Infection of wild and laboratory 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 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 . 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 oftolerant response was obtained by decreasing the virus dose or byphenotypic 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 ofenibryos.

INTRODUCTION , , and , the Muri- dae Mus and Rattus, and the echimyid Proechimys. This report presents a selective summary of our Attempts to isolate virus from the kidney or spleen of continuing work on the biology of Machupo and a representative number of each genus were negative. Latino viruses. Studies were pursued in two principal The search for Latino antibody has been hampered directions: (a) a search for laboratory animals that by the lack of an adequate neutralization test; might serve as models for human Bolivian haemor- however, in non-anticomplementary rodent sera, no rhagic fever, and (b) elucidation of the mechanisms complement-fixing antibody was detected in any by which Machupo virus is transmitted among Calo- wild-caught rodent, including Calomys callosus (the nmys callosus rodents. only species from which Latino virus was recovered).

NATURAL INFECTION IN ENZOOTIC FOCI EXPERIMENTAL INFECTION IN WILD, OF MACHUPO VIRUS DOMESTIC, AND LABORATORY ANIMALS Naturally occurring antibody to Machupo virus Antibody responses to experimental infection of has been found only in the small cricetine rodent various wild and domestic animals with Machupo Calomys callosus captured in localized areas of Boli- virus (1) are summarized in Table 1. None of the via. A survey of other rodents and marsupials in the animals became ill or died. The number of animals areas of Bolivia where Machupo virus is endemic used per genus was limited, but only in Proechimys and in nonendemic surrounding areas included mar- did all the rodents that were inoculated develop supials of the genera Caluromys, Didelphis, and neutralizing (N) antibody. Marmosa, other cricetines of the genera , Adult animals in general proved clinically resis- * From the Middle America Research Unit, Balboa tant to Machupo virus infection. As indicated in Heights, Canal Zone. Table 2, the list of susceptible adults thus far 1 Virologist, assigned by the National Institute of Allergy includes only guinea-pigs (strain C-13), the marmo- and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, USA. set (Saguinus geoffroyi), and the rhesus monkey. 2 Virologist, Gorgas Memorial Institute. Guinea-pig disease was nondescript; there was little 3 Director. or no viraemia and no evidence of haemorrhage.

3376 - 493 - BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 494 P. A. WEBB ET AL.

Table 1. Formation of specific neutralizing antibody cal lesions were cortical necrosis of lymph nodes following experimental Machupo virus infection of and splenic reticular hyperplasia with lymphoid de- animals pletion. Antibody No antibody Three strains of Latino virus apparently failed to infect the marmoset as measured by (a) failure to Domestic: horse pig isolate virus at sacrifice 3 months after inoculation, cat chicken rabbit (b) absence of complement-fixing (CF) antibody at 1 and 2 months after inoculation, and (c) complete Feral: susceptibility to Machupo virus challenge 3 months Rodentia Oryzomys Tylomys Sigmodon after inoculation. The arenaviruses, Tacaribe, Junin, Proechimys and Pichinde, do infect marmosets as evidenced by Rattus Capybara the appearance of CF or N antibody, or both, within Marsupilia Didelphus 2 months after inoculation, but all animals failed to resist Machupo virus challenge. Three marmosets Reptilia turtle inoculated with a higher dose of Pichinde virus died Primate Cebus albifrons Cebus capucinus Aotus 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 Detailed studies of pathogenesis have not been done. pathological changes recorded for Bolivian haemor- Marmosets developed anorexia, tremors, and clinical rhagic fever were observed (2). Rhesus monkeys are shock, and succumbed 8-20 days after inoculation. currently being used as the definitive host for work There was viraemia and virus multiplication in many on Machupo virus vaccine. tissues, excluding the brain. Prominent histologi- The responses of colonized rodents to infection

Table 2. Experimental infection of laboratory animals with Machupo and Latino viruses

Machupo virus Latino virus 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. MACHUPO AND LATINO VIRUS INFECTIONS 495 with Machupo or Latino virus are depicted in Table 3. Dose response in Calomys to Machupo virus Table 2. Suckling hamsters and mice were suscep- infection tible to Machupo virus infection with fatal outcome; No. of animals showing type A adult hamsters and mice developed N antibody. Dose or type B response Latino virus failed to infect either suckling or adult Ratio (PFU) Experiment 1 Experiment 2 A/B mice, but the hamster response pattern was similar to that of Machupo virus. A B A B

Calomys callosus has proved to be a fascinating 106 12 14 6 11 1:1.5 model, affording many as yet unsolved but intriguing 104 16 8 2:1 riddles. For example, Latino virus was found to produce chronic but nontolerant infection in suck- 102 24 1 12 2 12:1 ling animals, whereas all such Calomys inoculated with Machupo virus had persistent viraemia and no readily detectable antibodies. The split response of This virus also produced the expected split response Calomys older than 9 days of age to inoculation with in weaned Calomys. Machupo virus has been described previously (3, 4). Alternatively, Machupo virus might activate an- The " immunocompetent " response of half the ani- other agent, ordinarily latent in Caloniys rodents. mals is characterized by clearance of viraemia, pre- Although such a possibility can probably never be sence of N, CF, and immunofluorescent (IF) anti- eliminated, three types of experiment were done that body, minimal or absent viruria, and no anaemia or at least place certain limits on this hypothetical splenomegaly. Animals showing such a response will mechanism. Although histologically the spleens of hereafter be referred to as type B. By contrast, the type A rodents are somewhat similar to those of other half are " immunotolerant " responders, refer- mice infected with Rauscher virus (5), we found that red to subsequently as type A; they have persistent this virus was incapable of producing overt disease viraemia, little or no N or IF antibody, viruria, or splenomegaly in Calomys. Furthermore, we found anaemia, splenomegaly, and reduced fertility. that splenectomized Calomys also exhibited all the The anaemia associated with type A Machupo other parameters of the split response to Machupo virus infection was found to be microcytic and virus, thus eliminating this organ as the refuge of a hypochromic. Heavy accumulations of iron pigment presumed latent agent. Finally, we discovered, as were observed in phagocytic cells of the spleen and seen in Table 3, that progressive dilution of the liver, strongly suggesting a haemolytic process. But standard virus pool led to an increasing incidence of both direct and indirect Coombs tests done with type A (tolerant) infection. This is the reverse of anti-Calomnys globulins were uniformly negative. what would be expected were Machupo virus acting Histological examination of the enlarged spleens as a direct " helper " to an unknown latent agent revealed chronic reticular hyperplasia, maturational responsible for the observed phenomena. arrest of lymphoid elements, and a marked erythroid The role of host genetic factors in determining the hyperplasia. response to virus infection was examined. Prelimi- nary results of an inbreeding study using 52 families FACTORS INFLUENCING RESPONSE and their descendants are presented in Table 4. The OF WEANED CALOMYS RODENTS TO biggest hurdle in this study was the necessity to MACHUPO VIRUS INFECTION breed all the animals before knowing their Machupo virus response, since fertility is drastically reduced in What triggers the split response of an individual " immunotolerant " responders after virus infection Calomys is still speculative. Since the Machupo virus and since no infected animal could be permitted to strain used in our experiments had been passaged leave the high-security laboratory. In only 37 of the twice in hamsters, it seemed possible that a latent matings of the P1 generation were we able to deter- hamster agent might be responsible for some or all mine the phenotype of both parents. Twenty-two of of the responses seen in type A infection in Calomys. the sire/dam pairings were homotypic, A-A 10, B- To test this hypothesis, a Machupo virus strain from B 12; 15 were heterotypic. A 3: 1 ratio of offspring human splenic tissue was passaged and plaque- phenotype in favour of the parent phenotype was purified only in African green monkey (Vero) cells. found in the F1 generation of homotypic parents. 496 P. A. WEBB ET AL.

Table 4. Crude analysis of genetic breeding study causes both anaemia and splenomegaly in Calomys inoculated as sucklings. Group B received Latino No. of animals showing type A or type B response Ratio virus, an arenavirus that infects Calomys but pro- A/B duces neither anaemia nor splenomegaly. A nonvirus A a Bb control group C was sham-inoculated. Mean body weights were less in the Machupo-infected animals, Total Fi of 37 matings 167 187 1 :1 both at 4 months and at 9 months. The death rate Fl offspring of parents: was twice as high at the end of one year in the A-A 71 25 3:1 Machupo-infected animals, 65% versus 31% and 34% respectively. Latino virus had almost no effect B-B 27 i79 1 :3 upon either weight gain or longevity. The incidence All offspring of parents: of tumours did not exceed 4 % in any group. Unfor- A-A 1 3481 75 5:1 tunately, this study had to be terminated at the end of one year. B-B 213 12471 1:1

EFFECT OF MACHUPO VIRUS INFECTION a A = viraemia; no N antibody. ON THE FERTILITY OF CALOMYS RODENTS b B = N antibody; no viraemia. Fertility in Calomys females with immunocompetent response to Machupo virus However, when the phenotype of all offspring within Such animals, although they at times excreted a family pedigree was examined (and this might small amounts of virus in the urine, proved equally include up to 6 generations of brother-sister matings, as fertile as controls when bred with normal males. and at least three backcross matings), homotypic A Small amounts of Machupo N antibody were found offspring seemed easier to obtain than homotypic B in some of the offspring prior to suckling, and titres offspring. More precise analysis of these data is in rose rapidly to within fourfold of the maternal progress. Although the data suggest that genetic values in the first week of life. Animals weaned at 21 factors are important, particularly in terms of phe- days were never infected by their mothers, but within notypic immunotolerance, it is clear that a single the next 40 days became fully susceptible to Ma- gene locus does not account for the Calomys split chupo virus. This may represent one mechanism response. The explanation may be that complex responsible for the finding of normal mice in a polygenetic factors determine the outcome or that population where the virus is present. unknown nongenetic control of host-response occurs. Fertility in Calomys with immunotolerant response to Machupo virus LONG-TERM EFFECTS OF MACHUPO VIRUS Although anatomical and functional sexual devel- IN CALOMYS INFECTED AS SUCKLINGS opment was apparently normal in both male and female rodents infected as sucklings, virus and fluo- During our work with Calomys rodents we had rescent antigen were detected for many months in observed the appearance of spontaneous tumours in most tissues, including the reproductive organs. a number of the colonized animals. Fifty Calomys, Testicular localization of antigen was confined to one year of age, were studied to establish base-line intertubular connective tissue and the secretory epi- tumour rates in noninfected rodents. Tumours thelium of the epididymus. In females, however, occurred in 280% of the females and 400% of the antigen was found in the epithelium of the vagina males between the first and second year of life. The and uterus and in nearly every cell type of the ovary, tumours were principally sarcomas of various mor- occasionally including the ovum. When type A phological types. In order to see what effect males were mated with normal females a normal Machupo infection had upon longevity and rate of percentage of pregnancies occurred, but the average tumour appearance in Calomys, a study was initiated litter size was about half that among controls. About using three groups of 120 suckling Calomys each. 750 of females became infected after 3 days' contact Group A was infected with Machupo virus, which with such males, whereas no normal males exposed MACHUPO AND LATINO VIRUS INFECTIONS 497

Table 5. Viability and infection of embryos in normal Table 6. Machupo virus infection and viability of and Machupo-virus-infected female Calomys callosus embryos in pregnant female Calomys callosus infected mated with normal males intraperitoneally at varying times after mating with normal males a Duratinof umber Viable Dead or resorbing Duration of embryos Number ~embryos Virus Viable embryos Dead or resorbing gestatio autopsied inocu- No. with Vibeebys embryos (days) Virus Virus Virus Virus lated No. of dead or positive negative positive negative (days females infected after embryos Virus Virus Virus Virus mating) positive negative positive negative Infected females: 0 3 3 2 5 9 1 18-19 2 4 0 1 1 3 6 4 0 28 2 1 20-21 11 14 0 13 8 5-7 8 0 0 46 0 0 23-26 6 4 0 4 3 Normal females: a Animals autopsied at 17-19 days of gestation. 17-21 17 119 1

proportion of infected or dead embryos. Both virus- positive and virus-negative embryos were found in for a similar interval to type A infected males the same female. Live offspring were not infected, became infected. Thus, there is little doubt that but all soon became so from maternal milk, since all sexual transmission of Machupo virus among Calo- females were viraemic at this time. Finally, a similar mys is an important natural mechanism. type ofexperiment was performed using type A males In contrast, immunotolerant females, produced as the simultaneous source of both virus and sper- only 5 % of the expected number of viable offspring, matozoa. The results are depicted in Table 7. Again despite normal estrus cycles and direct evidence there was scattered embryonic infection and death. obtained by vaginal washings that copulation had From these data we infer that once implantation and occurred. Part of this near total sterility was due to placental closure have taken place, Machupo virus failure of fertilization or successful embryonic im- does not reach the fetus of Calomys callosus. The plantation. Only 10 of 17 successfully impregnated pattern of fetal infection observed when simultane- tolerant females showed evidence of pregnancy when ous infection and impregnation occur seems best autopsied 17-20 days after insemination, in compar- explained by either random infection of eggs before ison with 9 of 9 normal females. The remainder of or after fertilization or by local infection of implan- the fetal wastage was ascribable to Machupo virus tation sites on the uterine epithelium or both. 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- Table 7. Viability and infection of embryos from tive or negative for virus, depending on the degree of normal female Calomys callosus mated with Machupo- resorption. By 23-26 days the majority of embryos virus-infected males a were dead and parturition, normally occurring at or resorbing was No. with Viable embryos Dead 21-22 days, inhibited. Fluorescent antigen was No. of dead or embryos widely distributed in embryonic tissues, notably in femalesfea infected embryosteembryos Virus Virus Virus Virus the thymus, spleen, bone marrow, and brain. The positive negative positive negative placenta also contained Machupo antigen. Viraemic: Effect on the fertility of Calomys females of varying the time of virus infection and insemination 9 6 1 40 7 4 Non-viraemic: Female rodents were inseminated and inoculated intraperitoneally with Machupo virus at different 3 0 0 19 0 0 times as shown in Table 6. When virus was given within 3 days of insemination, we found a small a Animals autopsied at 20-21 days of gestation. A9 P. A. WEBB ET AL.

DISCUSSION AND CONCLUSIONS Finally, the dynamics of Machupo virus infection Several observations and speculations arise from in colonized Calomys rodents may provide a gener- these very incomplete data. One is the little-noted ally valid model of the ecology of all 4 arenaviruses fact that Latino virus was apparently incapable of that cause human disease. The split response to infecting laboratory mice. Without suckling Machupo virus observed in weaned Calomys has not hamsters this agent would not at present " exist ". yet been demonstrated for any of the other agents How many other arenaviruses await discovery? Will and, in fact, definitive studies employing field strains they necessarily all be parasites of rodents, and what of virus and appropriate nonselected rodent hosts detection systems will reveal their presence? have not been done. But the LCM-laboratory-mouse Although the marmosets and the rhesus monkeys model has many fundamental features in common provide reasonable models of Bolivian haemorrhagic with ours, including runting, anaemia, and reduced fever in man, neither represents the ideal host for life span, and reduced fertility in mice infected early future work on the pathogenesis of, and experimen- in life (11). The differences, such as the fact that tal protection against, Machupo virus infection. congenital LCM infection may be quite compatible Unless they can be colonized, marmosets are too with life (12) or that LCM can multiply its way heavily infected with other parasites to provide through the formed placenta to the fetus (13) may completely reliable data. Rhesus monkeys, although turn out to be related to host factors inadvertently superior in the biological sense, are now almost im- selected in the course of breeding laboratory Mus possible to obtain. Thus more mammalian species, musculus. Indeed, the data of Blumenthal et al. (14), particularly primates, should be examined as poten- obtained in feral Mus populations infected with tial model hosts. LCM, are quite similar to those predicted by the Chronic, viraemic infection of the principal Machupo-Calomys model, which suggests that virus natural rodent hosts of all 4 arenaviruses pathogenic infection would be more common in larger rather for man has now been demonstrated (6, 7, 8). But than smaller wild colonies of rodents and that this mechanism does not necessarily operate for all infected colonies would eventually pass through a known arenaviruses. Suckling Calomys were usually phase of reduced population with near complete, able to make immunocompetent responses to Latino tolerant infection. If this concept of viral mainten- virus (9) and a similar pattern obtained in the case of ance is correct, the epidemiological implications for Tamiami virus and Siginodon hispidus (10). 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 tolerants, l'infection est associee a une anemie hemoly- s'observe seulement chez le rongeur Cricetine, Calomys tique et une splenomegalie, lesions que l'on n'observe callosuis. Le premier virus entraine une infection fatale pas chez les animaux capables d'eliminer le virus de leur chez les souris et les hamsters A la mamelle, ainsi que sang et de produire des anticorps neutralisant humoraux. chez les cobayes, les ouistitis et les singes rhesus adultes. On a obtenu experimentalement un accroissement de la Le virus Latino tue seulement les hamsters A la mamelle; fraction donnant une reponse de tolerance, en abaissant il produit une infection chronique sans viremie chez la dose de virus ou bien par des croisements phenoty- Calomys. piques consanguins des rongeurs. Les effets eloignes de Au contraire, le virus Machupo entraine une infection l'infection

REFERENCES

1. WEBB, P. A. ET AL. Some characteristics of Machupo Mastomys natalensis. Bull. World Health Organ., 52: virus, causative agent of Bolivian hemorrhagic fever. 523-534 (1975) Amer. J. trop. Med. Hyg., 16: 535 (1967) 9. WEBB, P. A. ET AL. Behavior of Machupo and Latino, 2. TERRELL, T. G. ET AL. Pathology of Bolivian hemor- viruses in Calomys callosus from two geographic rhagic fever. Amer. J. Path., 73: 479 (1973) areas of Bolivia. In: Lehmann-Grube, F., ed. Lym- 3. JusTINEs, G. & JOHNSON, K. M. Immune tolerance in phocytic choriomeningitis virus and other arena- Calomys callosus infected with Machupo virus. Na- viruses, Berlin, Heidelberg, & New York, Springer, ture (Lond.), 222: 1090-1091 (1969) 1973, p. 318 4. JOHNSON, K. M. ET AL. Biology of Tacaribe-complex 10. MuRPHY F. A. ET AL. Early lymphoreticular viral viruses. In: Lehmann-Grube, F., ed. Lymphocytic tropism and antigenic persistence: Tamiami virus, choriomeningitis virus and other arenaviruses. Ber- infection in the cotton rat. Lab. Invest., in press lin, Heidelberg, & New York, Springer, 1973, p. 251 11. MiMs, C. A. Observations on mice infected con- genitally or neonatally with lymphocytic chorio- 5. RAUSCHER, F. J. A virus-induced disease of mice meningitis (LCM) virus. Arch. ges. Virusforsch., 30: characterized by erythrocytopoeisis and lymphoid 67-74 (1970) leukemia. J. nat. Cancer Inst., 29: 515-543 (1962) 12. POLLARD, M. ET AL. Congenital lymphocytic chorio- 6. TRAuB, E. The epidemiology of lymphocytic chorio- meningitis virus infection in gnotobiotic mice. Proc. meningitis virus in white mice. J. exp. Med., 64: 183- Soc. exp. Biol., 127: 755-761 (1968) 200 (1936) 13. MiMs, C. A. Effect on the fetus of maternal infection 7. SABATTNI, M. S. & MAIZTEGUI, J. I. Adelantos en with lymphocytic choriomeningitis (LCM) virus. J. medicina: Fiebre hemorragica argentina. Medicina infect. Dis., 120: 582-597 (1969) (B. Aires) 30, Suppl. 1: 111-128 (1970) 14. BLUMENTHAL, W. ET AL. Epidemic distribution of the 8. WALKER, D. H. ET AL. Comparative pathology of virus of lymphocytic choriomeningitis in an endemic Lassa virus infection in monkeys, guinea-pigs, and region. Dtsch. med. Wschr., 93: 944-948 (1968)

DISCUSSION

COLE: Do you have an explanation for the fact that low thing about the amount of antigen that he sees in type A doses of Machupo virus seem to give a high frequency and type B Calomys in the long term after infection as of carrier states in Calomys, whereas with LCM virus adults. it is the high doses that seem to produce a carrier state. JUSTINES: The amount of antigen in the type A animat WEBB: We have some hypotheses, but we have no answers. is high; if you do a fluorescent antibody test on a section In fact, when we first did the experiment we thought we from any organ you see specific fluorescence. When the had done it wrong, so we did it over again two or three type B animal develops antibody, it clears the viraemia times. and you do not find virus very easily. We have been able to recover virus using suckling hamsters, but not in EDDY: I wonder whether Dr Justines could tell us any- tissue culture.

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