Chemotherapy of a herpesvirus hominis infection in newborn mice

Infection of the newborn mouse with the genital (type 2) strain of Herpesvirus hominis (HVH), provides an experimental infection that closely resembles a disseminated HVH infection of the human newborn infant. This experimental infection was utilized to evaluate the therapeutic effectiveness of 5-iodo-2'-deoxyuridine (IUDR), 1-B-D arabino-furanosylcytosine (Ara-C), 9-B-D arabinofuranosyladenine (Ara-A), polyriboinosinic-polyribocytidylic acid (poly I:C), and exogenous interferon. Experiments designed to evaluate the effectiveness of these compounds included: 1) the effect of therapy on mortality of HVH type 2 infected mice, 2) the effect of treatment on the pathogenesis of the infection, 3) the relative sensitivity of the virus to each drug, and 4) the levels of drug activity obtained in target organs. The pathogenesis of HVH infection in suckling mice following intranasal inoculation involves primary replication of virus in the respiratory tract, followed by a low level viremia. Target organs such as liver, spleen, and brain are subsequently seeded with virus from the blood. The olfactory and trigeminal nerves served as alternate pathways for the virus from the nasopharynx to the central nervous system (CNS). Death of the animals appeared to result primarily from encephalitis. Therapy with IUDR had no effect on final mortality or on the mean survival time, although a significant effect on the pathogenesis of the infection was observed. Virus replication in the lung was reduced and the viremia as well as subsequent involvement of liver and spleen was completely inhibited. In contrast nerve route transmission to and replication of HVH in the CNS was not affected by therapy. The lack of inhibition of viral replication in the CNS appeared to be due to inadequate levels of IUDR in brain tissue, and is the likely explanation for the therapeutic failure of IUDR in this model infection. Therapy with ara-C did not reduce final mortality, but it did significantly increase the mean survival time by one day of those animals that died. The effects of ara-C treatment on the pathogenesis of the infection were characterized by a one day delay in the appearance of HVH in blood, liver, and spleen, and a reduction of virus replication in lung and brain, which also lasted for about one day. Treatment with ara-A also failed to affect final mortality, but increased the mean survival time by two days. This was highly significant. Therapy with ara-A markedly altered the pathogenesis of HVH infection. There was a delay or a suppression for two days of virus replication in blood, lung, liver, spleen and brain. The alteration of HVH pathogenesis in target organs of animals treated with either ara-C or ara-A coorelated with the increase observed in the mean survival time. In this experimental HVH infection of newborn mice, ara-A appeared to be more effective than was ara-C. Neither of the two drugs, however, was completely effective in inhibiting viral replication in target organs, or protecting the animals from a lethal HVH infection. Treatment with poly I:C resulted in only low levels of protection, a significant increase in the mean survival time, and a marked alteration of pathogenesis of infection. Virus replication was completely inhibited in all organs tested except brain. CNS involvement was delayed, however, for two days. Administration of 3000 units/day of exogenous interferon was not effective in decreasing final mortality, or in altering the mean survival time. The failure of interferon or interferon inducers to alter the mortality of newborn mice infected with HVH appears to be primarily due to insufficient levels of interferon in the CNS.

CHEMOTHERAPY OF A HERPESVIRUS HOMINIS INFECTION IN NEWBORN MICE by Earl Ray Kern A dissertation submitted 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 Earl Ray Kern has been approved July 1973 Me r . I ~)l~#J[wtJO ~ .• "d.~~ .... C~, Major tterPaitiilent ') --<I.#Jfi:Je lJfdu~1ts~/;;t ; 4~( I ACKNOWLEDGEMENT The author would like to express his appreciation to Dr. Lowell A. Glasgow for his support and guidance through­out the course of these investigations and the preparation of this dissertation. Gratitude is also extended to Dr. James C. Overall, Jr., for his helpful suggestions and contributions to these studies. He would also like to thank Dr. Douglas W. Hill, Dr. stanley Marcus, Dr. Arthur D. Broom, and Dr. John B. Hibbs, Jr., for their suggestions and criticisms and the faculty of the Department of Microbiology for their contributions to the author1s education. The author wishes to extend special appreciation and gratitude to his wife, Aloma, and our children Barry and Scott for their support, patience, and understanding throughout these studies. Further acknowledgement is given to Aloma for typing and proofreading this dissertation. The research reported herein was partially supported by Contract No. NIH 70-2133 from the Antiviral Substances Program of the National Institute of Allergy and Infectious Disease and by Grant No. AI-I02l7 from the National Institute of Allergy and Infectious Disease. iii TABLE OF CONTENTS ABSTRACT • • • • • • • • . . . . . . . . . . . · . vii INTRODucrrION I RATIONALE, OBJECTl YES . . . . . 1 REVIE~ OF LITERATURE · . . . . . . 4 I. Herpesvirus hominis (HVH) infections of humans • • • • • • • • • • • • • •• 4 A. Differentiation of HVH types 1 and 2 4 B. Mode of transmission to the human C. D. newborn · . . . . . Pathogenesis of infection Frequency and severity of neonatal infections ••••••••••• 5 6 · . 7 II. Treatment of HVH infections of humans. • 9 A. Gamma globulin · . . . . . . . . 10 B. IUDR . . . . . . . . . . . . . . . . 10 C. Ara-C. . . . . . 12 D. Ara-A • . . . . . . . . . . . . . 13 E. Interferon and interferon inducers 13 III. Experimental HVH infections in animal models •••••••••• • • • 14 A. Description of experimental systems. 14 B. pathogenesis of HVH infections in experimental animals •••••• · . 15 IV. Effect of anti viral agents aga.inst HVH in tissue culture and animal model infections 18 A. lUDR . . . . . . . . . . . . . . . . 18 page B. Ara-C · • • • 20 C. Ara-A • • • • 21 D. Interferon and interferon inducers 23 V. Mechanism of action and pharmacology of antiviral agents • • • • • • 25 A. IUDR • • • • • • • 25 B. Ara-C · • • • • 26 C. Ara-A • • • • • • • 28 D. Interferon • • 29 VI. Summary • • • • • 31 VII. Literature Cited • • • 32 ~XPERIP~NTAL RESULTS · • • • • 49 I. Herpesvirus hominis infection of newborn mice: I. Development of an experimental model and therapy with iododeoxyuridine 50 Abstract • • • • 51 Introduction • • • • • 52 Materials and methods · • 54 Results . • • • • • • 57 Discussion • • • • • • • 67 References • • • • 74 Tables • • • • 80 Figures • • 82 II. Herpesvirus hominis infection of newborn mice: II. A comparison of the thera-peutic efficacy of Ara-C and Ara-A 93 Abstract • • • 94 v III. VITA Introduction Materials and methods Results Discussion Literature cited Tables • Figures Herpesvirus hominis infection of newborn mice: III. The effectiveness of inter­feron and interferon inducers Abstract Introduction • :tvlaterials and methods Results Discussion Literature cited • Tables • • • vi Page 96 98 102 113 124 132 136 146 147 148 150 154 165 174 180 188 ABSTRACT Infection of the newborn mouse with the genital (type 2) strain of Herpesvirus hominis (HVH), provides an experi­mental infection that closely resembles a disseminated HVH infection of the human newborn infant. This experimental infection was utilized to evaluate the therapeutic effec­tiveness of 5-iodo-2 1-deoxyuridine (IUDR), l-B-O arabino­furanosylcytosine (Ara-C), 9-B-D arabinofuranosyladenine (Ara-A), polyriboinosinic-polyribocytidylic acid (poly I:C), and exogenous interferon. Experiments designed to evaluate the effectiveness of these compounds included: 1) the effect of therapy on mortality of HVH type 2 infected mice, 2) the effect of treatment on the pathogenesis of the in­fection, 3) the relative sensitivity of the virus to each drug, and 4) the levels of drug activity obtained in target organs. The pathogenesis of HVH infection in suckling mice following intranasal inoculation involves primary repli­cation of virus in the respiratory tract, followed by a low level viremia. Target organs such as liver, spleen, and brain are subsequently seeded with virus from the blood. The olfactory and trigeminal nerves served as alternate pathways for the virus from the nasopharynx to the central nervous system (CNS). Death of the animals appeared to result primarily from encephalitis. Therapy with IUDR had no effect on final mortality or on the mean survival time, although a significant effect on the pathogenesis of the infection was observed. Virus replication in the lung was reduced and the viremia as well as subsequent involvement of liver and spleen was completely inhibited. In contrast nerve route transmission to and replication of HVH in the CNS was not affected by therapy. The lack of inhibition of viral replication in the CNS appeared to be due to inadequate levels of IUDR in brain tissue, and is the likely explanation for the therapeutic failure of IUDR in this model infection. Therapy with ara-C did not reduce final mortality, but it did significantly increase the mean survival time by one day of those animals that died. The effects of ara-C treat­ment on the pathogenesis of the infection were characterized by a one day delay in the appearance of HVH in blood, liver, and spleen, and a reduction of virus replication in lung and brain, which also lasted for about one day. Treatment with ara-A also failed to affect final mortality, but in­creased the mean survival time by two days. This was highly significant. Therapy with ara-A markedly altered the patho­genesis of HVH infection. There was a delay or a suppression for two days of virus replication in blood, lung, liver, spleen and brain. The alteration of HVH pathogeneSis in target organs of animals treated with either ara-C or ara-A coorelated with the increase observed in the mean survival viii time. In this experimental HVH infection of newborn mice, ara-A appeared to be more effective than was ara-C. Neither of the two drugs, however, was completely effective in inhibiting viral replication in target organs, or pro­tecting the animals from a lethal HVH infection. Treatment with poly IIC resulted in only low levels of protection, a significant increase in the mean survival time, and a marked alteration of pathogenesis of infection. Virus replication was completely inhibited in all organs tested except brain. CNS involvement was delayed, however, for two days. Administration of 3000 units/day of exogenous inter­feron was not effective in decreasing final mortality, or in altering the mean survival time. The failure of interferon or interferon inducers to alter the mortality of newborn mice infected with HVH appears to be primarily due to insufficient levels of interferon in the CNS. ix INTRODUCTION, RATIONALE, OBJECTIVES Herpesvirus hominis infection of the human newborn infant constitutes a wide spectrum of disease including subclinical infections, encephalitis, and a disseminated form with or without central nervous system involvement. In the more severe forms of HVH infections it has been esti­mated that greater than 50% of untreated cases may terminate fatally or result in permanent neurological sequelae. These estimates point out the seriousness of these infections and the obvious need for effective therapy. While various types of treatment have been attempted in these infections, con­trolled studies have not been carried out to definitively prove the efficacy of any chemotherapeutic agent in the treatment of herpesvirus infections. The most widely used form of therapy for disseminated infections of the neonate and encephalitis in older individuals has been the use of IUDR, and while there have been some reported successes, its effectiveness has not been proven. Although IUDR has been efficaceous in the topical treatment of herpesvirus keratitis in animal model studies, it has generally failed in the treatment of encephalitis or generalized infections. There is virtually no information available, however, on why the drug is not effective, nor how chemotherapy can be made more effective. While the 2 newer more promising antiviral agents, ara-C and ara-A have been shown to be highly effective against HVH in vitro, there is little animal model data available demonstrating their therapeutic effectiveness, especially against a dis­seminated HVH infection. Since these two compounds are currently being utilized in human trial studies, additional data are needed in animal models which are similar to the human disease for which they are going to be used to com­plement that obtained in humans. While there are diffi­culties in transferring information obtained in experimental animals directly to the treatment of human infections, valuable information may be obtained which might be applied to make chemotherapy more effective. Data relating to the sensitivity of the virus to the drug, the distribution of the drug to infected target organs, and the effect of treat­ment on the pathogenesis of the infection can best be ob­tained and evaluated in tissue culture and in experimental animal infections. The objective of these studies was to attempt to de­lineate some of the factors involved in the success or fail­ure of the treatment of herpesvirus infections of the human newborn. These studies were performed utilizing the newborn mouse as an animal model which closely resembles the human newborn infection. Since the oral route is the most likely portal of entry for the virus in the human infection, new­born mice were infected with a strain of HVH type 2 (genital type) by the intranasal route. utilizing this model 3 infection we have determined the therapeutic efficacy of IUDR, ara-C, ara-A, poly I:C, and exogenous interferon. Experiments designed to test the efficacy of these compounds included (a) elucidation of the pathogenesis of the disease in newborn mice, (b) establishment of toxicity levels for each of the drugs, (c) evaluation of therapeutic efficacy using various drug concentrations and treatment schedules, (d) comparison of the sensitivity of the virus strain used with other virus strains, (e) determination of the effect of treatment on the pathogenesis of infection, and (f) measurement of the levels of drug activity in target organs. Based on the necessity for evaluation of many parameters and not just the effect of treatment on mortality, it is felt that the proposed animal model simulates the human newborn herpesvirus infection and data obtained from these studies potentially may elucidate some of the factors in­volved which determine the efficacy, or lack of efficacy, of antiviral agents in the treatment of HVH infections of humans. REVIE~v OF LITERATURE I. Herpesvirus hominis infections of humans Herpesvirus hominis (HVH) (Herpes simplex virus, HSV) is one of the most common viral agents affecting man. In­fections caused by this virus range from asymptomatic, sub­clinical infections to severe, often fatal diseases. The most frequent sites of infection are the skin and mucous membranes. Skin sites most often affected are the lips and mouth (24) and the digits of the hands (108). Mucous mem­branes most commonly involved are in the mouth (100), eyes (44), and the female genital tract (90). ~ihile disseminated herpesvirus infections of neonates with or without central nervous system (eNS) involvement (75), and HVH encephalitis of adults (79) occur less frequently, their severity makes them high priority candidates for chemotherapy. Since the disseminated form of HVH infection in newborn infants is to be the subject of this dissertation, the other types of in­fections will not be discussed further. A. Differentiation of HVH types 1 and 2 The major source of infection by HVH in the human newborn infant is the infected genital tract of the mother at the time of delivery (70, 76). Nahmias and coworkers have shown that over 95% of all genital herpesvirus isolates and more than 80% of all HVH isolations from newborn infants belong to the antigenic type 2 group (75). Type 1 strains have primarily been recovered from the mouth, eyes, lips, and eNS of older individuals. In general, type 1 strains are from oral or non genital sites and type 2 strains are 5 of genital origin (25). In addition to these epidemiologi­cal differences the two virus types can also be separated on the basis of serological tests (72, 82, 89), cytopathic effects and infectivity titers in tissue culture cells (72, 106, 89), plaque production in chick embryo cells (15, 16), pock production in embryonated eggs (72, 73), growth at different temperatures (95), neurovirulence in animals (89, 87, 88, 72), density gradient centrifugation (41, 89), and immunofluorescent tests (74, 37). Although each of these methods has demonstrated distinct differences between HVH types 1 and 2, it has been suggested that this classification into two groups is too rigid, and that a whole spectrum of variants may exist (96). B. Mode of transmission of HVH to newborn An analysis of 156 cases of herpesvirus infections of human neonates compiled by Nahmias and associates (75) has indicated that a maternal genital infection is the major, but probably not the only, source of HVH to the newborn. The virus can be acquired by the newborn from the mothers genital tract during passage through an infected birth canal (116, 117), by an ascending route, particularly if membranes have been ruptured (119, 103, 110, 107), or by transplacental infection (68, 103, 118, 36). The portals of entry for the virus when the newborn infant becomes in­fected from the mother's genital tract are the skin, the umbilical cord, the eye, and oral and respiratory orifices (75). C. pathogenesis of infection 6 Infection of the newborn infant with HVH can re­sult in one of three classifications of disease: 1) local­ized, or asymptomatic form of infectionr 2) a disseminated form without CNS infection; and 3) a disseminated form with CNS involvement. The localized infection is usually mani­fested in the skin, eyes, oral cavity, or the eNS (75). The disseminated form involves primarily the liver and adrenal glands, as well as the brain, lungs, kidneys, spleen, pan­creas, heart, and stomach (3, 13). Histopathologic and virologic studies of disseminated HVH infections of newborn infants have generally indicated that there are four pro­gressive stages during the course of infection (75). In the first stage, following initial replication of virus at the portal of entry, a primary viremia develops and the most susceptible organs become infected. In the second stage the virus disappears from the blood and replicates in tissues of infected organs. At this point there is histologic evi­dence of cell damage. In the third stage a secondary viremia develops due to the virus production in the initially infected organs and additional organs are then seeded. There can be extensive cell damage in this stage. In the final stage the host either dies, or virus is cleared from 7 the blood with diminishing amounts of virus being detectable in organs. Tissues then may undergo cellular regeneration and repair of damage. During the course of HVH infection the amount of cellular damage incurred and the number of organ systems involved may be quite variable, thus, the manifestations and outcome of clinical disease may also be variable. In addition to viremic spread of HVH, data from infections of experimental animals have indicated that her­pesvirus may also be spread by multiple neurogenic routes (52, 72, 87, Kern, E. R., J. C. Overall, Jr., and L. A. Glasgow, J. Inf. Dis., In Press). Although this mechanism of spread has not been documented in human disease, it could explain the existence of localized CNS infection where peripheral, but no disseminated, involvement could be demonstrated. D. Frequency and severity of neonatal HVH infections An estimate of the frequency of herpesvirus infec­tions of newborn infants has been calculated by determining the number of neonatal HVH infections per number of deliveries during a certain time period (75). These calculations from two hospitals in different parts of the country have yielded estimates of one clinically diagnosed case for every 7,500 deliveries. Based upon these estimates it has been suggested that a minimum of 120 cases of neonatal herpes­virus infections would be expected each year in this country. Neonatal HVH infection is frequently considered to result in a disseminated fatal disease. This concept is 8 probably the result of recognition of many cases at autopsy. In a review of 148 cases by Nahmias and coworkers (75) 98 patients had a disseminated form of disease. The total mortality rate of these infections was 96%. The 50 cases that had localized or asymptomatic forms of infection had a case fatality rate of only 22%. Approximately half of the survivors of this group, however, suffered from severe neurologic or ocular sequelae. The overall mortality rate of both groups was 71%. out of the total of 148 cases, 66 had been diagnosed during the course of the clinical illness. The mortality rate of this group was 35%, 24 had dissemi­nated disease with 20 of these patients terminating fatally (83%) and two additional patients suffered subsequent sequelae. There were 42 patients who had a localized form of H~i infection, 17 of these with eNS involvement, and 23 at other sites1 two were asymptomatic. Of those 17 having eNS infection, only three died, however an additional 12 suffered neurological damage. The 23 cases of localized infections at sites other than the eNS, showed that none died, and eight suffered sequelae. There was little difference in the mortality rates between the total number of cases reviewed (96%), and the cases diagnosed clinically (83%) for patients who had disseminated HVH infections. Likewise, cases of local­ized eNS infections in both groups had a poor prognosis in terms of either death or permanent neurological damage. These data suggest that with increased awareness of HVH 9 infections of newborns on the part of physicians and im­proved diagnostic procedures for isolation of HVH, the overall mortality rate for HVH infections of human neonates may be somewhat lower than previously thought. It is evi­dent, however, that disseminated infections or those involving the CNS will continue to have a poor prognosis. Based upon estimates of frequency and severity of HVH in­fections of humans, there is justification for the develop­ment of effective chemotherapeutic agents for treatment of these infections. II. Treatment of HVH infections of humans Herpesvirus hominis infections of the human newborn infant may be manifested by a wide spectrum of diseases. In the more severe form of infection, it has been estimated that greater than 50% of untreated cases may terminate fatally or result in permanent neurological damage. The severity of these infections clearly establishes the need for effective therapy. Various modes of treatment such as gamma globulin, 5-iodo-2 1 -deoxyuridine (IUDR), l-B-D arabi no­furanosylcytosine (Cytosine Arabinoside) (Ara-C), and poly­riboinosinic acid - polyribocytidylic acid (poly I:C) have been utilized for HVH infections of humans. Another drug, 9-B-D arabinofuranosyladenine (Adenine arabinoside) (Ara-A), is currently being utilized in controlled human trials, however, no reports of its effectiveness have been published at this time. 10 A. Gamma globulin Passive gamma globulin has been given to a number of neonates either prior to the appearance of clinical signs of HVH infection or after the onset of disease (36, 116, 117). There was evidence in only one case (36) that any alteration of the course of infection had occurred. Severe or fatal disease ensued in all others treated. In the case reported by Wheeler and Huffines (116) gamma globulin was administered both to the mother prior to delivery and to the infant after delivery, but failed to alter the infec­tion in either the newborn or the genital infection of the mother. Failure of gamma globulin to alter the course of infection in herpesvirus infections of humans has been attributed to late administration after infection was al­ready es~ablished, the lack of sufficient HVH type 2 anti­bodies in the pooled globulin, or an ineffective route of administration. An additional explanation based on data obtained in experimental animals is that antibody is not effective against spread of HVH along neural routes (20, 26). B. IUDR The most widely utilized form of chemotherapy for HVH infections of humans has been with IUDR and has resulted in reports of both successes and failures. In some cases complete recovery from infection with no apparent neurologi­cal damage has been achieved (40, 81, 86, Ill), while in others, survivors had residual brain damage (15, 40, Ill). 11 The third type of cases reported are those in which the drug was not effective and the patient died (32, 86). Some of these authors have suggested that neurological damage in those infants that survived was the result of late initiation of IUDR therapy, after involvement of the CNS had already occurred. An additional explanation could be that IUDR did not reach the CNS in sufficient concentration to completely inhibit HVH replication. This explanation is strengthened by one reported case where virus was recovered from brain tissue following IUDR therapy (32). One of the major pro­blems involved in evaluating the efficacy of an antiviral agent in HVH infections of the newborn is that the course of disease is quite variable and does not always result in death or neurological sequelae (75). Complete recovery has been documented in infants who did not receive treat­ment (38). IUDR has also been reported to be effective in the treatment of herpesvirus encephalitis (77), which is most often caused by the type 1 strain of HVH (72). These have also been individual case reports and efficacy has not been confirmed by controlled studies (55). In a number of the cases of encephalitis treated with IUDR, diagnosis has been by serological tests (54), and has not been substantiated by virus isolation from brain biopsy. An additional problem in interpreting these case reports is the variability of the untreated disease (51). Additionally a number of patients who survived encephalitis following IUDR therapy have had 12 significant neurological damage. Since no controlled studies have been carried out utilizing IUDR for the treatment of either herpesvirus infections of the newborn, or HVH encep­halitis, it has not been possible to evaluate its effective­ness against these infections in humans. C. Ara-C The apparent lack of effectiveness of IUDR, as well as its toxicity, has stimulated investigators to search for better compounds directed against herpesvirus infections. These efforts have resulted in the discovery of a group of compounds which interfere with herpesvirus nucleic acid synthesis. One of these compounds, ara-C, has been primarily used as an anti-tumor drug, but also exhibits antiviral activity against members of the herpesvirus group. The use of ara-C in the treatment of disseminated HVH infections of human newborn infants has not been reported, however Chow and coworkers (17) treated two neonates, and four adults suffering from herpesvirus encephalitis. Both neonates, and two of the four adults survived. Clinical improvement was reported to be closely related to the initiation of therapy with ara-C. In one of the two adults that died, no virus could be isolated from the brain following four days of ara-C therapy. Of the four patients who survived, one neonate and one adult suffered neurological sequelae. Only one other case of HVH encephalitis treated with ara-C has been reported (27). In this case clinical improvement was also temporally related to the ara-C therapy. Successful results have been reported in the treatment of disseminated varicella-zoster infections (16, 49), however the failure of ara-C to alter the course of these infections has also been documented (22). The first controlled study of the efficacy of ara-C 13 against varicella-zoster infections has demonstrated that the drug is not effective (Medical World News, June 8, 1973). Although ara-C is currently being utilized in the treatment of HVH infections of humans, its effectiveness has not yet been evaluated in controlled studies. D. Ara-A The purine nucleoside, ara-A, has been reported to be highly effective against HVH both in tissue culture and in experimental animals. Although ara-A is currently being tested against human herpesvirus infections in con­trolled trials, there have been no published reports on its effectiveness. E. Interferon and interferon inducers Although interferon and interferon inducers have been widely used in experimental animal virus infections, their use in human infections has been quite limited. They have been utilized for the prevention or treatment of vaccinia virus infections, herpesvirus keratitis (43), and upper respiratory virus infections. They have not been utilized for the treatment of disseminated HVH infections of the human neonate and there has been only one report of their use in herpesvirus encephalitis (4). Bellanti and his colleagues treated a young male infant with poly I:C and although the patient appeared to improve clinically, a therapeutic effect was not definitely established. III. Experimental HVH infections in animal models 14 It is generally accepted in this country that no new potential antiviral compound can be tested against human disease until its effectiveness has been documented in similar infections of experimental animals. Effective dosages, toxicity levels, and side effects of experimental drugs have usually been determined in animals prior to their use in humans. Animal models utilized for evaluation of new compounds to be used in the treatment of herpesvirus infections, have primarily been designed to simulate HVH encephalitis, or herpesvirus keratitis. There has not been an animal model, however, which closely simulates a dis­seminated HVH infection of the newborn. A. Description of experimental systems Animals utilized for the evaluation of antiviral agents in herpetic skin infections have primarily been mice (113, 114, Lieberman, L., T. W. Schafer, and P. E. Came, sept. 1971, personal communication), and rabbits (33). Animals viere infected by a cutaneous route, treated with the antiviral agent, and the inhibition of lesion progression analyzed in comparison with an untreated control animal. Hamsters and rabbits have both been utilized as experimental animals for herpesvirus keratitis or keratoconjunctivitis 15 (56, 80, 102, 112). The corneas of the animals eyes were scarified and inoculated with herpesvirus. Therapy was administered either locally or systemically and the magnitude of infections of treated and control animals was compared. The most popular model infection for evaluation of efficacy of compounds to be used for herpesvirus infections has been HVH encephalitis in adult mice (2, 12, 14, 67, 104, 105). Both type 1 and 2 herpesvirus encephalitis can be produced in these animals following intracerebral, subcutaneous, or intraperitoneal inoculation of virus. The endpoint has usually been death, and the effectiveness of antiviral agents was evaluated according to its ability to reduce final mortality or increase the mean survival time. B. pathogenesis of HVH infections in experimental animals The pathogenesis of herpesvirus infections of animals is varied depending upon the age of the animal, the route of inoculation, and possibly the strain of virus. Johnson (52), utilizing fluorescent antibody staining, in­vestigated the pathogenesis of HVH type 1 infection of suckling mice following varied routes of inoculation. In­tracerebral inoculation resulted in spread of virus by way of the cerebrospinal fluid following initial replication in cells of the meninges and ectodermal lining of the ven­tricles. Infection then spread into the eNS. There was no significant extraneural growth, however, a few virus particles were detected in blood, liver or spleen. 16 Following intraperitoneal inoculation, a viremia developed between two and five hours after injection and continued until death. Rapid virus multiplication occurred in liver and spleen, but virus was not detected in the eNS until three days following inoculation. Infection of the eNS was by hematogenous spread with virus antigen first appearing around small cerebral blood vessels. subcutaneous ino­culation in the foot pad resulted in infection of some cutaneous cells and the endoneural cells of subcutaneous nerve fibers. No viremia was detected and there was no infection of visceral organs. The virus reached the eNS solely by a neural route. When herpesvirus was inoculated intranasally, there was a rapid initial multiplication in the lungs followed by a low grade viremia. Virus was spread to the liver and spleen on the second day but did not appear in the eNS until the third day. Virus gained access to the eNS by multiple neural pathways and by blood borne infec­tion. Direct invasion of the subarachnoid space, infection of cells along both olfactory and trigeminal nerves and evidence of hematogenous spread were found. Neural spread of herpesvirus along trigeminal and olfactory nerves following intranasal inoculation of mice has also been reported by Rabin and associates (93) and Rajcani and coworkers (94). In neither of these infections, however was a viremia detected. The ability of herpesvirus to gain access to the eNS by peripheral nerves, without systemic involvement, following foot pad inoculation has also been confirmed (58, 99). Nasal-oral inoculation of newborn pups with Canine herpesvirus results in a pattern of pathogenesis similar to that seen in the newborn mouse infected with HVH. The major sites of involvement were lung, liver, spleen, brain, spinal and sympathetic gang­lions, and nerve trunks (10, 83, 84). In HVH infections, age is an important host determinant in the development of encephalitis. It has 17 been amply documented that mice develop reSistance only to virus inoculated by extraneural routes (57, 59, 97). In an attempt to determine at what level this resistance occur­red, Johnson (53) inoculated various age animals with herpesvirus by a variety of routes and determined the manner of virus spread for each age group and route of inoculation. Fluorescent antibody staining of brain tissues at inter­vals following intracerebral inoculation showed that the pattern of virus growth was the same as previously observed in suckling mice (52). Following extraneural inoculation of virus in adult mice, initial virus replication occurred in the same cells as were initially infected in suckling mice. However, this infection failed to spread to the eNS. Following intraperitoneal inoculation, there was only a minimal amount of virus recovered from liver and spleen at 24 hr and all had disappeared by 48 hr. In contrast to the rapid multiplication of virus in the lungs of suckling mice following intranasal inoculation, only small amounts of virus were recovered from the lungs in adult animals. 18 The replication of HVH in eNS and the tissues initially in­fected were similar in adult and suckling mice. In adult mice the subsequent spread of the virus was limited. The inhibition of spread of virus inoculated by peripheral routes increased with the age of the animal and could be coorelated with resistance of the macrophage. The role of the macro­phage in the development of resistance to herpesvirus in­fections was subsequently documented by other investigators (47, 109). It has been reported that there are differences in the pathogenesis of infections caused by type 1 and type 2 herpesviruses (72). Plummer found a higher incidence of paralysis in rabbits inoculated with a type 2 strain than in those that received type 1. Additionally mice receiving type 2 virus progressed to encephalomyelitis, while those inoculated with type 1 did not (87). Differences in viru­lence of the two HVH strains in mice have also been reported by other investigators (I, 71, 88). IV. Effect of antiviral agents against HVH in tissue culture and animal model infections A. IUDR One explanation for the variable results reported for IUDR treatment of human HVH infections is that some strains may be more resistant to the drug than others. It has also been proposed that there may be a difference in sensitivity to IUDR between type 1 and type 2 strains. Person and coworkers (85) have reported that in chick embryo 19 fibroblast and WI-38 cells, type 1 strains were more sen­sitive than type 2 strains. However, in HeLa cells they found little difference in sensitivity between the two strains. Lerner and Bailey (60) have also reported type 1 strains to be more sensitive to the action of IUDR than type 2 strains in BHK-2l cells. In contrast to these data Walker and associates (115) reported that IUDR was effective against type 1, but not against type 2 strains in ~I-38 cells. The therapeutic effectiveness of IUDR against herpesvirus infections in animals appears to be dependant upon the clinical manifestations of the infection being treated. Kaufman (56) and others have demonstrated the effectiveness of IUDR against herpes simplex keratitis in the rabbit and its efficacy against skin infections induced in rabbits has also been reported (33). Sidwell and co­workers (101) have reported that vaccinia virus infection of mice following intracerebral inoculation was not altered by IUDR therapy, however, if animals were inoculated intra­nasally, the drug was effective in prolonging the mean survival time. These authors suggested that the drug did not cross the blood-brain barrier, but was effective against infections in other parts of the body. IUDR has also been reported to be totally ineffective against Herpes simplex virus inoculated intracerebrally (104) and that it had no therapeutic effect on systemic herpesvirus infections of mice (45). Although this drug has been utilized for the 20 treatment of disseminated herpesvirus infections of the human newborn, data evaluating its effectiveness against systemic infection in animal models are lacking. Furthermore the effect of treatment on the pathogenesis of infection and the tissue distribution of the drug have not been adequately defined. B. Ara-C The use of gamma globulin, IUDR, and other ex-perimental compounds for treatment of herpesvirus infections of human newborns has proved to be unacceptable either be-cause of lack of efficacy, toxicity, or too few numbers of patients to demonstrate significant effectiveness. The dis-covery of a group of nucleoside analogues that possess pro- \ perties of being inhibitors of nucleic acid synthesis has led to increased use of these compounds as antiviral agents against herpesvirus infections. One member of this group of compounds, ara-C, has been used principally as an anti-tumor drug, but has also demonstrated antiviral activity in many experimental systems including those infected with members of the herpesvirus group. Ara-C has been reported to be highly inhibitory to HVH in a .variety of cell culture systems. Buthala (7) demonstrated its effectiveness in rabbit, mouse, rat, chicken, guinea pig, and human cells. Ara-C was as effective as IUDR and herpesvirus did not be-come increaSingly resistant to ara-C as it did to IUDR. utilizing monkey kidney cells and two human cell lines (WI-38, HeLa) Walker and coworkers (115) concluded that 21 ara-C was at least as effective as IUDR against both type 1 and type 2 herpesviruses, and also that it exhibited less cell toxicity. Fiala and his associates (28) have reported similar data with type 1 strains being slightly more sen­sitive to ara-C than type 2 strains. Although ara-C is highly effective against HVH in tissue culture, there are relatively little data in the literature elucidating its effectiveness against herpesviruses in experimental animals. Underwood (112) has reported that ara-C was as effective in the treatment of herpes simplex keratitis in rabbits, as was IUDR. It has also been reported that ara-C had no effect on cutaneous herpesvirus infections of mice (Lieberman, M., T. W. Schafer, and P. C. Came. Sept., 1971, personal communication). utilizing a mouse model for target organ treatment of herpesvirus inoculated intracerebrally, Allen and Sidwell (2) reported that ara-C increased the number of survivors among treated mice. However, the drug was also inoculated intracerebrally. C. Ara-]\... Another one of the nucleic acid synthesis inhi­bitors showing promise for use in chemotherapy of herpesvirus infections is ara-A. Experimental data from studies both in vitro and in vivo have demonstrated sufficient efficacy against these infections that controlled human trials for this drug are being carried out. In a variety of cell CUl­ture systems it has been reported that ara-A possesses as much antiviral activity against herpesviruses as ara-C and 22 may be less toxic to human cells. In a comparison of the effectiveness of the two drugs, Miller and coworkers (66) reported that ara-A and ara-C had similar antiviral effects in HVH infected KB and Hep-2 cells, and that drug resistance did not develop to either compound. When comparing the sen­sitivities of HVH types 1 and 2, Person and associates (85) found that the two strains were equally sensitive to ara-A in WI-38 cells. In chick embryo cells, type 1 strains were more sensitive than type 2, however, in HeLa cells type 2 strains were slightly more sensitive than were the type 1 strains tested. Data from in vivo experiments in mice have indicated that both antigenic types are of equal sensitivity to ara-A (105). Animal model studies have demonstrated that ara-A is effective against herpes simplex keratitis in hamsters (102), and that treated animals had a decreased incidence and severity of encephalitis following herpetic keratitis (98). Sloan and coworkers (104) have reported that ara-A was effective against intracerebral HVH infections of mice when administered intraperitoneally, subcutaneously, or perorally. Therapeutic activity could be demonstrated when the drug was administrated as late as 24 hr post infection. Under these conditions ara-A exhibited marked activity while IUDR was ineffective. Ara-A has also been reported to be effective in controlling lesion development in a cutaneous herpesvirus infection of hairless mice when ara-C was not effective (Lieberman, M., T. W. Schafer, and P. E. came, sept., 1971, personal communication). 23 Further evidence from in vitro experiments indicates that ara-A is as effective against HVH as either ara-C or IUDR. The few animal model studies that have been reported suggest that ara-A is more effective against herpesvirus infections than IUDR or ara-C and may become the drug of choice in the treatment of human herpesvirus infections. D. Interferon and interferon inducers For interferon or an interferon inducer to be effective against a virus infection in man, the particular virus should be relatively sensitive to the action of in­terferon. Members of the Herpesvirus group are generally regarded as being relatively insensitive to the effects of interferon (9, 39, 50), but the type 2 strains of HVH have been reported to be more sensitive to interferon than type 1 strains (78). The effectiveness of exogenous interferon in experimental herpesvirus infections has not been docu­mented, however, the interferon inducer, poly I:C, has been reported to be effective against herpetic keratitis in rabbits and HVH encephalitis in mice. Park and Baron (80) reported that administration of poly I:C resulted in com­plete recovery from severe herpesvirus keratoconjunctivitis in rabbits. Therapy was effective when treatment was begun as late as three days following virus inoculation. Similar results have been reported by Hamilton and coworkers (45)1 these investigators also reported that poly A:U complexes were effective against herpes keratitis in rabbits. They further reported that poly I:C given 2 hr prior to infection 24 was capable of reducing mortality and prolonging the sur­vival time of adult mice infected ip with 60 LDSO of HVH. Therapy was also effective when initiated 24 hr post infec­tion. If treatment was initiated at 48 hr following in­fection, no decrease in mortality was noted, but there was an increase in the average day of death. Catalano and Baron (12) reported that treatment with poly I,C of HVH encepha­litis in mice was effective only when a virus challenge of 1 TCIDSO was used. They also described a delay in the time of death of treated animals that did not survive. Subsequent experiments by these investigators (14) demon­strated that a complex of poly r,c and poly-D-lysine enhanced the in vivo induction of interferon in mice and afforded greater protection to mice with HVH encephalitis than poly I,C alone. This enhanced protection was still observed when treatment was begun as late as four days following inocula­tion at which time some of the animals were showing Signs of early CNS infection. Lindh and coworkers (61) have also reported the effectiveness of poly I:C against a herpesvirus infection of adult mice. Although HVH is relatively resis­tant to the action of interferon when compared to other viruses, the results from treatment with poly r,c of herpes­virus keratitis and encephalitis in experimental animals indicate that interferon or interferon inducers may be effective in treatment of human herpesvirus infections. 25 v. Mechanism of action and pharmacology of antiviral agents A. IUDR IUDR is a nucleoside analogue of thymidine and its mechanism of action in inhibiting viral replication is at the level of DNA synthesis. Its primary mechanism of action is the inhibition of enzymes, thymidine kinase, thy­midylate kinase, and DNA polymerase, involved in the syn­thesis of DNA. IUDR is also incorporated into DNA in place of thymidine (42). Following administration IUDR is absorbed rapidly into tissues where it is metabolized and excreted into urine as inactive metabolic products. In a study of patients with suspected HVH encephalitis who were receiving IUDR therapy, Lerner and Bailey (60) measured levels of IUDR activity in serum, urine, and cerebral spinal fluid (CSF). During slow intravenous infusion, little measurable activity was present in these samples. The rate of inacti­vation and/or removal of drug equaled its rate of adminis­tration. During rapid infusion of drug, however, significant quantities of IUDR activity were detected in serum, urine, and CSF. Calabresi (8) reported that following a 2 hr in­fusion of IUDR-I131 in human patients 50% of the radioactivity in blood was present at 7-10 hr, most of this material how­ever was free iodine: by 24 hr 90% of the radioactivity had been excreted in urine. The author speculates that tissues would be in contact with active IUDR for less than four hr. The metabolism of IUDR when injected into dogs has been re­ported by Clarkson and coworkers (19). They found that 26 IUDR-I125 injected directly in brain ventricles was meta­bolized rapidly to the inactive products iodouracil, uracil, and iodide. When IUDR-I125 was administered intravenously, the half life in plasma was about three hr, and 80% of the radioactivity recovered in the CSF was in the form of iodide. Following intracerebral inoculation of IUDR-I125 , the drug was rapidly inactivated by enzymes in the brain to iodo­uracil and iodide. From these studies the authors concluded that either IUDR does not cross the blood-CSF, or blood­brain barrier, or that it is rapidly metabolized after reaching the CSF or brain tissues. Injections of mice with radioactive labeled IUDR results in 75% of the radio­activity being excreted into urine by four hr and 80-90% at 24 hr. The concentration of the metabolic products in urine at 24 hr were 83% iodide, 3.8% iodouracil, and 6.6% IUDR. (91, 92). The metabolic breakdown of IUDR probably occurs in both the mouse and man according to the following scheme: IUDR~Iodouracil~Iodide + Uracil. The three breakdown products are all inactive against herpesviruses. The failure of IUDR to reach the CNS in active form as well as its rapid breakdown in tissues and excretion into urine, probably explains its lack of efficacy for herpesvirus in­fections of the CNS. B. Ara-C Cytosine arabinoside is a synthetic nucleoside which differs from the natural nucleosides cytidine and deoxycytidine in that the sugar moiety of the molecule is 27 arabinose rather than ribose or deoxyribose. Detailed studies on the mechanism of action of ara-C suggest that its primary action is the inhibition of deoxycytidine synthesis (18), and by inhibition of DNA polymerase (35). It is poorly, if at all, incorporated into DNA (69). In liver and kidney of humans there is an enzyme present which deaminates ara-C to arabinofuranosyluracil (Ara-u), an inactive metabolite. Pharmacologic studies performed in man with ara-C-H3 have demonstrated that there is a rapid decrease of ara-C in the plasma that was later recovered in urine primarily as ara-U (48). Eighty percent of the radioactivity was recovered in the urine at 24 hr, eight percent as ara-C and 72 percent as ara-U. The conversion time was dose dependant, and ara-C given intrathecally had a much longer half-life than that administered intravenously. This was explained on the basis of lower levels of deaminase activity in the CNS than those found in blood and liver. Other in­vestigators have reported that conversion of ara-C to ara-U as determined by urinary excretion was not appreciably affected by the route of administration of the drug (30). The half-life of the drug in vivo was less than one hr by all routes of administration, except intrathecal which was not tested. A similar pattern of ara-C metabolism in man has been reported by Creasey and coworkers (21). The half­life of drug in blood following intravenous administration was 30-60 min. The metabolism of ara-C following intravenous administration of rhesus monkeys was similar to the data 28 reported for humans (65). Intravenous injection of labeled ara-C in dogs also results in a short plasma half-life and rapid urinary excretion of the drug (23). Additionally only a small amount of the administered radioactivity was found in the CSF. Parenteral administration of tritiated ara-C to mice resulted in rapid urinary excretion with 85% of the radioactivity excreted in 24 hr. From all the data available it appears that the metabolism of ara-C is similar in man and animals. The rapid breakdown of ara-C to inactive ara-U and excretion into urine indicates that the method of administration may have a great effect upon the efficacy of ara-C as an effective antiviral agent. C. Ara-A The mechanism of action of the purine nucleoside, ara-A, is probably due to its inhibition of DNA polymerase (34,35). In both in vitro and in vivo biological systems ara-A is rapidly deaminated to the arabinofuranosylhypoxan­thine (Ara-HX) analogue (5, 6). When 200 ug of ara-A-cl4 was given intravenously to mice 84% of the drug was removed after one min, after 30 min, 95% of the administered dose had been cleared from the blood. If one Mg of ara-A-cl4 per mouse was injected intraperitoneally, maximum drug levels appeared in the blood at 30 min. A comparison of the drug levels in blood after intravenous or intraperito­neal injection showed that the same concentration was reached in serum by 30 min when administered by either route. At 30 min following injection of ara-A-Cl4 35% of the 29 radioactivity was recovered in the urine. All the radio­activity was in the form of ara-HX. Labeled drug was in­corporated into all tissues tested with the greatest amounts in liver and intestine and the smallest concentration in brain (6). One of the explanations for the failures in the use of IUDR and ara-C against HVH infections has been their conversion to inactive products. In contrast, ara-A is deaminated to ara-HX which has been reported to retain antiviral activity. Although the activity of ara-HX against HVH in tissue culture was shown to be only one­tenth that of ara-A (66), it was quantitatively as effec­tive as ara-A against HVH encephalitis in mice (104). D. Interferon Interferon are a group of antiviral proteins produced by cells in response to a virus infection or ex­posure to certain nonviral substances. Treatment with in­terferon confers upon cells resistance to multiplication of a variety of viruses. Although there are variations in many of the properties of interferon according to the inducer utilized, and the host in which the particular interferon was produced, there are a number of physical-chemical and biological criteria that an antiviral substance should meet in order to be classified as an interferon (31). Some of these properties are: 1) species or tissue specificity, 2) stability at pH 2.0 for several days, 3) loss of activity when cells are treated with protein synthesis inhibitors, 4) non-dializable, 5) not sedimented at 100,000 X g for 4 30 hr, 6) inactivation of activity by proteolytic enzymes, but not nucleases, and 6) do not inactivate virus directly. The precise mechanism of how interferon interferes with viral replication is not completely understood although data are available from a number of laboratories from which a working hypothesis can be formulated (46). The mechanism of action of an interferon inducer involves two distinct aspects. In the first phase the virus, usually an RNA containing virus, or a synthetic inducer must interact with the host cell. In the case of a single stranded RNA virus, the virion penetrates the cell, is uncoated, and releases its RNA. As part of the viral replication cycle, a com­plementary RNA strand is probably produced. Double­stranded RNA is foreign to the cell and is thought to activate the cellular formation of interferon mRNA followed by the production of interferon. In the second phase interferon is transported to other cells where it is thought to derepress a second host cistron to form mRNA for an intermediate protein which then acts as an inhibitor of viral replication. It has been reported by Marcus and Salb (63) that this protein inhibits the translation of viral mRNA. other investigators, however, have been unable to confirm these observations. Carter and Levy (11) have suggested that the primary site of interferon action was not on viral RNA translation, but on the binding of viral RNA to cellular ribosomes. This work, likewise, has not been confirmed. More recently Marcus and associates (64) have 31 postulated that the action of interferon may not be at the translation level at all, but at the level of transcription by binding to RNA polymerase. VI. summary Herpesvirus hominis is the causative agent of a wide variety of infections in man. These infections range from a mild localized lesion on the skin to a severe, often fatal encephalitis. Treatment of these infections with a number of antiviral agents has been attempted in numerous individual cases, however, efficacy has not been established for any of these experimental compounds. Evaluation of the effect­iveness of these drugs must await the results of controlled human trials. Although any new potential chemotherapeutic agent for viral infections is usually initially tested in tissue culture it is well recognized that activity in vitro does not necessarily result in a new "wonder drug". Animal models utilized to determine the effectiveness of these com­pounds should correspond as closely as possible to the natural infection seen in man. Additionally, parameters other than just effect on mortality should be evaluated. 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In vitro determinations of viral susceptibility to drugs for possible clinical use. Ant1microb. Ag. Chemother. 1970. p. 380-384. 116. \~lheeler, C. E., Jr., and W. D. Huffines. 1965. Pri­mary disseminated Herpes simplex of the newborn. J. Amer. Med. Ass. 191:455-460. 117. '.vhi te, J. G. 1963. Fulminating infection wi th Herpes simplex virus in premature and newborn infants. N. Engl. J. Hed. 269:455-460. 118. 'Witzleben, C. L., and S. G. Driscoll. 1965. Possible transplacental transmission of Herpes simplex infection. Pediat. 36:192. 119. Yen, S. S. e., J. W. Reagan, and M. S. Rosenthal. 1965. Herpes simplex infection in female genital tract. Obstet. and Gyneco1. 25:479. EXPERIMENTAL RESULTS section I Herpesvirus hominis infection in newborn mice: section II I. Development of an experimental model and therapy with iododeoxyuridine Herpesvirus hominis infection in newborn mice: II. A comparison of the therapeutic efficacy of ara-C and ara-A Section III Herpesvirus hominis infection in newborn mice: III. The effectiveness of interferon and inter­feron inducers Please Note: Pages 50-92, I'Herpesvi rus Homi ni s Infection in Newborn Mice: I: Development of an Experimental Model and Therapy with Iododeoxyuridine,1I copyright 1973 by University of Chicago, not micro­filmed at request of author. Available for consultation at the University of Utah Library. University Microfilms. SECTION I Herpesvirus Hom1n1s Infection in Newborn Mice: I. Development of an experimental model and therapy with iododeoxyurid1ne Earl R. Kern, James C. Overall, Jr. and L. A. Glasgow Manuscript "In Press ll J. Infect. 015. 51 Abstract Although treatment of disseminated Herpesvirus hominis (HVH) infections of the human newborn infant has resulted in case reports of both success and failure, there have been no controlled studies to evaluate the effect of therapy on either the outcome or the pathogenesis of the disease. In­fection of the newborn mouse with the genital type 2 strain of HVH by the intranasal route provides an experimental model for the human disease. Following inoculation of mice, the virus multiplies in the lung and is disseminated through the blood to the liver and spleen and to the brain by both a viremia and nerve route transmission. Therapy with iodo­deoxyuridine (IUDR) had no effect on mortality, although a significant effect on pathogenesis was observed. Virus re­plication in the lung was reduced and the viremia as well as subsequent involvement of liver and spleen was completely inhibited. In contrast, nerve route transmission to and replication of virus in the central nervous system (CNS) was not affected by therapy. Lack of inhibition of viral re­plication in the CNS appeared to be due to inadequate levels of IUDR in brain tissue and is the likely explanation for the therapeutic failure of IUDR in this model infection. These observations suggest possible reasons for the variable results reported in the treatment with IUDR of disseminated herpesvirus infections of human newborn infants. 52 Introduction Herpesvirus hominis (HVH) infections of the human new­born infant may be manifested by a wide spectrum of diseases including subclinical infection, encephalitis, and a dis­seminated visceral form with or without central nervous system (eNS) involvement (1, 2). In the more severe forms of HVH infections, it has been estimated that greater than 50% of untreated cases may terminate fatally or result in permanent neurological sequelae. The severity of neonatal herpesvirus infections clearly establishes the need for effective therapy. While various modes of treatment such as gamma globulin (3), cytosine arabinoside (4), and poly I:C (5), have been attempted on a very limited scale, the most widely used form of chemotherapy has been 5-iodo-2 1 - deoxyuridine (IUDR). In some reports, treatment of herpes­virus infections with IUDR has yielded promising results (6, 7, 8). In other instances, however, IUDR treatment failed to prevent neurological sequelae or death (9, 10). The reported experience with IUDR to date has been in indi­vidual cases and controlled studies have not been carried out to definitively prove the efficacy of any chemothera­peutic agent in the treatment of this disease. Furthermore, the optimal dosage of IUDR, the effect of treatment on the pathogenesis of the infection, and the tissue distribution of the drug have not been adequately defined. Our studies, utilizing an animal model, were designed to investigate some of the factors determining the success 53 or failure of therapy in herpesvirus infections of the new­born. Experimental infection of the newborn mouse provides a model which appears to closely simulate the human infec­tion. Since the nasopharyngeal cavity is the most likely portal of entry for the virus (1), newborn mice were infected with the MS strain of HVH type 2 (genital strain) by the in­tranasal route. utilizing this experiment:al infection, we have: 1) elucidated the pathogenesis of herpesvirus in­fection in the newborn mouse, 2) establistled the maximum tolerated dosage of IUDR, 3) evaluated the therapeutic efficacy of this drug utilizing various tI~eatment schedules, 4) compared the sensitivity to IUDR of the MS strain with other strains of HVH, 5) determined the effect of treatment on the pathogenesis of the infection, and 6) attempted to correlate levels of lUDR activity with virus titers in tissues of target organs. These studies demonstrate the similarity of the natural course of disease in neonatal mice to that observed in some human neonates and suggest possible explanations for the lack of uniform success with lUDR therapy in human newborns. 54 Materials and Methods Animals. Newborn mice (3-7 days of age) from pregnant CD-l females (Charles River Breeding Laboratories) were in­oculated intranasally by allowing each mouse to inhale 6 drops (approximately 0.01 ml) of herpesvirus type 2 from a 26 gauge needle. Each animal received approximately 1000 pfu of HVH (8 LDSO) which resulted in a 90-100% mortality. Viruses. The MS strain of HVH type 2 was obtained from Dr. Andre Nahmias, Emory University, Atlanta, Georgia. The virus pool used in these studies was propagated in primary rabbit kidney cells and titered 2.0 X 106 pfu/ml. other type 2 strains, including Alabama, Curtis, Ellison, and Lovelace, and three type 1 strains were also obtained from Dr. Nahmias. Viruses isolated in our own laboratory from genital lesions include Holt, Jensen, and Turner. Al­though these isolates have not been completely characterized, preliminary data suggest that are type 2. Cell cultures. primary rabbit kidney cells were pre­pared by aseptically removing both kidneys from a four-week old rabbit. The capsule and cortex were removed and the remaining tissue was minced and washed with phosphate-buf­fered saline (PBS), deficient in calcium and magnesium, to remove as many red blood cells as possible. After washing, individual cells were extracted in 0.25% trypsin for one hour. The resulting suspension was centrifuged, resuspended in minimal essential medium (MEM: Auto-Pow, Flow Labs) con­taining 10% fetal calf serum, and seeded into 32 ounce prescription bottles. Primary mouse embryo fibroblasts (MEF), human foreskin fibroblasts (HFF), and fetal lamb kidney (FLK) cells were prepared in a similar manner. HFF and FLK cells were maintained as continuous passage in Blake bottles containing MEM with 10% fetal calf serum at 37°C. Virus assays. Assays for herpesvirus were performed using the plaque assay technique on a confluent monolayer of FLK cells in 10 x 30mm Falcon plastic petri dishes (Falcon PlastiCS, Oxnard, California). The medium was aspirated from the plates, the cells washed once with PBS 55 and a 0.2 ml inoculum of an appropriate virus dilution was added to the cell monolayer. The virus was allowed to adsorb at 37°C for 60-90 minutes with occaSional agitation. Fol­lowing adsorption, a semisolid overlay medium consisting of MEM and 0.5% agarose (Van Waters and Rogers, San FranCiSCO, California) was applied. Following 48 hour incu­bation, a second overlay consisting of MEM, 0.75% noble agar (Difco Laboratories, DetrOit, Michigan) and neutral red (MCB, Cincinnati, Ohio) was added. Plaques were counted 24 hours later. Assays for IUDR activity. The sensitivity of the HVH strains to IUDR was determined by mixing two-fold dilutions of the drug in twice-concentrated MEM with an equal volume of 1.0% agarose. The overlay mixture was applied to con­fluent monolayers of MEF or HFF cells one hour after in­fection with 50-100 pfu of herpesvirus. The inhibitory 56 level was defined as that concentration of drug which re­sulted in a 50% reduction of the number of plaques in the control plates. Activity of drug in mouse serum or tissues was measured by performing two-fold dilutions of serum or 10% tissue homogenates in twice-concentrated MEM and pro­ceeding as described above. The MS strain of virus was utilized and serial dilutions of known drug concentrations were run with each assay. In both assay systems plaques were stained and counted as described above. Antiviral drug. 5-iodo-2 1-deoxyuridine was obtained from calbiochem, La Jolla, California. The drug was pre­pared each day by dissolving in PBS with the aid of sodium hydroxide and administered to mice in a volume of 0.05 ml by the ip route. 57 Results pathogenesis of infection. Following administration of an inoculum of HVH type 2 by the intranasal route calcu­lated to produce approximately 90% mortality (1000 pfu), suckling mice developed an illness characterized by lethargy and decreased spontaneous movement. On days 3-4 of the infection they had difficulty righting themselves if turned over and often developed rapid repetitive movements of the extremities if stimulated. Death usually occurred by day five. To define the pathogenesis of the infection, suckling mice were inoculated with virus and ten animals sacrificed at 12 hour intervals until all animals had succumbed to the infection. At each time period, heparinized blood (less than 10 units/ml blood) was collected by cardiac puncture. Samples of whole brain, lung, liver, and spleen were pooled by organ group and prepared as a 10% homogenate (w/v) in MEM with 10% fetal calf serum. Samples were assayed for the presence of virus on FLK cells. The results are illustrated in Figure 1. Following intranasal inoculation, primary virus replication occurred in the lung where virus titers reached levels of 105 pfu/gm of tissue by 24 hours post in­fection and greater than 107 pfu/gm of tissue by 96 hours. A viremia, probably initiated from the lung, was first de­tected at 36 hours following inoculationr at this time the virus titer in the lung was 3 x 106 pfu/gm. Relatively low levels of virus, 102-103 pfu/ml , were found in the blood throughout the course of infection. The viremic phase was followed by the seeding of liver, spleen, and brain by 48 hours~ virus titers in these organs reached levels of 105- 106 pfu/gm of tissue by 96 hours. Johnson (11) and Rabin, et ale (12) have documented that, following intranasal inoculation of mice with HVH type 1, transmission of virus to the eNS occurs by way of 58 the olfactory and trigeminal nerves as well as by the blood-borne route. To determine if nerve route trans­mission of virus occurs in this model infection, a separate series of experiments were performed. Olfactory bulb, cerebellum-brain stem, and cervical spinal cord were harvested and pooled from groups of 10 animals at 12 hour intervals following virus inoculation. The titers of virus in the various eNS tissues are illustrated in Figure 2. Virus was first detected in the olfactory bulb and the cerebellum-brain stem at 48 hours, but was not found in the cerebrum or cervical spinal cord until 72 hours post infection. If virus was transmitted to the eNS exclusively by the blood, one might expect simultaneous appearance of virus in all parts of the brain. The fact that virus first appeared in those parts of the brain where the olfactory and trigeminal nerves enter, coupled with the experimental data of Johnson and Rabin et ale (11, 12) on nerve route transmission of herpesvirus type 1, would suggest that access to the eNS by peripheral nerves occurs in this model 59 infection. In summary, the pathogenesis of HVH infection in suck­ling mice following intranasal inoculation involves primary replication of virus in the respiratory tract. Viremia then ensues, probably initiated from the lung when titers of virus reach a critical threshold (approximately 106 pfu/ gm). Subsequently the liver, spleen, and brain are seeded with virus from the blood. The olfactory and trigeminal nerves serve as alternate pathways for the virus from the nasopharynx to the eNS. Death appeared to result primarily from encephalitis1 however, pneumonia, hepatitis, and other factors could be contributory. Infection in the newborn mouse appears to closely simulate neonatal herpesvirus infections of humans (13, 14), and provides a model for the controlled evaluation of antiviral agents prior to their use in the treatment of human herpesvirus infections. Treatment of IUDR. The effect of IUDR on the mortality of mice in this model infection was evaluated using various dosages of IUDR. Therapy was initiated on day a (1-2 hours post infection), or day one following infection. Treatment was administered daily for a period of five days or until the animals exhibited terminal symptoms of disease. The highest concentration of IUDR that could be administered daily for five days without lethal toxicity for suckling mice was 200 mg/kg/day. The therapeutic efficacy of this compound was evaluated, therefore, at dosages of 200 and 100 mg/kg/day. The results of treatment utilizing 200 60 mg/kg in groups of 40 mice are illustrated in Figure 3. The untreated group had a mortality of 95% while the group that received treatment immediately following infection had a final mortality of about 80%. This difference was not significant. Furthermore, no delay in the mean day of death was observed in the treated animals. The group in which treatment was initiated one day following infection also had a mortality of approximately 80%. Similar results were ob­served using a dosage of 100 mg/kg of IUDR. In order to eliminate the possibility that animals were receiving an overwhelming infection and to develop a more sensitive model, animals were inoculated with a concentration of virus that would result in approximately a 50% mortality. The treatment schedule was also altered to include groups of 40 animals treated immediately after infection with either 200 mg/kg once a day or 100 mg/kg twice daily for five days. Neither regimen reduced mortality nor resulted in an in­crease in the mean survival time. Under the conditions employed, rUDR was not effective in significantly reducing mortality of HVH infected mice. Sensitivity of HVH type 2 to lUDR. One possible ex­planation for the variable results of therapy reported in human neonates is that some strains could be relatively re­sistant to lUDR. To determine if the MS herpesvirus strain used in this experimental model was resistant and if there was significant variation in sensitivity to lUDR among genital strains of HVH, the sensitivity of the MS strain was 61 compared to four other known type 2 strains and three recent clinical isolates from genital lesions. Sensitivity to IUDR was determined for each virus strain in both mouse (MEF) and human (HFF) cells utilizing a plaque reduction assay. These results are summarized in Table 1. In both cell lines, the MS strain was as sensitive as the other passaged strains tested and the clinical isolates. Approximately 0.5 ug/ml of IUDR reduced the number of plaques by 50% in the mouse cell system, while approximately 1.0 ug/ml was required in the human cells. These levels of sensitivity of herpes­viruses to IUDR are comparable with those reported by Buthala (15) for type 1 strains. Since most of the sen­sitivity data in the literature are concerned with type 1 strains, we compared the relative sensitivity of three type 1 and five type 2 strains. In our system, type 1 strains are about two-fold more sensitive than the type 2 strains. These data confirm the results of Person and co-workers (16) who reported that in chick embryo fibroblast and WI-38 cells, type 1 strains were more sensitive than type 2 strains. However, in BeLa cells these authors found little difference between the two. Lerner and Bailey (17) have also reported type 1 st~ains to be more sensitive than type 2 herpes­viruses in BHK-2l cells. In contrast, Walker and co-workers (18) reported that IUDR was effective against type 1 but not against type 2 strains in WI-38 cells. Although there appears to be variation in the sensitivities of HVH strains to IUDR depending on the type of assay system, the particular cell lines utilized and the passage history of the virus, our data suggest that most strains of both types demon­strate a comparable degree of sensitivity. 62 ~ffect of therapy on pathogenesis. To determine why IUDR did not significantly reduce mortality in herpesvirus­infected mice in spite of the sensitivity of the virus to the drug, studies were initiated to define the effects of drug therapy on the pathogenesis of infection. Animals were inoculated with virus, then given daily doses of 200 mg/kg of IUDR ip beginning immediately after virus challenge. At 24 hour intervals following infection, 10 animals from the treated group and 10 animals from an untreated control group were sacrificed. Organs were removed, homogenized, and assayed for virus. As can be seen in Figure 4a and 4b, IUDR administration markedly altered the pathogenesis of the infection. The pattern of virus replication and dissemination in the untreated control group was similar to that illustrated in Figure 1. In the treated group, however, virus titers in the lung were reduced approximately 99% when compared to the control (from 107 to 105 pfu/gm of tissue). Detectable viremia and seeding of liver and spleen were completely inhibited, however, progression of the virus to the brain and replication in the central nervous system were not significantly altered. These data demon­strate that the failure of IUDR to reduce mortality in the herpesvirus infected animals is due to the lack of complete inhibition of virus replication in the brain and 63 and lung. These data also provide additional evidence for the importance of nerve route transmission to the CNS since detectable viremia was suppressed in treated animals. To further examine this possibility of nerve route transmission, the effect of IUDR therapy on neuropatho­genesis was investigated. Animals were inoculated with virus and treated as described above. Tissues assayed for virus at intervals following infection were olfactory bulb, cerebrum, cerebellum-brain stem, and cervical spinal cord. In both untreated control mice (Figure Sa) and IUDR treated mice (Figure Sb) high levels of herpesvirus were detected in olfactory bulb, cerebellum-brain stem, and cervical spinal cord by 72 hours and in cerebrum at 96 hours. Re­sults obtained in the above experiments provide further evidence that infection of the CNS was not exclusively de­pendant upon viremic spread of virus, but could also be transmitted by multiple neural pathways. Although IUDR was effective against the visceral component of the infec­tion, there was no effect on virus replication in nervous tissue. These data also suggest that either adequate levels of IUDR did not reach the central nervous system or that the drug was not effective at that site. Distribution of IUDR in target organs. One possible reason for the failure of a chemotherapeutic agent to con­trol virus replication is inadequate drug concentrations in target organs. Essentially no information has been reported on the levels of IUDR achieved in infected tissues during therapy. In a study of patients with suspected HVH encep­halitis who were receiving IUDR therapy, Lerner and Bailey (17) measured levels of IUDR activity in serum, urine, and cerebral spinal fluid (CSF). During slow intravenous in­fusion, little measurable activity was present in these samples~ however, during rapid infusion of drug, signifi­cant quantities of IUDR were detected in serum, urine, and CSF. The rate of inactivation, excretion into urine, 64 or removal of IUDR into tissues was shown to occur very rapidly following administration. Similar results utiliz­ing 131I-labeled IUDR have been reported (19, 20). studies to determine drug levels in brain and other organs, however, have not been carried out. This type of information would appear to be essential if a rational approach to chemother­apy of virus infections is to be developed. To investigate the possibility that effective levels of IUDn were not reaching all target organs, the following studies were initiated. Uninfected newborn mice were given 200 mg/kg of IUDR by the ip route and groups of animals were sacrificed at time intervals from 30 minutes through 12 hours. Samples of blood and 10% tissue homogenates of brain, lung, and liver were assayed for antiherpesvirus activity utilizing a plaque reduction assay on r~F cells. These results are presented in Table 2. Low levels of antiviral activity were detected in the blood during the first 60 minutes following IUDR administration. At 20-30 minutes following IUDR administration, there was approxi­mately 25 ug of rUDR/rnl of blood and at 60 minutes, 5-10 65 ug/ml. The fact that this inhibitory activity was not found in control samples and was reversible by thymidine indicates that it was due to the presence of IUDR. Low levels of antiviral activity could be detected in liver tissue for about two hours following administration and were also reversible by thymidine. The concentration of IUDR in liver throughout the first two hours was about 75-100 ug/gm of tissue. Approximately 20 ug/gm of tissue was found in the lung at 30 minutes, however no activity was present in any of the brain samples. The concentration of IUDR that resulted in a 50% plaque reduction endpoint in the assay system used was 0.5 ug/ml. Since a 1:5 dilution of serum was the highest concentration tested, the minimum level of IUDR activity that could be detected in blood would be approximately 2 ug/ml. The assay for rUDR activity in tissues was performed utilizing a 10% homogenate begin­ning with a one to five dilution1 therefore, the minimum detectable level of drug in tissue was approximately 20 ug/ gm. Although subdetectable levels of drug could have been present in tissue samples, it should be noted that an effective level of antiviral activity was not achieved in eNS tissue as indicated by the failure to reduce titers of HVH in the experiments on pathogenesis. In summary, the data concerning IUDR levels in tissues correlate with the results obtained from the effect of the drug on pathogenesis. In lung tissue where minimal amounts of drug were detected, virus replication was Significantly 66 reduced, but not completely eliminated. This reduction was sufficient, however, to eliminate detectable viremia. Re­plication in the liver was eliminated either because seeding of virus from the blood was blocked or because significant amounts of drug were present in that organ. In contrast, in brain tissue where no antiviral activity was demonstrated, virus growth was not altered. 67 Discussion The important role of maternal genital infections with HVH type 2 in the pathogenesis of the neonatal infection has been well documented (1). There is strong circumstan­tial evidence that most infants with disseminated neonatal disease acquire infection in the nasopharynx on passage through the birth canal, although alternate portals of entry could be the eye, the skin, the umbilicus (21), or the transplacental route (22). From the initial sites of replication in the nasopharynx, virus can reach the lungs, cause pneumonia, and spread through the blood to involve many organs (1). Herpesvirus has been isolated in several instances from the blood of infected infants, thus docu­menting the role of viremia in the pathogenesis (3, 13, 14, 23). Although there is no direct evidence in humans that herpesvirus is transmitted to the brain by peripheral nerves, this route is suggested by the occurrence of several cases of neonatal herpesvirus infection where symptomatology has been confined to the eNS (1). In addi­tion, the frequent occurrence of reactivated latent herpes facialis following neurosurgical procedures on the trigemi­nal nerves would seem to indicate the association of HVH with human neural tissue (24). Histopathologic studies of tissues from infants expiring with HVH infections have usually demonstrated greatest involvement in the brain, liver, adrenal glands, and lungs, although other tissues may also be involved. In those instances where virus 68 titrations were carried out on tissues obtained at autopsy, the highest levels of virus were present in lung, liver, spleen, and adrenals (3, 13, 14, 23). Death is probably the result of encephalitis, pneumonia, fulminant hepatitis, metabolic derangements, disseminated intravascular coagu­lation, or a combination of these (1, 13, 25, 26, 27). The animal model described in this paper appears to be similar in many respects to disseminated herpesvirus in­fections in human newborn infants. Suckling mice were inoculated with HVH type 2 by a natural route, the naso­pharyngeal cavity. The initial rapid multiplication of virus in the lung was followed by a low-grade viremia when virus titers in the lung exceeded 106 pfu per gram of tissue. The liver and spleen were infected by hematogenous spread. Virus appeared to reach the brain either by the blood stream or by direct nerve route spread. This latter mode of transmission was suggested by the initial appearance of virus in those portions of the brain where the olfac­tory and trigeminal nerves enter. In addition, virus was transmitted to the CNS in animals treated with IUDR, where no viremia was detectable. Other experimental models have demonstrated the im­portance of nerve route transmission of HVH following in­tranasal inoculation. Johnson (11) demonstrated by fluores­cent antibody techniques that HVH type 1 was transmitted to the CNS by the olfactory and trigeminal nerves as well as the blood-borne route. Similarly, Rabin and coworkers (12) 69 also demonstrated transmission to the eNS by the trigeminal nerve in suckling mice infected intranasally with type 1 herpesvirus. The type 2 virus utilized in our neonatal model has also been reported to be capable of spreading by neural routes (I, 28, 29). These studies strongly suggest that following intranasal inoculation of newborn mice, H\~ type 2 can be transmitted to the eNS by both blood and neural routes. The therapeutic efficacy of IUDR against herpesvirus infections in animal models appears to be dependant upon the clinical manifestations of the infection being treated. Kaufman (30) and others have demonstrated the effectiveness of IUDR against Herpes simplex keratitis in the rabbit, and its efficacy against skin infections induced in rabbits has also been reported (31). Sidwell and coworkers (32) have presented evidence that IUDR was inactive against vaccinia virus infections in mice inoculated by the intracerebral route, but was effective in prolonging the mean survival time when the animals were infected by the intranasal route. These authors suggested that the drug does not cross the blood-brain barrier, but is effective against infections in other parts of the body. IUDR has also been reported to be totally ineffective against H~i inoculated intracerebrally in mice (33). Although IUDR has been utilized in the treatment of generalized H~i infections of the human new­born, no controlled studies of its efficacy have been carried out. In our animal model of disseminated neonatal HVH in­fection, IUDR clearly had no therapeutic efficacy when utilized at maximum tolerated doses. Neither significant protection against mortality nor a delay in the mean day of death was observed. In spite of failure to reduce mor­tality, a significant alteration in the pathogenesis was demonstrated. Even though viremic spread and replication 70 of virus in liver and spleen were inhibited, there was no effect on neural transmission of virus to and replication within the eNS. Virus replication in the lung, though re­duced, was not totally suppressed. In reviewing data from these experiments it appeared that virus titers of 105 pfu per gram of lung tissue in treated animals were insufficient to result in a detectable viremia. The observed lack of effect on virus growth in the brain was associated with absence of detectable levels of IUDR in eNS tissue from treated animals. These data lend additional support to the concept that IUDR does not effectively cross the blood-brain barrier. It should be pointed out, however, that because of the relatively low sensitivity of our assay system IUDR could have been present in low concentrations ( <20 ug/gm) in tissues and not be detected. Nevertheless, IUDR therapy failed to appreciably alter mortality or pathogenesis of infection in the eNS. These factors, including lack of inhibition of rapid multiplication of virus in the lung, inability to suppress neural transmission of virus to the eNS, and ineffective levels of drug in critical target organs, 71 may also explain why IUDR has not been uniformly successful in the treatment of disseminated HVH infections in human newborn infants. In contrast to the results of treatment in our experi­mental animal model, IUDR has, in some instances, been re­ported to be effective in the treatment of disseminated neonatal HVH infections (6, 7, 8). However, treated in­fants who survived have frequently had residual brain damage (7, 8, 34). It was postulated that the sequelae resulted because of late initiation of therapy, after significant involvement of the CNS had already occurred. Although there are no data available to provide direct confirmation, an additional contributory factor could be the lack of suf­ficient IUDR in CNS tissue to appreciably inhibit virus replication. It should be further noted that the neonatal disease does not inevitably result in death or neurologic sequelae and that recovery has been documented in infants who have not received antiviral chemotherapy (35). In addition, the efficacy of IUDR in limiting replication of HVH in target organs other than the CNS suggests that therapeutic success might be predicted in infants who do not have CNS involvement. IUDR has been reported to be effective in the treat­ment of HVH encephalitis (36) which is most frequently caused by the type 1 strain of virus (28). Caution should be advised, however, in the interpretation of results of treatment based on uncontrolled studies (37). In a number of the reported cases of encephalitis treated with IUDR, the diagnosis has not been substantiated by isolation of 72 H~l from brain biopsy material and current serological tests alone may not provide a reliable means of diagnosis (38). More recently, however, Lerner and coworkers (39) have described the identification of specific passive hemagglu­tinating antibodies in the cerebral spinal fluid of patients with HVH encephalitis. If this finding is confirmed by other laboratories, it may improve the reliability of the serological diagnosis of central nervous system infection with this virus in the future. An additional problem in interpreting previous case reports is the variability of untreated disease (40). A number of patients who survived encephalitis following treatment with IUDR have had significant neurological sequelae. In one reported case, virus was recovered from brain tissue following a course of IUDR therapy (10). These data would suggest that IUDR may not reach adequate levels in eNS tissue. Although data regarding concen­trations of IUDR in brain tissue are not available, current evidence suggests that levels of drug can transiently be detected in the cerebrospinal fluid following rapid in­travenous administration (17). 'rhere are limitations in the extrapolation from the results of the studies in our animal model system to the treatment of human disease. The clinical pharmacology of the drug might be quite different in the mouse as compared 73 with the human. Furthermore, the drug has been administered to humans by continuous intravenous therapy, while the mice were given intraperitoneal doses once or twice a day. Therapeutic efficacy may well be achievable with IUDR in infants with less severe involvement and in infected organs other than the CNS. The results do suggest, however, that the lack of uniform success of IUDR in the treatment of human disease may be due to inadequate levels of drug in CNS tissue. Future efforts should be directed toward ob­taining documentation of whether adequate levels of antiviral activity are in fact achievable in CNS tissue in humans. Controlled studies should be carried out before IUDR is accepted as standard therapy for disseminated neonatal HVH infections. Finally, these results indicate the importance of de­fining the pathogenesis of infections in animal models simulating human viral diseases in order to evaluate efficacy of antiviral compounds. Furthermore, those para­meters which have been routinely used in the evaluation of antibacterial drugs (i.e., sensitivity of the organism to the drug, and the presence of inhibitory levels of drug at the site of infection) should be utilized in the evalua­tion of antiviral chemotherapeutic agents. References 1. Nahmias, A. J., Alford, C. A., Korones, S. B. tion of the newborn with Herpesvirus hominis. in Pediat. 17:185-226, 1970. Infec­Adv. 74 2. Overall, J. C., Jr., Glasgow, L. A. Virus infections of the fetus and newborn infant. J. Pediat. 77:315- 333, 1970. 3. Wheeler, C. E., Jr., Huffines, W. D. Primary dis­seminated Herpes simplex of the newborn. JAMA 191: 455-460, 1965. 4. Hrynuik, W., Foerster, J., Shojania, M., Chow, A. cytarabine for herpesvirus infections. JAMA 219:715- 718, 1972. 5. Bellanti, J. A., Catalano, L. w., Jr., Chambers, R. W. Herpes simplex encephalitis: Virologic and serologic study of a patient treated with an interferon inducer. J. Pediat. 78:136-145, 1971. 6. Partridge, J. W., Millis, R. R. Systemic Herpes sim­plex infection in a newborn treated with intravenous Idoxuridine. Arch. Dis. Childh. 43:377-381, 1968. 7. Tuffli, G. A., Nahmias, A. J. Neonatal herpetic infec­tion. Report of two premature infants treated with systemic use of idoxuridine. Amer. J. Dis. Child. 118:909-914, 1969. 8. Charnock, E. L., Cramblett, H. F'. 5-iodo-2 1 -deoxy­uridine in neonatal Herpesvirus hominis encephalitis. J. Pediat. 76:459-463, 1970. 9. pettay, 0., Leinikki, P., Donner, M., Lapinleimu, K. Herpes simplex virus infection in the newborn. Arch. Dis. Childh. 47:97-103, 1972. 10. Fishman, M. A., Haymond, M. W., Middlekamp, J. N. 75 Failure of idoxuridine treatment in Herpes simplex encephalitis. AIDer. J. Dis. Child. 122:250-252, 1971. 11. Johnson, R. T. The pathogenesis of herpesvirus encep­halitis. I. Virus pathways to the nervous system of suckling mice demonstrated by fluorescent anti­body staining. J. Exp. Med. 119:343-356, 1964. 12. Rabin, E. R., Jenson, A. B., Melnick, J. L. Herpes simplex virus in mice: Electron microscopy of neural spread. Science 162:126-127, 1968. 13. Catalano, L. w., Jr., Safley, G. H., Museles, M., Jarzynski, D. J. Disseminated herpesvirus infection in a newborn infant. J. Pediat. 79:393-400, 1971. 14. Becker, W. B., Kipps, A., MCKenzie, D. Disseminated Herpes simplex virus infection. Amer. J. Dis. Child. 115:1-8, 1968. 15. Buthala, D. A. Cell culture studies on antiviral agents. I. Action of cytOSine arabinoside and some comparisons with 5-iodo-2 1 -deoxyuridine. Proc. Soc. Exp. Biol. Med. 155:69-77, 1964. 16. Person, D. A. , Sheridan, P. J., Herrmann, E. C., Jr. sensitivity of types 1 and 2 Herpes simplex virus to 5-iodo-2 1 -deoxyuridine and 9-B-D arabinofuranosyla­denine. Infect. Immun. 2:815-820, 1970. 17. Lerner, A. M., Bailey, E. J. Concentrations of idox­uridine in serum, urine, and cerebrospinal fluid of pat,ients wi th suspected diagnoses of Herpesvirus hominis encephalitis. J. Clin. Invest. 51:45-59, 1972. 18. V/alker, W. E., waisbren, B. A., Martins, R. R., Batayias, G. E. In vitro determinations of viral susceptibility to drugs for possible clinical use. Antimicrob. Agents Chemother. 1970:380-384, 1971. 76 19. Prusoff, W. H. A review of some aspects of 5-iodo­deoxyuridine and azuridine. Cancer Res. 23:1246-1259, 1963. 20. Ca1abresi, P. Current status of clinical investiga­tion with 6-azuridine, 5-iodo-2 t -deoxyuridine, and related derivatives. Cancer Res. 23:1260-1267, 1963. 21. Nahmias, A. J., Josey, w. E., Naib, Z. M., Freeman, 22. M. G., Fernandez, R. J., Wheeler, J. H. Perinatal risk associated with maternal genital Herpes simplex virus infection. Aroer. J. Obstet. Gynec. 110:825- 837, 1971. Mitchell, J. E., McCall, F. C. tion by herpes simplex virus. 106:207-209, 1963. Transplacental infec­Amer. J. Dis. Child. 23. White, J. G. Fulminating infection with Herpes simplex virus in premature and newborn infants. New Eng. J. Med. 269:455-460, 1963. 24. Paine, T. F., Jr. Latent Herpes simplex infection in man. Bacteriol. Rev. 28:472-479, 1964. 77 25. Bahrani, M., Boxerbaum, B., Gilger, A. P., Rosenthal, M. S., Teree, T. M. Generalized Herpes simplex and hypoadrenocorticism. AIDer. J. Dis. Child. 111:437- 445, 1966. 26. Haynes, R. E., Azimi, P. H., Cramb1ett, H. G. Herpesvirus hominis infections in children. pathologic, and virologic characteristics. 312-319, 1968. Fatal Clinical, JAMA 206: 27. Miller, D. R., Hanshaw, J. B., O'Leary, D. S. Hnilicka, J. V. Fatal disseminated Herpes simplex virus infec­tion and hemorrhage in the neonate. J. Pediat. 76: 409-415, 1970. 28. Nahmias, A. J., Dowdle, W. R. Antigenic and biologic differences in Herpesvirus hominis. Prog. Med. Virol. 10:110-159, 1968. 29. Plummer, G., Hackett, S. paralysis of animals. 1966. Herpes simplex virus and Brit. J. Exp. Path. 47:82-85, 30. Kaufman, H. E. Clinical cure of Herpes simplex kera­titis by 5-iodo-2'-deoxyuridine. Proc. Soc. Exp. Biol. Med. 109: 251-252, 1962. 31. Force, E. E., stewart, R. e., Haff, R. F. Herpes sim­plex skin infection in rabbits. I. Effect of 5- iodo-2'-deoxyuridine. Virology 23:363-369, 1964. 78 32. Sidwell, R. W., Dixon, G. J., sellers, S. M., Schabel, F. M., Jr. In vivo antiviral properties of biolo­gically active compounds. II. Studies with influenza and vaccinia viruses. Applied Microbiol. 16:370- 392, 1968. 33. Sloan, B. J., Miller, F. A., Ehrlich, J., McLean, I. W., MacHamer, H. E. Antiviral activity of 9-B-D arabino­furanosyladenine. IV. Activity against intracerebral Herpes simplex virus infections in mice. Antimicrob. Agents Chemother. 1968:161-171, 1969. 34. Golden, B., Bell, W. E., MCKee, A. P. Disseminated Herpes simplex with encephalitis in a neonate: Treatment with idoxuridine. JAMA 209:1219-1221, 1969. 35. Gershon, A. A., Fish, I., Brunell, P. A. Herpes simplex infection of the newborn. Amer. J. Dis. Child. 124: 739-741, 1972. 36. Nolan, D. e., Carruthers, M. M., Lerner, A. M. Her­pesvirus hominis encephalitis in Michigan. Report of thirteen cases, including six treated with idoxuri­dine. New Eng. J. Med. 282:10-13, 1970. 37. Johnson, R. T. Treatment of Herpes simplex virus en­cephalitis. Arch. Neurol. 27:97-98, 1972. 38. Johnson, R. T., Olson, L. C., Buescher, E. L. Herpes simplex virus infections of the nervous system: problems in laboratory diagnosis. Arch. Neurol. 18:260-264, 1968. 79 39. Lerner, A. M., Lauter, C. B., Nolan, D. e., Shippey, M. J. Passive hemagglutinating antibodies in cerebrospinal fluids in Herpesvirus hominis encepha­litis. Prac. Soc. Exp. Biol. Med. 140:1460-1466, 1972. 40. Johnson, K. P., Rosenthal, M. 9., Lerner, P. I. Herpes simplex encephalitis: The course in five virologi­cally proven cases. Arch. Neuro1. 27:103-108, 1972. 80 Table 1. Sensitivity of reference type 2 strains and genital isolates of Herpesvirus hominis to iododeoxyuridine. The inhibitory level is that concentration of drug which reduces the control virus plaque count by 50% in either mouse embryo fibroblast (MEF) or human foreskin fibroblast (HFF) cells. Strain of HVH Reference Strains MS Alabama Curtis Ellison Lovelace Genital Isolates Holt Jensen Turner 50% Inhibitory Levels (ug/ml) Cell Culture MEF 0.39-0.78 0.78-1.6 0.78 0.39-0.78 0.39-0.78 0.39-0.78 0.39 0.39-0.78 HFF 0.78-1.6 0.78-1.6 0.78-1.6 0.78-1.6 0.78-1.6 0.39-0.78 0.39-0.78 0.78 81 Table 2. Presence (+) or absence (-) of antiherpesvirus activity (reversible with thymidine) in tissues of suckling mice obtained at the indicated times following treatment with a single dose of iododeoxyuridine (200 mg/kg ip). The lowest level of activity which could be detected in the blood was 2 ug/mlr in the tissues 20 ug/gm. Approximate concentrations of drug detected are provided in the text. Time in Tissues f-tlinutes Blood Brain Lung Liver 30 + + + 60 + + 90 - + 120 + 240 82 Figure 1. pathogenesis of Herpesvirus hominis type 2 infection following intranasal inoculation of suckling mice. 'l'iters of virus (lo910 pfu/gram of tissue or ml of blood) in lung, blood, spleen, liver, and brain are shown at 12 hour intervals following inoculation. 6.0 0' 5.0 -"~'" Q. :ff4.0 o ...J r.n ~ 3.0 > 2.0 1.0 <1.0 ·n ..... LUNG :-:::: ~~~~N -- LIVER --BRAIN ••••• ~ ~. ~, •.+ l II! III It III III III .I..t ; ! It w It III II! .w. It := : : II! J .,. .. 11 ..... ,..t: •• .. -""-""'. -. ... . ....... ... ......... . 0---­o 12 24 36 45 60 72 84 96 HOURS POST INFECTION Figure 1 83 84 Figure 2. Neuropathogenesis of Herpesvirus hominis type 2 infection following intranasal inoculation of suckling mice. Titers of virus (10910 pfu/gram of tissue) in olfactory bulb, cerebellum-brain stem, cervical cord, and cerebrum at 12 hour intervals following inoculation. 85 0--0 OLFACTORY BULB 6.0 0-0 CEREBELLUM-BRAIN STEM -l:'I---ll CERVICAL CORD CEREBRUM 5.0 c::::rt "'- -:::J 4.0 a. 0 (!) .0.. .J 3.0 en ::::::> a:: > 2 .0 1.0 <1.0 12 24 36 48 60 72 84 96 HOURS POST INFECTION Figure 2 86 Figure 3. The effect of iododeoxyuridine therapy (200 mg/ kg/day for five days administered ip) on the mortality of Herpesvirus hominis type 2 infection of suckling mice. Treatment was initiated either immediately following (day 0) or the day after infection (day 1). The cumulative percent mortality is shown for each day following infection. 100 >- 80 t:: ..J ~ ~ 60 =- 20 -, VIRUS CONTROL ---THERAPY INITIATED DAYO ······THERAPY INITIATED DAY I --DRUG CONTROL r' I I I I I I , ~,--. *.--............. ~ (f---- ;;1 O~--~~~~~~----~--~-------------- o 2 4 6 8 10 12 14 16 DAYS POST INFECTION Figure 3 87 88 Figure 4. The effect of iododeoxyuridine treatment (200 mg/kg/day for five days administered ip) on the pathogenesis of Herpesvirus hominis type 2 infection following intranasal inoculation of suckling mice compared with untreated controls. a = untreated control group b = treated group Titers of virus (10910 pfu/gram of tissue or ml of blood) are shown in lung, blood, spleen, liver, and brain at 24 hour intervals following inoculation. 7.0 ()o--o() LUNG A---l::J. BLOOD 0-0 SPLEEN e--e LIVER 6.0 --- BRAIN ...0... .. 5.0 -~ Q. 52 4.0 <.!) 0 ..J V) ::l 3.0'" 0::: > 2.0 1.0 <1.0 6.0 5.0 E 0'1 ~ 4.0 ..... 0- o g 3.0 -1 ::::E:. > ::t: 2.0 1.0 24 •••••• LUNG -'-BLOOD ••••• SPLEEN .... ~~LIVER --BRAIN 48 72 96 HOUR S POST IN FECTI ON Figure 4a <I.O~.n&n. ..~ ~an. .r .r.&n. .r w.n..n"~~__" I__1_ _'_ _____~ . '._...~ ,_-I. r..__ ~ ._-l- ~-_"~ _.'. ",_- ~_' "_~ _I" _"U;_I ____ ....... .-,-. •••••••••••••••••••• 24 48 72 96 HOURS POST INFECTION Figure 4b 89 90 Figure 5. The lack of effect of iododeoxyuridine treatment (200 mg/kg/day for five days administered ip) on the neuro­pathogenesis of Herpesvirus hominis type 2 infection follow­ing intranasal inoculation of suckling mice compared with untreated controls. a = untreated control group b = treated group Titers of virus (loglO pfu/gram of tissue) are shown in olfactory bulb, cerebelllu'TI-brain stem, cervical cord, and cerebrum at 24 hour intervals following inoculation. C" ......... -~ 0.. 0 (!) (.f) ::::> a-: > 0-0 OLFACTORY BUL B 6.0 0--0 CEREBELLUM-BRAIN STEM ~ CERVICAL CORD e--e CEREBRUM 5.0 4.0 3.0 2.0 1.0 91 <1.0 ' ................. ... 11......... .. ................... . ;0......... .. ....... .•... .........•.•.•.. . .•.••..•..................•.•......•........ 48 72 96 HOURS POST INFECTION Figure Sa 6.0 5.0 ..c..::.r.o ::::J 0. 4.0 0 (!) 0 3.0 -' U) ::::> a:: 2.0 > 1.0 <1.0 0--0 0-0 l:r--l:l. e--e OLFACTORY SU LS CEREBELLU M - BRAI N STE M CERVICAL CORD CEREBRUM --I 24 I 48 I 72 HOURS POST INFECTION Figure 5b I 96 92 SECTION II Herpesvirus Horninis Infection in Newborn Mice: II. A comparison of the therapeutic efficacy of ara-C and ara-A !1anuscript to be submitted to Antimicrobial Agents and Chemotherapy ABSTRACT Infection of the newborn mouse with the genital type 2 strain of HVrl by the intranasal route provides an ex­perimental infection that closely resembles a disseminated herpesvirus infection of the human newborn infant. Follow­ing inoculation of mice, the virus multiplies in the respiratory tract and is disseminated through the blood to the liver and spleen, and to the brain by both a viremia and nerve route transmission. Therapy with l-B-D arabino­furanosylcytosine (l~a-C) did not reduce mortality, but it did significantly increase the mean survival time. The effects of Ara-C treatment on the pathogenesis of the infection were characterized by a one day delay in the appearance of HVH in blood, liver and spleen, and a reduc­tton of virus replication in lung and brain, which also lasted for about one day. Treatment with 9-B-D arabino­furanosyladenine (Ara-A) had no effect on final mortality, but increased the mean survival time by two days. This was highly signtflcant. rrherapy with Ara-A markedly altered the pathogenesis of HVH infection. There was a delay or a suppression of virus replication for two days in blood, lung, liver, spleer.l. and brain. rrhe alteration of HVH pathogenesis in or9ans of animals treated with Ara-A correlated Hi th the tw(,) increase observed in the mean 95 survival time. In this experimental HVH infection of new­born mice, Ara-A appeared to be more effective than was Ara-C. Neither of the two drugs, however, was completely effective in inhibiting viral replication in target organs, or protecting animals from a lethal HVH infection. INTRODUCTION Herpesvirus horoinis (HVH) is the causative agent of a variety of human diseases including encephalitis in the adult (35), and a generalized infection of the neonate with or without central nervous system involvement (eNS) (33, 36). The high mortality rate associated with these infections clearly justifies the search for an effective chemotherapeutic agent. Although the most widely utilized form of therapy has been 5-iodo-2 1 -deoxyuridine (IUDR), the effectiveness of this compound has not been clearly established (8, 16, 38, 40, 49). In a detailed evaluation of the effectiveness of IUDR in an HVH type 2 infection of newborn mice we have previously reported that IUDR was relatively ineffective in controlling virus replication in the eNS (Kern, E. R., J. C. Overall, Jr., and L. A. Glasgow, J. Infect. Dis., In press). In recent years, two inhibitors of DNA synthesis have been shown to be highly effective against HVH both in vitro and in vivo. l-B-D arabinofuranosylcytosine (cytosine arabinoside, Ara-C) is inhibitory to both antigenic types of HVH in a variety of cell cultures (5, 14, 51), is as effective as IUDR in the treatment of Herpesvirus keratitis (25, 50), and has been used in the treatment of herpes­zoster (9, 17, 22, 27) and herpesvirus encephalitis (10, 22) with promising results. The efficacy of Ara-C in these latter two diseases has not, as yet, been documented in controlled studies (12). The purine nucleoside 9-B-D arabinofuranosyladenine (adenine arabinoside, Ara-A) is as effective against H~l types 1 and 2 as Ara-C and IUDR in vitro (28, 39) and may be less toxic to human cells. Animal model studies have demonstrated that Ara-A is effective against herpes sim­plex keratitis in hamsters (45), and herpesvirus encepha­litis in hamsters and mice (1, 29, 43, 46, 47). Although Ara-A is being utilized for human herpesvirus infections on a trial baSiS, there have been no published reports on its effectiveness. The purpose of our studies was to compare the thera­peutic efficacy of Ara-C and Ara-A in an experimental 97 animal infection. Intranasal inoculation of the newborn mouse with H~rl type 2 provides a model which appears to closely simulate disseminated HVH infections of the human neonate. Experiments designed to compare the effective­ness of the two drugs in this experimental infection in­clude: a comparison of the sensitivity to Ara-C and Ara-A of the MS strain utilized in ·these studies with other strains of HV.E t determinati.on of the maximum tolerated dosages of each drug, evaluation of the therapeutic effi­cacy of each compound ut.ilizing various treatment schedules, determination of the of treatment on the pathogenesis of infection, and correlation of drug activity with virus titers in tissues of target organs. MATERIALS AND METHODS Animal model. Newborn mice (S-7 days of age) from pregnant Swiss Webster females (Simonson Laboratories, Gilroy, Calif.) vlere inoculated intranasally by allowing each mouse to inhale six drops (approximately 0.01 ml) of H~1 type 2 from a 26 gauge needle. Each animal received approximately 1000 pfu (8 LDSO) of herpesvirus resulting in a 90-100% mortality. When lower mortality rates were desired appropriate dilutions of the inoculum were utilized. Viruses. The type 2 MS strain of HVH utilized in these studies was obtained from Dr. Andre Nahmias, Emory University, Atlanta, Georgia. The virus pool used was pro­pagated in primary rabbit kidney cells and titered 1.0 X 106 pfu/ml. other type 2 strains, including Alabama, Curtis, Ellison, and Lovelace, and three type 1 strains, shealey, Tyler, and VR3 were also obtained from Dr. Nahmias. Viruses isolated in our own laboratory from genital lesions include Holt, Turner, and Jensen. Although these isolates have been only partially characterized, they are probably type 2. ,i\1edia. Eagle' s minimum essential medium (MEM: Auto- 1:ow, :F'low Laboratories, Rockville, l'1d.) containing 10% fetal calf serum (Grand Island Biological Co., Grand Island, N. Y.), 100 units/ml of penicillin, and 50 ug/ml of streptomycin was used for all cell cultures and diluent medium. Cell cultures. Primary rabbit kidney cultures were prepared by aseptically removing both kidneys from a four­week old rabbit. The capsule and cortex were removed and the remaining tissue was minced and then washed with phosphate-buffered saline (PBS), deficient in calcium and magnesium, to remove as many red cells as possible. After washing, individual cells were extracted from the tissue 99 in 0.25% trypsin for I hr at 37C. The resulting suspension was centrifuged, resuspended in MEM and seeded into 32 ounce prescription bottles. primary mouse embryo fibroblasts (MEF), human foreskin fibroblasts (HFF), and fetal lamb kid­ney (FLK) cells were prepared in a similar manner. Human embryonic lung ('vvI-38) cells were obtained from Dr. Leonard Hayflick, Stanford University, palo Alto, Calif.. HFF, FLK, andwI-38 cells were maintained as continous passage in 32 ounce bottles containing r-1EM with 10% calf serum at 37C. Antiviral drugs. Cytosine arabinoside (Ara-C, Cytosar, Cytarabine) was kindly supplied by the Upjohn Company, Kalamazoo, Michigan. The lyophilized powder was reconstituted with the diluent supplied, and diluted in PBS in order to obtain the desired concentrations. The drug was administered in a volume of 0.05 ml by the in­traperitoneal route. Adenine arabinoside (Ara-A) was prepared by Parke Davis and Co., Detroit, Michigan, and supplied through the Antiviral Substances Program of the 100 NIH, Bethesda, Md. 'rhe drug 'VIas stored as a powder at 4C and "vas prepared just prior to use. Since the drug is quite insoluble, it was administered as a suspension in 0.4% carboxymethylcellulose (Sigma Chemical Co., st. Louis, MO.) in a volume of 0.05 ml intraperitoneally. Virus assays. Assays for herpesvirus were performed using the plaque assay technique on a confluent monolayer of FLK cells in 10 x 30 mm plastic tissue culture dishes (Falcon Plastics, Oxnard, Calif.). The medium was aspirated from the plates, the cells washed once with PBS and a 0.2 ml inoculum of an appropriate virus dilution was added to the cell monolayers. The virus was allowed to adsorb at 37C for 60-90 min with occasional agitation. Following adsorption, a semisolid overlay medium consisting of .ME!,! and 0.5% agarose (Van Waters and Rogers, San Francisco, Calif.) was applied. Following 48 hr of incubation, a second overl