Experimental Infection of the New World Owl Monkey (Aotus trivirgatus) with Hepatitis A Virus

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1 INFECTION AND IMMUNITY, May 1983, p /83/ $02.00/0 Copyright , American Society for Microbiology Vol. 40, No. 2 Experimental Infection of the New World Owl Monkey (Aotus trivirgatus) with Hepatitis A Virus JAMES W. LEDUC,1 STANLEY M. LEMON,2* CHARLOTTE MILLER KEENAN,3 R. ROSS GRAHAM,4 RUTH H. MARCHWICKI,2 AND LEONARD N. BINN2 Medical Division' and Division of Animal Resources,4 U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701, and the Division of Communicable Diseases and Immunology2 and the Division of Pathology,3 Walter Reed Army Institute of Research, Washington, D.C Received 2 September 1982/Accepted 13 January 1983 Epidemiological studies have demonstrated the susceptibility of the owl monkey (Aotus trivirgatus) to hepatitis A virus, but have not shown an association between infection and histopathological or chemical evidence of liver disease. Therefore, 12 seronegative, colony-bred monkeys were inoculated intravenously with a fecal suspension containing either PA33 strain hepatitis A virus (a strain recovered from a naturally infected Aotus sp.) or HM-175 virus (recovered from a human). Viral antigen was detected by radioimmunoassay in the feces of six monkeys 6 to 17 days after inoculation with PA33 virus, and by 9 to 21 days serum aminotransferase activities were significantly elevated in each. Antibody to the virus developed in each monkey by 28 days after inoculation. Similar findings were noted in five of six monkeys inoculated with HM-175 virus, although the incubation period preceding aminotransferase elevations was somewhat longer (25 to 39 days). Liver biopsies obtained from the 11 infected monkeys demonstrated mild to moderate portal inflammation, as well as random areas of focal necrosis and inflammation extending outward from the portal region. These data confirm the susceptibility of Aotus sp. to hepatitis A virus and indicate that the infection of this primate provides a useful animal model of human hepatitis A. Chimpanzees (Pan troglodytes) and several species of marmosets (Saguinus sp.) are susceptible to hepatitis A virus (HAV) and have proven valuable as animal models of human hepatitis A (2). Until recently, these primates provided the only available means for the amplification of virus stocks and were the only animals which could be utilized in studies of pathogenesis. The chimpanzee, however, is endangered and in short supply. In addition, susceptibility to infection with HAV varies among the various species of marmosets (2, 9), and their availability is also limited. The recent development of techniques for the in vitro cultivation of HAV (1, 8) has not eliminated the need for animal studies, and as vaccine development efforts proceed, this need will continue and probably even increase. For these reasons, the development of alternative animal models of hepatitis A is of major importance. We recently documented a sustained epizootic of HAV infection among newly captured New World owl monkeys (Aotus trivirgatus) held within a primate colony in Panama (5). Almost all monkeys admitted to this primate colony became infected with HAV, although fewer than 2% had antibody to the virus (anti-hav) on 766 arrival. HAV antigen recovered from these naturally infected Aotus sp. was serologically indistinguishable from the MS-1 strain of human HAV. However, it was unclear from these studies whether or not infected Aotus sp. developed significant abnormalities in hepatic function or morphology. To resolve this question and to ascertain whether Aotus sp. might, indeed, be useful as a model of hepatitis A, we inoculated 12 seronegative, colony-bred owl monkeys with one of two different strains of HAV (including the strain recovered from Aotus sp. in Panama). Our results indicate that the HAV infection of Aotus sp. is associated with significant hepatic disease and that this primate will be a useful model for studies of hepatitis A. MATERIALS AND METHODS A. trivirgatus. Twelve colony-bred Aotus monkeys, ranging in age from 2 to 5 years, were selected for study. Seven were males, and five were females; three were karyotype I, one was karyotype II, and eight were hybrids (crosses of karyotypes I, II, III, IV, and IX). All lacked detectable anti-hav. The monkeys were divided into two groups of six, and each group received a different virus strain. These groups were housed in separate rooms and were inoculated several months apart.

2 VOL. 40, 1983 HEPATITIS A IN OWL MONKEYS 767 Two to three months before infection, six monkeys were placed together in a single room from which other laboratory animals or agents were excluded. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transpeptidase (GGTP) activities; bilirubin (Centrifichem 500; Baker Instruments, Pleasantville, N.Y.); and total and differential leukocyte counts were determined weekly. Four to seven weeks before infection, an incisional biopsy of the liver was taken from each monkey under ketamine anesthesia. Each monkey was subsequently inoculated intravenously (i.v.) with 1.0 ml of a fecal suspension containing HAV. Daily fecal specimens were collected from each monkey beginning 1 week before inoculation and continuing through the period of infection. After inoculation, monkeys were bled twice weekly for the determination of serum chemistries and leukocyte counts. At the earliest evidence of ALT elevation, each monkey underwent a second incisional liver biopsy. HAV inocula. A 0.2% filtered suspension containing PA33 strain HAV was prepared as previously described (4) from feces collected from a naturally infected Aotus sp. held in a colony in Panama (5). This material contained virus particles which were morphologically and antigenically indistinguishable from human HAV and had previously been shown to be infectious in primary African green monkey kidney cell cultures (S. M. Lemon, L. N. Binn, R. R. Redfield, R. H. Marchwicki, N. L. Gates, and W. H. Bancroft, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 21st, Chicago, Ill., abstr. no. 214, 1981). The suspension was bacteriologically sterile and contained no other viral agent detectable upon prolonged incubation with OMK-210 cells, an owl monkey kidney cell line which supports the replication of many agents infectious for Aotus sp. (7). In addition, a 0.2% filtered suspension was prepared from a human fecal specimen containing HM-175 strain HAV, recovered during an outbreak of hepatitis A in TABLE 1. Australia (1). This material, which was the generous gift of Stephan M. Feinstone, Bethesda, Md., has been shown to contain infectious virus both by the experimental inoculation of susceptible marmosets and by successful isolation of virus in primary African green monkey kidney cells (1). Histopathology. Liver biopsies were fixed in 10% neutral buffered Formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. All liver sections were examined under code. Virological studies. HAV antigen was detected by testing 10% fecal suspensions by a solid-phase radioimmunoassay described previously (5). Specimens generating a sample/negative-control ratio (P/N) greater than 2.1 ratio units in this assay were considered positive, provided that the reaction could be specifically blocked (greater than 50% reduction in counts per minute) by postinfection, but not homologous preinfection, reference chimpanzee sera (5). Preinfection fecal specimens collected from the Aotus sp. were nonreactive in this radioimmunoassay (P/N less than 2.1). Anti-HAV was determined by radioimmunoassay (Havab; Abbott Laboratories, North Chicago, Ill.). RESULTS Biochemical changes associated with HAV infection in Aotus sp. At 9 to 21 days after the i.v. inoculation of the Aotus fecal suspension containing PA33 strain HAV, the six monkeys developed significant elevations (>3 standard deviations above base-line values) of serum ALT (Table 1). These abnormal elevations, which ranged up to almost nine times the base-line value, persisted for 7 to 14 days. In five of these monkeys, AST levels also became significantly elevated during approximately the same period. The one monkey (WR-149) which did not dem- ALT, AST, and GGTP activities in HAV-inoculated A. trivirgatus ALT (U/liter) AST (U/liter) GGTP (U/liter) Monkey Base linea Maximum Days Base line Maximum Days Base line Maximum Days Baseline Maxmumelevatedb elevated elevated PA33 inoculum WR (6.3) (14.4) (4.0) WR (17.3) (39.2) (3.6) WR (11.4) (26.0) (3.2) WR (10.8) (19.9) (2.2) WR (30.6) (188) (6.5) WR (6.8) (24.3) (2.9) HM-175 inoculum E (5.7) (4.1) (7.8) 24 N (10.6) (33.2) (2.1) N (11.2) (17.7) (1.5) WR (21.4) (44.9) (1.0) 13 WR (6.0) (11.8) (6.6) WR (18.0) (29.1) c 21 - a Base-line values are the mean of 5 to 12 weekly pre-inoculation determinations (standard deviation). b Days post-inoculation when values were elevated more than 3 standard deviations above the base line. None. c Single base-line value.

3 768 LEDUC ET AL. onstrate a significant AST elevation had a high AST level during this period (543 U/liter); however, it also had a relatively high and fluctuating AST level during the period of base-line observations. GGTP levels became significantly elevated in all six PA33-infected monkeys, although the levels of this enzyme appeared to rise somewhat later, i.e., 16 to 37 days after inoculation. Serum ALT and AST elevations also developed in five of six monkeys inoculated with the human fecal suspension containing HM-175 strain HAV (Table 1). Compared with the PA33- infected monkeys, the ALT elevations occurred somewhat later in HM-175-infected monkeys (25 to 39 days after inoculation), but were of equal or greater magnitude (up to 16 times preinfection values) and persisted for a similar length of time (4 to 21 days). Significant elevations of GGTP were noted in only three of the HM-175-inoculated monkeys, 32 to 56 days after inoculation. One monkey, WR-204, had no detectable alterations in serum enzyme activities. No significant changes were noted in serum bilirubin levels or blood leukocyte counts in any monkey. Virus excretion and development of anti-hav. HAV antigen was detected by solid-phase radioimmunoassay in the feces of 11 of the 12 monkeys (Table 2). Antigen was detected as early as 6 to 7 days after inoculation with PA33 strain HAV and 8 to 9 days after inoculation with HM- 175 virus. In general, the presence of viral antigen in feces appeared to precede the development of liver enzyme abnormalities (Fig. 1). INFECT. IMMUN. Viral antigen was shed for 4 to 11 days during infection with PA33 virus and for 7 to 29 days during infection with HM-175 strain HAV. Sera collected before and after inoculation with virus were tested for anti-hav (Table 2). Five of six monkeys infected with PA33 virus demonstrated substantial increases in serum blocking activity by 14 days after inoculation, and all six were seropositive by 28 days. The one monkey (WR-148) which lacked antibody at 14 days also appeared to have had a somewhat longer incubation period than the other PA33- infected monkeys in terms of fecal shedding of antigen and elevation of serum ALT. After inoculation with HM-175 virus, anti-hav developed in 5 of 6 monkeys, although its appearance was somewhat delayed compared with the development of antibody in the PA33-infected monkeys (Table 2). Only one monkey (WR-204) failed to develop anti-hav, even when tested as late as 100 days after the inoculation of HM-175 virus. WR-204 was also the only monkey not to develop significant ALT elevations or shed detectable viral antigen and was, therefore, apparently not infected. Histopathological changes associated with HAV infection. The histopathological findings are summarized in Table 3. Preinfection liver biopsies were normal with only a few minor changes noted. These included the minimal infiltration of mononuclear cells and a few polymorphonuclear leukocytes into the portal areas and occasional small, random foci of similar cells in the parenchyma. In contrast, liver biopsies taken during TABLE 2. Virological and serological findings in HAV-inoculated A. trivirgatus Fecal HAV antigen Anti-HAV antibodya (%) at (day): Monkey Days Maximum Preinfection present PIN' PA33 inoculum WR NDd WR ND WR ND WR ND WR ND WR ND HM-175 inoculum E N N WR WR WR e a The percent reduction in counts b per minute in the Havab test (>50% is positive). Days post-inoculation. -, None. 'P/N, Radioimmunoassay ratio units (except where noted, all values were reduced >50% by the addition of reference anti-hav serum [5]). d ND, Not done. enonspecific reaction, not inhibited by reference anti-hav serum.

4 VOL. 40, 1983 HEPATITIS A IN OWL MONKEYS 769 m I.- a 50 -A In,, I*I I 1 1A L1 0 P oc 0 s * s n --- a PRMM a 5 B kw Ca I , , 09!-2 k DAYS POST-INOCULATION 500 Acidophilic or ballooning degeneration of indi- 400 vidual hepatocytes or small clusters of hepatocytes was observed in 9 of 10 monkeys and was MO present throughout the parenchyma, extending outward from the portal areas (Fig. 2B). Some 200 X areas of focal hepatic necrosis were surrounded - by inflammatory cells, including lymphocytes, < macrophages, and polymorphonuclear leuko- 0 cytes. These changes were not noted in one, monkey (N-2), biopsied 4 days before, or in 500 another (WR-149), biopsied 4 days after, the first elevation of ALT. Cholestasis was not noted in 400!zany liver biopsy. Pathological changes in the liver were similar in the two groups of monkeys. 3 X Despite these chemical and histopathological 200, changes, the infected Aotus sp. demonstrated little change in their appearance or behavior. At loo0 15 days after inoculation with the PA33 strain, two monkeys (WR-130 and WR-148) had moder- 40 ately enlarged livers, and three transiently developed diarrhea (WR-80, WR-130, and WRinfection in 148). There were no clinical findings in Aotus sp. FIG. 1. Course of PA33 strain HAV i two seronegative, colony-bred A. trivirgat us monkeys infected with HM-175 virus. No changes in (WR-153 [A] and WR-130 [B]). The arrows indicate the feeding patterns were noted, and all survived the day of i.v. inoculation. The solid bars repiresent indi- experiment. vidual ALT determinations. The open ciircles represent fecal HAV antigen as determinedi by radio- DISCUSSION immunoassay (RIA); values > 2.1 raticd units are The experimental infections described here positive. In both monkeys, peak fecal an tigen shedconfirm and extend our previous observations of ding preceded maximal serum enzyme ele vations. HAV in the Aotus monkey (5). We had previously noted that newly captured Aotus sp., which were almost always anti-hav negative, rapidly the period of ALT elevation had hiistological developed antibody to the virus, including changes characteristic of viral hepatitiis in 9 of 10 immunoglobulin M (IgM) anti-hav, after ad- infiltrat- mission to the colony in Panama. The develop- monkeys. Portal areas were moderately ed with inflammatory cells, predominiantly lym- ment of antibody to the virus was accompanied phocytes, but with some macrophage' s and poly- or preceded by a period of fecal viral shedding, morphonuclear leukocytes (F ig. 2A). and the transmission of the virus between mon- TABLE 3. Histological changes in liver biopsies from HAV-infected A. trivirgatus Pre-inoculation biopsy/post-inoculation biopsya Monkey inoculation Portal Focal Focal inflammation inflammation necrosis PA-33 inoculum WR /+++ +l++ 0/+++ WR /+ + 0/+ + 0/+ WR /+ + +l+ 0/+ WR I+++ +l++ 0/+++ WR l+ +/+ 0/0 WR /+++ 0/+ + 0/+ HM-175 inoculum E I+++ 0/+++ 0/+++ N-2b 28 +l+ +/0 0/0 N /+++ 0/+++ 0/+++ WR /+ + 0/+ 0/+ WR /+ 0/+ 0/+ a Interpretation of paired pre-inoculation and acute-phase liver biopsies: 0, absent; +, minimal; + +, mild; + + +, moderate. b Biopsied 4 days before the first ALT elevation.

5 770 LEDUC ET AL. ~~~~~~~~~~~~~~~~~~ ;(*; 9 i 2a- a X '.W 4'0. 'N',\ANA'9' *~~~~~~~~~~~~~~~~~~~~~~~~ kv*~~~~~~~~~~ a INFECT. IMMUN. FIG. 2. Liver biopsy taken 17 days post-inoculation of PA33 strain HAV (A. trivirgatus WR-68) showing (A) a dense infiltrate of mononuclear cells, predominantly lymphocytes, in a portal area and a few small foci of necrotic hepatocytes in the surrounding parenchyma (hematoxylin and eosin, 250x) and (B) multiple foci of hepatocellular necrosis with accumulation of mononuclear inflammatory cells (arrows) (hematoxylin and eosin, 250x). keys in the colony was presumed to be due to fecal-oral spread. The PA33 and PA21 strains of HAV which were identified in the feces and liver of infected Aotus sp. were morphologically identical to human HAV and were antigenically indistinguishable from human HAV (MS-1 strain) by conventional radioimmunoassay methods (5). In the present study, the experimental infection of Aotus sp. with HAV was associated with significant liver dysfunction and histological changes which were similar to, but somewhat milder than, those associated with hepatitis A in human adults (12). After an incubation period of 9 to 39 days, liver enzymes, including ALT, AST, and GGTP, became significantly elevated.

6 VOL. 40, 1983 The elevation of GGTP appeared to occur somewhat later than abnormalities in the aminotransferases, a pattern we have observed previously in HAV-infected chimpanzees (S. M. Lemon and W. H. Bancroft, unpublished data). Liver enzyme abnormalities were associated with mild to moderate histological changes in liver biopsies, including portal infiltration with mononuclear cells and polymorphonuclear leukocytes and areas of focal hepatic necrosis. Cholestasis, an occasional feature of hepatitis A in humans, was not noted. Peak serum enzyme elevations followed the shedding of HAV antigen in feces and occurred approximately coincidentally with the development of anti-hav antibody. Similar findings were noted in monkeys whether inoculated with PA33 strain or HM-175 strain HAV, although the incubation periods to the elevation of liver enzymes and development of antibody were somewhat longer in monkeys infected with HM-175. This difference could have been related to differences in the titer of infectious virus in the two inocula and does not necessarily indicate a specific difference in the response of Aotus sp. to these two HAV strains. Altogether, the course of events after the infection of Aotus sp. with either strain of HAV closely resembled that associated with human hepatitis A (3). These data thus indicate that the HAV infection of Aotus sp. provides an accurate model of the infection in humans. Previous attempts to infect Aotus sp. with HAV have resulted in only limited success (10). One monkey inoculated i.v. with MS-1 strain HAV developed anti-hav, whereas two which received the virus orally did not. In no case was fecal shedding of virus or significant liver dysfunction noted. The lack of success in this earlier study may have been related to the strain of HAV employed. The PA33 strain employed in our study has clearly had the opportunity to become adapted to Aotus sp. during multiple passages in this species in the primate colony in Panama. The adaptation of human HAV to a nonhuman primate species has been described by Provost et al. (9), who noted that multiple passage of CR326 strain HAV in the marmoset S. mystax resulted in a shortening of the incubation period and an increase in the amount of antigen found in the liver. However, we also observed striking chemical and histological changes in Aotus sp. which had been inoculated i.v. with human fecal material containing the HM-175 strain of HAV. Aotus sp. are therefore clearly susceptible to at least this one human strain of HAV. Although it is now possible to propagate HAV in primate cell cultures (1, 8), the need for animal models of this virus infection has not lessened. Future efforts at vaccine development HEPATITIS A IN OWL MONKEYS 771 are certain to require multiple experimental infections, and important questions concerning the pathogenesis of HAV-induced hepatitis remain totally unresolved. Although the chimpanzee, because of its size and demonstrated susceptibility to the virus, remains the ideal subject for such experimental infections, this species is endangered, and the availability of colony-bred individuals is very limited. Relatively speaking, the Aotus sp. is more available and less expensive. The species has been utilized extensively in antimalarial drug development programs, and several successful breeding colonies have been established (11). Compared with chimpanzees and marmosets, the owl monkey appears to be approximately as susceptible to infection with HAV both in terms of the degree of liver pathology and the quantity of antigen produced. Whereas the measurement of serum isocitrate dehydrogenase has generally been employed to detect significant liver involvement in infected marmosets (9), the Aotus sp. responds to HAV infection with significant elevations of ALT and AST (enzyme markers commonly employed in clinical testing for hepatitis) and thus provides a model which is perhaps more analogous to human hepatitis A. In addition, the Aotus sp. is larger than the marmoset and thus better able to withstand certain experimental procedures, such as incisional liver biopsies. For these reasons, the Aotus sp. appears to offer several substantial advantages over the marmoset for use in experimental hepatitis A infections. Finally, a recent report indicating that the stumptailed macaque (Macaca speciosa) is also susceptible to HAV, along with the finding of anti- HAV in other nonhuman primates, should challenge previous assumptions that only a small number of primate species are susceptible to this virus (6). ACKNOWLEDGMENTS We acknowledge K. G. Ishak, W. H. Bancroft, G. A. McNamee, C. J. Peters, N. L. Gates, and J. L. Pfalser for their advice and assistance with these studies. LITERATURE CITED 1. Daemer, R. J., S. M. Feinstone, I. D. Gust, and R. H. Purcell Propagation of human hepatitis A virus in African green monkey kidney cell culture: primary isolation and serial passage. Infect. Immun. 32: Deinhardt, F Hepatitis in primates. Adv. Virus Res. 20: Dienstag, J. L Hepatitis A virus: identification, characterization and epidemiologic investigations, p In H. Popper and F. Schaffner (ed.), Progress in liver diseases. Grune and Stratton, New York. 4. Dienstag, J. L., S. M. Feinstone, R. H. Purcell, J. H. Hoofnagle, L. F. Barker, W. T. London, H. Popper, J. M. Peterson, and A. Z. Kapikian Experimental infection of chimpanzees with hepatitis A virus. J. Infect. Dis. 132: Lemon, S. M., J. W. LeDuc, L. N. Binn, A. Escajadillo, and K. G. Ishak Transmission of hepatitis A virus

7 772 LEDUC ET AL. among recently captured Panamanian owl monkeys. J. Med. Virol. 10: Mao, J. S., Y. Y. Go, H. Y. Huang, P. H. Yu, B. Z. Huang, Z. S. Ding, N. L. Chen, J. H. Yu, and R. Y. Xie Susceptibility of monkeys to human hepatitis A virus. J. Infect. Dis. 144: Melendez, L. V., M. D. Daniel, H. H. Barahona, C. E. 0. Fraser, R. D. Hunt, and F. G. Garcia New herpesviruses from South American monkeys. Lab. Anim. Sci. 21: Provost, P. J., and M. R. Hilleman Propagation of human hepatitis A virus in cell culture in vitro. Proc. Soc. Exp. Biol. Med. 160: INFECT. IMMUN. 9. Provost, P. J., V. M. Villarejos, and M. R. Helleman Suitability of the rufiventor marmoset as a host animal for human hepatitis A virus. Proc. Soc. Exp. Biol. Med. 155: Purcell, R. H., and J. L. Dienstag Experimental hepatitis A virus infection, p In T. Oda (ed.), Hepatitis viruses. University Park Press, Baltimore, Md. 11. Schmidt, L. H Infections with Plasmodium falciparum and Plasmodium vivax in the owl monkey. Trans. R. Soc. Trop. Med. Hyg. 67: Teixeira, M. R., I. V. D. Weller, A. Murray, M. Bamber, H. C. Thomas, S. Sherlock, and P. J. Scheuer The pathology of hepatitis A in man. Liver 2:53-60.