Bertram Flehmig

Acid Stability of Hepatitis A Virus

The acid stability of unpurified and highly purified hepatitis A virus (HAV) was tested and compared with that of poliovirus type 1, coxsackievirus types A9 and B1 and echovirus type 9. Only HAV had a high residual infectivity after 2 h of exposure to pH 1 at room temperature, remaining infectious for up to 5 h. At 38 degrees C, pH 1, HAV remained infectious for 90 min. Highly purified HAV was found to be infectious for 8 h at pH 1 and room temperature. This indicates that the increased stability is not due to protection by cellular material attached to the virus, but is a virus-specific marker. Under the same conditions, at pH 1 and room temperature, unpurified and highly purified HAV antigens were traceable for 5 and 4 h respectively.

Hepatitis A-virus in cell culture: I. Propagation of different hepatitis A-virus isolates in a fetal rhesus monkey kidney cell line (Frhk-4)

A fetal rhesus monkey kidney cell line (Frhk-4) was infected with different hepatitis A-virus (HAV) isolates GBG, GBM, GJA. The time-dependent adsorption of the HAV isolates to Frhk-4 cells was measured. Replication of all three isolates in these cells could be demonstrated intracellularly 8–10 weeks after infection, and release of HAV into the supernatant some 10–15 weeks after infection could be shown. The specificity of the virus determination by RIA from supernatants of HAV-infected cells from passages 1,2, and 3 in Frhk-4 cells was shown with sera that were collected from a chimpanzee infected with MS-1 both before infection as well as during convalescence. These results were subsequently compared with sera collected from human patients before the onset of hepatitis as well as during convalescence. With immunofluorence microscopy a cytoplasmic fluorescence could be shown in HAV-infected Frhk-4 cells and finally the release of 27 nm HAV particles into the supernatant of HAV-infected Frhk-4 cells could be demonstrated by immune electron microscopy.

Persistent Infection of Human Fibroblasts by Hepatitis a Virus

Infection of human embryo fibroblasts with hepatitis A virus (HAV), a picornavirus, leads to an inapparent, persistent infection; cultures can be passed serially with consistent recovery of the virus in the supernatant. All of the cells of a HAV carrier culture are infected and proliferate. Subcultivation under HAV-immune serum cannot achieve a cure or even a reduction in the number of infected cells in HAV carrier cultures. No interferon activity can be detected during HAV infection and persistence. Addition of exogenous interferon eliminates HAV infection in vitro. Persistence of HAV in vitro appears to contradict the clinical course of HAV infection in vivo. The system presented offers the possibility of evaluating the role of immunological injury of HAV-infected cells, an injury which may lead to damage of these cells and to elimination of HAV during an HAV infection in vivo.

Immunogenicity of a killed hepatitis A vaccine in seronegative volunteers

32 volunteers who lacked antibody to hepatitis A virus (HAV) received, at intervals of one month, three injections of a killed vaccine made from HAV propagated in diploid human fibroblast cell cultures. In 16 the vaccine was coupled with aluminium hydroxide as adjuvant. The serconversion rates measured by radioimmunoassay, with and without adjuvant respectively, were 13% and 25% at four weeks, 81% and 89% at eight weeks, and 94% and 100% at twelve weeks. Four weeks after the third injection all volunteers had high neutralising antibody titres. Twelve weeks later the titres of anti-HAV and anti-HAV neutralising antibodies had not declined.

Correlation of pathogenicity and gene constellation of influenza A viruses. II. Highly neurovirulent recombinants derived from non-neurovirulent or weakly neurovirulent parent virus strains.

Abstract Mixed infections with various nonneurovirulent or weakly neurovirulent influenza A strains yielded recombinants that were highly neurovirulent for mice. A correlation was detected between gene constellation and neurovirulence of these recombinants. For the recombination pair FPV/A2-England the Pol 1 and Ptra genes had to be derived from the human strain; while the HA and/or M gene has to be from FPV in order to obtain highly neurovirulent recombinants. For the FPV/PR8 pair only the Pol 1 gene needs to be derived from PR8 while the HA gene had to be from FPV in order to get highly neurovirulent recombinants. The derivation of the other genes does not appear to be important in this respect. Recombinants between FPV and the A2-Singapore influenza strain do not exhibit significant neurovirulence suggesting that at least one parent strain should be adapted to grow in mice in order to obtain neurovirulent recombinants. In addition, there is a correlation between pneumovirulence and growth in mouse kidney cells, but neurovirulence for mice and pathogenicity for chickens was not correlated. The data presented here demonstrate in principle the possiblity of an increase in pathogenicity in recombinants derived from less or nonpathogenic parent viruses.

Hepatitis A-virus in cell culture. V. Neutralizing antibodies against hepatitis A-virus

A test system for the detection of neutralizing antibodies against hepatitis A-virus (anti-HAV-Nt) is presented. The anti-HAV-Nt assay is performed with Frhk-4/R cells and the hepatitis A virus (HAV) strain GBM/Frhk-4/R which has been adapted to these cells. Non-neutralized HAV is demonstrated 14 days after infection of Frhk-4/R cells using a radio-immunoassay for detecting newly grown HAV.The influence of differing amounts of HAV on the anti-HAV-Nt titre and the effect of variations in incubation time of virus-serum mixtures are described. The time course of anti-HAV-Nt is shown in sera from a hepatitis-A patient which were taken at different stages of the disease. Anti-HAV-Nt is compared with anti-HAV and anti-HAV-IgM. It is shown that anti-HAV-Nt correlates closely with anti-HAV and separated anti-HAV-IgG, but only slightly with anti-HAV-IgM.The test system presented makes possible the demonstration of neutralizing antibodies against HAV in gamma-globulin preparations and during vaccination studies.

IDENTIFICATION OF GBV-C HEPATITIS G RNA IN CHRONIC HEPATITIS C PATIENTS

Sera from patients with chronic hepatitis C were examined for the presence of GBV‐C/HGV RNA by RT‐PCR. The amplified products, derived from the 5′ non‐coding, NS3, and NS5a regions, were detected in 19 (19%) of the 100 HCV RNA‐positive samples. Analysis of GBV‐C/HGV prevalence rates revealed that dual infections are related to shared parenteral risk factors. Intravenous drug abuse and multiple transfusions were the factors clearly associated with a simultaneous HCV and GBV‐C/HGV infection. Apart from this, patients with dual infections had a statistically significant lower mean age compared to those patients infected solely by HCV. Determination of HCV genotypes involved in GBV‐C/HGV coinfection by RFLP analysis showed no correlation between the presence of GBV‐C/HGV and a distinct HCV genotype. The study demonstrates that, based on the assessment of risk criteria, GBV‐C/HGV is transmitted efficiently parenterally and is frequently linked to hepatitis C coinfection, regardless of HCV genotype.

Hepatitis A virus in cell culture. II. Growth characteristics of hepatitis A virus in Frhk-4/R cells.

The propagation of hepatitis A virus (HAV) is shown in a rapidly growing fetal rhesus monkey kidney cell line (Frhk-4/R). In the course of ten passages through Frhk-4/R cells the HAV, which was released in the supernatants of the cell cultures, was adapted to these cells. In the tenth passage 6 days after infection, it was possible to detect low amounts of HAV in the supernatants by radioimmunoassay (RIA). With indirect immunofluorescence 3 days after infection, a specific granular fluorescence was demonstrated in the cytoplasm of cells infected with HAV from the tenth passage. A persistent infection of Frhk-4/R cells with HAV is shown over a period of 6 months. The HAV produced in Frhk-4/R cells appeared with a main peak at a density of 1.32 g/cm3 in CsCl density gradients. In the 1.32 g/cm3 fractions, typical HAV particles are shown by electron microscopy. Furthermore the HAV produced in Frhk-4/R cells is shown to be a useful antigen for diagnostic tests.

Hepatitis A virus protein 2B suppresses beta interferon (IFN) gene transcription by interfering with IFN regulatory factor 3 activation

Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of retinoic acid-inducible gene I-mediated and melanoma differentiation-associated gene 5-mediated beta interferon (IFN-beta) gene expression. This study showed that this is due to an interaction of HAV with mitochondrial antiviral signalling protein (MAVS)-dependent signalling, in which the viral non-structural protein 2B and the protein intermediate 3ABC recently suggested in this context seem to be involved, cooperatively affecting the activities of MAVS and the kinases TANK-binding kinase 1 (TBK1) and the inhibitor of NF-kappaB kinase epsilon (IKKepsilon). In consequence, interferon regulatory factor 3 (IRF-3) is not activated. As IRF-3 is necessary for IFN-beta transcription, inhibition of this factor results in efficient suppression of IFN-beta synthesis. This ability might be of vital importance for HAV, which is an exceptionally slow growing virus sensitive to IFN-beta, as it allows the virus to establish infection and maintain virus replication for a longer period of time.

Recombination of influenza a strains with fowl plague virus can change pneumotropism for mice to a generalized infection with involvement of the central nervous system

Abstract Recombination between fowl plague virus FPV/Rostock (Hav1N1), an influenza A strain which produces a generalized fatal infection with involvement of the central nervous system in chickens, and mouse lung-adapted human strains England/1/61 (H2N2) or PR/8/34 (H0N1 results in recombinants which produce a generalized infection in suckling mice with involvement of the central nervous system. A combination of the hemagglutinin and M protein from FPV/Rostock (Hav1N1) with certain polymerase proteins from the mouse lung-adapted strains produces generalized infections in mice. Infectious virus was present in lung, brain, and blood even after intranasal infection. A correlation between the ability to induce a generalized infection of suckling mice in vivo and the replication in mouse embryo fibroblasts in vitro could be demonstrated.