Infection of wild and laboratory animals with Machupo and Latino viruses*

Infection of wild and laboratory animals with Machupo and Latino viruses* P. A. WEBB,1 G. JUSTINES,2 & K. M. JOHNSON 3 Natural infection with Machupo and Latino viruses occurs only in the cricetine rodent Calomys callosus. Machupo virus inducesfatal infection in suckling mice and hamsters, and in adult guinea-pigs, marmosets, and rhesus monkeys. Latino virus kills only suckling hamsters; it produces chronic but non-viraemic infection in Calomys rodents. Machupo virus, in contrast, induces a viraemic immunotolerant infection in suckling Calomys, and a split response in animals more than 9 days of age. Tolerant infection is associated with haemolytic anaemia and splenomegaly, lesions not observed in animals able to clear viraemia andproduce circulating neutralizing antibodies. Experimental increase in the fraction of tolerant response was obtained by decreasing the virus dose or by phenotypic inbreeding of rodents. Long-term effects of tolerant infection included mild runting, decreased survival time, and almost total sterility among females, largely caused by fatal virus infection of enibryos. INTRODUCTION This report presents a selective summary of our continuing work on the biology of Machupo and Latino viruses. Studies were pursued in two principal directions: (a) a search for laboratory animals that might serve as models for human Bolivian haemor- rhagic fever, and (b) elucidation of the mechanisms by which Machupo virus is transmitted among Calo- nmys callosus rodents. NATURAL INFECTION IN ENZOOTIC FOCI OF MACHUPO VIRUS Naturally occurring antibody to Machupo virus has been found only in the small cricetine rodent Calomys callosus captured in localized areas of Boli- via. A survey of other rodents and marsupials in the areas of Bolivia where Machupo virus is endemic and in nonendemic surrounding areas included mar- supials of the genera Caluromys, Didelphis, and Marmosa, other cricetines of the genera Oryzomys, * From the Middle America Research Unit, Balboa Heights, Canal Zone. 1 Virologist, assigned by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, USA. 2 Virologist, Gorgas Memorial Institute. 3 Director. Zygodontomys, Holochilus, and Nectomys, the Muri- dae Mus and Rattus, and the echimyid Proechimys. Attempts to isolate virus from the kidney or spleen of a representative number of each genus were negative. The search for Latino antibody has been hampered by the lack of an adequate neutralization test; however, in non-anticomplementary rodent sera, no complement-fixing antibody was detected in any wild-caught rodent, including Calomys callosus (the only species from which Latino virus was recovered). EXPERIMENTAL INFECTION IN WILD, DOMESTIC, AND LABORATORY ANIMALS Antibody responses to experimental infection of various wild and domestic animals with Machupo virus (1) are summarized in Table 1. None of the animals became ill or died. The number of animals used per genus was limited, but only in Proechimys did all the rodents that were inoculated develop neutralizing (N) antibody. Adult animals in general proved clinically resis- tant to Machupo virus infection. As indicated in Table 2, the list of susceptible adults thus far includes only guinea-pigs (strain C-13), the marmo- set (Saguinus geoffroyi), and the rhesus monkey. Guinea-pig disease was nondescript; there was little or no viraemia and no evidence of haemorrhage. 3376 - 493 - BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 P. A. WEBB ET AL. Table 1. Formation of specific neutralizing antibody following experimental Machupo virus infection of animals Antibody No antibody Domestic: horse pig cat chicken rabbit Feral: Rodentia Oryzomys Tylomys Sigmodon Proechimys Rattus Capybara Marsupilia Didelphus Reptilia turtle Primate Cebus albifrons Cebus capucinus Aotus Detailed studies of pathogenesis have not been done. Marmosets developed anorexia, tremors, and clinical shock, and succumbed 8-20 days after inoculation. There was viraemia and virus multiplication in many tissues, excluding the brain. Prominent histologi- cal lesions were cortical necrosis of lymph nodes and splenic reticular hyperplasia with lymphoid de- pletion. Three strains of Latino virus apparently failed to infect the marmoset as measured by (a) failure to isolate virus at sacrifice 3 months after inoculation, (b) absence of complement-fixing (CF) antibody at 1 and 2 months after inoculation, and (c) complete susceptibility to Machupo virus challenge 3 months after inoculation. The arenaviruses, Tacaribe, Junin, and Pichinde, do infect marmosets as evidenced by the appearance of CF or N antibody, or both, within 2 months after inoculation, but all animals failed to resist Machupo virus challenge. Three marmosets inoculated with a higher dose of Pichinde virus died 27-30 days after inoculation. The disease produced by Machupo virus in rhesus monkeys resembled even more closely that seen in humans. Haemorrhagic signs and many of the pathological changes recorded for Bolivian haemor- rhagic fever were observed (2). Rhesus monkeys are currently being used as the definitive host for work on Machupo virus vaccine. The responses of colonized rodents to infection Table 2. Experimental infection of laboratory animals with Machupo and Latino viruses Machupo virus Latino virus Animal Viraemia N antibodies Death Viraemia CF antibodies Death Suckling: hamster + + + + white mouse + + - - - Calomys callosus ex. San Joaquin + - - a -+ ex. Juan Latino +- -1+ - +- + - Adult: hamster NT c + - NT + - white mouse NT + - NT Calomys callosus ex. San Joaquin +/- -/+ - +/- -/+ ex. Juan Latino +/- -1+ - - + guinea-pig (C-13) - + NT NT NT marmoset (S. geoffroyi) + + rhesus monkey (M. mulatta) d + + - ? a = split response. b -/+ antibody response corresponds to indicated viraemic split response. c NT = not tested. d Data from Terrell et al. (2) and from G. Eddy, personal communication. 494 MACHUPO AND LATINO VIRUS INFECTIONS with Machupo or Latino virus are depicted in Table 2. Suckling hamsters and mice were suscep- tible to Machupo virus infection with fatal outcome; adult hamsters and mice developed N antibody. Latino virus failed to infect either suckling or adult mice, but the hamster response pattern was similar to that of Machupo virus. Calomys callosus has proved to be a fascinating model, affording many as yet unsolved but intriguing riddles. For example, Latino virus was found to produce chronic but nontolerant infection in suck- ling animals, whereas all such Calomys inoculated with Machupo virus had persistent viraemia and no readily detectable antibodies. The split response of Calomys older than 9 days of age to inoculation with Machupo virus has been described previously (3, 4). The " immunocompetent " response of half the ani- mals is characterized by clearance of viraemia, pre- sence of N, CF, and immunofluorescent (IF) anti- body, minimal or absent viruria, and no anaemia or splenomegaly. Animals showing such a response will hereafter be referred to as type B. By contrast, the other half are " immunotolerant " responders, refer- red to subsequently as type A; they have persistent viraemia, little or no N or IF antibody, viruria, anaemia, splenomegaly, and reduced fertility. The anaemia associated with type A Machupo virus infection was found to be microcytic and hypochromic. Heavy accumulations of iron pigment were observed in phagocytic cells of the spleen and liver, strongly suggesting a haemolytic process. But both direct and indirect Coombs tests done with anti-Calomnys globulins were uniformly negative. Histological examination of the enlarged spleens revealed chronic reticular hyperplasia, maturational arrest of lymphoid elements, and a marked erythroid hyperplasia. FACTORS INFLUENCING RESPONSE OF WEANED CALOMYS RODENTS TO MACHUPO VIRUS INFECTION What triggers the split response of an individual Calomys is still speculative. Since the Machupo virus strain used in our experiments had been passaged twice in hamsters, it seemed possible that a latent hamster agent might be responsible for some or all of the responses seen in type A infection in Calomys. To test this hypothesis, a Machupo virus strain from human splenic tissue was passaged and plaque- purified only in African green monkey (Vero) cells. Table 3. Dose response in Calomys to Machupo virus infection No. of animals showing type A Dose or type B response Ratio (PFU) Experiment 1 Experiment 2 A/B A B A B 106 12 14 6 11 1:1.5 104 16 8 2:1 102 24 1 12 2 12:1 This virus also produced the expected split response in weaned Calomys. Alternatively, Machupo virus might activate an- other agent, ordinarily latent in Caloniys rodents. Although such a possibility can probably never be eliminated, three types of experiment were done that at least place certain limits on this hypothetical mechanism. Although histologically the spleens of type A rodents are somewhat similar to those of mice infected with Rauscher virus (5), we found that this virus was incapable of producing overt disease or splenomegaly in Calomys. Furthermore, we found that splenectomized Calomys also exhibited all the other parameters of the split response to Machupo virus, thus eliminating this organ as the refuge of a presumed latent agent. Finally, we discovered, as seen in Table 3, that progressive dilution of the standard virus pool led to an increasing incidence of type A (tolerant) infection. This is the reverse of what would be expected were Machupo virus acting as a direct " helper " to an unknown latent agent responsible for the observed phenomena. The role of host genetic factors in determining the response to virus infection was examined. Prelimi- nary results of an inbreeding study using 52 families and their descendants are presented in Table 4. The biggest hurdle in this study was the necessity to breed all the animals before knowing their Machupo virus response, since fertility is drastically reduced in " immunotolerant " responders after virus infection and since no infected animal could be permitted to leave the high-security laboratory. In only 37 of the matings of the P1 generation were we able to deter- mine the phenotype of both parents. Twenty-two of the sire/dam pairings were homotypic, A-A 10, B- B 12; 15 were heterotypic. A 3: 1 ratio of offspring phenotype in favour of the parent phenotype was found in the F1 generation of homotypic parents. 495 P. A. WEBB ET AL. Table 4. Crude analysis of genetic breeding study No. of animals showing type A or type B response Ratio A/B Aa Bb Total Fi of 37 matings 167 187 1 :1 Fl offspring of parents: A-A 71 25 3:1 B-B 27 i79 1 :3 All offspring of parents: A-A 1 3481 75 5:1 B-B 213 12471 1:1 a A = viraemia; no N antibody. b B = N antibody; no viraemia. However, when the phenotype of all offspring within a family pedigree was examined (and this might include up to 6 generations of brother-sister matings, and at least three backcross matings), homotypic A offspring seemed easier to obtain than homotypic B offspring. More precise analysis of these data is in progress. Although the data suggest that genetic factors are important, particularly in terms of phe- notypic immunotolerance, it is clear that a single gene locus does not account for the Calomys split response. The explanation may be that complex polygenetic factors determine the outcome or that unknown nongenetic control of host-response occurs. LONG-TERM EFFECTS OF MACHUPO VIRUS IN CALOMYS INFECTED AS SUCKLINGS During our work with Calomys rodents we had observed the appearance of spontaneous tumours in a number of the colonized animals. Fifty Calomys, one year of age, were studied to establish base-line tumour rates in noninfected rodents. Tumours occurred in 280% of the females and 400% of the males between the first and second year of life. The tumours were principally sarcomas of various mor- phological types. In order to see what effect Machupo infection had upon longevity and rate of tumour appearance in Calomys, a study was initiated using three groups of 120 suckling Calomys each. Group A was infected with Machupo virus, which causes both anaemia and splenomegaly in Calomys inoculated as sucklings. Group B received Latino virus, an arenavirus that infects Calomys but pro- duces neither anaemia nor splenomegaly. A nonvirus control group C was sham-inoculated. Mean body weights were less in the Machupo-infected animals, both at 4 months and at 9 months. The death rate was twice as high at the end of one year in the Machupo-infected animals, 65% versus 31% and 34% respectively. Latino virus had almost no effect upon either weight gain or longevity. The incidence of tumours did not exceed 4% in any group. Unfor- tunately, this study had to be terminated at the end of one year. EFFECT OF MACHUPO VIRUS INFECTION ON THE FERTILITY OF CALOMYS RODENTS Fertility in Calomys females with immunocompetent response to Machupo virus Such animals, although they at times excreted small amounts of virus in the urine, proved equally as fertile as controls when bred with normal males. Small amounts of Machupo N antibody were found in some of the offspring prior to suckling, and titres rose rapidly to within fourfold of the maternal values in the first week of life. Animals weaned at 21 days were never infected by their mothers, but within the next 40 days became fully susceptible to Ma- chupo virus. This may represent one mechanism responsible for the finding of normal mice in a population where the virus is present. Fertility in Calomys with immunotolerant response to Machupo virus Although anatomical and functional sexual devel- opment was apparently normal in both male and female rodents infected as sucklings, virus and fluo- rescent antigen were detected for many months in most tissues, including the reproductive organs. Testicular localization of antigen was confined to intertubular connective tissue and the secretory epi- thelium of the epididymus. In females, however, antigen was found in the epithelium of the vagina and uterus and in nearly every cell type of the ovary, occasionally including the ovum. When type A males were mated with normal females a normal percentage of pregnancies occurred, but the average litter size was about half that among controls. About 750 of females became infected after 3 days' contact with such males, whereas no normal males exposed 496 MACHUPO AND LATINO VIRUS INFECTIONS Table 5. Viability and infection of embryos in normal and Machupo-virus-infected female Calomys callosus mated with normal males Duratinof umber Viable embryos Dead or resorbingDuration ofNumber ~embryosgestatio(days) autopsied Virus Virus Virus Virus positive negative positive negative Infected females: 18-19 2 4 0 1 1 20-21 11 14 0 13 8 23-26 6 4 0 4 3 Normal females: 17-21 17 119 1 for a similar interval to type A infected males became infected. Thus, there is little doubt that sexual transmission of Machupo virus among Calo- mys is an important natural mechanism. In contrast, immunotolerant females, produced only 5% of the expected number of viable offspring, despite normal estrus cycles and direct evidence obtained by vaginal washings that copulation had occurred. Part of this near total sterility was due to failure of fertilization or successful embryonic im- plantation. Only 10 of 17 successfully impregnated tolerant females showed evidence of pregnancy when autopsied 17-20 days after insemination, in compar- ison with 9 of 9 normal females. The remainder of the fetal wastage was ascribable to Machupo virus infection as shown in Table 5. At 18-19 days of gestation, all viable embryos were virus-positive, and there were many dead and resorbing embryos, posi- tive or negative for virus, depending on the degree of resorption. By 23-26 days the majority of embryos were dead and parturition, normally occurring at 21-22 days, was inhibited. Fluorescent antigen was widely distributed in embryonic tissues, notably in the thymus, spleen, bone marrow, and brain. The placenta also contained Machupo antigen. Effect on the fertility of Calomys females of varying the time of virus infection and insemination Female rodents were inseminated and inoculated intraperitoneally with Machupo virus at different times as shown in Table 6. When virus was given within 3 days of insemination, we found a small Table 6. Machupo virus infection and viability of embryos in pregnant female Calomys callosus infected intraperitoneally at varying times after mating with normal males a Virus Viable embryos Dead or resorbinginocu- No. with Vibeebys embryos lated No. of dead or (days females infected after embryos Virus Virus Virus Virus mating) positive negative positive negative 0 3 3 2 5 9 1 3 6 4 0 28 2 1 5-7 8 0 0 46 0 0 a Animals autopsied at 17-19 days of gestation. proportion of infected or dead embryos. Both virus- positive and virus-negative embryos were found in the same female. Live offspring were not infected, but all soon became so from maternal milk, since all females were viraemic at this time. Finally, a similar type of experiment was performed using type A males as the simultaneous source of both virus and sper- matozoa. The results are depicted in Table 7. Again there was scattered embryonic infection and death. From these data we infer that once implantation and placental closure have taken place, Machupo virus does not reach the fetus of Calomys callosus. The pattern of fetal infection observed when simultane- ous infection and impregnation occur seems best explained by either random infection of eggs before or after fertilization or by local infection of implan- tation sites on the uterine epithelium or both. Table 7. Viability and infection of embryos from normal female Calomys callosus mated with Machupo- virus-infected males a No. with Viable embryos Dead or resorbing No. of dead or embryos females infectedfeaembryoste Virus Virus Virus Virusembryos positive negative positive negative Viraemic: 9 6 1 40 7 4 Non-viraemic: 3 0 0 19 0 0 a Animals autopsied at 20-21 days of gestation. 497 P. A. WEBB ET AL. DISCUSSION AND CONCLUSIONS Several observations and speculations arise from these very incomplete data. One is the little-noted fact that Latino virus was apparently incapable of infecting laboratory mice. Without suckling hamsters this agent would not at present " exist ". How many other arenaviruses await discovery? Will they necessarily all be parasites of rodents, and what detection systems will reveal their presence? Although the marmosets and the rhesus monkeys provide reasonable models of Bolivian haemorrhagic fever in man, neither represents the ideal host for future work on the pathogenesis of, and experimen- tal protection against, Machupo virus infection. Unless they can be colonized, marmosets are too heavily infected with other parasites to provide completely reliable data. Rhesus monkeys, although superior in the biological sense, are now almost im- possible to obtain. Thus more mammalian species, particularly primates, should be examined as poten- tial model hosts. Chronic, viraemic infection of the principal natural rodent hosts of all 4 arenaviruses pathogenic for man has now been demonstrated (6, 7, 8). But this mechanism does not necessarily operate for all known arenaviruses. Suckling Calomys were usually able to make immunocompetent responses to Latino virus (9) and a similar pattern obtained in the case of Tamiami virus and Siginodon hispidus (10). Finally, the dynamics of Machupo virus infection in colonized Calomys rodents may provide a gener- ally valid model of the ecology of all 4 arenaviruses that cause human disease. The split response to Machupo virus observed in weaned Calomys has not yet been demonstrated for any of the other agents and, in fact, definitive studies employing field strains of virus and appropriate nonselected rodent hosts have not been done. But the LCM-laboratory-mouse model has many fundamental features in common with ours, including runting, anaemia, and reduced life span, and reduced fertility in mice infected early in life (11). The differences, such as the fact that congenital LCM infection may be quite compatible with life (12) or that LCM can multiply its way through the formed placenta to the fetus (13) may turn out to be related to host factors inadvertently selected in the course of breeding laboratory Mus musculus. Indeed, the data of Blumenthal et al. (14), obtained in feral Mus populations infected with LCM, are quite similar to those predicted by the Machupo-Calomys model, which suggests that virus infection would be more common in larger rather than smaller wild colonies of rodents and that infected colonies would eventually pass through a phase of reduced population with near complete, tolerant infection. If this concept of viral mainten- ance is correct, the epidemiological implications for human arenavirus disease are evident. UMJ2 INFECTION PAR LES VIRUS MACHUPO ET LATINO CHEZ LES ANIMAUX SAUVAGES ET DE LABORATOIRE L'infection naturelle par les virus Machupo et Latino s'observe seulement chez le rongeur Cricetine, Calomys callosuis. Le premier virus entraine une infection fatale chez les souris et les hamsters A la mamelle, ainsi que chez les cobayes, les ouistitis et les singes rhesus adultes. Le virus Latino tue seulement les hamsters A la mamelle; il produit une infection chronique sans viremie chez Calomys. Au contraire, le virus Machupo entraine une infection viremique avec immunotolerance chez les Calomys A la mamelle, alors que les animaux ages de plus de 9 jours presentent deux types de reponses. Chez ceux qui sont tolerants, l'infection est associee a une anemie hemoly- tique et une splenomegalie, lesions que l'on n'observe pas chez les animaux capables d'eliminer le virus de leur sang et de produire des anticorps neutralisant humoraux. On a obtenu experimentalement un accroissement de la fraction donnant une reponse de tolerance, en abaissant la dose de virus ou bien par des croisements phenoty- piques consanguins des rongeurs. Les effets eloignes de l'infection <immunotol6rante comprenait: une legere hypotrophie, la diminution du temps de survie, et une sterilite presque totale parmi les femeilles, due en grande partie A l'infection fatale des embryons par le virus. A9 MACHUPO AND LATINO VIRUS INFECTIONS 499' REFERENCES 1. WEBB, P. A. ET AL. Some characteristics of Machupo virus, causative agent of Bolivian hemorrhagic fever. Amer. J. trop. Med. Hyg., 16: 535 (1967) 2. TERRELL, T. G. ET AL. Pathology of Bolivian hemor- rhagic fever. Amer. J. Path., 73: 479 (1973) 3. JusTINEs, G. & JOHNSON, K. M. Immune tolerance in Calomys callosus infected with Machupo virus. Na- ture (Lond.), 222: 1090-1091 (1969) 4. JOHNSON, K. M. ET AL. Biology of Tacaribe-complex viruses. In: Lehmann-Grube, F., ed. Lymphocytic choriomeningitis virus and other arenaviruses. Ber- lin, Heidelberg, & New York, Springer, 1973, p. 251 5. RAUSCHER, F. J. A virus-induced disease of mice characterized by erythrocytopoeisis and lymphoid leukemia. J. nat. Cancer Inst., 29: 515-543 (1962) 6. TRAuB, E. The epidemiology of lymphocytic chorio- meningitis virus in white mice. J. exp. Med., 64: 183- 200 (1936) 7. SABATTNI, M. S. & MAIZTEGUI, J. I. Adelantos en medicina: Fiebre hemorragica argentina. Medicina (B. Aires) 30, Suppl. 1: 111-128 (1970) 8. WALKER, D. H. ET AL. Comparative pathology of Lassa virus infection in monkeys, guinea-pigs, and Mastomys natalensis. Bull. World Health Organ., 52: 523-534 (1975) 9. WEBB, P. A. ET AL. Behavior of Machupo and Latino, viruses in Calomys callosus from two geographic areas of Bolivia. In: Lehmann-Grube, F., ed. Lym- phocytic choriomeningitis virus and other arena- viruses, Berlin, Heidelberg, & New York, Springer, 1973, p. 318 10. MuRPHY F. A. ET AL. Early lymphoreticular viral tropism and antigenic persistence: Tamiami virus, infection in the cotton rat. Lab. Invest., in press 11. MiMs, C. A. Observations on mice infected con- genitally or neonatally with lymphocytic chorio- meningitis (LCM) virus. Arch. ges. Virusforsch., 30: 67-74 (1970) 12. POLLARD, M. ET AL. Congenital lymphocytic chorio- meningitis virus infection in gnotobiotic mice. Proc. Soc. exp. Biol., 127: 755-761 (1968) 13. MiMs, C. A. Effect on the fetus of maternal infection with lymphocytic choriomeningitis (LCM) virus. J. infect. Dis., 120: 582-597 (1969) 14. BLUMENTHAL, W. ET AL. Epidemic distribution of the virus of lymphocytic choriomeningitis in an endemic region. Dtsch. med. Wschr., 93: 944-948 (1968) DISCUSSION COLE: Do you have an explanation for the fact that low doses of Machupo virus seem to give a high frequency of carrier states in Calomys, whereas with LCM virus it is the high doses that seem to produce a carrier state. WEBB: We have some hypotheses, but we have no answers. In fact, when we first did the experiment we thought we had done it wrong, so we did it over again two or three times. EDDY: I wonder whether Dr Justines could tell us any- thing about the amount of antigen that he sees in type A and type B Calomys in the long term after infection as adults. JUSTINES: The amount of antigen in the type A animat is high; if you do a fluorescent antibody test on a section from any organ you see specific fluorescence. When the type B animal develops antibody, it clears the viraemia and you do not find virus very easily. We have been able to recover virus using suckling hamsters, but not in tissue culture. 6