Current Topics in Microbiology 92 and Immunology Edited by w. Henle, Philadelphia· P.H. Hofschneider, Martinsried P. KoldovskY, DUsseldorf· H. Koprowski, Philadelphia O. Maa10e, Copenhagen· F. Melchers, Basle . R. Rott, GieBen HG. Schweiger, Ladenburg/Heidelberg . L. Syrucek, Prague P.K. Vogt, Los Angeles Natural Resistance to Tumors and Viruses Edited by Otto Haller With 22 Figures Springer- Verlag Berlin Heidelberg N ew York 1981 Dr. Otto Haller Institut fur Immunologie und Virologie der Universillit ZUrich, GloriastraBe 32 B CH-8028 ZUrich, Switzerland ISBN-I3: 978-3-642-68071-7 e-ISBN -13: 978-3-642-68069-4 DOl: 10.1007/978-3-642-68069-4 This work is subject to copyright All rights are reserved, whether the whole or part of the ma terial is concerned, specifically those of translation, reprinting, re-use of illustration broadca sting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to 'Verwertungsgesellschaft Wort', Munich. © by Springer-Verlag Berlin Heidelberg 1981. Library of Congress Catalog Card Number 15-12910. Softcover reprint of the hardcover 1st edition 1981 The use of registered names, trademarks, etc. in this publication, does nor imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Fotosatz Service Weihrauch, Wiirzburg Table of Contents Preface . . . . . . . . . . . . . . . . . VI Margo A. Brinton: Genetically Controlled Resistance to Flavivirus and Laptate-Dehydrogenase-Elevating Virus- Induced Disease . . . . . . . . . . .. 1 C. Lopez: Resistance to Herpes Simplex Virus - Type 1 (HSV-l) ............... 15 O. Haller: Inborn Resistance of Mice to Orthomyxoviruses. 25 J.-L. Virelizier: Role of Macrop hages and Interferon in Natu- ral Resistance to Mouse Hepatitis Virus Infection . . 53 V. Kumar and M. Bennett: Genetic Resistance to Friend Vi rus-Induced Erythroleukemia and Immunosuppression 65 R.M. Welsh: Natural Cell-Mediated Immunity During Viral Infections. . . . . . . . . . . . . . . . 83 R. Kiessling and H. Wigzell: Surveillance of Primitive Cells by Natural Killer Cells . 107 Subject Index . . . . .125 Indexed in Current Contents Preface Natural resistance is now coming to be recognized as a potentially important phenomenon in host defense against infection and ma lignancy. Genetically controlled resistance mechanisms are usUally effective early in infection and before conventional immune responses are generated. Comparisons of experimental systems where natural resistance plays a prominent role demon strate the complexities of the host defense mechanisms involved, as evidenced in the present volume. Nevertheless, some com mon components of genetic resistance are discernible and largely comprise natural killer cells, macrophages, and interferon These and additional factors would seem to constitute a first line of de fense in host resistance against both viruses and tumors. It is evi dent that considerable variation in the relative importance of di stinct mechanisms may be found among various resistance sy stems and that, most likely, additional effector functions will be discovered. Resistance to tumors and most viruses is under polygenic control, has a complex mode ofi nheritance, and depends on appro priately complex effector mechanisms. Instances, however, whe re a single gene locus determines resistance or susceptibility to a virus, as in the case of resistance to flaviviruses or influenza viru ses, would seem to offer good prospects for elucidating the basic factors involved. Resistance to influenza virus would indeed seem to represent a comparatively simple situation: resistance is expressed at the host cell level, and interferon is its main media tor. The present volume provides insight into current concepts of such resistance mechanisms. It contains contributions from di stinguished laboratories presently engaged in relevant research in this field. A variety of experimental systems are analyzed cove ring genetic resistance in mice to flaviviruses, herpes simplex virus, lactate dehydrogenase elevating virus, influenza viruses and mouse hepatitis virus. Other chapters deal with interesting aspects of resistance to leukemogenesis and immunosuppression by Friend virus, with the biological significance of natural cell me diated immunity in viral infection and with tumor resistance and immune regulation by natural killer cells. Zurich, Apri11981 Otto Haller Genetically Controlled Resistance to Flavivirus and Lactate Dehydrogenase-Elevating Virus-Induced Disease MARGO A. BRINTON* 1 Introduction . . . . . 2 Genetically Controlled Resistance to Flaviviruses. . . . . . . . . . . . . 3 Genetically Controlled Resistance to Lactate-Dehydrogenase-Elevating Virus-Induc- ed Paralysis 9 4Sunnnary 12 References 12 1 Introduction The ftrst demonstrations that a host gene could control resistance to disease induced by an animal virus were reported independently by Lynch and Hughes (1936) and Webster and Clow (1936). Subsequently this resistance was found to be speciftcally directed againstflaviviruses. A number of other genes which confer resistance to other types ofv i rus infections have since been identifted (Pincus and Snyder1975; Bang 1978). Different classes of viruses vary greatly in their mode and site ofr eplication, and it would be expect ed that the mechanisms of action of various resistance gene products would also differ signiftcantly. The strict virus speciftcity of host genetically controlled resistance indicates that the resistance gene products interact with unique molecular events characteristic of only one type of virus. Such a speciftc resistance mechanism acting at the cellular level constitutes a ftrst-line host defense mechanism. However, the phenotypic expression of resistance genes on the whole-animal level can certainly be modifted by the degree of functioning of other types of host defense mechanisms. 2 Genetically Controlled Resistance to Flaviviruses More than 50 different flaviviruses have been identifted to date by serologic means, and several of these are the cause of signiftcant human morbidity and mortality worldwide. However, many of the molecular details of the flavivirus replication cycle are still poorly understood. Flaviviruses belong to the togavirus family, which also consists of alphaviru ses, pestiviruses, rubivirus, and several unclassifted viruses, including lactate-dehydroge nase-elevating virus (SchlesingerI980). In general, togaviruses are characterized by a lipid envelope and an infectious single-stranded RNA genome. The various genera represent ed within the togavirus family are distinguished by differences in their modes of replica tion, by their fme morphological detail, and by their interaction with host resistance genes. *The Wi star Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104 2 Margo A. Brinton Webster (1923), working with Bacillus enteriditisinfection in a stock of randomly bred Swiss mice found that susceptibility varied greatly among individual mice. By selection and inbreeding, Webster (1933) developed bacteria-resistant (BR) and bacteria-suscepti ble (BS) strains. Subsequent studies indicated that mouse strains resistant (VR) or sus ceptible (VS) to Louping ill virus could also be selected, but that virus and bacterial resist ance were inherited independently (Webster and Fite 1933, 1934). Mice resistant to Lou ping ill virus were also resistant to St Louis encephalitis virus (Webster 1937) and Russian spring-summer encephalitis virus (Casals and Schneider 1943). Since at the time of Web ster's studies these viruses had not yet been classified (Casalsand Brown 1954), the flavivi rus-specific nature of the observed host-controlled resistance was not at frrst realized (Sa bin 1953). Genetically controlled resistance to yellow fever virus (YFV), another flavivirus, was independently observed by Smt.yer and Lloyd (1931) among randomly bred Rockefeller Institute mice, by Lynch and Hughes (1936) among randomly bred mice of the "Def' strain, and by Sabin (1952a, b, 1954) in Princeton Rockefeller mice (PR!). Within all known resistant mouse strains, "Def', BRV R, BSVR, and PRI, the flavivi rus resistance is inherited as a simple autosomal dominant allele (Lynch and Hughes 1936; Webster 1937; Sabin 1952b). No other inbred mouse strains commonly used in laborato ries have been found to possess the flavivirus resistance gene (Darnell et al. 1974). Using PRI mice as a source for the gene, another inbred resistant strain, C3H1RV, was created congenic to C3H1HE (Groschel and Koprowski 1965). C3H1RVand C3H1HE mice share common red blood cell antigens, and skin grafts are interchangeable between them. The development of the congenic C3H strains has allowed comparative studies of genetically controlled resistance to flaviviruses to be carried out against a low background of unrelat ed variables. A study of wild mice caught in California and Maryland demonstrated the presence of the flavivirus resistance gene among wild mouse populations (Darnell et al. 1974) (Table 1). The finding that this resistance gene has continued to segregate within wild mice populations indicates that the gene may actually convey a selective advantage under natural conditions. However, which flaviviruses exert selective pressure on wild Mus musculus populations, other than possibly Powassan, is not yet known. Factors such as the age of the host, its immune status, the degree of virulence ofthe infecting flavivirus, and the route of infection have been observed to influence the phe notypic expression of the flavivirus resistance gene. However, no evidence has been re ported which indicates that these factors are involved in the specific mechanism of resist ance mediated by the product of the gene. Resistant mice do support the replication of flaviviruses, but virus yields are lower and the spread of the infection is slower and usually selflimiting as compared to suscept ible mice (Goodman and Koprowski 1962a; Darnell et al. 1974). For instance, C3H1RV mice survive intracerebral injection of undiluted 17D-YFV, whereas 100% of C3H1HE mice die. The phenotypic expression of resistance can be overwhelmed by large doses of a virulent flavivirus given by the intracerebral route (Goodman and Koprowski 1962a). West Nile virus (WNV) can kill resistant mice after being injected intracerebrally, but 100-1000 times more virus is required to produce disease and death in resistant mice as compared to susceptible controls (Vanio et al. 1961; Hanson and Koprowski 1969; Darnell et al. 1974). The day of onset of disease symptoms is delayed in resistant mice, and the maximum virus titer in the brain is 2 to 3 logs lower than in comparable susceptible mice (see Fig. 1). Genetically Lactate-Dehydrogenase-Elevating Disease 3 Table 1. Inheritance of resistance to yellow fever virus (YFV, strain 17D) in wild Mus Musculus Wild (?J X C3H1He (rr) Fl Wild parent Surviving:deadb G31c 0:9c G32 0:14 G33 9:2 G41 13:0 G42 4:6 051 7:10 G52 17:0 F I Survivors t Resistant Fl (Rr) X Resistant Fl (Rri t F2 7:3 9:1 7:2 5:1 6:0 Total 34:7 Reproduced with permission from Darnell et al. 1974 a Genotype b Ratio of mice surviving challenge to those dying from it Two litters of progeny from each parent were tested c Two-month-old Fl mice were given an intracerebral injection of 0.03 m1 undiluted 17D-YFV d Survivors were mated brother to sister approximately 2 months after their original infection In susceptible animals the levels of neutralizing antibody and interferon which are produced in response to WNV infection are higher than in resistant ones; this corre sponds to the higher titers of virus synthesized by susceptible animals (Jacoby et al. 1980; Hanson and Koprowski 1969; Darnell and Koprowski 1974). The in vivo expression of ge netic resistance to flaviviruses requires an intact lymphoreticular system (Goodman and Koprowski 1962a; Jacoby and Bhatt 1976a, b; Jacoby et al. 1980). Immunosuppression of resistant animals with cyclophosphamide, sublethal X-ray irradiation, or thymus cell de pletion converts a normally asymptomatic flavivirus infection into a lethal one. However, under such conditions the onset of sickness in resistant mice is delayed several days as compared to susceptible control mice, and the virus titers in moribund resistant brains are lower than in comparable susceptible brains. Jacoby and Bhatt (1976b) demonstrated that even though T-cell-depleted, flavivirus-infected resistant mice did produce detect able levels of hemagglutination-inhibiting antiviral antibody, they were not protected from lethal infections. 4 Margo A. Brinton 9 8 ~ <tz 0::- m<t 7 0:: :!Em ~:!E (!)<t 6 ....... 0:: (/)(!) 1- ....... -=> Zu.. 5 =>a. !:(!) (!)o 4 0...J ...J 3 <2.5 2 3 4 5 6 7 DAYS AFTER INJECTION Fig. I. Growth of West Nile virus (WNV) in the brains of susceptible C3H mice (e-e) and resistant C3H1RV mice (0-0). Interferon levels were also measured in brains of the C3H (11-11) and C3H/RV (0-0) mice. The intracerebral inoculum (105 PFU in 0.03 m1) was a WNV pool produced in MK2 cells. (Reproduced with permission from Hanson and Koprowski 1969) The clearance of WNV from the blood of resistant and susceptible mice has been found to be essentially complete by 10 to 12 h after an intraperitoneal injection of virus (Goodman and Koprowski 1962b). In susceptible mice, this clearance was immediately followed by a rise in the titer of infectious virus in the blood; virus levels in the blood re mained high for at least 2 days. No such secondary viremia was detectable in blood from resistant mice (Goodman and Koprowski 1962b; Groschel and Koprowski 1965). Cell cultures derived from various tissues of resistant mice produce lower yields of flaviviruses than do comparable cultures of cells from congenic susceptible mice. This phenomenon was ftrst observed by (Websterand Johnson (1941), who studied the replica tion of St Louis encephalitis virus in brain cell cultures from resistant and susceptible animals. Goodman and Koprowski (1962a) reported a similar difference in yield with WNV. Resistant brain cell cultures infected with WNV yielded loo-fold less infectious virus than did susceptible cultures. Cultures of spleen cells, peritoneal exudate cells, and embryoftbroblasts from resistant animals displayed a similar diminished ability to support flavivirus replication when infected with either 17D-YFV or WNV (Goodman and Koprowski 1962a; Vanio 1963a, b; Hanson and Koprowski 1969; Darnell and Koprow ski (1974). The differential ability to replicate flaviviruses was maintained in established cell lines developed from SV4 0-transformed resistant and susceptible embryoftbroblasts (Darnell and Koprowski 1974). WNV adsorption and penetration occur normally in resist ant cells, since the same percentage of cells show virus-positive immunofluorescence in resistant and susceptible cultures by 6 to 8 h after infection (Darnell and Koprowski 1974). Recent studies indicate that the level of flavivirus RNA and protein synthesis is signif- Genetically Lactate Dehydrogenase-Elevating Disease 5 icantly lower in resistant cells as compared to susceptible ones, indicating that inhibition of flavivirus replication within resistant cells occurs at an early step in virus replication (Brinton 1981b). We have isolated a number of temperature-sensitive mutants ofWNV from persistently infected cultures of resistant and susceptible cells and are currently using these as tools for gaining a further understanding of the steps involved in flavivirus replication, as well as the differences in the virus-host interaction in resistant and suscept ible cells. The genesis of defective interfering (Dl) virus particles, one type ofv iral deletionmu tant, has been found to occur in cells infected with virtually any animal virus (Huangand Baltimore 1976) and seems to be controlled by host cell factors. Tissue culture experi ments indicate that flavivirus DI particles are produced more readily and/or interfere with standard infectious virus replication more efficiently in resistant cells than in sus ceptible one (Darnell and Koprowski 1974). Assessment of the ability of serially passaged culture fluids from infected resistant and susceptible cell cultures to interfere with the replication of infectious homologous standard virus revealed that detectable inter ference was only observed with samples from resistant cells (Table 2). Since the ratio of defective to infectious particles determines the extent of interference, the lack of an observable interference by samples from susceptible cultures apparently was due to the presence of an insufficient number of DI particles to cause a detectable interference in the test Susceptible cultures probably do produce DI particles as indicated by the cycling titer of infectious virus observed during serial undiluted passage in susceptible cells (Fig. 2). An identical decline in infectious vesicular stomatitis virus (VSV) was observed after serial undiluted passage in either flavivirus-resistant or -susceptible cell cultures, indicat ing that similar numbers of defective VSV particles are synthesized by both cultures and that the extent of interference is similar (Huang, personal communication). The reduced yield of flaviviruses observed in resistant cultures and animals was not accompanied by an earlier or enhanced production of interferon (Vanio et al. 1961). As Table 2. Interference between serial undiluted passage WNV and brain-produced WNV" 48 h WNV yield (loglo PFU/ml) Cell Moiof bBrain- bBrain-produced WNV bBrain-produced WNV type brain- produced plus 3rd passage plus 3rd passage produced WNV T-MEF-HE WNV T-MEF-RV WNV WNV T-MEF-RV 1.82 2.7 1.9 0.18 2.35 2.2 0.9 0.Q18 1.0 1.75 Undet~ctable T-MEF-HE 1.42 7.3 6.3 0.14 6.6 6.5 5.75 0.014 5.5 6.25 4.35 a WNV, West Nile virus. Reproduced with permission from Darnell and Koprowski 1974 b Titers of different virus preparations used for infection: brain-produced WNV = 108.9 PFU/ml; 3rd passage T-MEF-HE WNV = 104.5 PFU/ml; 3rd passage T-MEF-RVWNV = 102.5 PFU/ml; T-MEF-RV = SV40 transformed resistant C3M1RV embryofibroblasts; T-MEF-HE = SV40 transformed susceptible C3M/HE embryofibroblasts