Biomedical subjects

L N Binn

Publications and source records attributed to L N Binn.

At least 37 records · Page 2Linked to original sources

PubMed1990

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.

Animals↗

PubMed1990

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.

Animals↗

PubMed1988

Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli.

We have constructed a recombinant plasmid producing, in bacteria, a hepatitis A virus (HAV) capsid protein that may be useful as a subunit vaccine. HAV VP1 coding sequences were fused in-frame to NH2-terminal Escherichia coli TrpE coding sequences under the control of the tryptophan promoter. In the absence of exogenous tryptophan, E. coli containing this recombinant plasmid produced high levels of an 88-kilodalton fusion protein that was recognized in immunoblots by antibodies to TrpE and HAV. The TrpE/HAV VP1 protein was gel-purified and used to immunize rabbits. The resulting antiserum reacted with denatured HAV VP1 in immunoblots but did not react with intact virus. However, subsequent inoculation of an immunized animal with a subimmunogenic dose of inactivated, whole HAV resulted in the rapid appearance of a stable, virus-neutralizing antibody response.

Animals↗

PubMed1988

Characterization of minute virus of canines (MVC) and its pathogenicity for pups.

Minute virus of canines (MVC, canine parvovirus-1), originally isolated in 1970 from the feces of normal dogs, was compared with canine parvovirus type-2 (CPV-2). The two viruses, which differ in their host cell ranges and spectra of hemagglutination, also were found distinct in their antigenic and genomic properties. We demonstrated that the MVC replicates in dogs and is capable of producing pathologic changes that were most prominent in oronasally-exposed neonatal pups. Macroscopic and microscopic lesions were most prominent in the thymus and lymph nodes; minor changes were found in the duodenal crypts. The MVC strain used had been passaged 13 times in cell cultures and it may not represent the true virulence of naturally occurring virus.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

Genomic heterogeneity among human and nonhuman strains of hepatitis A virus.

Cloned cDNA probes derived from the P1 and P2 regions of the genome of HM175 virus, a reference strain of human hepatitis A virus (HAV), failed to hybridize under standard stringency criteria with RNA from PA21 and PA33 viruses, two epizootiologically related HAV strains recovered from naturally infected New World owl monkeys. Hybridization of these probes to PA21 RNA was only evident under reduced stringency conditions. However, cDNA representing the 5' nontranslated region of the HM175 genome hybridized equally to HM175 and PA21 RNA under standard stringency conditions, while a probe derived from the 3' 1,400 bases of the genome yielded a reduced hybridization signal with PA21 RNA. In contrast, no differences could be discerned between HM175 virus and three other HAV strains of human origin (GR8, LV374, and MS1) in any region of the genome, unless increased stringency conditions were used. These results suggest that PA21 and PA33 are unique among HAV isolates and may represent a virus native to the owl monkey. Despite extremely poor homology within the P1 region, which encodes capsid polypeptides, monoclonal antibody analysis confirmed that the immunodominant neutralization epitopes of HAV were highly conserved between HM175 and PA21 viruses. Furthermore, experimental challenge of the owl monkey with successive PA33 and HM175 inocula confirmed a high but incomplete degree of cross-protection. Only one of six monkeys previously infected with PA33 developed recurrent hepatitis 28 days after intravenous HM175 challenge, while none of six monkeys previously infected with HM175 had demonstrable hepatitis following PA33 challenge. These data provide molecular evidence for the existence of HAV strains unique to nonhuman primate species and indicate that strict conservation of antigenic function may accompany substantial genetic divergence in HAV.

Animals↗

PubMed1987

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.

Administration, Oral↗

PubMed1986

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.

Animals↗

PubMed1985

Incomplete neutralization of hepatitis A virus in vitro due to lipid-associated virions.

Hepatitis A virus (HAV) released from infected BS-C-1 cells was incompletely neutralized when incubated with a variety of convalescent sera (non-neutralizable fraction of 17 to 32%). Chloroform extraction of virus resulted in a substantial reduction of the non-neutralizable fraction (to less than 1%), suggesting that non-neutralizable virions might be associated with lipids. Non-neutralizable HAV recovered from untreated cell culture supernatant fluids sedimented heterogeneously and less rapidly than normal virus in rate-zonal sucrose gradients and also banded at a lower density in CsCl (1.14 to 1.18 g/ml) than normal, neutralizable virus (1.32 g/ml). This bimodal distribution of HAV in CsCl gradients was confirmed by cDNA-RNA hybridization. Together, these observations suggest that a substantial proportion of HAV particles released from infected cells are lipid-associated and imply an important role for cell membranes in the assembly and release of HAV in vitro.

Animals↗

PubMed1985

Human lymphocyte responses to hepatitis A virus-infected cells: interferon production and lysis of infected cells.

Peripheral blood mononuclear cells (PBMC) from nonimmune healthy donors who did not have antibody to hepatitis A virus lysed hepatitis A virus-infected BS-C-1 cells to a greater degree than uninfected BS-C-1 cells. The predominant effector cells were contained in the nonadherent peripheral blood lymphocyte (PBL) fraction, although some lytic activity was associated with adherent cells. Characterization of the PBL with monoclonal antibodies showed that the responsible effector lymphocytes were contained in Leu-11+ and M1+ subsets, but not in the T3+ or T4+ subsets. The phenotypes of the effector cells active in the lysis of hepatitis A virus-infected cells are similar to those of human natural killer cells that lyse K562 cells. Human PBL produced high titers of interferon-alpha (IFN-alpha) when exposed to hepatitis A virus-infected cells. These results imply that hepatitis A virus infection may be controlled by lymphocyte responses in the liver, i.e., by lymphocyte-mediated lysis of the hepatitis A virus-infected cells, and by the production of high titers of IFN-alpha by lymphocytes exposed to hepatitis A virus-infected cells. Furthermore, these results, along with the observations that hepatitis A virus infection results in a persistent noncytocidal infection in vitro, support the hypothesis that lysis of hepatocytes infected with hepatitis A virus is by lymphocyte-mediated cytotoxicity and not by virus-induced destruction of the liver cell.

Antibodies, Monoclonal↗

PubMed1984

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.

Animals↗

PubMed1984

Pathology of hepatitis A infection in the owl monkey (Aotus trivirgatus).

Sequential liver biopsies of owl monkeys that had been experimentally infected with one of two strains of hepatitis A virus (HM-175 or PA-33) were examined for histopathologic alterations. Preinoculation biopsies were normal with only occasional minimal mononuclear cell infiltrates in portal tracts and hepatic lobular parenchyma. Histopathologic features that were present in biopsies taken during the period of elevated serum alanine aminotransferase activity (16-43 days after the intravenous inoculation of virus) included infiltration of predominantly mononuclear inflammatory cells into portal tracts and surrounding parenchyma, degeneration and necrosis of hepatocytes, and hypertrophy of Kupffer cells. Changes were similar in monkeys infected with either HM-175 or PA-33 virus strains. Convalescent biopsies (147-186 days after inoculation) showed resolving lesions with mild portal inflammation and occasional focal collections of inflammatory cells in the parenchyma. These histologic changes are similar to those associated with hepatitis A infection in man, chimpanzees, and several species of marmosets, and support the further use of the owl monkey as a model of human hepatitis A.

Acute Disease↗

PubMed1983

Serum neutralizing antibody response to hepatitis A virus.

Serum neutralizing antibody to hepatitis A virus (HAV) was measured in experimentally infected primates and naturally infected humans by means of an assay based on the autoradiographic detection of viral replication foci in vitro. Infection of primates with either PA-33 or HM-175 strains of HAV elicited antibody capable of neutralizing either strain. Sequential testing of two monkeys showed that neutralizing antibody correlated closely with antibody detected by immunoassay, developed before liver enzyme elevations, and was associated with a substantial reduction in fecal shedding of viral antigen. In tests performed on human subjects involved in an outbreak of hepatitis A, neutralizing antibody was present three to five days before the onset of symptoms and was found in both 19S and 7S immunoglobulin fractions. Immunity to HAV is probably due primarily to neutralizing antibody, and the ability to quantitate this antibody will be helpful in the evaluation of new HAV vaccines.

Animals↗

PubMed1983

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.

Animals↗

PubMed1983

Antigenic relatedness of two strains of hepatitis A virus determined by cross-neutralization.

Cell culture-adapted HM-175 (Australia) and PA-21 (Panama) strains of hepatitis A virus were compared in a cross-neutralization radioimmunofocus inhibition assay. The ratio of antibody titers achieved with two sera against the two viruses differed by less than twofold, indicating a high degree of antigenic relatedness between epidemiologically disparate strains of virus.

Antibodies, Viral↗

PubMed1983

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.

Animals↗

PubMed1982

Transmission of hepatitis A virus among recently captured Panamanian owl monkeys.

The presence of antibody to hepatitis A virus (anti-HAV) in 60% of procured owl monkeys (Aotus trivirgatus) held within the United States prompted a study of recently captured A trivirgatus in Panama. Only 2 of 145 newly captured monkeys, but all of 35 A trivirgatus held within a colony for over 100 days, were found to have anti-HAV. Of 41 sero-negative, newly captured monkeys followed prospectively, 25 became infected with hepatitis A virus (HAV) as evidenced by seroconversion or demonstration of virus in the liver at death. Only one monkey that survived over 60 days within the colony was not infected. HAV was identified in the feces of most infected monkeys prior to the development of antibody and was antigenically indistinguishable from human HAV in cross-blocking radioimmunoassays. This colony-centered epizootic provides strong evidence that A trivirgatus is susceptible to HAV and should be investigated further as a potential model of human hepatitis A.

Animals↗

PubMed1982

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.

Animals↗