Hepatitis E virus infection in chimpanzees: a retrospective analysis.

Different patterns of disease were observed among 11 chimpanzees who were inoculated intravenously with hepatitis E virus (HEV) positive fecal specimens from four different outbreaks (Nepal 1981, Uzbekistan 1981, Pakistan 1985, and Mexico 1986). Five chimpanzees had marginal or no liver enzyme elevations within 70 days of inoculation. Two of the chimpanzees had limited viremia, but did not produce detectable antibody. The four remaining chimpanzees had liver enzyme elevations, viral shedding, viremia, seroconversion to anti-HEV, and detectable HEV antigen in liver biopsy specimens. These results may reflect the range of infection patterns that develop in humans after natural exposure to the HEV.

Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus.

We have sequenced the complete genome of a hepatitis B virus (HBV) strain that was transmitted from a gibbon with chronic hepatitis B to a chimpanzee that subsequently developed acute hepatitis B. The genome was 3,182 nucleotides long and had a genetic organization identical to and including the characteristics of other mammalian hepadnaviruses. Thus, the regulatory elements, the direct repeats, and the four open reading frames (ORFs) of this virus were all maintained, although there were amino acid substitutions affecting all the ORFs. Within the S gene encoding for the hepatitis B surface antigen (HBsAg), the subtype could be deduced as ayw3 in accordance with previous serological results. There were 25 amino acid substitutions affecting the P gene, 12 of which were within the spacer region. This region, which was the most divergent part of the genome compared to other HBV strains, also encodes for the pre-S proteins. A comparison with sequences of other hepadnaviruses revealed that the genome of gibbon HBV was unique as compared to previously described HBV genotypes. It was most similar to the chimpanzee HBV strain with which it shared 90.3% nucleic acid homology at the level of the complete genome and 96.3% homology at the level of the S-gene region corresponding to HBsAg, although being a distinct genotype as compared to the latter virus. Analyses performed using five different algorithms for phylogenetic tree construction showed more than 99% bootstrap support for the gibbon and the chimpanzee HBV to be grouped within the human HBV strains and that they represented later offshoots than the HBV strains of genotype F. However, in most of the dendrograms both the gibbon and the chimpanzee strains represented early lineages, indicating that these viruses are indigenous to their respective hosts and not recent acquisitions from man.

Unique preS sequence in a gibbon-derived hepatitis B virus variant.

A unique hepatitis B virus (HBV) variant has been identified in a gibbon (Hylobates lar) which could be passed to a chimpanzee by experimental inoculation. This HBV variant had been shown to have no reactivity to a monoclonal anti-preS2 antibody (preS2 mAb 116-34) differentiating it from all human HBV specimens tested. This gibbon sera also was not recognized by an anti-preS1 mAb which binds the preS1 hepatocyte receptor region, amino acids 27-35. In this paper, we report the DNA sequence of the gibbon HBV PreS gene. The lack of preS2 mAb (116-34) binding can be explained by a unique nucleotide substitution of A for C in the second codon of the preS2 region leading to the replacement of glutamine with lysine. Two other unique changes were observed at the seventh and 24th amino acid positions in the preS2 gene leading to a substitution of a valine for threonine and alanine, respectively. Unlike all human derived HBV sequences in the preS1 region, the gibbon HBV had a glutamic acid instead of an aspartic acid at amino acid residue 27. Another unique substitution was a leucine for alanine at preS1 position 33. These amino acid changes in the gibbon HBV may explain its unique preS mAb reactivity.

Parenterally transmitted non-A, non-B hepatitis: virus-specific antibody response patterns in hepatitis C virus-infected chimpanzees.

An established chimpanzee model of parenterally-transmitted non-A, non-B hepatitis was used to define virus-specific immune response patterns in acutely and persistently infected animals. Serial bleedings were obtained from 23 chimpanzees that had been experimentally infected with an isolate of hepatitis C virus, originally recovered from contaminated lots of factor VIII (antihemophilic) materials. Sera were assayed for the presence of antihepatitis C virus by a newly developed radioimmunoassay procedure that incorporated recombinant DNA-expressed viral antigen as a reagent. Twenty-one of 23 hepatitis C virus infected animals were shown to acquire antihepatitis C virus, most within 2-8 weeks after the major peak of alanine aminotransferase activity. All chimpanzees with biochemical, electron microscopic, and histological evidence of chronic disease clearly acquired antibody; 14 of 16 animals observed through the acute phase of disease were also shown to acquire antibody. A booster effect or anamnestic response was noted in two chimpanzees (one of which was negative for antihepatitis C virus following the acute phase of disease) after challenge with hepatitis C virus. Antihepatitis C virus was not neutralizing, because some animals with high levels of antibody were also shown to have high titers of circulating hepatitis C virus. The development and maintenance of anti-hepatitis C virus appears to reflect concomitant virus replication and high potential for infectivity.

Appearance of immune complexes during experimental hepatitis A infection in chimpanzees.

Circulating immune complexes (CICs) were detected during the course of experimental hepatitis A virus (HAV) infection in 8 of 9 chimpanzees. In all cases, the predominant class of antibody detected in the CIC was IgM. The appearance of IgM-CIC usually preceded the onset of liver enzyme elevations, and in all instances, the appearance of IgM-CIC correlated with the presence of IgM anti-HAV. Six of 8 animals tested had significant depression of C3 concentrations during the course of infection, and this depression occurred at the peak of CIC activity. Immunohistologic studies demonstrated granular deposits of IgM localized in sinusoidal cells during peak of IgM-CIC activity. IgM-CICs appear to be a fairly consistent finding during HAV infection and probably represent the viremic phase of the disease. However, they do not appear to mediate hepatocellular injury by direct action on hepatocytes.

Experimental transmission of the delta virus to a hepatitis B chronic carrier chimpanzee with the development of persistent delta carriage.

A delta virus (DV) infection was experimentally transmitted to a hepatitis B chronic carrier chimpanzee. The infection was monitored by the examination of liver biopsy materials with the use of FITC-labeled human anti-delta IgG or by an indirect immunoperoxidase staining technique and by measurement of the serum alanine aminotransferase (ALT) activity. Three different antibody enzyme immunoassays (EIAs) were developed for assessment of the serologic response: 1) blocking assay, 2) IgM-specific capture assay, and 3) IgG-specific capture assay. An antigen-specific EIA was also developed for monitoring delta antigen in the serum. The results indicate that a superinfection with the DV produced a biphasic ALT response concomitant with the appearance of the delta antigen in biopsy materials. The persistence of these markers over the observation period (350 days) indicates the development of a persistent DV infection.

Posttransfusion non-A, non-B hepatitis in chimpanzees. Physicochemical evidence that the tubule-forming agent is a small, enveloped virus.

Posttransfusion non-A, non-B hepatitis associated with the formation of hepatocyte cytoplasmic tubules was experimentally transmitted to chimpanzees by intravenous inoculation of a proven-infectious plasma that had been pelleted and microfiltrated, or purified by a combination of pelleting and rate-zonal banding. The results of these studies indicate that a factor VIII-derived non-A, non-B tubule-forming agent will pass through an 80-nm membrane filter and that it can be recovered from infected plasma by use of a purification procedure that assumes the non-A, non-B tubule-forming agent is a small, enveloped virus. Our findings, in combination with the known sensitivity of the non-A, non-B tubule-forming agent to chloroform and its apparent lack of nucleic acid homology with hepatitis B virus, further suggest that at least one etiologic agent of human posttransfusion non-A, non-B hepatitis may be a small, enveloped RNA virus.

Hepatitis A virus: growth characteristics of in vivo and in vitro propagated wild and attenuated virus strains.

Serial passage of the MS-1 strain hepatitis A virus (HAV) in marmosets was shown to increase the yield of virus and to shorten the incubation period from approximately 55 days in the first passage to 3-7 days in the ninth and higher passages. Intravenous inoculation of susceptible chimpanzees with MS-1 HAV was found to result in a typical course of disease in two animals who had received eighth marmoset-passage virus, including the occurrence of elevated ALT activity, presence of HAV antigen in liver and stool, and seroconversion to anti-HAV. Two chimpanzees inoculated with 20th passage MS-1 HAV (M001 liver homogenate) exhibited normal or nearly normal ALT activity and had no demonstrable or significant HAV in weekly liver biopsy specimens or in serial stool suspensions obtained during 64 days of observation. However, both animals seroconverted to anti-HAV within 2 weeks after inoculation, as did the animals who had received eighth passage MS-1 HAV. These findings suggest that subpassage of the MS-1 strain of HAV in marmosets resulted in the generation of an attenuated virus strain that was still capable of inducing a vigorous antibody response in intravenously infected chimpanzees. Serial propagation of wild and attenuated strains of HAV (HAS-15 and MS-1/M001, respectively) in FRhK-4 cells was associated with a significant decrease in the growth period for both viruses. Our studies have also shown that HAS-15 HAV can be recovered in maximum yield in later passages as early as 2 to 3 days after inoculation.

Inactivation of hepatitis B virus by intermediate-to-high-level disinfectant chemicals.

In five separate tests, hepatitis B virus in dried human plasma was exposed for 10 min at 20 degrees C to disinfectant chemicals having activity levels ranging from intermediate (e.g., 70% isopropyl alcohol) to high (e.g., 2% aqueous glutaraldehyde at pH 8.6). Five chimpanzees (one animal per disinfectant chemical) received treated material intravenously, and none showed signs of infection after post-inoculation periods of 9 months. Two animals were rechallenged with inoculum treated in the same manner, except that saline was used instead of a disinfectant chemical; both were infected within 4 weeks. Our results showed that hepatitis B virus was not as resistant to disinfectant chemicals as once thought and suggested that chemicals with similar activity levels (intermediate to high) might possibly be used on hepatitis B virus contamination with a margin of safety.

Posttransfusion non-A, non-B hepatitis: physicochemical properties of two distinct agents.

Two separate and distinct episodes of non-A, non-B hepatitis were induced in each of two chimpanzees by two inocula: one containing a chloroform-resistant agent and the other containing a chloroform-sensitive agent. Both agents were recovered from liver tissue and plasma obtained from a single chimpanzee during the acute and chronic phases of infection with a factor VIII concentrate, respectively. The chloroform-resistant agent did not cause unique changes in hepatocytes; in contrast, the chloroform-sensitive agent did induce the formation of cytoplasmic tubules, convoluted endoplasmic reticulum, and dense reticular inclusion bodies. The latter changes are similar in character to those induced in infected cells by some enveloped mammalian RNA viruses.

Non-A, non-B hepatitis in chimpanzees: interference with acute hepatitis A virus and chronic hepatitis B virus infections.

Two chimpanzees with persistent non-A, non-B (NANB) hepatitis were superinfected with marmoset-passaged MS-1 HAV. Two control chimpanzees were also infected with marmoset-passaged HAV. Neither animal with persistent NANB hepatitis developed elevated alanine aminotransferase (ALT) activity, whereas both control chimpanzees exhibited ALT elevations within 3 weeks after inoculation. In addition, both NANB-infected chimpanzees demonstrated a delayed anti-HAV antibody response in which one animal failed to produce detectable IgM anti-HAV. With the exception of one stool, all serial liver biopsy specimens and daily stool suspensions from the superinfected chimpanzees were negative for HAV antigen. One chimpanzee with a chronic HBV infection was superinfected with non-A, non-B hepatitis and was shown to develop elevated ALT activity and hepatocyte ultrastructural alterations accompanied by a marked reduction in the titer of serum HBsAg. Our combined findings indicate that acute and persistent non-A, non-B hepatitis infections are capable of interferring with two distinctly different hepatotropic viruses. These results also suggest that in vitro detection of non-A, non-B hepatitis infection or virus(es) may be achieved by antibody-independent methodologies that employ the basic principle of viral interference.

Survival of hepatitis A virus in feces after drying and storage for 1 month.

Hepatitis A virus in feces remained viable after being dried and then stored at 25 degrees C and 42% relative humidity for 30 days, as evidenced by infection of two marmosets inoculated with 1 ml of the treated fecal material. The virus was excreted in stool specimens from these animals, and seroconversion to antibody to hepatitis A virus occurred 28 and 35 days post-inoculation.

Transmission of type B viral hepatitis via eye inoculation of a chimpanzee.

To simulate a splash of infectious material in the eyes, plasma from a hepatitis B patient was placed on the corneal surfaces of a chimpanzee. The animal became infected 9 weeks later. This result indicates a need for the use of eye protection in high-risk areas, such as clinical laboratories, hemodialysis units, and dental operatories.

Pathogenetic aspects of hepatitis A virus infection in enterally inoculated marmosets.

Experimental hepatitis A virus (HAV) infection was studied in marmosets after enteral (intragastric) inoculation with special reference to the primary sites of HAV replication and immunopathology of the disease. The experiment was carried out using 28 Saguinus mystax negative for antibody to HAV (anti-HAV) and with statistically uniform baseline values of serum isocitrate dehydrogenase (SICD) activity. Each animal was infected with 1 ml of a 15% w/v stool suspension that was derived from marmosets infected with the third or fourth passage of the MS-1 strain of HAV. The incubation period measured by the first significant SICD elevation was 32 days in 11 of 13 marmosets. The animals were sacrificed 2, 4, 7, 11, 14, 18, 23, 28, and 32 days after inoculation and 1, 4, 8, 14, 21, 28, and 35 days after SICD elevation. HAV antigen, immunoglobulins, complement, and fibrin were identified in the liver, eight segments of the gastrointestinal tract, lymphoid system, and kidneys. HAV antigen was found only in the cytoplasm of hepatocytes and in gallbladder bile. These findings indicated that the liver was the sole and primary site of virus replication. Combined immunomorphologic and histopathologic observations also revealed that HAV antigen localization was associated with the sites of hepatocellular damage. There was no immunomorphologic evidence for humoral immune clearance of HAV antigen in the liver, lymphoid system, or kidneys.

Persistent non-A, non-B hepatitis in experimentally infected chimpanzees.

Non-A, non-B (NANB) hepatitis was transmitted to six chimpanzees by intravenous inoculation of antihemophilic (factor VIII) materials, acute-phase chimpanzee liver, and chronic-phase plasma obtained from two NANB hepatitis-infected chimpanzees 10 and 16 months, respectively, after their inoculation. Five of six experimentally infected chimpanzees observed for more than one year demonstrated persistent or intermittent elevations in levels of serum alanine aminotransferase (ALT) indicative of continuing liver dysfunction. Liver biopsy specimens obtained from three chimpanzees with persistent elevations in levels of ALT were positive for hepatocyte cytoplasmic structures associated with NANB hepatitis for as long as 27 months after inoculation. Liver biopsy specimens obtained from four infected animals 13-30 months after inoculation also showed mild but persistent histopathologic lesions of undefined character. The detection of circulating immune complexes in one chimpanzee with persistent elevations in levels of ALT suggests that these complexes may be involved in the pathogenesis of NANB hepatitis.

Non-A/non-B hepatitis in experimentally infected chimpanzees: cross-challenge and electron microscopic studies.

Inoculation of eight chimpanzees with factor VIII, factor IX, or "H" strain plasma resulted in enzymatic and histopathologic evidence of non-A/non-B hepatitis in all eight animals. Challenge of two chimpanzees convalescent from factor VIII-induced disease with either factor IX or "H" strain plasma resulted in non-A/non-B hepatitis only in the animal inoculated with factor IX materials. Reciprocal cross-challenge of a chimpanzee convalescent from factor IX-induced disease with factor VIII also produced unequivocal enzymatic and histopathologic evidence of non-A/non-B hepatitis. Cross-challenge of a chimpanzee convalescent from "H" strain-induced non-A/non-B hepatitis with factor VII did not cause a second bout of non-A/non-B hepatitis. These findings suggest the factor VIII materials and "H" strain plasma used in these studies share a common etiologic agent (or agents), but that factor VIII and factor IX may contain two distinct agents. Electron microscopic (EM) examination of thin-sectioned, acute-phase liver biopsies from all but one of the chimpanzees receiving the primary inocula revealed the presence of abnormal hepatocyte cytoplasmic structures previously shown to be associated with non-A/non-B hepatitis. Crystalline structure containing 25 to 30 nm particles were visualized by EM in the cytoplasm of endothelial or Kupffer cells in acute-phase liver biopsies obtained from three chimpanzees inoculated with either factor VIII materials or "H" strain plasma.

Experimental infection of chimpanzees with antihemophilic (factor VIII) materials: recovery of virus-like particles associated with non-A, non-B hepatitis.

Non-A, non-B viral hepatitis was transmitted to four colony-born chimpanzees by infusion of three lots of antihemophilic factor (factor VIII) implicated in the transmission of non-A, non-B hepatitis to two human recipients. All four inoculated animals showed histopathological evidence of viral hepatitis, and all demonstrated significant ALT elevations between seven and one-half weeks after inoculation. Acute-phase plasma from one of the infected chimpanzees (no. 771) was shown to induce non-A, non-B hepatitis in two other chimpanzees approximately three weeks after their inoculation. In addition, an acute-phase open liver wedge biopsy obtained from animal no. 771 was processed and examined by immune electron microscopy (IEM) for virus-like particles with convalescent serum from a serologically confirmed case of non-A, non-B hepatitis. Twenty-five to 30 nm (mean = 27 nm) diameter virus-like particles that were either "full" or "empty" were identified in this liver preparation by IEM. Two additional chimpanzees inoculated with a cesium chloride gradient fraction of an isopycnically banded liver homogenate (animal no. 771) also developed elevated ALT activity two to two and one-half weeks later. Our findings have experimentally verified that commercially produced factor VIII materials can induce non-A, non-B hepatitis in champanzees and that the disease can be subpassaged in these animals by inoculation of either acute-phase plasma or liver. These results also provide evidence for the association of 27 nm-diameter virus-like particles with non-A, non-B viral hepatitis.

Immunofluorescence of hepatitis A virus antigen in chimpanzees.

Chimpanzee liver biopsies and necropsy tissues were examined by immunofluorescence for hepatitis A virus antigen. Results further indicate that the liver may be the sole site of replication for the virus.

Experimental infection of marmosets with hepatitis A virus.

Saguinus mystax marmosets were experimentally infected with two strains of human hepatitis A virus. One of these strains of HAV was successfully subpassaged in this species of marmosets. In another experiment, the 1.32 and 1.41 g/cm3 buoyant density species of HAV derived from an infected chimpanzee stool were shown to be infectious in three species of marmosets. The value of the marmoset as an experimental model for hepatitis A infection was demonstrated by these studies.