for publication). supplied by Dr. P. Russell '~'/ashington, ('~"'al ter CV, originally Reed Army Medical Center, D. C. ), was propagated in BHK-21/C13 cells and titered at 3 x 10 7 PFU/ml on CV-l cells. Semliki Forest virus (SFV), obtained from Dr. S. Baron (NIH, Bethesda, I'1d.) was propagated in an identical manner and titered at 1 x 10 8 PFU/ml on CV-l cells. The MS strain of HVH type II (HVH-II) was supplied by Dr. A. Nahmias (Emory University, Atlanta, Ga.), prepared in primary rabbit kidney cells, and titered at 2 x 10 6 PFU/ml on MEF cells. Vaccinia virus, obtained from the NIH, was grown in primary chick embryo cells and titered at 7 x 10 6 PFU/ml on MEF cells. The Herts strain of Newcastle disease virus (NDV), donated by Dr. S. Baron, ¥1as propagated in embryonated chicken eggs which had been injected via the allantoic route, and titered at 3.5 x 10 9 PFU/ml when assayed on primary chick embryo cells. preparation of leukocyte~. Peripheral blood leukocytes from fetal and adult sheep were prepared in the manner previously described (Rinaldo, et al., tion). submitted for publica- Briefly, the leukocytes were obtained from the buffy coat fraction and residual erythrocytes were lysed by treatment with NH4Cl at 4 C. Cell viability, as detected by trypan blue dye exclusion, routinely ranged from 95-98% in adult leukocyte preparations, and from 70-90% in white cell 97 cultures derived from fetal lambs. Differential counts made with Wright's stain demonstrated that leukocytes of both the blood and final preparations from adult ewes ranged between 55-65% lymphocytes, 25-35% neutrophils, 5-10% monocytes, 5-10% eosinophils, and less than 2% basophils. The blood and final culture preparations from second and third trimester fetuses contained approximately 67-85% lymphocytes, 5-23% neutrophils, 5-15% monocytes, and less than 2% eosinophils and basophils. Both the blood and leukocyte cultures from second and early third trimester fetuses contained a substantial population of nucleated erythrocytes. Preparation of spleen cells. Spleens from adult ewes and from the same donor fetal lambs used in the leukocyte studies were removed aseptically. The inner portions of spleens from adult and late trimester fetal lambs, containing both the red and white pulp areas, were separated from the capsule and finely minced in a phosphate buffered saline (PBS) solution. Due to their relatively small size, whole spleens from second and early third trimester fetuses were used in these investigations. The spleen cell suspensions were centrifuged at 15 x g for ten min at room temperature to remove tissue debris. The cell-rich supernatant was then centrifuged for ten min at 250 x g, and the cell pellet was treated with 0.83% NH Cl at 4 C for 15 min to lyse residual 4 erythrocytes. Differential counts made with Wright's stain showed that final preparations of spleen cells from adult ewes contained 80-90% lymphocytes, 5-8% neutrophils, and 98 4-12% macrophages. Cells from spleens of fetal sheep were (~90% of total), the majori ty also predominatly mononuclear of which were lymphocytes. Several varieties of immature cells of the type involved in hematopoiesis were also observed in the fetal preparations. Interferon induction and viral replication. For pur- poses of these studies, the normal lSO-day gestational period of the ovine has been separated into 50-day trimesters. Adult and fetal leukocytes were suspended to a final concentration of 2 x 10 6 cells/ml (unless otherwise noted) in MEM without FCS, as serum was found to enhance the clumping of the white blood cells. Spleen cells from fetal and adult sheep were treated in a similar manner, except MEM with 10% FCS was used as it did not enhance spleen cell clumping. The cell cultures were inoculated with the parti- cular virus at a specified multiplicity of infection (MOl), dependent upon the titer of the viral pool utilized. The MOl of the various viruses used in the experiments, as well as the number of donors of different ages from which the leukocyte and spleen cells were obtained, are presented in Table 1. Following a one hr adsorption at 37 C in S% C02 and humidity, the cells were washed twice to remove residual, unadsorbed virus by centrifuging at 250 x g for ten min and resuspending the cell pellets in five ml of cold MEN. After the final wash, the infected cell pellets were resuspended to the appropriate volume in ~mM with 10% FCS. The infected cell cultures were distributed in one ml aliquots into 99 siliconized glass culture tubes, and incubated at 37 C in 5% C02 and humidity. Timed samples were resuspended, and a portion of the infected cell suspension was frozen at -70 C for virus. The remainder of the sample was centrifuged at 400 x g for ten min at room temperature and the cell-free supernatant was stored at -20 C for interferon. Cell-free aliquots conSisting of virus in MEM with 10% Fes were incubated concurrently with the virus-infected cell samples to measure thermal inactivation of virus. Iro examine for the presence of autogenous or spontaneous interferon production, aliquots of uninfected leukocyte and spleen cell suspensions at a concentration of 2 x 10 6 cells/ ml were included in the experiments. Following centrifu- gation, cell-free supernatants from timed samples were stored for interferon. Prior to assaying, viral samples were rapidly freezethawed to release intracellular virus. Viral plaque counts were made in the apPI"opriate cell system previously indicated for each viral species. Interferon was assayed by the 50% viral plaque reduction technique on FLK cells with vsv as the challenge virus (Rinaldo, et al., submitted for publication). RESULTS Absence of viral replication in leukocyte and spleen cell cultures. peripheral blood leukocytes and spleen cells from fetal and adult sheep, as listed in Table I, were examined for their ability to support replication of a variety of viral agents. A one log or greater rise in viral titer in the infected cell samples, in contrast to a concurrent decrease in virus in the thermal inactivation control, was taken as evidence of viral replication. No detectable mul- tiplication of BTV, HVH-II, or CV occurred in either leukocyte or spleen cell cultures derived from adult sheep, second trimester, or third trimester fetal lambs. Results of representative experiments showing a lack of, in this case, BTV replication in leukocyte cultures are displayed in Figure 1. Note that BTV titers in the infected leuko- cyte cultures decreased essentially in parallel with the drop in virus in the thermal inactivation control over the four day incubation. Similarly, peripheral blood leukocytes from adult and third trimester animals were unable to support replication of SFV, VSV, NOV, or vaccinia virus as witnessed by a lack of increase in viral titers over a four to seven day incubation period. Interferon production in fetal and adult leukocytes and spleen cell cultures. Leukocyte and spleen cell cultures 101 derived from adult and fetal sheep were examined for their ability to produce interferon in response to viral infection. There appeared to be no difference between adult or second and third trimester fetal leukocytes and spleen cells in the levels of interferon induced by each particular viral agent. Interferon production induced by BTV in cells from adult and fetal sheep is displayed in Figure 2 and 3. Although assays of samples from several experiments remain to be done, the interferon response elicited by CV, SFV, NDV, and HVH-II in fetal and adult cells was shown to be essentially equivalent to that illustrated in Figure 2 and 3 for BTV. It can be observed that mean titers of interferon induced by BTV in both fetal and adult cells reached peak levels of 1200 to 3000 units by 24 hr post-virus inoculation. The range in interferon titers induced by BTV and the other viral agents appeared to be influenced by animal-to-animal variation and the viral inducer used, and was not significantly altered by the age of the cell donor or the cell type employed. The similarity between fetal and adult interferon production induced by CV in vitro (Table 2) is in direct contrast to the 100- to 1000-fold differences in the serum interferon response induced by CV following i.v. inoculation of fetal and adult sheep (39). There was also no difference in the amount of interferon induced by VSV or vaccinia virus in .adult and third trimester fetal leukocyte cultures. The average interferon titers induced, however, were of lower magnitude ( <40-550 102 units/ml) than those elicited by the five other viral agents. Peak interferon levels, furthermore, were not reached in either fetal or adult cell cultures until 48 hr post-virus inoculation. Figure 4 illustrates the kinetics of the in- terferon response and the mean interferon titers induced by vaccinia virus in cells from fetal and adult animals. The induction of interferon by VSV followed the same pattern as that shown for vaccinia virus. In summary, peripheral blood leukocytes and spleen cells from second and third trimester fetal lambs were as competent as cells from adults to produce interferon in response to the seven viral inducers employed. Variation among the interferon titers induced in cells from the same donor age group. Although there was no appar- ent difference between the average levels of interferon produced by cells from each separate age group, considerable animal-to-animal and experiment-to-experirnent variation in interferon levels occurred between the same age cell donors. The interferon titers induced by CV in adult and fetal leukocytes are listed in Table 2 as a typical example of the variation observed between animal donors among the same age group. Notice that interferon levels produced by 24 hr following CV inoculation ranged from 70-2500 units/ml in adult leukocyte cultures, 350-3800 units/ml in third trimester fetal cells, and 300-3700 units/ml in leukocytes from second trimester fetuses. Fluctuation of interferon levels induced by the same 103 virus in cells from the same animal donated on separate occasions has also been documented. Table 3 displays the variation in interferon production induced by BTV in adult leukocytes derived from the same ewe on four different occasions. It can be observed that, for example, the inter- feron titers ranged from 400-5400 units/ml at 24 hr post virus inoculation, and that these variations did not appear to correlate with the MOl of virus used. A similar varia- tion in response has been recognized with the six other viral agents employed in these studies. Characterization of interferon. The antiviral substance induced in fetal and adult leukocyte and spleen cell cUltures was identified as interferon by the following criteria: a) inactivation by trypsin treatment, b) resistance to pH 2.0 for 24 hr at 4 C, c) no detectable activity in mouse L cells, d) no activity in FLK cells treated with actinomycin D, and e) activity that could not be removed from cells by washing. DISCUSSION The present studies demonstrate that peripheral blood leukocytes and spleen homogenate cells derived from adult ewes and second or third trimester fetal lambs are equally capable of producing interferon following viral infection in vitro. These data also provide evidence that cells from fetal animals are as capable as those from adult to cope with viral infection in vitro. The results presented also corroborate the previous reports from this laboratory (39, 41) and others (32) which suggested that lack of interferon production may not be the basis of enhanced susceptibility of the intact fetal host to viral infections. It is signi- ficant, however, that CV has been shown to induce 100- to lOaD-fold greater levels of circulating interferon in the fetal lamb than in the adult ewe following i.v. inoculation (39, 41). This is in direct contrast to the similar titers of interferon induced by CV in both fetal and adult ovine cell cultures. terferon in YiY2 Further studies on the production of inby several other inducers are in progress to determine whether there is a similar lack of correlation. The data with CV suggest, however, that it may be erroneous to assume that in vitro studies with peripheral blood leukocytes or spleen cells will accurately reflect the ability of the intact fetal host to produce interferon. In this regard, interferon production by fetal leukocytes 105 has been utilized by several authors as a measure of the ability of the human fetus to mount an interferon response. Cantell and associates (5) challenged whole, heparinized blood samples from adult, child, neonatal, and fetal donors with Sendai virus. Supernatant fluids harvested 24 hr post infection exhibited a correlation between slightly increasing mean interferon titers and rise in donor age. ~{hen ex- pressed as units of interferon per lymphocyte rather than units/ml, however, interferon yields appeared to be the same for each donor age. There may have been no actual difference between fetal and adult cells, therefore, if one considers the lymphocyte to be the major interferon-producing cell of the blood (57). These results have been confirmed by Ray (46) who found no consistent differences in interferon levels induced by Sendai virus in fetal and adult human blood lymphocyte cultures. These observations have been extended further by Banatvala and colleagues (1) to include examination of interferon induction by Sendai and rubella viruses in whole, heparinized blood suspensions and tissue cultures derived from cells from ten to 23 week gestation human fetuses. Sendai virus was capable of inducing considerably higher levels of interferon by 24 hr post inoculation than rubella virus in each type of fetal cells except placental tissue. This correlates well with the present observations that certain viral agents (e.g., BTV) induce greater amounts of interferon than others (e.g., VSV) in fetal and adult ovine 106 cells. It also appeared that comparable levels of inter- feron \vere produced by different cell cultures (e.g., lung, brain, spleen, blood) derived from the same fetus, as has also been demonstrated with ovine leukocytes and spleen cells in this report. Fetal cell cultures, furthermore, were capable of producing interferon levels in response to Sendai or rubella virus which were not influenced by gestational age. It is apparent that the kinetics of the interferon response in the ovine leukocyte cultures were dependent upon the particular inducer used. Both VSV and vaccinia virus elicited peak interferon levels as late as 48 hr post inoculation, in contrast to a 24 hr peak induced by the remaining viruses. In future studies employing virus-infected leukocytes to determine the capacity to produce interferon, multiple samples should be obtained in order to examine the complete kinetic curve. Interpretations about com- parative interferon response of different leukocyte populations based on, for example, a single 24 hr sampling time could well be fallacious. Variations in interferon production by cells from the same adult ewe taken on different occasions have also been demonstrated. 'rhis phenomenon is not unusual, however, as similar variation in virus-induced interferon production by human leukocytes obtained from the same donor on separate occasions has been observed by others (43). It is conceivable that cells from fetal lambs less than 107 70 days gestational age may have a decreased capacity to produce interferon as compared with older fetuses and adults. Extensive investigations of the interaction of viruses with cells from first and early second trimester animals will be quite difficult, however, considering the small number of cells available. For example, the spleen and blood from the youngest, 70-day gestation fetus used in these studies each yielded an average of only 5 x 10 6 cells. Methods requiring a smaller amount of cells than that needed for the present studies may have to be employed in future investigations of similar virus-cell interactions. There is the possibility that at a certain gestational age the cells of the fetal host are unresponsive to the antiviral effects of interferon, regardless of their capacity to produce it. Hanson and colleagues (15) have reported that peritoneal macrophage cultures from mice genetically susceptible to ~'lest Nile virus were three times less sensi- tive to the effects of interferon than were cells from resistant mouse strains. This decreased sensitivity to inter- feron appeared specific for group B arboviruses. It has also been demonstrated that fibroblastic cell lines derived from the least mature human embryos were less sensitive to interferon than those derived from neonatal tissues (49). ',mether the antiviral action of interferon plays a significant role in age-related host resistance of the fetal lamb will, of course, require a model wherein a virus has the ability to replicate in ovine cells. 108 The observation that several different DNA and RNA viruses failed to replicate to detectable levels in adult or fetal ovine cells is of significance. This would suggest that enhanced susceptibility of the fetus to viral infection may not be based on an increase in viral multiplication in fetal spleen and blood cells. The literature contains numerous reports that many viruses are intimately associated with blood, lymphatic, and RES tissue during host infection (13, 33, 59). In relation to age-dependent antiviral host resistance, rubella virus has been isolated from the spleen and thymus of congenitally infected fetuses (34) and blood leukocytes of infants with congenital rubella (20, 31). Suggestive evidence of rubella virus replication in normal, non-transformed lymphocytes has also been presented (36). It has been postulated that such virus-lymphoid cell interactions may play a key role in the pathogenesis of the congenital rubella syndrome (7, 45, 52). Cytomegalovirus has also been recovered from lymphocytes of adult donors (9) and infants following congenital infection (26), suggesting a major involvement of leukocytes in this disease. Apparently the major cell type from the blood, lymphoid and RE systems which is capable of supporting viral replication is the lymphocyte undergoing blastogenesis (10, 59). Blood monocytes (58) and tissue macrophages (15, 56), however, have also been shown to support viral replication. Although polymorphonuclear leukocytes are known to harbor viral antigens during host infection (53), they may not be 109 capable of supporting viral growth (57, 58, 59). Regarding the present studies, it is reasonable to postulate that inducers of lymphocyte transformation, such as phytohemagglutinin (FHA), may not activate adult lymphocytes to the same degree as fetal, thereby leading to enhanced replication of virus in the fetal cells. Supportive evidence for this con- cept is in the observation that human cord blood lymphocytes were more responsive to PHA than adult cells (27), although conflicting data exists (24, 42). Assuming that a direct relationship exists between cell-mediated immunity and lymphocyte transformation (3), and that the fetal lamb has the ability to reject skin homografts after day ao of gestation (51), it follows that post-aO day gestation fetal lymphocytes are at least as competent as adult cells to undergo blastic transformation. In order to examine these concepts, future studies may investigate FHA-induced transformation and its effects on viral replication in lymphocytes from fetal and adult sheep. As the fetal and adult ovine cell cultures used in these studies contained predominantly lymphocytic cells, the role of the macrophage in age-related antiviral resistance remains to be elucidated. Preliminary studies in this laboratory have shown that peritoneal exudate cells from two adult ewes and a 98-day gestation fetus did not support viral replication and produced equivalent levels of interferon following BTV infection. Further investigations of virus-macrophage interactions in the ovine model will be 110 limited based on the relatively small yield of cells obtainable from the peritoneal cavity of fetal animals (e.g., 1 x 10 6 cells from a 98 day gestation fetal lamb). The importance of the macrophage to increased host resistance with age has been indicated in studies involving HVH-I infection of the mouse. Johnson (23) demonstrated that peritoneal macrophage cultures from suckling mice mirrored the immature host's enhanced susceptibility to HVH-I i.p. infection. Although the initial infection of both adult and suckling mouse macrophages occurred to an equal degree, cells from the newborn displayed cytopathic effects and allowed HVrl-I to spread ,to neighboring, uninfected macrophages, whereas adult cells controlled the infection. In an exten- sion of these studies, Hirsch and associates (17) found that adult peritoneal macrophages produced slightly higher amounts of interferon and more efficiently inhibited release of complete infectious virus t.han did cells from the immature mice. Apparently HVH-I components are inefficiently assembl- ed within macrophages from adult mice due to an error in viral DNA metabolism (54). others have shown that macro- phage cultures derived from mouse liver explants displayed increased resistance with age to mouse hepatitis virus (12), which paralleled the ontogeny of resistance of the mice to i.p. viral infection. The literature contains contrasting reports, however, which demonstrate that the ability of virus to replicate in macrophage cultures may not be of significance in age-related resistance of rodents to vaccinia III (22) and cowpox (55) virus infections. It is evident that the relationship of macrophage maturation to enhanced susceptibility of the immature host to viral infection may vary depending on the virus utilized. The results of the studies with BTV vaccine virus are of particular interest. BTV, an arthropod-borne, double- stranded RNA virus (4, 35), is a well-known pathogen of sheep (18). In contrast to the relatively severe disease caused by BTV in naturally-infected ovines, with the experimental infection both the attenuated vaccine and virulent strains usually result in mild symptoms characterized by elevated body temperature, leukopenia, and oral lesions (29, 30). During acute infection of adult sheep, virus has been recovered mainly from the buffy coat, spleen, and mesenteric lymph nodes (44) and leukopenia has been observed (28). These observations would indicate a close association of BTV with blood and lymphoid cells. Of greater importance is the observation that experimental infection of adult ewes with BTV vaccine has been shown to result in severe eNS disease in the fetus (60). Osburn and associates (37, 38) have demonstrated that congenital anomalies such as hydraencephaly and porencephaly developed in fetal lambs infected in utero with the BTV vaccine prior to 100 days gestation. Serum-neutralizing antibodies to BTV, however, could not be detected until 122 days of gestation. The most intense cellular response, consisting predominantly of macrophage infiltration, occurred 112 between 50 and 58 days gestation and appeared less pronounced with increasing fetal age. This would suggest that functions of host resistance other than humoral immunity and the cellular inflammatory response are of greater significance in the fetal ovine response to BTV infection. The present results indicate that BTV is quite capable of inducing interferon, but is unable to replicate in blood leuKocytes or spleen cells derived from either adult ewes or 70 to 145 day gestational fetal lambs. Data on the in vivo interferon response and sites of viral replication are needed for proper interpretation of these studies. They do suggest, however, that interferon production and viral replication in leukocytes and spleen cells may not be of paramount value to antiviral resistance of the ovine fetus to BTV. LrrERATURE CITED 1. Banatvala, J.E., J.E. potter, and J.M. Best. 1971. In- terferon response to Sendai and rubella viruses in human foetal cultures, leucocytes and placental cultures. 2. J. Gen. Virol. 13:193-201. Baron, S., and A. Isaacs. 1961. Mechanism of recovery from viral infection in the chick embryo. Nature (London) 191:97-98. 3. Bloom, B.R. 1971. In vitro approaches to the mechanism of cell-mediated immune reactions. Advan. Immunol. 13:101-208. 4. Borden, E.C., R.E. Shope, and F.A. Murphy. 1971. Phy- siochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus - a new taxonomic group. studies. 5. Physicochemical and serological J. Gen. Virol. 13:261-271. Cantell, K., H. Strander, L. Saxen, and B. Meyer. 1968. Interferon response of human leukocytes during intrauterine and postnatal life. J. Immunol. 100:1304- 1309. 6. Catalano, L.v'v., and J.L. sever. 1971. The role of viruses as causes of congenital defects. Microbial. 25:255-282. Annu. Rev. 114 7. Dent, P.B., and rubella: ~.E. Rawls. 1971. Human congenital the relationship of immunologic aberration Ann. N. Y. Acad. Sci. 181:209- to viral persistence. 221. 8. Desmyter, J., F.F. Barrett. la: \~.E. Rawls, J.L. Melnick, 1-1.D. Yow, and 1967. Interferon in congenital rubel- response to live attenuated measles vaccine. J. Immunol. 99:771-777. 9. Diosi, P., E. Moldovan, and N. rromescu. 1969. cytomegalovirus infection in blood donors. Latent Brit. Med. J. 4:660-662. 10. ~ustatia, J.!vl., and J. VanDerVeen. 1971. Viral repli- cation in cultures of phytohemagglutinin - treated mouse lymphocytes. Proc. Soc. Exp. BioI. Hed. 137: 424-428. 11. Fenner, F.J. 1968. tions, p. 606-612. Vol. 2. 12. The pathogenesis of viral infecIn The biology of animal viruses, Academic Press, New York. Gallily, R., A. 'vvarwick, and F.B. Bang. 1967. onto- geny of macrophage resistance to mouse hepatitis in vivo and in vitro. 13. J. Exp. Med. 125:537-548. Gresser, I., and D.J. Lang. 1966. tween viruses and leucocytes. Relationships be- Progr. Med. Virol. 8: 62-130. 14. Hansha\~, tion: J.B. 1971. Congenital cytomegalovirus infec- a fifteen year perspective. 123:555-561. J. Infec. Dis. 115 15. Hanson, B., H. Koprowski, S. Baron, and C.E. Buckler. 1969. Interferon - mediated natural resistance of mice to arbo B virus infection. 16. ,t..1icrobios IB:51-68. Heineberg, Hit, E. Gold, and F.C. Robbins. 1964. Differences in interferon content in tissues of mice of various ages infected with Coxsackie Bl virus. Proc. Soc. Exp. BioI. l"1ed. 115:947-953. 17. Hirsch, N.S., B. Zisman, and A.C. Allison. 1970. Hac- rophages and age-dependent resistance to herpes simplex virus in mice. 18. J. Immunol. 104:1160-1165. Howell, P.G., and D.W. Verwoerd. virus, p. 35-74. 1971. Bluetongue In Virology monographs No.9. Springer-Verlag, New York. 19. Isaacs, A., and S. Baron. 1960. interferon in embryonic cells. 20. Jack, I., and J. Grutzner. Antiviral action of Lancet 2:946-947. 1969. Cellular viraemia in babies infected with rubella virus before birth. Br it. 1'·'1e d • J. 1: 289- 292 • 21. Jandasek, L. 1970. Influence of anti-leucocyte serum on intraperitoneal vaccinia virus infection in rats. Acta Virol. 14:467-473. 22. Jandasek, L., and M. votava. 1970. macrophages to the vaccinia virus. sensitivity of rat Immun. - Forsch. 139:439-444. 23. Johnson, R.T. encephalitis. 1964. II. The pathogenesis of herpes virus A cellular basis for the davelop- ment of resistance with age. J. Exp. Med. 120:359-374. 116 24. Jones,~·v .R. 1969. lymphocytes. 25. In vitro transformation of fetal Amer. J. Obst. Gynecol. 104:586-592. Kono, Y., and M. Ho. 1965. The role of the reticulo- endothelial system in interferon formation in the rabbit. 26. Virology 25:162-166. Lang, D.J., and B. Noren. 1968. lowing congenital infection. 27. Luedke, A.J. 1969. and viremia. 29. 1964. Effects Lancet 2:591. Bluetongue in sheep: viral assay AIDer. J. Vet. Res. 30:499-509. Luedke, A.J., J.G. Bowne, M.M. Jochim, and C. Doyle. 1964. Clinical and pathologic features of bluetongue in sheep. 30. J. Pediat. 73:812-819. Lindahl-Kiessling, K., and J.A. Book. of PHA on leucocytes. 28. Cytomegalovirus fol- AIDer. J. Vet. Res. 25:963-970. Luedke, A.J., and M.M. Jochim. 1968. Clinical and seriologic responses in vaccinated sheep given challenge inoculation with isolates of bluetongue virus. z.~er. 31. J. Vet. Res. 29:841-852. Harshall, \·v.C., iN.A. Cope, J.F. Soothill, and J.A. Dudgeon. 1970. In vitro lymphocyte response in some immunity deficiency diseases and in intrauterine virus infections. 32. Proc. Roy. Soc. Mad. 63:351-354. Mendelson, J., R. Kapusta, and V. Dick. 1970. nal and fetal interferon production in the rat. MaterAmer. J. Obst. Gynecol. 107:902-907. 33. t·ams, C.A. diseases. 1964. Aspects of the pathogenesis of virus Bacterial. Rev. 28:30-71. 117 34. Monif, G.R.G., G.B. Avery, S.B. Korones, and J.L. Sever. 1965. Postmortem isolation of rubella virus from three children with rubella-syndrome defects. Lancet 1:723-725. 35. Hurphy, P'.A., E.C. Borden, R.E. Shope, and A. Harrison. 1971. Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus a new taxonomic group. Electron microscopic studies. J. Gen. Virol. 13:273-288. 36. Olson, G.B., P.D. Dent, \·v.E. Rawls, Iv1.A. South, J.R. Hontgomery, J.L. lv1elnick, and R.A. Good. 1968. Ab- normalities of in vitro lymphocyte responses during rubella infections. 37. J. Exp. Med. 128:47-68. Osburn, B.I., A.M. Silverstein, R.A. Prendergast, R.T. Johnson, and C.J. parshall, Jr. 1971. Experi- mental viral-induced congenital encephalopathies. I. pathology of hydraencephaly and porencephaly caused by bluetongue vaccine virus. 38. Lab. Invest. 25:197-205. Osburn, B.l., R.T. Johnson, A.M. Silverstein, R.A. Prendergast, H.M. Jochim, and S.E. Levy. 1971. Experi- mental viral-induced congenital encephalopathies. II. 'rhe pathogenesis of bluetongue vaccine virus in- fection in fetal lambs. 39. Lab. Invest. 25:206-210. Overall, J.C., Jr., and L.A. Glasgow. response to viral infection: in sheep. 1970. Fetal interferon production Science 167:1139-1141. 118 40. 1970. overall, J.C., Jr., and L.A. Glasgow. Virus infections of the fetus and newborn infant. J. Pediat. 77:315-333. 41. 1971. Overall, J.C., Jr., and L.A. Glasgow. Host resistance to virus infection in the fetus. Interferon (IF) production. 42. young people. 43. 1969. Pentycross, C.R. I. Pediat. Res. 5:403. Lymphocyte transformation in Clin. Exp. Immunol. 5:213-216. Pidot, A.L.R., G. O'Keefe, III, N• .McI.JIanus, and O.R. f1clntyre. 1972. Human leukocyte interferon: the variation in normals and correlation with FHA transformation. 44. Proc. Soc. Exp. BioI. Hed. 140:1263-1269. Pini, A., W. Coackley, and H. Ohder. 1966. Concen- tration of bluetongue virus in experimentally infected sheep and virus identification by immune fluorescence technique. 45. Ravlls, 'd.E. Arch. Gesamte Virusforsch. 18:385-390. 1968. Congenital rubella: cance of virus persistence. the signifi- Progr. r'1ed. Virol. 10: 238-285. 46. Ray, C.G. 1970. The ontogeny of interferon production by human leukocytes. 47. J. Pediat. 76:94-98. Rinaldo, C.R., Jr., J.C. Overall, Jr., and L.A. Glasgow. 1973. The interaction of viruses with fetal and adult ovine leukocytes and spleen cells in vitro. II. Viral infection in a mixed cell system, this journal. 119 48. sawicki, L. 1961. Influence of age of mice on the recovery from experimental Sendai virus infection. Nature (London) 192 :1258-1259. 49. Siewers, C.M.F., C.E. John, and D.N. Medearis, Jr. 1970. Sensitivity of human cell strains to interferon. Proc. Soc. Exp. BioI. Med. 133:1178-1183. 50. Sigel, H.M. 1952. Influence of age on susceptibility to virus infections with particular reference to laboratory animals. 51. Annu. Rev • .r.'~icrobiol. 6: 247-280. Silverstein, A.M., R.A. Prendergast, and K.L. Kraner. 1964. Fetal response to antigenic stimulus. IV. Rejection of skin homografts by the fetal lamb. J. Exp. Hed. 119:955-964. 52. Simons, 1·1. J • 1968 • gical paradox? 53. Congenital rubella: an immunolo- Lancet 2:1275-1278. Sommerville, R.G. 1968. Rapid diagnosis of viral in- fections by immunofluorescent staining of viral antigens in leucocytes and macrophages. Progr. Med. Viro1. 10:398-414. 54. stevens, J.G., and M.L. Cook. 1971. herpes simplex virus by rnacrophages. the cell-virus interaction. 55. Subrahmanyan, T.P. 1968. Restriction of An analysis of J. Exp. Med. 133:19-38. A study of the possible basis of age-dependent resistance of mice to poxvirus disease. Aust. J. Exp. BioI. !'-1ed. Sci. 46:251-265. 120 56. Tompkins, '~v.A.F., J.1'1. Zarling, andV;.E. Rawls. 1970. In vitro assessment of cellular immunity to vaccinia virus: contribution of lymphocytes and macrophages. Infec. Immun. 2:783-790. 57. dheelock, E.F. 1966. Virus replication and high-titered interferon production in human leukocyte cultures infected with Newcastle disease virus. J. Bacteriol. 92:1415-142. 58. ~'\lneelock, E.F., and R. Edelman. 1969. Specific role of each human leukocyte type in viral infections. III. l7D yellow fever virus replication and inter- feron production in homogeneous leukocyte cultures treated with phytohemagglutinin. J. Immunol. 103:429- 436. 59. 0fueelock, E.F., and S.T. Toy. 1973. lymphocytes in viral infections. participation of Advan. Immunol. 16: 123-184. 60. Young, S., and D.R. Cordy. 1964. An ovine fetal encephalopathy caused by bluetongue vaccine virus. J. Neuropathol. Exp. Neural. 23:635-659. 121 TABLE 1. Viral r101 and number of different aged donors used to examine viral replication and interferon production in ovine cell cultures Number of leukocyte donors Number of spleen cell donors 0.05-1.70 0.03-0.50 6 9 4 2 0.03-5.00 7 7 0.06-0.30 0.05-0.15 6 7 4 2 0.06-1.00 7 7 1.50-5.00 1.50-6.00 7 8 4 2 1.00-4.00 7 7 SEV 2.00-2.50 2.50-4.00 3 6 o o Adult ewes 'rhird trimester fetuses vsv 4.00-5.00 2.00-6.00 3 5 o o Adult ewes Third trimester fetuses 3 o 5 o ewes Third trimester fetuses 3 o Adult ewes Third trimester fetuses Virus BTV EVIl-II cv HOI vacciniao.30-0.50 0.30-0.70 NDV 2.00-2.50 2.00-3.50 3 a. 107-145 days of gestation b. 70-98 days of gestation o Donor Adul t e'l.'le s Third trimester fetuses a Second trimester fetuses b Adult ewes Third trimester fetuses Second trimester fetuses Adult ewes Third trimester fetuses Second trimester fetuses L~dult 122 TABLE 2. Variation in interferon production induced by CV in ovine leukocyte cultures Interferon (Hours post-virus inoculation) Donor Age £.101 24 48 72 96 t 5.0 5.0 5.0 1.5 1.0 1.5 750 a 2000 2500 350 350 70 800 1100 2000 500 650 80 850 1500 2400 500 700 90 900 1700 2300 900 600 80 22 145 b 145 138 135 130 124 2.5 1.5 3.0 2.0 3.0 2.0 3800 2200 1800 3000 350 350 3800 2000 2500 3250 400 350 3800 2800 2000 3000 850 300 2600 2300 3250 3000 600 __ c 33 33 44 35 98 98 82 70 1.0 1.0 4.0 3.0 700 900 3700 300 300 800 3800 450 250 700 4000 350 650 Expt. 4 15 18 21 33 35 28 19 10 25 8 }~du1 II 1/ II II II a. Units/ml b. Estimated gestational age of the fetus c. No sample taken 123 r.rli.BLE 3. Variation in interferon production induced by BTV in leukocytes derived from the same donor on different occasions Interferon Hours Post-Virus Inoculation Expt. gOI 24 48 72 96 18 0.05 2500 a 2500 3400 3300 23 0.25 5400 6500 6500 5400 30 0.30 400 400 350 350 32 0.30 3400 3200 3400 3200 a. Units/ml 124 Fig. 1. Absence of viral replication in BTV-infected leukocyte cultures derived from adult, third trimester, and second trimester fetal sheep, as compared with a thermal inactivation control. 125 o ADULT 6. THIRD TRIMESTER 7 o SECOND TRIMESTER • THERMAL INACTIVATION 6 • - - - - . ----______ 5 ------.----. o f}~~----~ ~ ~O~ ~ V) :::> 0:: ;: 3 -0 o 0 H 0 ~::----- -0 2 l~-----__--~--______~----------~------~ I ., 96 24 48 72 o HOURS POST VIRUS INOCULATION 126 :Fig. 2. Interferon induced by BTV in ovine leukocyte cultures derived from adult, third trimester, and second trimester fetal sheep. 127 10,000 o ADULT (n=5) ~ THIRD TRIMESTER (n=7) TRIMESTER (n=4) o SECOND ----0 O~O o 1000 -... r-- E ........... U') ..-z :::> z 0 0::: w I.J.. 0::: W ..-Z 100 <40 - - - - - - - - - - - - - - - - - - - - - - - - 10~------~--------~------~1------~ o 24 48 72 HOURS POST VIRUS INOCULATION 96 128 Fig. 3. Interferon induced by BTV in ovine spleen cell cultures derived from adult and second trimester fetal sheep. 129 10,000 o ADULT (n=3) o SECOND TRIMESTER (n=3) ____-----0 ~::::::::::..- -= 0 8 1000 .-E .......... V') I- ...... Z 0 0:0: w w.... 0:0: W IZ ...... 100 <40 - - - - - - - - ... - - - - - - - - - - - - - - 10'~-------~----------~-------~---------J o 24 48 72 96 HOURS POST VIRUS INOCULATION 130 Fig. 4. Interferon induced by vaccinia virus in ovine leukocyte cultures derived from adult and third trimester fetal sheep. 131 1000 o ADULT en=3) THIRD TRIMESTER (n=5) ~ ,.... E ........ V) l- I-! z: :::::> 10 z: 0 e:::: L..LJ lJ... e:::: L..LJ I- z: I-! <40 - - a - - - - - - .- - 24 - - - - - - - - - - - - 48 72 HOURS POST VIRUS INOCULATION 96 EXPERIMENTAL RESULTS SECTION II THE INTERACTION OF VIRUSES WITH FETAL AND ADULT OVlNE LEUKOCYTES AND SPLEEN CELLS IN VITRO II. VIRAL INFECTION IN A MIXED CELL SYSTEM In preparation for submission to Infection and Immunity ABSTRi\C'r Blood leukocytes and spleen cells from adult sheep and 70 to 145 gestation fetal lambs were found equally capable of inhibiting Herpesvirus hominis type II (I-IVH-II) and bluetongue virus (BTV) infection in fetal lamb kidney (FLX) cell rnonolayers. Decreased cytopathic effect as Hell as diminished viral replication was demonstrated in the mixed cell cultures as compared with control FLK cultures. Increasing the total number of leuKocytes or spleen cells from 0.5 x 10 6 to 2.0 x 10 6 resulted in enhanced antiviral protection and \-'las accompanied by a concurrent increase in interferon production in mixed leukocyte-FLK cell cultures but not in mixed cultures of spleen and FLK cells. leuKocytes did not inhibit the progression of HVl~-II Ovine in- fection in mouse embryo fibroblast cultures, indicating that the antiviral effect was species specific. An in- terferon-like substance V\Tas also detectable in mixed cultures containing leukocytes or spleen cells and uninfected FLK cell rnonolayers. Similar levels of the interferon-like substance \lere found when cul tures of ovine leukocytes \V'ere added to either mycoplasma-contaminated FLK cells or antibio·tic-·treated FLK cells free of detectable mycoplasma. Uninfected ovine leukocyte, spleen cell, or FLK cell cultures did not produce interferon. INTRODUCTION L'Jumerous studies have described an age-associated increase in resistance to a number of viruses during experimental (11, 32) and natural (6, 29) infections. Several re- ports have suggested that enhanced susceptibility of the fetus to viral infections may be partially based on altered interactions of viruses with cells of the blood, lymphoid tissue, and reticuloendothelial system. It has been post- ulated, for example, that virus-lymphoid cell interactions may play a key role in the pathogenesis of congenital rubella (30, hUinans. 34) and cytomegalovirus (20, 26) infections of 11he importance of the macrophage to age-dependent host resistance has been indicated in Herpesvirus hominis type I (HV::I-I) (21, 24), vaccinia virus (23), and mouse hepatitis virus (13) infections of rodents. 'rhe previous report (31) has shown, however, that peripheral white blood cell and spleen cell cultures derive d from 2nd and 3rd trimester fetal lambs are as capable as adult cells in the inhibition of viral replication and the production of interferon. In order to examine other aspects of the role of leukocytes and spleen cells in agerelated host-resistance, a mixed cell system based on that devised by Glasgow (15) was developed in this laboratory. using this model, the ability of fetal and adult leukocytes 135 and spleen cells to inhibit viral infection in kidney cell monolayers was examined. Bluetongue virus (BTV), an etiologic agent of congenital ovine disease (22), and HVH-II, which can result in severe congenital (35) and neonatal (28) disease of humans, were chosen as model viruses for use in this in vitro system. Adult, 2nd trimester, and 3rd tri- mester fetal cells were compared for their ability to inhibit viral infection in fetal lamb kidney (FLK) cell monolayers by monitoring cytopathic effect (ePE) and viral titers in the mixed cell cultures. Interferon levels in the cell supernatants were also measured as further indication of the relative antiviral activity in the mixed cell systems. MATERIALS AND METHODS Cells, media, and viruses. Cells and cultivation pro- cedures have been previously described (31, Rinaldo, C.R., Jr., J.C. Overall, Jr., B. Cole, and L.A. Glasgow, submitted for publication). All cell cultures were maintained on Eagle·s minimum essential media (~~M) supplemented with 10% (vol/vol) fetal calf serum and antibiotics. BTV vaccine, vesicular stomatitis virus (VSV), and HVH-II used in these experiments were assayed as the number of plaque forming units (PFU) per ml under 0.5% (wt/vol) agarose in MEM containing double concentrated amino aCids and vitamins. Peri- pheral blood leukocytes and spleen cells were prepared in the manner previously described (31, Rinaldo, et al., submitted for publication). Viral infection in a mixed cell system. BTV or HVH-II were added to FLK cell mono layers grown in 35 rom plastiC tissue culture dishes (Falcon PlastiCS, Oxnard, Calif.) at a low inoculum (0.2 ml containing 20-30 PFU/plate), and allowed to adsorb for 1-1 1/2 hr at 37 C in 5% C02 and humidity. Following the adsorption period, 2 ml of adult or fetal leukocytes or spleen cells at a concentration of 2.5 x 105 (0.5 x 10 6 total cells) or 1.0 x 10 6 cells/ml (2.0 x 10 6 total cells) were added to the infected kidney cell monolayers. Control cultures consisted of a) virus 137 infected FLK cell rnonolayers, b) leukocytes or spleen cells added to uninfected cell monolayers, c) uninfected FLK cell monolayers, and d) virus-infected spleen cells or leukocytes in si1iconized glass culture tubes, with appropriate controls as detailed in the previous report (31). Supernatants from the mixed cell cultures were removed at daily intervals and stored for assay of both interferon and virus. The cells were then fixed with a 20% (vol/vol) aqueous formaldehyde solution for 30 min, rinsed thoroughly with tap water, and stained with methylene blue. Viral mediated CPE was visualized on a scale ranging from countable plaque numbers to a +1 (25% destruction of the monolayer) through +4 (100% cell destruction) system. The assay procedures for virus and interferon have been described in detail (31, Rinaldo, et al., submitted for publication). RESULTS Inhibition of vir~l infection in FLK cell monolayers by adult and fetal ovine leukocytes. Peripheral blood leukocytes from five adult ewes, three 3rd trimester fetuses (107-140 days), and seven 2nd trimester gestational age fetal lambs (70-98 days) were examined for their ability to inhibit BTV and HVH-II infection in FLK cell monolayers. For purposes of these studies, the normal ovine gestational period of 150 days has been separated into 50 day trimesters. The progression of the viral infection was measured by daily counts of viral plaque formation and CPE, as well as titration of supernatant samples for virus. Results listed in Tables 1 and 2 show that in a representative experiment blood leukocytes from a 98-day gestation fetus and its parental ewe were equally capable of inhibiting BTV infection in the kidney cells. Viral titers reached peak levels of 10 6 • 7 PFU/ml by three days post-adsorption in BTV-infected FLK cell cultures in the absence of leukocytes. Viral re- plication in these cultures resulted in complete destruction of the monolayer by two days, as is illustrated in Figs. 1 and 2. In sharp contrast, the addition of a total of 0.5 x 10 6 or 2.0 x 10 6 adult leukocytes to infected FLK cells limited BTV replication to only 10 3 • 3 and 10 2 • 0 PFU/ml respectively by three days (Table 1). Viral mediated CPE was 139 suppressed concurrently with the decrease in BTV titers in the mixed cell system (Fig. 1). Fetal lamb leUKocytes re- sulted in a similar decrease in BTV replication, as titers of virus reached only 10 3 • 3 - 10 3 • 7 PFU/ml by seven days (Table 2) and CPE was inhibited accordingly in the cell monolayers (Fig. 2). As with BTV, there appeared to be no appreciable differences between the effect of adult and 98 day gestation fetal leUKocytes on the progress of HVH-II infection (Tables 3 and 4, Figs. 3 and 4). The pattern of herpesvirus in- fection differed from that of BTV in controls, however, as in the absence of leukocytes, viral titers rose slowly to a peak of 10 3 • 9 PFU/ml and completely destroyed the monolayer by seven days post-inoculation. The infection was effectively limited by the addition of adult or fetal leukocytes, as peak levels of virus ranged from 101 • 9 to 10 2 • 2 PFU/ml in the adult leukocyte-FLK cell system and 101 • 5 to 10 2 • 2 with fetal leukocytes by two days, and decreased to low or undetectable amounts by seven days. The drop in viral titer from day two to day seven may have been aided by thermal inactivation of this highly labile virus. It was also observed that increasing the total number of leukocytes (0.5 x 10 6 to 2.0 x 10 6 ) resulted in a more effective control of the HVH-II infection. Role of interferon in the antiviral response of ovine leukocytes in the mixed cell model. The antiviral effect of the ovine leukocytes which was demonstrated appeared to 140 correlate directly with the production of interferon. Al- though FLK cells alone failed to produce interferon in response to BTV or HVH-II infection, the antiviral substance was ~roduced by fetal or adult white blood cells following their addition to the infected monolayers (Tables 1-4). Note that interferon titers produced in the presence of 2.0 x 10 kocytes during STV or H~~-II 6 leu- infection ranged from two to ten-fold higher than levels induced with the lower number of leukocytes (0.5 x 10 6 ). The appearance of enhanced inter- feron levels, furthermore, seemed to correlate with the protective effect of ovine leukocytes in the FLK cell cultures as evidenced by lower viral titers and depressed CPE in the mixed cell systems. The greater replication of BTV may have accounted for the higher levels of interferon induced in BTV-infected leukocyte-FLK cell cultures (Tables 1 and 2) as compared with HVH-II (Tables 3 and 4), since these agents have been shown to be equally potent interferon inducers when ovine white blood cell cultures were challenged directly with a similar inoculum (31). Further experiments were performed to define the relative contribution of interferon to the observed antiviral effect. The lack of activity of ovine interferon in mouse tissue has previously been established (31). Therefore, a mixed ovine leukocyte-mouse cell monolayer system was chosen to eliminate the antiviral effect of sheep interferon. Data from two experiments in mouse embryo fibroblast (MEF) cultures are summarized in Table 5. Following the adsorption of 141 a low inoculum (20-30 PFU/ml) of HVH-II to !'lEF monolayers, a total of 2 x 10 6 adult ovine leukocytes in two ml was added to the infected cultures. The progression of viral infection in the i''1EF cells was not affected by the presence of sheep leukocytes, as titers of H~~-II and viral-mediated CPE in control and mixed cell cultures rose concurrently. These data suggest that interferon production by ovine leukocytes plays a more significant role than uptake and intracellular inactivation of virus in the suppression of viral infection in the mixed leukocyte-FLK cell cultures. It is apparent that production of an interferon-like substance occurred in cultures of uninfected FLK cells to which fetal or adult ovine leukocytes had been added (Tables l-4). Levels of interferon present in these uninfected mixed cell cultures ranged from 350 to 1000 units/mI. BTV infection resulted in a two to six-fold enhancement of interferon over these amounts, while HVH-II infection did not yield significant increases in interferon above these levels. Neither uninfected FLK cells or uninfected leukocyte cUltures contained detectable amounts of interferon. super- natants from the FLK cells were found to induce low levels (20-90 units/ml) of interferon in adult ovine white blood cultures. A mycoplasma contaminant subsequently isolated from the FLK cells proved capable of inducing similar low levels of interferon in sheep leukocytes (Rinaldo, et al., submitted for publication). Treatment of the FLK cells with 25 ug/ml of tylosin, an anti-mycoplasma antibiotic, 142 resulted in the complete elimination of detectable mycoplasma, but the induction of high levels (500-1000 units/ml) of interferon still occurred upon addition of leukocytes to the "cured tl FLK cells. This phenomenon appeared to be a per- sistent variable in all of these studies with mixed cell cultures. Inhibition of viral infection in FLK cell monolayers by adult and fetal ovine spleen cells. Spleen cells derived from three adult ewes, two 3rd trimester fetuses (107-110 days), and seven 2nd trimester fetal lambs (70-98 days) vlere examined for their ability to inhibit BTV and HVH-II infection. Data from a representative experiment (Tables 6 and 7) demonstrate that spleen cells from fetal and adult animals were equally capable of controlling BTV infection in mixed cell cultures. In this experiment, cells were derived from a 98-day gestation fetus and its parental ewe. As with ovine blood leukocytes, increasing the total number of spleen cells resulted in an enhanced antiviral effect, as quantitated by suppression of viral replication (Tables 6 and 7) and spread of CPE (not shown). fection, By day three of in- BTV titers reached only 10 3 • 2 and 10 2 • 7 PFU/ml in mixed cultures containing a total of 2.0 x 10 6 spleen cells from adult and fetal animals respectively, as compared to 10 6 • 7 PFU/ml in the control cells. Control of HVH-II in- fection by ovine spleen cells resembled that shown for the blood leukocytes (Tables 3 and 4), in respect to both inhibition of viral replication and spread of CPE. It is 143 apparent, therefore, that there was little difference in the observed antiviral effect between mixed FLK-Ieukocyte or FLK-spleen cell cultures. As shown in Tables 6 and 7, there was no direct correlation between an increase in interferon production and an inhibition of viral infection in mixed cultures of fetal or adult spleen cells and FLK cell monolayers. Production of interferon (100-400 units/ml) was also detected in uninfected FLK cell cultures following addition of either adult or fetal spleen cells. These results differ from those ob- served for the interferon response in mixed cultures of leukocytes and FLK cells (Tables 1-4), where interferon levels increased concomitantly with enhanced suppression of viral replication and ePEe It is pOSSible, therefore, that factors other than interferon may playa significant role in the antiviral response of spleen cells in the mixed culture model. DISCUSSION The important contribution of the interaction of viruses with lymphoid, reticuloendothelial, and peripheral blood cells to host resistance has been well documented (1, 16, 19, 27, 33, 38). In addition to their paramount role in the immune response to viral infection (3, 16) lymphoid cells have been described as significant producers of interferon (7) and sites of viral replication (8, 10, 38) and persistent infection (30). Evidence has also been presented that fixed and mobile macrophages may be major determinants in preventing spread of virus from primary sites of infection (1, 27). Mechanisms involved in this response may include replication of certain viruses in macrophages of susceptible hosts (2, 18, 27) and the production of interferon (17). Polymorphonuclear cells of the blood have also been reported to be intimately associated with viruses during several infectious processes (19). As has been discussed in the preceding paper (31), several studies have suggested that these cells have a role in age-related host resistance to viral infection. In an attempt to define more fully this concept, blood leukocytes and spleen cells from adult and 70-145 day gestational fetal lambs were studied using a mixed cell culture model. Cells from fetal and adult sheep were shown to be equally competent 145 to suppress the progression of BTV and HVH-II infection in FLK cell monolayers as evidenced by decreased CPS and viral replication. These data provide additional evidence to that presented in the accompanying article (31) that the agedependent antiviral response of the fetal lamb may not be related to an inability of fetal blood or spleen cells to control viral infection in vitro. It is possible that cells from earlier gestational age fetal lambs do not possess the antiviral capabilities shown by cells of older animals. As discussed in the preceding report (31), however, studies with fetuses earlier than 70 days gestation will be 'limited based on the relatively small amount of blood, reticuloendothelial, and lymphoid cells available. These data have demonstrated that interferon may be a primary mediating factor in the antiviral effect of blood leukocytes in the mixed cell cultures. The results further support the concept that interferon may be of significance in the host defense against certain viral infections (4, 15). The inhibition of BTV and HVH-II infection in vitro by spleen cells, however, may involve additional factors (e.g., uptake of virus by splenic macrophages) which remain to be elucidated. Using a similar mixed cell model, Ennis (9) has recently reported an immune specific control of HVtI-I in cell cultures by sensitized murine spleen cells and antibody. Apparently interferon was not a significant factor in this response, as the sensitized mouse cells were able to 146 inhibit HVH-I in both murine and human cell monolayers. The presence of an antiviral substance in the supernatants of mixed cultures of blood leukocytes or spleen cells and uninfected F'LK cell monolayers must be considered in the interpretation of these results. As this phenomenon occurred throughout these experiments, it is unlikely that conclusions regarding competence of fetal and adult cells would be erroneous. The mechanism of induction of the in- terferon-like substance is at present unknown. Supernatant from the FLI< cells was found to induce relatively 10\-1,1 levels of interferon in ovine leukocytes, suggesting the presence of an adventitious agent (Rinaldo, et al., submitted for publication). A mycoplasma contaminant \..ras subsequently isolated from the FLI< cells and proved capable of inducing similar low levels of interferon in ovine leukocytes. Levels of interferon induced by mycoplasma in leukocytes, however, were five to ten-fold lower than those detected in the mixed leukocyte-FLK cell cultures. Mixture of leukocytes with FLK cells in which antibiotic-treatment had eliminated all detectable mycoplasma, furthermore, resulted in the production of interferon titers equal to those induced with untreated FLK cells. It is possible that mycoplasma are being sequestered 'Ylithin cytoplasmic processes (12, 36, 39) and are effectively protected from the action of antibiotics, as well as being undetectable by the usual procedures. Apparently factors other than mycoplasma may be involved in the induction of the interferon-like substance in 147 non-viral infected mixed cell cultures. A similar pheno- menon has been observed in mixed cultures of mouse L cells and lymphocytes from L cell-immunized mice (5, 25), although conflicting dat.a have been reported (37). Studies tvi th lymphocytes from non-immune animals were not presented. It was suggested that interferon induction was stimulated by a cellular immune response of the lymphocytes upon contact with foreign L cell histocompatibility antigens. In sup- port of this concept, mixed lymphocyte cultures from mice with marked differences in histocompatibili ty antigens \V'ere found to produce interferon (14). These investigations did not consider the effect of contaminating mycoplasmas in their systems. Further studies in this laboratory will compare the ability of mycoplasma contaminated, antibiotic-cured, and non-contaminated non-antibiotic treated FLK cell lines to induce interferon in the mixed cell system. Preliminary evidence has shown that an interferon-like substance was induced in mixed cell cultures of adult leukocytes and nonantibiotic treated, low passage FLK cells which did not contain detectable mycoplasma before or during the experiment. LITERATURE CITED 1. 1970. Allison, A.C. On the role of macrophages in some pathological processes, p. 422-440. (ed. ), Mononuclear phagocytes. In R. van Furth F .A. Davis, Philadelphia. 2. Bang, F.B., and A. Warwick. 1960. Mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility. Proc. Nat. Acad. Sci. USA 46:1065-1075. 3. Baron, S. 1967. p. 77-110. Host defenses during virus infection, In R.B. Heath and A.P. waterson (eds.), Nodern trends in medical virology. Butterworths, London. 4. Baron, S. 1970. The biological significance of the interferon system. 5. Arch. Intern. Med. 126:84-93. Borecky, L., N. Fuchsberger, J. Zemla, and V. Lackovic. 1971. Properties of the interferon-like virus inhi- bitor released during interaction of mouse sensitized lymphocytes with their target cells. Eur. J. Immunol. 1:213-218. 6. catalano, L.v~., and J.L. Sever. 1971. rrhe role of viruses as causes of congenital defects. r·tlcrobiol. 25 :255-282. Annu. Rev. 149 7. DeHaeyer-Guignard, J., and E. DeMaeyer. 1971. Effect of antilymphocytic serum on circulating interferon in mice as a function of the inducer. Nature, New BioI. 229:212-214. 8. Edelman, R., and E.F. Wheelock. 1968. Specific role of each human leukocyte type in viral infections. II. Phytohemagglutinin-treated lymphocytes as host cells for vesicular stomatitis virus replication in vitro. 9. J. Virol. 1:1139-1149. EnniS, F.A. 1973. Host defense mechanisms against herpes simplex virus. I. Control of infection in vitro by sensitized spleen cells and antibody. Infec. Immun. 7:898-904. 10. Eustatia, J.M., and J. Van Der Veen. 1971. Viral replication in cultures of phytohemagglutinin-treated mouse lymphocytes. Proc. Soc. Exp. BioI. Med. 137:424- 428. 11. Fenner, F.J. 1968. tions, p. 606-612. Vol. 2. 12. The pathogenesis of viral infecIn The biology of animal viruses, Academic Press, New York. F'ogh, J., and H. Fogh. 1967. Morphological and quantitative aspects of mycoplasma-human cell relationships. 13. Proc. Soc. Exp. BioI. !-'led. 125:423-430. Ga11i1y, R., A. ~'larwick, and F.B. Bang. 1967. onto- geny of macrophage resistance to mouse hepatitis in vivo and in vitro. J. Exp. Med. 125:537-548. 150 14. Gifford, C.E., A. Tibor, and D.L. Peavy. 1971. Inter- feron production in mixed lymphocyte cultures. Infec. I~~un. 15. 3:164-166. Glasgow, L.~. 1965. Leukocytes and interferon in the host response to viral infections. I. Nouse leukocytes and leukocyte-produced interferon in vac- J. Exp. Med. 121: cinia virus infection iri vitro. 1001-1018. 16. G1asgo\v, L.A. 1970. Cellular immunity in host resis- tance to viral infections. Arch. Intern. Ned. 126: 125-134. 17. Glasgovl, L.A., and K. Habel. 1963. Interferon pro- duction by mouse leukocytes in vitro and in vivo. J. Exp. IvIed. 117:149-160. 18. Goodman, G.T., and H. Koprowski. 1962. liiacrophages as a cellular expression of inherited natural resistance. 19. Proc. Nat. Acad. Sci. USA 48:160-165. Gresser, I., and D.J. Lang. 1966. tween viruses and 1eucocytes. Relationships be- Progr. Med. Virol. 8: 62-130. 20. Hanshav/, J .B. fection: 1971. Congenital cytomegalovirus in- a fifteen year perspective. J. Infec. Dis. 123:555-561. 21. Hirsch, !<l.S., B. Zisman, and A.C. Allison. 1970. 1,lacrophages and age-dependent resistance to herpes simplex virus in mice. J. Immuno1. 104:1160-1165. 151 22. Howell, P.G., and D.~·l. virus, p. 35-74. Voewoerd. 1971. Bluetongue In Virology monographs No.9. Springer-Verlag, New York. 23. Jandasek, L. Influence of anti-leucocyte serum 1970. on intraperitoneal vaccinia virus infection in rats. Acta Virol. 14:467-473. 24. Johnson, R.T. encephalitis. 1964. II. The pathogenesis of herpes virus A cellular basis for the develop- ment of resistance with age. J. Exp. Ned. 120:359- 374. 25. Lackovic, V., and L. Borecky. 1970. Release of an interferon-like virus inhibitor during contact of mouse leukocytes with target cells. Acta Virol. 14: 178. 26. Lang, D.J., and B. Noren. 1968. following congenital infection. Cytomegalovirus J. Pediat. 73:812- 819. 27. X1ims, C.A. diseases. 28. 1964. Aspects of the pathogenesis of virus Bacteriol. Rev. 28:30-71. Nahmias, A.J., C •.l\. Alford, and S.B. Korones. 1970. Infection of the newborn with Herpesvirus hominis. Advan. Pediat. 17:185-226. 29. overall, J.C., Jr., and L.A. Glasgow. 1970. infections of the fetus and newborn infant. Pediat. 77:315-333. Virus J. 152 30. Rawls, ~.E. 1968. Congenital rubella: cance of virus persistence. the signifi- progr. riled. Virol. 10: 238-285. 31. Rinaldo, C.R., Jr., J.C. Overall, Jr., and L.A. Glasgow. 1973. The interaction of viruses with fetal and adult ovine leukocytes and spleen cells in vitro. I. Interferon production and viral replication, this journal. 32. sigel, M.M. 1952. Influence of age on susceptibility to virus infections with particular reference to laboratory animals. 33. Silverstein, S. Annu. Rev. Microbiol. 6:247-280. 1970. Macrophages and viral immunity. Gemin. Hemat. 7:185-214. 34. Simons, I1.J. 1968. gical paradox? 35. South, 1'1.A., Ravv-ls. an immunolo- Lancet 2:1275-1278. i~\J.A.F. 1969. Congenital rubella: Tompkins, C.R. Horris, and i/\l.E. Congenital malformation of the cen- tral nervous system associated with genital type (type 2) herpesvirus. 36. stanbridge, E. 1971. J. Pediat. 75:13-18. ~~coplasmas and cell cultures. Bacteriol. Rev. 35:206-227. 37. Svet-!-101davsky, G.J., and I. Ju. Chernyakhovskaya. 1967. Interferon and the interaction of allogeneic normal and immune lymphocytes \vith L-cells. (London) 215:1299-1300. Nature 153 38. Nheelock, E.F., and S.T. Toy. 1973. of lymphocytes in viral infections. participation Advan. Immunol. 16:123-184. 39. Zucker-Franlclin, D.N. Davidson, and L. Thomas. 1966. The interaction of mycoplasmas with mammalian cells. I. HeLa cells, neutrophi1s, and eosinophils. Exp. £-'led. 124:521-532. J. 154 'rABLE 1. Inhibition of BTV infection in FLI< cell monolayers by adult ovine leukocytes Interferon and Virus Days Total number of leukocytes added None a 0.5 x 10 6 a 2.0 x 10 6 a 2.0 x 10 6 b a. 1 2 3 4 Interferonc <10 <10 <10 <10 As sal: Virus d 5.2 Interferon 350 Virus 2.8 Interferon 6.0 6.7 1900 3.3 6 7 -e 5.6 300 3.2 5 650 3.9 3100 4.4 1100 4.7 2150 4.8 1800 Virus 1.7 1.7 2.0 2.1 2.8 3.5 Interferon 800 850 1000 550 550 350 3.5 20-30 PFU of BTV adsorbed to FLK cell monolayer prior to addi tion of leul<:.ocytes b. Leukocytes added to uninfected FLK cell monolayer c. units/m1 d. LoglO PFU/ml e. No sample taken 155 TABLE 2. Inhibition of BTV infection in FLK cell monolayers by 98 day gestation fetal ovine leukocytes Interferon and Virus Days Total number of leul<.ocytes added None a 0.5 x 10 6 a 2.0 x 10 6 a 2.0 x 10 6 b a. 2\Ssay 1 2 3 4 5 Interferon c <10 <10 <10 <10 -e Virus d 5.2 6.0 6.7 5.6 Interferon 450 Virus 1.2 Interferon 250 1.3 0.7 Interferon 550 3.2 2500 3500 Virus 2.4 0.7 0.7 1.3 6 7 300 450 3.5 3.2 3.7 30,00 - 2150 1.7 2.2 3.3 900 20-30 PFU of BTV adsorbed to FLK cell monolayer prior to addition of leukocytes b. Leukocytes added to uninfected FLK cell monolayer c. units/ml d. LoglO PFU/ml e. No sample taken 156 TABL,3 3. Inhibition of HVH-II infection in FLK cell monolayers by adult ovine leukocytes Interferon and Virus Total number of leuJ<.ocytes added None a 0.5 x 10 6 a 2.0 x 10 6 a 2.0 x 10 6 b a. Days AssaX 1 2 3 4 Interferon c <10 <10 <10 <10 <0.7 1.3 2.3 3.0 Virus d 5 7 6 3.2 3.8 3.9 175 100 2.0 1.7 1050 750 Interferon 200 Virus 1.8 Interferon 700 Virus 1.9 1.9 1.0 2.0 1.4 O. 7 <0. 7 Interferon 800 850 1000 550 550 550 350 200 2.2 2.1 2.2 750 20-30 PFU of EVE-II adsorbed to FLK cell monolayer prior to addition of leukocytes b. LeuJeocytes added to uninfected FLK cell monolayer c. units/ml d. Log lO PFU/ml e. No sample taken 157 T1"\BLE 4. Inhibition of HVH-II infection in FLK cell mono1ayers by 98 day gestation fetal ovine leukocytes Interferon and Virus 'rota1 number of leukocytes added Hone a 0.5 x 10 6 a 2.0 x 10 6 a 2.0 x 10 6 b a. Days Assa:i 1 Interferon c <10 Virus d <0.7 Interferon 200 Virus 1.7 Interferon 850 Virus 1.3 Interferon 550 5 7 2 3 4 <10 <10 <10 e 1.3 2.3 3.0 3.2 3.8 3.9 200 250 1.4 0.7<0.7 500 550 250 1.5 1.4 1.3 500 2.2 6 1.2 <0.7 <0.7 <0.7 0.7 900 20-30 PFU of BYE-I-II adsorbed to FLK cell monolayer prior to addition of leukocytes b. Leukocytes added to uninfected Fr...I< cell monolayer c. Units/ml d. Log10 PFU/ml e. No sample tal<en 158 TABLf; 5. Failure of ovine leukocytes to inhibit EVIl-II infection in i'1EF cell monolayers Virus EXEeriment Total number of leukocytes added Days 1 2 3 4 5 None 1.7 a 3.3 3.9 5.3 5.3 2.0 x 10 6 2.0 3.3 4.3 5.3 5.3 None 1.0 3.1 2.3 4.6 -b 2.0 x 10 6 1.4 3.1 4.2 4.8 29 34 a. Log 10 PFU/ml b. No sample taken 159 TABLE 6. Inhibition of BTV infection in FLK cell monolayers by adult spleen cells Interferon and Virus Total number of spleen cells added None a 0.5 x 10 6 a 2.0 x 10 6 a 2.0 x 10 6 b a. Days 2 3 4 5 Interferonc <10 <10 <10 <10 -e Virus d 5.2 6.0 6.7 5.6 Interferon <40 50 300 Virus 3.7 4.4 5.0 Interferon 200 200 300 Virus 2.5 2.8 3.2 Interferon 100 Assa:l 1 5.2 3.0 6 7 900 600 5.8 5.8 6.0 350 500 4.2 4.3 4.2 250 20-30 PFU of BTV adsorbed to FLK cell monolayer prior to addition of spleen cells b. Spleen cells added to uninfected FLK cell monolayer c. Units/ml d. LoglO PFU/ml e. No sample taken 160 TABLE 7. Inhibition of BTV infection in FLK cells monolayers by 98 day gestation fetal spleen cells Interferon and Virus Days Total number of spleen cells added None a 0.5 x 10 6 a 2.0 x 10 6 a 2.0 x 10 6 b a. Assa:L 1 2 3 4 5 Interferonc <10 <10 <10 <10 -e Virus d 5.2 6.0 6.7 5.6 50 50 250 Virus 3.1 4.0 4.3 Interferon 150 150 150 Virus 2.0 2.3 2.7 Interferon 700 4.7 3.0 6 7 - 1200 5.1 5.4 5.6 150 350 3.9 3.7 4.3 --Interferon 400 200 20-30 PFU of BTV adsorbed to FLK cell monolayer prior to addition of spleen cells b. Spleen cells added to uninfected FLK cell monolayer c. units/ml d. La910 PFU/ml e. No sample tak.en 161 Fig. 1. Inhibition of CPE in BTV-infected FLK cell monolayers by adult peripheral blood leukocytes. 162 (" x i LEUKOCYTES [)tY 1 DAY 3 DAY 5 DAY 7 2. U x: ; r./ LElJKOCnCS , ! I,r t rt CONTROL 163 Fig. 2. Inhibition of CPE in BTV-infected FLK cell monolayers by 98-day gestation fetal peripheral blood leukocytes. 164 x I (,. LE.UKOC YHS L. U x I rjC LEUKOCYTt~S utj It, r- E CTE D CmnRO L 165 Fig. 3. Inhibition of CPE in HVH-II infected FLK cell monolayers by adult peripheral blood leukocytes. 166 it s l:,YlTROL DAY 3 DAY 5 DAY 7 f ~. O. LEUKOCYTES .0 x I LEUKOCYT[S UNINFECTED CONTROL 167 Fig. 4. Inhibition of CPE in HVH-II infected FLK cell monolayers by 98-day gestation fetal peripheral blood leukocytes 168 F T E~i<DCl I L S [)ty 1 DAY 3 DAY 5 DAY 7 CONTROL EXPERII·1ENTAL RESULTS SECTION III HYCOPLASi1A-ASSOCIATED INDUCTION OF INTERFERON IN OVlNE LEUKOCYTES Charles R. Rinaldo, Jr. James C. overall, Jr. Barry C. Cole Lowell A. Glasgow Submitted to Infection and Immunity ABSTRACT A mycoplasmal species, Acholeplasma laidlawii, isolated as a contaminant from a fetal lamb kidney cell line, was shown to be associated with the induction of interferon in cultures of ovine peripheral blood leukocytes. Broth cul- tures of the mycoplasma induced between 20 and 230 units/ml of interferon in leukocytes from two adult ewes. The amount of interferon produced correlated with the inoculum size of mycoplasma. Interferon production was associated with replication of the mycoplasma in the leukocyte cultures. Interferon was not induced by sterile mycoplasmal broth, a cell-free filtrate of the mycoplasmal cultures, or heatinactivated mycoplasmas. The antiviral substance was char- acterized as interferon by the usual criteria. INTRODUCTION !~ wide spectrum of agents have been shown to be capable of stimulating the production of interferon in animals and in cell cultures. Interferon has been reported to be induced by active and partially inactivated viruses, natural and syn- thetic polynucleotides and polysaccharides, synthetic polymeric and non-polymeric compounds, mitogenic agents, and a series of microbial agents and products (8, 15, 31). Ho\"ever, attempts at inducing interferon with mycoplasmas in cell cultures have so far been unsuccessful (2, 33, 34, 40). During the course of investigations to determine the role of ovine peripheral blood leukocytes in resistance to viral infection ~ vitro, an interferon-like antiviral sub- stance was recovered from the media of uninfected fetal lamb (FLK) cells to which leukocytes had been added. FLK cells had been previously shown to be unable to produce interferon in response to a variety of inducers. A search for an ad- ventitious agent in the kidney cells which might have been responsible for the induction of interferon in the sheep leukocytes led to the discovery of a mycoplasmal contaminant, i~cholepl~ laidlawii. This report describes the associa- tion of £!. laidlawi1 with interferon induction in ovine peripheral blood leukocyte cultures. f''1!\TZRli·\LS AND rtlETHODS Cells and media... A semi-continuous line of FLK cells vias prepared by aseptically removing kidneys from fetal lambs of pregnant ewes obtained through Dr ....?\..E. Larsen, Univ. of utah... The capsule and cortex were removed and the remaining tissue was minced and washed \'lith phosphate-buffered saline (P3S). Cells were extracted in 0.25% (v/t/vol) trypsin sodium citrate-potassium chloride solution for one hour, centrifuged, and resuspended in Eagle's minimum essential medium (J·E!:r-·'l) consisting of Hanks balanced salt solution (Microbiological Associates, Bethesda, Nd.) supplemented "lith single concentrated }'1EB vitamins (North Pl.merican Biologicals, Rockville, 11d.) and amino acids (Grand Island Biological, Grand Island, N.Y.): 1% (vol/vol) of a 300 rng/ liter solution of glutamine: penicillin, 100 units/ml: streptomycin, 100 ug/ml: and 10% (vol/vol) fetal calf serum (Grand Island 3iological). NaHC03. The pH was adjusted to 7.4 ,\",i th The FLK cells were maintained through semi-con- tinuous passage in Roux bottles... After isolation of the mycoplasmal contaminant, tylosin tartrate (Grand Island Biologicals) was added to the MEN (final concentration of 25 ug/ml). This resulted in elimination of detectable mycoplasma from the FLK cell cultures. Primary chick em- bryo cells, derived from eight to ten day old chick embryos, 173 were prepared in a similar manner as the FLK cell cultures. irhe baby hamster kidney (BHK) and L cells utilized in the experiments originated from cloned continuous lines (mouse BI-IK-21/C13 and L929) obtained from the American Type Culture Collection Cell Repository (Rockville, tld.). rrhe semi- continuous line of human embryonic lung WI-38 cells used was supplied by Dr. L. Hayflick, Stanford University. Viruses. The Indiana strain of vesicular stomatitis virus (VSV) was obtained from the American rrype Culture Collection. VSV pools were prepared in primary chick embryo cells and titered at 1.5 x 10 7 plaque forming units (PFU) per ml in FLK cells. VSV and other viruses used in these experiments were assayed utilizing the plaque titration method under 0.5% (wt/vol) agarose (Van waters and Rogers, San Francisco, Calif.) in t~M concentrated amino acids and vitamins. containing double The bluetongue virus (BTV) preparation, a modified live lyophilized vaccine (Blucine~ Cutter Labs, Berkeley, Calif.), containing two immunologically identical isolates, strain 8 and 11, was reconstituted in sterile distilled water. stock pools were pr~pared in BHK-21/C13 cells and titered at 4 x 10 6 PFU/ml in mouse L cells. Dr. P. Russell Pools of Sindbis virus, obtained from (~valter Reed Army Medical Center, v"lashington, D.C.) were grown in WI-38 cells and titered at 5 x 10 6 in FLK cells. ~ucoplasma. Tissue culture cells were tested for myco- plasmal contamination using Difco mycoplasma agar or broth 174 supplemented to final concentrations of 20% (vol/vol) horse serum, 5% (vol/vol) fresh yeast extract, and 1000 units/ml of penicillin (5, 16). The Acholeplasma laidlawii isolate, designated the U 2 Strain, was subsequently grown in the tryptose broth medium described by Maniloff (25) or modified serum-free rabbit infusion broth (38) supplemented to final concentrations of 5% (vol/vol) fresh yeast extract, 0.5% (wt/vol) glucose and 1000 units/ml penicillin. Mycoplasmal suspensions were prepared and titered as colony forming units (CFU) per ml (11). Heat-killed mycoplasmal suspensions vIera prepared by total emersion for one hour in a water bath at 60 o C. MYcoplasmal filtrates were prepared by two succes- sive passages through a 0.22 u .MF Millipore membrane filter supported in a Swinny holder (E-1illipore Corporation, Bedford, l'·lass. ). Treated suspensions were cultured to confirm the absence of viable organisms. Growth inhibition (GI) and metabolic inhibition (MI) antibody tests were performed as previously described (36, 37), but using serum-free tryptose medium (25). ~. Donkey antiserum (Cat. #M728-50l-57l) against laidla'>lii type A (Natl. Inst. Allergy and Infectious Diseases, Bethesda, Nd.) was utilized to identify the U 2 strain. Preparation of leukocytes. Venous blood was collected from adult ewes in plastic syringes containing five units of heparin (Lipo-Hepinr Riker Labs, Northridge, Calif.) per ml of blood. All glassware used in the preparation of the leukocytes was siliconized. Following centrifugation of the 175 blood at 1800 x 9 at 4 0 C for 20 min, the topmost layer, consisting predominantly of leukocytes, was carefully removed. Residual erythrocytes were lysed by adding 30 ml of a 0.83% (\,rt/vol) concentration of NH 4 Cl to the buffy coat for 15 min, and the leukocyte pellet was resuspended to the appropriate volume in cold PBS for counting in a hemocytometer. Cell viability, as detected by trypan blue dye ex- clusion, routinely ranged from 95 to 98%. Differential counts made on ':lright I s stained preparations demonstrated that both the blood and final cultures contained between 55-65% lymphocytes, 25-35% neutrophils, 5-10% monocytes, 5-10:;G eosinophils, and less than 2% basophils. Interferon induction and assa~. Sheep leukocyte pre- parations ylere suspended to a final concentration of 2 x 10 6 cells/ml in MEN with 10% (vol/vol) fetal calf serum. Leukocyte cultures were inoculated with 0.02 to 2.0 CFU/cell of the U 2 strain of of infection of 0.25. ~. ~lawii, or BTV at a multiplicity The infected cell cultures vlere distributed in two ml aliquots into glass culture tubes and incubated at 37 0 C in 5% C02 and humidity. Samples \'lere harvested each day from single culture tubes. 1;; 0.2 ml aliquot, containing both supernatant and leukocytes, vIas collected and assayed for mycoplasma. The remainder of each sample was centrifuged at 400 x 9 for 10 min and the supernatant was stored for assay of interferon. The interferon samples were treated at pi:-! 2 fc·r 24 hr, a procedure \-lhich was shown to inactivate mycoplasma or BTV. 176 Aliquots (one ml) of the appropriate dilution of the interferon samples made in XvlEl\'! with 2% (vol/vol) fetal calf serum were a.dded to FLK cell monolayers in 35 nun plastic petri dishes (F'alcon Plastics, Oxnard, Calif.). FoIl o\.lli ng overnight incubation at 37 o C, the cell monolayers were washed vIi th PBS, challenged \-li th approximately 50 PFU of VSV, and overlayed ~..,i th two ml ofO. 5% agarose. Tvlenty-four hours later a O. 75% (wt/vol) agar-NEl"1 solution containing neutral red at a final concentration of 1:40,000 was added. counts \-lere made the following day. Plaque The ti ter of interferon was considered as the reciprocal of the highest dilution which inhil~i ted cul tures. I:\~ 50% of the plaques in the control cell laboratory standard ovine interferon prepara- tion was included in each assay as a control for the variation in the assay system. ReSULTS Interferon induction by media from FLK cultures and identification of a mycoplasmal contaminant. After the dis- covery of substantial levels of an antiviral substance in mixed cultures of ovine leukocytes and FLK cell monolayers, investiga'tions were undertaken to determine whether an adventitious agent in the kidney cell cultures played a role in this phenomenon. The medium from the F'LK cells was found to contain a high titer of mycoplasma, which was subsequently identified as Acholeplasma laidlawi!, a member of the nonsterol requiring mycoplasmal family Acholeplasmataceae (9), by the GI and 111I antibody tests utilizing specific hyperimmune serum. The mycoplasmal isolate was designated as the U 2 strain after the line of FLK cells it was isolated. (F~K-U 2) from t.,rhich Portions of the contaminated supernatants from the FL:r: cells were added to ovine leukocyte cultures and observed for interferon production. In Table 1 are illus- trated the titers of mycoplasma obtained from the conta~i­ nated media of FLK cells on two separate occasions (experiments 36 and 39). In addition, the final concentration of mycoplasma in the inoculated leukocyte cultures at the beginning of the experiment as well as the titers of interferon induced in the ovine luekocytes over a four day period are shovln. 178 Interferon induction and mycoplasmal replication. Table 2 displays the results of a series of experiments which demonstrate that broth cultures of ~. laidlawii, U 2 strain, were associated with the induction of between 20 and 140 units of interferon in ovine leukocytes derived from one donor animal (ewe #7). Interferon was not detectable in the media until at least 48 hours post inoculation and the titer appeared to correlate with the number of CFU of laidlawii. mycoplas~a ~. In each experiment a 100-fold dilution of resulted in the induction of less interferon. It appears that the presence of approximately 10 4 CFU/ml of A. laidlawii was required to induce detectable interferon and that higher titers of mycoplasma--10 6 CFU/ml or greater--did not result in appreciably higher levels of interferon. Furthermore, it is apparent that the inter- feron production is assoCiated with replication of the mycoplasma over the four-day sampling period. These data further indicate that there was a variation in the interferon response in leukocyte cultures obtained from the same animal (ewe #7) on different occasions. This phenomenon is not unusual as such variations with other inducers and other species of leukocytes have been observed in our laboratory. Pidot and colleagues (28) have demon- strated a similar variation in interferon production by human leukocytes obtained from the same donor on different occasions following exposure to Newcastle disease virus. 179 Requirement of viable A. laidlawii for interferon induction. The next series of experiments were undertaken to determine \vhether viable ~. laidlawii was required for the induction of interferon in ovine leukocytes. Cultures containing 3 x 10 7 CFU/ml of ~. laidlawii, U 2 strain, were subjected to the following treatments: (1) heat inacti- vation at 60 0 C for one hour, and (2) passage twice through a 0.22 u millipore filter. Separate leukocyte cultures were challenged with an aliquot of each of the preparations as well as an aliquot of the untreated pool of viable mycoplasma. In addition, the leukocytes were challenged with a similar portion of the sterile mycoplasmal broth. As displayed in Table 3, results from a typical experiment show that viable mycoplasma were necessary to induce interferon (25-75 units). Although in this experiment some viable mycoplasma survived the heat inactivation, interferon was not detected in the leukocyte cultures. In three subsequent heat inactivation experiments neither viable mycoplasma nor interferon were detected. The fact that the mycoplasma-free filtrate failed to induce interferon would suggest that a filterable agent (e.g., a mycoplasma virus) was not responsible for the interferon induction. Finally, the sterile mycoplasmal broth did not contain substances capable of inducing interferon and the leukocytes did not produce interferon "spontaneously". These data indicate that the induction of interferon in ovine leukocytes required the presence of a relatively high titer (10 4 CFU/ml) 180 of viable mycoplasma. Interferon production and mYcoplasmal replication in leukocytes from different ovine donors. Cells derived from different sheep donors were shown to produce varying levels of interferon in response to the U 2 strain of as illustrated in Table 4. ~. laidlawii, Leukocytes from ewe #7, the same animal used in the previous experiments, produced 140 units of interferon in experiment 39 and over 200 units of interferon in experiment 40 within 48 hours following inoculation with mycoplasma. Although much less or undetect- able levels of interferon were produced by leukocytes from ewe +1=46, the ti ters of mycoplasma in the cultures 'Vlere similar. Again, interferon production was associated with replication of the mycoplasma in the leukocyte cultures. BTV, a double-stranded RNA virus (4, 27) and a natural pathogen of sheep (19), has previously been shown to induce relatively high levels of interferon in ovine leukocyte cultures (C.R. Rinaldo, J.C. Overall, Jr., and L.A. Glasgow, Abst. Z\mer. Soc. l1icrobiol., p. 196, 1972) and, therefore, vlas used as a control interferon inducer. As with ~. laidlawii, BTV elicited higher levels of interferon in leukocytes from ewe 4*7 than in cell cultures from etve The interferon induced by ~. ~*46. laidlawii was usually not de- tectable until 48 hours post inoculation, whereas that induced by BTV was apparent at 24 hours. Characterization of mycoplasma-induced interferon. The mycoplasma-induced antiviral substance was characterized as 181 interferon by the following criteria: trypsin treatment, (a) inactivation by (b) resistance to pH for 24 hours at 4 0 C, (c) no detectable activity in mouse L cells, at 56 0 C for 30 min, Cd) stability (e) activity against both VSV and Sindbis viruses in FLK cells, (f) inability to inactivate VSV directly, and (g) no activity in FLK cells treated with actinomycin D. Further experiments were performed to eliminate the possibility that antiviral interference of non-interferon origin was mediated directly by ~. laidlawii present in the preparations being assayed for interferon. Portions of sam- ples from experiment 43 (Table 2) were filtered through a 0.22 u millipore membrane, resulting in the elimination of 99-100% of the detectable mycoplasmas. Filtered and unfil- tered portions from the same samples were then acid-treated and assayed for interferon. No significant difference in interferon titers between the two portions was observed, indicating that the antiviral activity in the infected leukocyte samples was filterable and was not mediated directly by the presence of mycoplasma. To further eliminate the possibility that A. laidlawii present in the media of the leukocyte cultures following acid treatment mediated interference, the following experiments were performed. Serial dilutions of viable mycoplasma and serial dilutions of acid-treated mycoplasma were incubated overnight with FLK cells. The cell monolayers were washed and challenged with VSV in a manner identical to that 182 of the interferon assay. In several experiments, a concen- tration of 2 x 10 6 CFU/ml or greater of viable mycoplasma produced evidence of interference at a 1/30 dilution even in the presence of tylosin containing media. an acid-treated aliquot of an 1 x 10 (50% ~. In contrast, laidlawii pool titering at 7 CF'U/ml failed to induce significant interference reduction in number of plaques in the control plates) at a 1/20 dilution. These experiments strongly indicate that the antiviral activity present in the media of the leukocyte cultures is not non-specific interference mediated by acid-treated mycoplasma. DISCUSSION The data presented indicate that inoculation of ovine peripheral blood leukocyte cultures with Acholeplasma laidla\vii (U 2 strain) was associated with both the replication of the mycoplasma and the induction of interferon. 'ro our knowledge, this is the first reported instance of a mycoplasmal infection of mammalian cell cultures being associated with the production of interferon. Supportive evidence has been obtained by Stinebring and Youngner (personal communication), who found that pure concentrates of l1ycoplasma pneumo~ induced low but definite levels of circulating interferon in mice following intravenous injection. The failure of other investigators to demonstrate interferon induction by mycoplasmas may be related to the cell type employed. Yershov and Zhdanov (40) could not detect interferon induction in monolayers of chick embryo cells infected with ~. laidlawii or ~. workers (34) have shown that although gallisepticum, and ~. agalactiae. ~. Other laidlawii, M. hominis were capable of replication in chick embryo cells, detectable interferon was not induced. Armstrong and Paucker (2) also demonstrated that, despite mycoplasmal multiplication, neither Negroni strain of !1. hominis nor the !1. pulmonis elicited interferon production 184 in mouse L cells, and that £1. pneumoniae and £1. hominis failed to induce interferon in human embryonic kidney cells. In a more recent study (33) neither ~. arginini nor ~. hyorhinis induced interferon in monolayers of hamster embryo fibroblasts although they were able to replicate in the cell cultures. It is reasonable to postulate that leukocytes and macrophages may interact with mycoplasmas in a different manner than do other cell types. It is now generally ac- cepted that, although some species of mycoplasma may reside intracellularly (21), most exist predominantly in the extracellular environment and/or in association with the plasma membrane of mammalian cells (I, 10, 21). The concept that mycoplasmal interaction with leukocytes is different from that with fibroblastic cells is supported by the electron microscopic studies of Zucker-Franklin and associates (41, 42). Their evidence suggested that within a few minutes of incubation, and ~. ~. gallisepticum, ~. Eneumoniae, neurolyticum were avidly ingested and degraded within the phagosomes of human peripheral blood neutrophils, eosinophils, and monocytes. These authors also emphasized that a significant portion of cells morphologically indistinguishable from lymphocytes were able to endocytose, although not degrade, each of these mycoplasmal species. In contrast, mycoplasmas were rarely seen within HeLa cells, although the organisms proliferated on the cell membrane. Other mycoplasmal species appear to react differently 185 with leukocytes. Simberkoff and Elsbach (32) demonstrated no measurable decrease of ~. hominis from the supernatant during two hours of incubation with rabbit polymorphonuclear leukocytes or human peripheral blood leukocytes. Even though direct uptake of mycoplasma could not be detected, a marked enhancement of C02 production (an indirect measure of phagocytosis) occurred. iilhereas mouse peritoneal mac- rophages exposed to immune serum are capable of ingesting N. pulmonis (22), Cole and Ward (6) failed to detect uptake of H. arthritidis by murine peritoneal macrophages even in the presence of homologous convalescent rat or mouse sera. An interaction of the mycoplasma with the cell membrane would appear to be a necessary prerequisite for interferon induction. Following attachment to the leukocytes, at least two mechanisms for induction of interferon are possible. First, mycoplasmas have been shown to alter the morphology of the plasma membrane of mammalian cells (41). Thus,~. laidlawii may act at the cell surface to initiate interferon production, as has been postulated in one model for interferon induction by poly I:C (3). Second, if the process is similar to that of animal Viruses, ingestion of the organism by the leukocytes with subsequent release of nucleic acid may be required (15). In either case, the particular species of mycoplasma and cell type utilized may determine whether or not interferon is induced. It is also possible that an adaptation of the particular mycoplasma, such as the U 2 strain, to grow in 186 tissue culture is required to enhance its ability to infect leukocytes. If this is so, A. laidlawii and other common mycoplasmal contaminants of tissue culture (35) may be expected to possess greater interferon-inducing capacities than the usual non-contaminating species. The ability of mycoplasma to induce interferon in leukocyte cultures may further depend upon the environmental conditions provided. Jones and Hirsch (22) have observed, for example, that ~. pulmonis vlas able to proliferate in mouse macrophage CUltures only if the ~1EN \-Tas supplemented \vi th 5-20% heart infusion broth. Interferon induction by mycoplasma in leukocyte cultures :nay be mediated by immunologic mechanisms. Green and associates (14) have reported that relatively low levels of interferon can be induced by 96 hours in lymphocytes obtained from human donors and challenged with specific antigens to which the subjects were shown to be i~~une. 1ne possibility that lymphocytes from sheep specifically sensitized to ~. laidlawii (as a result of prior natural in- fection) played a role in the production of interferon should be considered. Although in our studies peak levels of mycoplasma-induced interferon were usually reached by 48 hours, the interferon titers generally ranged at comparably 10vl levels. Since A. laidlawii is a common soil organism and has been frequently isolated from cattle (7), it is conceivable that the sheep used in these investigations had been naturally infected with this mycoplasmal species. 187 preliminary evidence in this laboratory has indicated, ho\vever, that sheep used in these experiments did not have prior antigenic exposure to A. laidlawii as evidence by the absence of serum GI and 1·1I antibodies. ~lternatively the interferon induction observed in these studies may be due to the presence of a mycoplasma-associated product. It has recently been demonstrated that many, if not all, strains of A. laidlawii are infected with viral agents (12, 13, 24). Our observation that mycoplasma-free ~. laidlawii U 2 cultures did not induce supernatants from interferon suggests that, if a virus is responsible for interferon induction, it requires the presence of mycoplasmas in order to interact with the leukocytes. Alternatively, the viral agent, if present, may have no significant role in the interferon response. Studies designed to define the possible role of mycoplasmal viruses in interferon induction are in progress. Relatively high doses of endotoxin (200 ug) have been sho\'1n to induce low levels (two to eight uni ts) of interferon three hours after addition to bovine leuKocyte culture s (23). Z:;.l though ~. laidlawi i has never been reported to produce an endotoxin, a galactan isolated from l\l. mycoides has been shown to exhibit endotoxin-like properties (20, 39). However, the differences in the kinetics of production and the higher levels of interferon induced by ~. laidlawii, as well as our preliminary finding that bacterial endotoxin induces little or no interferon in ovine 188 leukocytes, would tend to negate a role for mycoplasmal endotoxin in interferon induction by ~. laidlawii. Numerous reports reviewed by Stanbridge (35) have dernonstrated that mycoplasmas may enhance, depress, or have no effect on the induction of interferon by various agents in tissue culture. Similar diverse effects of mycoplasmas on viral replication are \vell documented. The ability of .!\. laidlav-lii to induce interferon in mammalian cells should be viewed with some concern due to increasing frequency of isolation of this species from cell cultures (35). The possibility that unsuspected contamination with mycoplasmal species may influence results of studies concerning interferon production in cell cultures, particularly those with leukocytes or macrophages, should be considered. Studies are in progress to determine the ability of several different I mycoplasmal species Ito induce interferon in cells from sheep and other animal species. rrhe preliminary report of stinebring and Youngner indicates that mycoplasmas may be capable of inducing interferon in vivo. It is reasonable to postulate that in vivo induction of interferon by mycoplasmas could alter the susceptibility of the host to viral infection. However, data concerning the effects of mycoplasmas on viral pathogenesis are limited. Studies have shown that 1'1. gallisepticum induced a more severe disease during dual infection \V'ith viruses in avian hosts (18, 29). l~. ~dlavlii Pertinent to our results, has been isolated directly from the human oral 189 cavity (30), leukemic bone marrow (17), and human burns (26). However, this organism has not as yet been examined with regard to its interaction with viral infections in humans and other animals. LITERATURE CITED 1. Anderson, D.R., and R.A. Nanaker. 1966. Electron microscopic studies of Hycoplasma (PPLO strain 880) in artificial medium and in tissue culture. J. Nat. Cancer Inst. 36:139-154. 2. L-U'mstrong, G., and K. Paucker. 1966. Effect of myco- plasma on interferon production and interferon assay in cell cultures. 3. Bausek, G.B., and T.C. I1erigan. ~'li th 1969. Cell interaction a synthetic polynucleotide and interferon pro- duction in vitro. 4. J. Bacteriol. 92:97-101. Virology 39:491-498. Borden, E.C., R.E. Shope, and F.}\ • .Hurphy. 1971. Phy- siochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus--a new taxonomic group. studies. 5. Physicochemical and serological J. Gen. Virol. 13:261-271. Chanock, R.M., L. Hayflick, and M.F. Barile. 1962. Growth on an artificial medium of an agent associated with atypical pneumonia and its identification as a PPLO. 6. Proc. Nat. Acad. Sci. USA 48:41-49. Cole, B.C., and J.R. Ward. l~ycoplasma 1973. arthritidis and other mycoplasmas \'I]i th murine peritoneal macrophages. 699. Interaction of Infec. Immun. 7:691- 191 7. cottew, G.S., and R.H. Leach. i vlycoplasmas 1969. cattle, sheep, and goats, p. 527-570. of In L. Hayflick (ed.), The Mycoplasmatales and the L-phase of bacteria. X~ppleton-Century-Crofts, 8. New York. DeClercq, E., and T.C. lvlerigan. 1970. Current con- cept of interferon and interferon induction. Annu. Rev. r,led. 21: 17-46. 9. ~d\'lard, D.G. ff., and E.A. Freundt. 1970. Amended nomenclature for strains related to ,(I'Tycoplasma laidlawii. 10. J. Gen. Microbiol. 62:1-2. Fogh, J., and H. Fogh. 1967. Morphological and quan- tita'tive aspects of mycoplasma-human cell relationships. 11. Proc. Soc. Exp. BioI. r·1ed. 125:423-430. Goli~Jhtly-Howland, Niley. 1970. L., B.C. Cole, J.R. \;'Jarc1, and B.B. Effect of animal passage on arthrito- genic and biological properties of 0¥coplasma arthritidis. 12. Gourlay, R.N. Infec. Immun. 1:538-545. 1970. Isolation of a virus infecting a strain of Nycoplasma laidlawii. Nature (London) 225:1165. 13. Gourlay, R.N. 1971. Nycoplasmatales virus-laidlavlii 2, a new virus isolated from Acholeplasma laidlawii. J. Gen. Virol. 12:65-67. 14. Green, J.A., S.R. Cooperband, and S. Kibrick. 1969. Immune specific induction of interferon production in cultures of human blood lymphocytes. 1415-1417. Science 165: 192 15. Grossberg, S.E. ducers: The interferons and their in- 1972. molecular and therapeutic considerations. 11. Sngl. J • .Hed. 287:13-19, 79-85, 122-128. 16. Hayflick, L. Tissue culture and mycoplasmas. 1965. Tex. Rep. Biol. flied. 23 (1) :285-303. 17. Hayflick, L., and E. Stanbridge. 1967. Isolation and identification of mycoplasma from human clinical materials. 18. ~.{eisllJ:nan, Ann. N.Y. Acad. Sci. 143:608-621. J.O., N.O. Olson, and C.J. Cunningham. ""\t."'" 1969. rrransmission of l':1ycoplasma gallisepticu!TI, Nevlcastle disease, infectious bronchitis and combinations in a three-phase broiler house. 19. l·lo~'!(;:ll, P.G., and virus, p. 35-74. D.:.'l. Avian Dis. 13:1-6. Ver~'loerd. 1971. Bluetongue In Virology monographs No.9. Springer-Verlag, Ne\\T York. 20. 3udson, J.R., S.H. Buttery, and G.S. cottew. 1967. Investigations into the influence of the galactan of .l,lycoplasma ~ides that organism. 21. on experimental infection ·""ith J. Pathol. Bacteriol. 94:257-273. Humrneler, K., and D. Armstrong. Observations 1967. on mycoplasma strains in tissue cultures. ..:~:..cad. 22. Ann. N.Y • Sci. 143:622-625. Jones, rr. C., and J.G. Hirsch. 1971. The interaction in vitro of r.:ycoplasma pulmonis with mouse peritoneal macrophages and L-cells. J. :~xp. r·:ed. 133 :231-259. 193 23. !'Zono, Y. 1967. Interferon-like inhibitor produced in bovine leukocyte cultures after inoculation with endotoxin. 24. Liss, l'~., ~;'rch. Gesamte Virusforsch. 21 :277-281. and J. Haniloff. 1971. Isolation of Hyco- plasmatales viruses and characterization of nVL52 , and 1,·1\1G 51. 25. iianiloff, J. 1969. Science 173: 725-727. Use of blood agar plates to study ::!ycoplasma physiology. 26. Hicrobios 1 :125-135. I":ar}cham, J.G., and :N. P. IJIarkham. laidlaHii in human burns. 27. ~NLl, 1969. I'1ycoplasma J. Bacteriol. 98:827-828. J.:urphy, P.A., E.C. Borden, R.E. Shope, and A. Harrison. 1971. Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus-a new taxonomic group. Electron microscopic studies. J. Gen. Virol. 13:273-288. 28. Pidot, I-'... L.R., G. O'Keefe, III, N. !··lcManus, and O.H.• .l,lclntyre. 1972. Human leukocyte interferon: the variation in normals and correlation with PHA transformation. 29. Proc. Soc. Exp. BioI. ~~d. 140:1263-1269. Ranck, R.I·!., L.C. Grumbles, C.F.Hall, and J.E. Grimes. 1970. Serology and gross lesions of turkeys ino- culated vJ'ith avian influenza A Virus, a pararnyxovirus and :',1ycoplasma gallisepticum. 30. Avian Dis. 14:54-65. Hazin, 3., J. Hichmann, and Z. Shimshoni. 1964. The occurrence of ;'1.ycoplasma (PPLO) in the oral cavity of dentulous and edentulous subjects. 43:402-405. J. Dent. Res. 194 31. Hodgers, R., and T.e. Merigan. its inducers: 1972. Interferon and antiviral and other effects. eRC Crit. Rev. Clin. Lab. Sci. 3:131-162. 32. Simberkoff, N.S., and P. Elsbach. 1971. The interac- tion in vitro between polymorphonuclear leukocytes and mycoplasma. 33. J. Exp. Hed. 134:1417-1430. 1969. Singer, S.H., M.F. Barile, and R.L. Kirschstein. ~nhanced virus yields and decreased interferon pro- duction in mycoplasma-infected hamster cells. Proc. Soc. :Gxp. BioI. Hed. 131:1129-1134. 34. Smirnova, 'r.D., and G. Ya. Kagan. 1971. The effect of mycoplasma-viral infection of the primary culture of chick embryo cells on interferon production induced by Langat virus (in Russian, English abstract). ~,:icrobiol. 35. 2h. Epidemiol. Imrnunobiol. 48 (12): 54-58. stanbridge, E. 1971. Mycoplasmas and cell cultures. Bacteriol. Rev. 35:206-227. 36. stanbridge, R., and L. Hayflick. 1967. Growth in- hibition test for identification of Nycoplasma species utilizing dried antiserum-impregnated paper discs. J. Bacteriol. 93:1392-1396. 37. Taylor-Robinson, D., R.B. Purcell, D.C. Chanock. 1966. ~\long, and R.H A colour test for the measurement of antibody to certain mycoplasma species based upon the inhibition of acid production. J. Hyg. 64:91-104. 195 38. Taylor-Robinson, D., N.L. Somerson, H.C. Turner, and R .l~. Chanock. Serological relationships among 1963. hUf.1an mycoplasmas as shown by complement-fixation and gel diffusion. 39. J. Bacteriol. 85:1261-1273. Villemot, J.M., A. Provost, Endotoxin from l~coplasma ~nd R. Queval. mYcoides. 1962. Nature (London) 193:906-907. 40. Yershov, F.I., and V.r1. Zhdanov. 1965. Influence of PPLO on production of interferon in virus-infected cells. 41. Virology 27:451-453. '[;ucker-FranJ<lin, D., H. DaVidson, and L. Thomas. 1966. 'rhe interaction of mycoplasmas with mammalian cells. I. £leLa cells, neutrophils, and eosinophils. J. EXp. Ned. 124:521-532. 42. Zucker-Franklin, D., ~1. Davidson, and L. Thomas. 1966. The interaction of mycoplasmas with mammalian cells. II. 542. Honocytes and lymphocytes. J. Exp. Med. 124:533- 196 TABL3 1. Induction of interferon in ovine leukocyte cultures by media from FLK contaminated with ~. laidlawii Interferon titers (Hours post-inoculation) lZxperiment number Mycoplasma in media from l"LK cells 36 8.23 a 39 7.08 Inoculum of m:lco.elasma 7.l0 b 5.28 24 48 72 96 50 c 90 55 60 20 35 30 20 a. Log lO CFU/ml b. Log lO CFU/ml final concentration of mycoplasma in the inoculated leukocyte cultures c. Unit s/:nl 197 'r.:'l. . BLE 2. Interferon production and mycoplasmal replication in ovine leukocyte cultures Hours post-inoculation .sxperiment number Inoculum of A. laidlavlii 4.57 a 24 48 72 96 r'1ycoplasma 2.48 b 3.25 3.52 4.23 Interferon <20 c <20 20 100 37 11ycoplasma 1.70 1.70 3.23 3.41 Interferon <20 <20 <20 < 20 [i!ycoplasma 4.25 4.34 4.78 5.95 Interferon <20 140 40 85 i"Iycoplasma 2.50 2.84 3.93 4.60 Interferon <20 <20 25 <20 :'lYcoplasma 6.72 6.60 5.90 5.08 Interferon <20 75 25 30 nycoplasma 4.32 4.60 5.08 5.90 Interferon <20 30 45 40 2.57 6.18 39 4.18 6.57 43 4.57 a. LoglO CFU/ml final concentration in the inoculated leUkocyte cultures b. LoglO Cl"U/ml c. units/ml 198 T,~':..}3LJ.: 3. n.eC:~l.lirement of viable ~. laidla~ for induction of interferon in ovine leukocyte cultures Hours post-inoculation Inducers l~. l~ssa:i 24 48 72 96 1'1ycoplasma 6.72 a 6.60 5.90 5.08 Interferon < 20 b 75 25 30 laidlawii ;ieat-treated l-:ycoplasma 1.30 < 1.30 <1.30 2.48 A. Interferon <20 <20 <20 <20 rrycoplasma <1.30 <1.30 <1.30 <1.30 Interferon <20 <20 <20 <20 nycoplasma -c <1.30 <1.30 Interferon <20 <20 ,LiIycoplasma <1.30 <1.30 Interferon <20 <20 laidla~'lii ----....' -.... Fil'trate of laidla\'liiA• .-......-.. sterile mycoplasma broth Done b. 10 CF'U/ml Units/reI c. ~Jo a. L09 sample tal(en 1"1_~.:.BLS 4. Interferon production and mycoplasmal re?lication in leukocytes from different ovine donors Hours post-inoculation Experiment number Leukocyte donor Inducer ~. Bluetongue Virus 24 48 72 96 I1ycoplasma 4.25 a 4.34 4.38 5.95 Interferon < 20 b 140 40 85 Interferon 600 700 600 700 Hycoplasma 4.18 4.48 5.00 5.50 Interferon <20 <20 <20 <20 Interferon 100 250 350 250 Mycoplasma 4.90 5.15 5.70 Interferon <20 1100 210 230 1000 900 Laidlawii S'tve :ft:7 39 Assay A. laidlawii Ewe #46 Bluetongue Virus A. laidlawii Evle ~47 Bluetongue Virus 40 A. laidlawii E\ve :fl:46 Bluetongue Virus a. h. c. Log CFU/rnl Uni s/ml No sample taken Eo Interferon rvIycoplasma 4.93 6.18 5.74 Interferon 25 40 45 Interferon 550 300 500 -c ...... \.0 \.0