Pathogenesis of hepatitis A in orally inoculated owl monkeys (Aotus trivirgatus).

The pathogenesis of hepatitis A virus (HAV) infection was studied in owl monkeys following oral administration of the wild-type HM-175 strain of HAV. Stools were collected daily and blood and pharyngeal swabs twice weekly for viral isolation, and animals were necropsied at various intervals after inoculation. Organs were examined for the presence of virus by isolation in cell culture and for viral antigens by immunofluorescence. Monkeys excreted HAV in the stools for 1-4 days after inoculation, presumably due to the residual unabsorbed inoculum. No virus was found in stools for the next 2-3 days. HAV re-appeared on days 4-7 and then persisted through day 39. Viremia occurred on the 10th day and continued until day 35. Virus was isolated occasionally from throat swabs 1 or 2 weeks after it was detected in stools and blood, and there was no evidence that HAV replicated in the pharyngeal tissues. Animals acquired anti-HAV antibody by the 4th week, and alanine aminotransferase (ALT) was elevated 5-5.5 weeks after inoculation. HAV was isolated from liver 5 days after inoculation; however, viral antigens were first detected in Kupffer cells of the liver at 14 days and in hepatocytes at 21 days. HAV antigen was detected in epithelial cells of the intestinal crypts and in the cells of the lamina propria of the small intestine 3 days postinoculation and thereafter until the 5th week, suggesting that these cells might represent an additional site of HAV replication.

Laboratory tests and reference reagents employed in studies of inactivated hepatitis A vaccine.

Procedures to evaluate inactivated hepatitis A vaccines in volunteers have been examined. Solid-phase immunoassays were standardized with reference preparations and have been tested to measure antibody response to immunization and antigen content of vaccines. Following immunization, there was a good correlation between antibody response, determined with commercial immunoassays, and neutralization titres, as measured by the radioimmunofocus inhibition test. However, at lower titres of neutralizing antibody, the commercial immunoassay often yielded negative results. To improve the sensitivity of the immunoassay, the serum volume was increased. A fourfold increase of test serum resulted in greater sensitivity, increasing from 54 to 94%, while retaining 100% specificity. Further increases in the volume of test serum resulted in a loss of specificity. In a comparison of neutralization tests, similar titres of postvaccination sera were obtained by using the HM175/18f cytopathic strain of hepatitis A virus in a plaque reduction assay or the HM175 parental virus in the radioimmunofocus inhibition test. Use of the cytopathic virus obviates the need for radioactively labelled serum and reduces the time taken to conduct neutralization tests. The current laboratory procedures can meet the needs of large field trials of inactivated hepatitis A vaccines.

Preparation of noninfectious hepatitis A virus hemagglutinin for detecting hemagglutination inhibition antibodies.

Hepatitis A virus (HAV) harvested from infected MRC-5 cells can hemagglutinate various species of erythrocytes at acid pH (Eckels et al., 1989). Further studies revealed that the majority of the hemagglutinin (HA) in MRC-5 and BS-C-1 cells was cell-associated. A simplified procedure for preparing HAV-HA consisted of collecting infected cells in phosphate-buffered saline followed by three cycles of freeze-thawing and sonication. The fluids were clarified and stored at 4 degrees C. The analysis of HA by rate-zonal sucrose gradient centrifugation indicated that the majority of HA co-migrated with infectious virus. Complete inactivation of infectious HAV with 0.03% beta-propiolactone (BPL) did not affect HA activity, while inactivation with 0.05% formalin caused a 16-fold reduction in titer. There was no difference in HAI antibody titers when BPL-treated HA was compared to untreated HA in the hemagglutination inhibition (HAI) test.

In vivo replication and reversion to wild type of a neutralization-resistant antigenic variant of hepatitis A virus.

Six seronegative owl monkeys were intravenously inoculated with an antigenic variant (S18) of hepatitis A virus that is highly adapted to growth in cell culture and resists neutralization by monoclonal antibodies due to replacement of aspartic acid 70 of capsid protein VP3 with histidine. Each developed hepatitis 22-33 days after inoculation. Virus in feces, serum, and liver was quantified by radioimmunofocus assay. Viremia developed 7-11 days after inoculation, in parallel with fecal shedding of virus, and persisted for a mean of 20.5 days. Although the antigenic variant was recovered from feces or liver of three animals, virus in liver at the time of enzyme elevations was predominantly wild-type antigenic phenotype. Virus was not recovered from liver 96 days after challenge. These studies further define virologic events in hepatitis A and show that in vivo replication of an antigenic variant was restricted compared with that of wild-type virus.

Demonstration of hepatitis A virus in cell culture by electron microscopy with immunoperoxidase staining.

Virus-like particles were demonstrated by electron microscopy in BS-C-1 cells infected with hepatitis A virus (HAV). Particles were usually enclosed within vesicles and accompanied by myelin-like membranous structures. Less often they were seen free in the cytoplasm. They were never observed in the nucleus. By immunoperoxidase staining particles were found to contain HAV antigens. These antigens were also found in the membrane of the vesicles surrounding the masses of particles and adjacent parts of the mitochondrial membranes. Our results demonstrate the usefulness of an electron microscopic immunocytochemical technique to study replication of HAV.

Induced oral infection of the owl monkey (Aotus trivirgatus) with hepatitis A virus.

Several species of nonhuman primates have served as animal models for hepatitis A virus (HAV) infection and disease. This study was to determine the suitability of Aotus trivirgatus as an orally induced model for HAV infection and to reconfirm the owl monkey's susceptibility to the intravenous route of inoculation. Animals were inoculated, either orally or intravenously, with varying concentrations of PA-33 strain of HAV. Serum enzymes ALT, AST and GGTP levels were monitored and liver biopsies performed when values exceeded three standard deviations above individualized mean baseline values. All animals had postinoculation elevations of serum ALT and AST values, shed virus in their feces, and were seropositive to HAV by 60 days after inoculation. Eight of the ten postinoculation biopsy specimens had histologic lesions compatible with acute viral hepatitis. We conclude that the owl monkey is a useful and valuable model for the study of HAV disease.

Preparation of a prototype inactivated hepatitis A virus vaccine from infected cell cultures.

Studies were conducted on the preparation, inactivation, safety, and immunogenicity of a prototype hepatitis A virus vaccine prepared from infected cell cultures. BS-C-1 cells maintained in medium 199 without serum were infected with the HM175 strain of hepatitis A virus and harvested after 21-28 days. The harvested virus preparation contained 6.8-7.4 (log 10) cell culture infectious doses/ml. After exposure to 1:4,000 formalin at 35 C, the infectivity titer decreased 10(6)-fold in 30 hr at an exponential rate, although virus was detected in 5.0-ml vaccine samples for up to three days. Three separate vaccine lots elicited antibody in all the guinea pigs given three doses. Owl monkeys given three doses of vaccine did not have any evidence of HAV infection but developed antibodies identifiable by radioimmunoassay and serum neutralization tests. After either oral or intravenous challenge with at least 10(6) monkey infectious doses of a virulent field strain of hepatitis A virus, none of the vaccinated monkeys shed virus in their feces or had elevated serum levels of alanine aminotransferase. The findings suggest that an effective inactivated whole virus hepatitis A vaccine can be prepared from cell culture.

Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures.

Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum.

Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures.

A new method is described for the quantitation of hepatitis A virus in cell cultures, based on the immune autoradiographic detection of foci of infected cells (radioimmunofoci) developing beneath an agarose overlay 14 days after the inoculation of petri dish cultures of continuous African green monkey kidney cells (BS-C-1). The number of foci developing in each culture was linearly related to the dose of hepatitis A virus (either HM-175 or PA-21 strain) inoculated. Focus development was prevented by prior incubation of virus with specific antisera, and the specificity of the radiolabeled antibody reaction was confirmed in competitive blocking experiments. This new assay method retains many of the advantages of conventional plaque assays for virus. Compared with existing end-dilution methods for the quantitation of hepatitis A virus, the radioimmunofocus assay offers greatly improved accuracy and comparable sensitivity, yet is relatively rapid and highly conservative of reagents.

Experimental infection of the New World owl monkey (Aotus trivirgatus) with hepatitis A virus.

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.

Parvovirus enteritis in vaccinated juvenile bush dogs.

Parvovirus enteritis developed in 10 of 17 vaccinated juvenile bush dogs (Speothos venaticus) from 4 litters in a 5-month period. Nine dogs died. The first outbreak involved 6 of 9 bush dogs from 2 litters. Each had been vaccinated with a killed feline-origin parvovirus vaccine at 11 and 14 weeks of age. The 6 affected dogs became ill at 29 weeks of age and died. The second outbreak involved a litter of 6 bush dogs. Each had been vaccinated every 2 weeks starting at 5 weeks of age. Two were isolated from the colony at 16 weeks of age for treatment of foot sores. Three of the 4 nonisolated dogs developed parvovirus enteritis at 20 weeks of age; 2 died at 6 and 8 days, respectively, after onset of signs. The 3rd outbreak involved a litter of 2 bush dogs. Both had been vaccinated every 2 to 3 weeks, starting at 6 weeks of age. One of these dogs became ill at 17 weeks and died 13 days later. A litter of 6 maned wolves (Chrysocyon brachyurus) and a litter of 3 bush dogs were isolated from their parent colonies at 13 and 15 weeks of age, respectively. Each animal had been vaccinated weekly, beginning at 8 weeks of age, using an inactivated canine-origin parvovirus vaccine. None of the isolated animals developed the disease. Serologic testing during isolation did not reveal protective titers (greater than or equal to 1:80) against canine parvovirus in the bush dogs until they were 23 weeks old, whereas protective titers developed in the maned wolves when they were 14 to 18 weeks old. One hand-raised bush dog was vaccinated weekly, beginning at 8 weeks of age, and a protective titer developed by 21 weeks of age. It was concluded that the juvenile bush dogs went through a period during which maternal antibodies interfered with immunization, yet did not protect against the disease. When the pups were isolated from the colony during this period, then vaccinated repeatedly until protective titers developed, the disease was prevented.

Epizootic of viral enteritis in dogs in Thailand.

An epizootic of enteritis occurred in dogs in Thailand during 1979. Observations were made on 44 dogs that had clinical signs of enteritis or had a recent history compatible with a clinical diagnosis of enteritis. Eight of the 44 dogs died. Gross and histopathologic examinations performed on these dogs revealed that the lesions were similar to those described for canine viral enteritis. Antigens that agglutinated rhesus macaque RBC were detected in feces from 4 of 20 dogs. Cytopathic effects were observed in canine A-72 cells after their inoculation with fecal suspensions from these 4 dogs and with a fecal suspension from another dog. Cell cultures inoculated with each of the suspensions produced antigens that agglutinated RBC. All hemagglutinating antigens were inhibited in the presence of feline panleukopenia virus antiserum. Using electron microscopy, parvovirus-like virions were observed in a fecal suspension from 2 dogs (1 dog that had antigen that agglutinated rhesus macaque RBC and 1 dog that was negative for feline panleukopenia virus). Canine parvovirus hemagglutination inhibition antibody was detected in sera from 33 of the 40 dogs examined, and canine coronavirus (CV) neutralizing antibody was found in 29 of 30 dogs. Antibody titer increases indicative of recent canine panleukopenia virus (CPV)-like virus and CV infections were observed in paired sera for 2 of 35 and for 5 of 30 of the dogs in Thailand were infected with CPV-like virus and a CV, and these viruses were most likely the cause of the epizootic of viral enteritis.

Measles virus antibodies in a laboratory colony of owl monkeys (Aotus trivirgatus).

Serologic testing revealed that 17/84 (20.2%) of bought-in Aotus and 1/31 (3.2%) of colony-born Aotus had haemagglutination-inhibition antibody. Clinically-inapparent measles infections were detected in 3 monkeys by increased antibody titres. Following the detection of a recent infection, antibody titre persisted at a high level for at least 240 days. Although 84% of the monkeys were sero-susceptible, no further serological evidence of measles infection occurred.

Viral antibody studies of laboratory dogs with diarrheal disease.

Viral antibody studies were done on laboratory dogs in an epizootic of gastrointestinal disease. Increased hemagglutination-inhibition antibody titers to a parvovirus (PV) antigenically related to feline panleukopenia virus were found in convalescent serum specimens of 78% (20/26) of the affected dogs and in 83% (5/6) of apparently healthy dogs. With one exception, all dogs tested had significant levels of hemagglutination-inhibition antibody to this PV. Similar increased antibody titers were found to feline panleukopenia virus. Also, neutralizing antibody responses were detected to the canine coronavirus in 24% (6/25) and canine herpesvirus in 45% (10/22) of the affected dogs. However, antibody titers did not increase to canine distemper virus, infectious canine hepatitis virus, canine parainfluenza virus, or minute virus of canines. Subsequent serotesting of the colony provided evidence that additional PV infections occurred in pups from each of 8 litters born 3 to 8 months after the epizootic. These findings indicated the continued presence of the PV for more than 1 year in the infected colony. Of 19 laboratory personnel who worked with the affected dogs, none, including 4 with a concurrent diarrheal disease, developed or had antibodies to the PV or canine coronavirus.

Establishment of a canine cell line: derivation, characterization, and viral spectrum.

A cell line, designated A-72, for virus studies was established from a tumor surgically removed from a female, 8-year-old Golden Retriever dog. Following explant culture, the cells were serially passaged 135 times. The A-72 cells maintained a fibroblastic appearance and, at the 123rd passage, had a population doubling time of approximately 27 hours. Karyotypic analysis of the high passage cells showed the modal 2n chromosome number was 92 to 93. Using starch gel electrophoresis for enzyme characterization, the electrophoretic mobilities of enzymes extracted from A-72 cells were identical with those of canine peritoneal fibroblasts and primary canine kidney cells. The A-72 cells were susceptible to infection with infectious canine hepatitis virus, canine adenovirus type II, canine herpesvirus, canine parainfluenza virus, and canine coronavirus, but were not susceptible to canine distemper virus or the minute virus of canines. These cells have been particularly useful for studies of the fastidious canine coronaviruses, as the commonly used primary canine kidney cells exhibit varied susceptibility to these viruses.

Enzyme-linked immunosorbent assay for evaluation of antibody to canine distemper virus.

An enzyme-linked immunosorbent assay (ELISA) for canine immunoglobulin (Ig) G antibodies to canine distemper virus (CDV) was developed and its test results were compared with those of the serum-neutralization (SN) test. The sera of 273 random-source adult dogs were examined. The two tests had a high degree of correlation. Very few discrepancies occurred and most of these were at the lower limits of each test. When the sera were tested at 1:100 dilution, there was a 98% agreement between the ELISA and SN test. Titrated SN and IgG ELISA tests also were performed on sera from 77 dogs whose lifetime medical histories were known. The results showed excellent agreement between the tests. Only five of the 77 sera showed any discrepancies and these were at detection-threshold levels. To follow antibody development in two dogs experimentally infected with CDV, it was necessary to use an ELISA which detected both canine IgM and IgG. The ELISA detected CDV-specific IgM a week before the SN test results became positive. The IgM titers rose for 3 weeks and descended to lower levels about the 4th and the 5th weeks. The SN titers closely followed the IgM titers. The ELISA detected IgG antibody about the 5th and the 6th weeks of infection. Results of both the SN test and the IgG ELISA remained elevated through the 70th day of testing.

An enzyme-linked immunosorbent assay for the detection of canine antibodies to canine adenoviruses.

An enzyme-linked immunosorbent assay was used to detect canine immunoglobulin G antibodies specific for infectious canine hepatitis virus and the serologically related canine adenovirus Type 2. The sequential development of homologous and heterologous antibodies was measured by the enzyme-linked immunosorbent assay and serum neutralization tests in two groups of dogs which were experimentally infected with either infectious canine hepatitis virus or canine adenovirus Type 2. Both tests were comparable in their abilities to detect the development of homologous and heterologous antibodies. Homologous antibodies were detected earlier and to a higher titer in both tests. There was a 98% agreement between the serum neutralization test and the enzyme-linked immunosorbent assay when sera from 224 random-source dogs were examined for infectious canine hepatitis virus antibodies. The enzyme-linked immunosorbent assay was found to be a highly efficient and rapid test to determine the immune status of dogs to infectious canine hepatitis virus and canine adenovirus Type 2.

Studies of respiratory disease in random-source laboratory dogs: viral infections in unconditioned dogs.

A prospective study was conducted to identify the viruses causing respiratory diseases in unconditioned, random-source dogs. During the quarantine period, respiratory disease occurred in 86 of 167 (52%) dogs, and 34 (21%) died. Most affected dogs had a distemper-like illness which required extensive and prolonged care. Histopathologic studies confirmed the diagnosis of canine distemper in 10 of 12 (83%) fatal infections examined. Sixty-seven of 91 (74%) dogs which arrived without canine distemper antibody became ill, and 30 (32%) died. In contrast, only 16 of 67 (24%) dogs with canine distemper antibody had respiratory disease, and only 3 (4%) died. Parainfluenza SV5 and canine adenovirus--type II were recovered from 27 of 54 and 22 of 54 sick dogs, respectively. Canine herpesviruses, canine coronaviruses, and canine parvoviruses were less frequently isolated. Increased antibody titers to SV5 were found consistently, and rises in titer to the other viruses were demonstrated. Many of the sick dogs were infected with two or more viruses. Although several viral agents were detected during these epizootics, prevention of canine distemper appeared to be the key to controlling severe, prolonged, and often fatal respiratory disease.

Recovery of reovirus type 2 from an immature dog with respiratory tract disease.

Two similar cytopathic agents were recovered from the throat and rectal swab specimens of an immature dog with upper respiratory tract disease. The reference isolate, 14-72R, was shown to be a member of the reovirus group by its physicochemical properties, cytopathic effects in cell cultures, and appearance when examined in the electron microscope. Both isolates hemagglutinated human type O erythrocytes and antigenically were closely related to reovirus type 2. The affected pup had an increase in antibody titer to reovirus types 2 and 3. The latter findings provide evidence for possible heterotypic antibody responses in dogs to reovirus infection.