A novel hepatovirus identified in wild woodchuck Marmota himalayana.

Hepatitis A virus (HAV) is a hepatotropic picornavirus that causes acute liver disease worldwide. Here, we report on the identification of a novel hepatovirus tentatively named Marmota Himalayana hepatovirus (MHHAV) in wild woodchucks (Marmota Himalayana) in China. The genomic and molecular characterization of MHHAV indicated that it is most closely related genetically to HAV. MHHAV has wide tissue distribution but shows tropism for the liver. The virus is morphologically and structurally similar to HAV. The pattern of its codon usage bias is also consistent with that of HAV. Phylogenetic analysis indicated that MHHAV groups with known HAVs but forms an independent branch, and represents a new species in the genus Hepatovirus within the family Picornaviridae. Antigenic site analysis suggested MHHAV has a new antigenic property to other HAVs. Further evolutionary analysis of MHHAV and primate HAVs led to a most recent common ancestor estimate of 1,000 years ago, while the common ancestor of all HAV-related viruses including phopivirus can be traced back to 1800 years ago. The discovery of MHHAV may provide new insights into the origin and evolution of HAV and a model system with which to explore the pathogenesis of HAV infection.

Detection of viral hepatitis type A.

Several technics are currently being used to detect hepatitis type A antigen or its antibody. These include immunoelectronmicroscopy, immune adherence, and complement fixation. This paper describes another promising technic, a microtiter solid-phase immunoradiometric assay, in which hepatitis A antigen and antibody are detected. Such a method can be utilized for biochemical and biophysical analysis of purified particles, for the seroepidemiology of type A hepatitis, and as a means for monitoring hepatitis A antigen in cell cultures.

Isolation and characterization of hepatitis A virus.

This paper presents current isolation technics of hepatitis A virus (HAV) from human and chimpanzee stool, liver, and bile specimens, as well as comparative characterizations of HAV buoyant density properties of human and chimpanzee stool-derived particles. In addition, methods designed for the extraction and purification of HAV from large samples of stool and liver tissues, including agar gel filtration, are discussed in detail.

Small virus particles in faeces of patients with infectious hepatitis (hepatitis A).

Forty-one faecal samples from infectious-hepatitis patients and their contacts were investigated for the presence of hepatitis-A-associated viral particles. Of these, 16 gave a positive result by immune electronmicroscopy or caesium-chloride density-gradient centrifugation. The latter method proved invaluable in detecting small numbers of virus particles. The particles found had buoyant density of 1-34-1-35 and a size range of 21-28 nm. Epidemiological evidence suggested that they might be the causative agent of hepatitis A.

Large-scale purification of inactivated hepatitis A virus by centrifugation in non-ionic gradients.

Formalin-inactivated hepatitis A virus (HAV) can be purified for vaccine preparation by centrifugation in Renografin-76 (diatrizoate meglumine and diatrizoate sodium) gradients. Both continuous-flow rate-zonal and isopycnic methods were used for the separation of a major antigen component from minor antigen and host protein. The major antigen component, which appeared to contain complete virions by electron microscopy, could be recovered from gradients and accounted for approximately one third of the total antigen in the starting material. The HAV-specific purified antigen could be enriched 200-300-fold by either centrifugation procedure. The purified HAV antigen, when adsorbed to alum and inoculated into mice, was found to be highly immunogenic.

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.

Identification of enterically transmitted hepatitis virus particles by solid phase immune electron microscopy.

Small 'featureless' viruses (less than 50 nm) are difficult to identify by routine immune electron microscopy techniques, particularly when they are mixed with debris from stool or cell culture extracts. A combination of conventional immune electron microscopy (IEM) and solid phase IEM (SPIEM) methodologies was used to identify hepatitis A virus (HAV) in stool and cell culture extracts and non-A non-B hepatitis (hepatitis E) in stool extracts. Compared with conventional IEM, the modified SPIEM method resulted in a significant increase in the number of particles observed. Several small aggregates, each containing 2-20 particles, were observed scattered randomly within most grid squares. Similar results were seen with stool extracts from hepatitis E (HEV) infections. The SPIEM method is a simple, highly sensitive specific assay that facilitates rapid identification of enteric hepatitis viruses. Several experiments were done to characterize the effects of altered physical environment within the assay and to evaluate potential modifications.

Prediction of secondary structure, spatial organization and distribution of antigenic determinants for hepatitis A virus proteins.

On the basis of the secondary structure calculations from the known amino acid sequence we came to the conclusion that hepatitis A virus capsid proteins have the typical antiparallel beta-sheet bilayer structure. The predicted secondary structure of the HAV proteins can be well aligned with those of the poliovirus (type 1 Mahoney) and human rhinovirus (type 14). It enabled us to use the X-ray structure of the PV-1M and HRV-14 proteins as a template and then, firstly, to localize the positions of alpha and beta regions in the architecture of the HAV protein molecules and, secondly, to discover the amino acid homologies of the secondary structure regions aligned. The obtained model of the three-dimensional structure for HAV proteins helped us to indicate the exposed regions of the polypeptide chains and to pinpoint the potential neutralizing antigenic sites.

Biophysical and biochemical properties of CR326 human hepatitis A virus.

CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 mmu spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests by infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, identical 27 mmu viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The buoyant density of the virus in CsCl was 1.34 and it was heat (60 C), ether, and acid stable but was destroyed by heat (100 C), formalin (1:4000), and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the enterovirus family and not to hepatitis B virus.

Relationship of hepatitis A antigen to viral hepatitis.

Progress in research on hepatitis type A has begun to accelerate because of the recent discovery of an antigen associated specifically with hepatitis type A infection and the development of tests for antibody to the antigen. Hepatitis A antigen is associated with 27 nm virus-like particles found in the liver and stool of animals experimentally infected with hepatitis type A and in the stool of humans experimentally or naturally infected with the virus. The density of the particulate antigen when isolated from the liver is 1.34, but antigen particles with densities ranging from 1.32 to 1.40 have been detected in stool. However, antigens from the liver and from the stool appear to be antigenically related. Using immune electron microscopy as a serologic tool for detecting antibody to hepatitis A antigen, we detected antibody in convalescent sera from 100 per cent of patients experimentally or naturally infected with hepatitis type A. In contrast, patients with hepatitis type B or non-B hepatitis not epidemiologically compatible with a diagnosis of hepatitis type A did not have a serologic response to hepatitis A antigen. Antibody was found in approximately 50 per cent of normal individuals tested; the frequency was directly related to age. By the use of immune electron microscopy for the detection of hepatitis A antigen and antibody, the temporal relationship of antigen, antibody and liver damage was determined in experimentally infected humans and chimpanzees. On the basis of serologic comparisons, hepatitis type A does not appear to be related to experimental hepatitis caused by the GB agent of Deinhardt, nor is the hepatitis A antigen serologically related to the fecal antigen of Cross.

Biophysical validation of Epaxal Berna, a hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes (IRIV).

Inactivated vaccines usually contain an adjuvant which potentiates the immune response to the antigen. During the last 70 years aluminium salts have been the only adjuvant licensed for human use. The adjuvanting activity is based on their serving as an antigen depot and inducing a localized inflammatory response. Our efforts to develop a potent and well tolerated adjuvant has focussed on the use of immunopotentiating reconstituted influenza virosomes (IRIV). The IRIV base is a liposome with a mean diameter of 150 nm, comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These mammalian phospholipids are virtually non-immunogenic and have enjoyed a long history of use in human pharmaceutical preparations. The haemagglutinin (HA) and trace quantities of viral neuraminidase and phospholipids from the A/Singapore 6/86 virus strain are intercalated within the phospholipid bilayer. The presence of the HA is necessary to enhance the immunopotentiating effect to antigen associated with IRIV The excellent characteristics of IRIVs as adjuvants have been demonstrated in several systems. IRIVs as alternative adjuvant system for human use are registered by most European, Asian and American countries in commercial hepatitis A and influenza vaccines. IRIVs were first used in the manufacture of a hepatitis A vaccine. This contains formalin-inactivated and highly purified hepatitis A virus (HAV) of strain RG-SB, cultured in human diploid cells, which is coupled to the IRIV vesicle. For a new influenza vaccine the surface spikes (HA and NA) of three currently circulating influenza strains were jointly inserted into the vesicle membrane of the IRIVs and successfully tested clinically. In Epaxal Berna, the first commercially available liposomal vaccine is expected to be the inactivated hepatitis A virus adsorbed to IRIV particles. In the virosomal hepatitis A vaccine, the antigen is believed to be attached to the IRIV by interacting with phospholipids which are considered to correspond to its natural receptor on hepatocytes. The present investigation includes data based on light scattering measurements which show the binding of the virions to vesicles.

[Detection of hepatitis A virus by means of electron microscopy (author's transl)]. / Elektronenoptischer Nachweis von Hepatitis-A-Virus.

During an outbreak of hepatitis A in an institution, 34 stools of children were investigated by means of electron microscopy. Hepatitis A virus was found in four samples. All four children had previously received gammaglobulin injections and did not develop any clinical symptoms.

Propagation of hepatitis A virus in human diploid fibroblast cells.

Human hepatitis A virus (HAV) was propagated in human diploid fibroblast cultures (2BS cells) in vitro. Replication of the virus was followed by immunofluorescent staining (IF), indirect ELISA, and by immune electron microscopy. When 2BS cells were inoculated with faecal extracts containing HAV, synthesis of hepatitis A antigen (HAAg) could be detected in the cytoplasm by IF. Its concentration reached a maximum at four weeks post-inoculation. Measured by solid-phase indirect ELISA, the positive/negative (P/N) ratio for HAAg reached values of up to 7.7. The identity of newly synthesized virus particles with HAV was established by immune electron microscopy, IF-blocking, and neutralization with human convalescent serum. Infected cells showed no signs of a specific cytopathic effect. Two of the virus strains propagated in 2BS cells may prove useful as a source of antigen for serologic tests; one of them might be a candidate strain for HAV vaccine.

[Comparative study of the effectiveness of immunoelectron microscopic methods for detecting rotaviruses and the hepatitis A virus]. / Sravnitel'noe issledovanie éffektivnosti immunoélektronno-mikroskopicheskikh metodov vyiavleniia rotavirusov i virusa gepatita A.

Different variants of immune electron microscopy method used for the detection of rotaviruses and hepatitis A virus in specimens from patients were compared. Immune electron microscopy using filtration into agar was shown to be the optimal method for diagnosis of such prevalent infections as rotavirus gastroenteritis and viral hepatitis A.

[Combination of radioimmunological and immunoelectron microscopic methods for detecting hepatitis A virus]. / Kombinatsiia radioimmunologicheskogo i immunoélektronno-mikroskopicheskogo metodov dlia obnaruzheniia virusa gepatita A.

The possibility of combined performance of radioimmunoassay (RIA) and immune electron microscopy (IEM) in one preparation using protein A of Staphylococcus aureus for hepatitis A virus (HAV) detection in fecal specimens from hepatitis A patients within a short time (40-50 min) has been demonstrated. In the examinations of one preparation by RIA and IEM, their sensitivity was found to be approximately similar. According to RIA, a high content of HAV antigen was observed in those preparations where in addition to typical particles there were structures resembling individual fragments of empty HAV particles coated with antibody.

[The adaptation of an isolate of the hepatitis A virus to reproduction in cell cultures]. / Adaptatsiia izoliata virusa gepatitia A k reproduktsii v kletochnykh kul'turakh.

A HAV strain derived from a patient in Moscow multiplied in a continuous cell line PLC/PRF/5 at 32 degrees C (variant MI-1) and at 37 degrees C (variant MI-1.1). These two variants of the strain MI retained the ability for reproduction both at 32 degrees C and 37 degrees C after passages in cell culture. The strain MI also replicates in MK cells. Negative results on HAV cultivation were obtained in HEF-240 and FRhK-4 cell cultures. The MI-1 and MI-1.1 variants are typical of hepatitis A viruses by their physicochemical properties, the results of ELISA, protein electrophoresis and dot hybridization tests.

[Viruses in the feces of patients with viral hepatitis and other enterovirus infections]. / Virusy v fekaliiakh bol'nykh virusnym gepatitom i drugimi énterovirusnymi infektsiiami.

Direct and immune electron microscopy was used to determine the frequency of finding of hepatitis A virus (HAV) and other viral agents in feces of patients in relation to the diagnosis and epidemiological situation. HAV-containing excretions from patients were analysed ultrastructurally. The highest frequency of HAV detection was established in patients in a water-borne and food-borne outbreak of hepatitis A (HA) and was 40.9% and 36.9%, respectively. In patients with HA diagnosis in the period of a seasonal rise of HA incidence HAV particles were found in 11.8%, and in the interseasonal period in 5%. Apart from HAV particles, in a small per cent of patients with HA diagnosis adenovirus and enterovirus particles were found. In patients with the disease diagnosed as hepatitis B (HB) only adenovirus and enterovirus particles were found. In contrast to the patients with HAV and HB diagnosis, the patients who were combined into a conditional "non-hepatitis" group were found to have, in addition to HAV (in the period of seasonal rise of HA incidence), adenovirus and enterovirus particles, also particles of astrovirus, coronavirus, and rotavirus. In fecal specimens of patients containing typical HAV particles structures were found resembling individual fragments of empty HAV particles, antibody-covered 17-22 nm and 27 nm spherical structures poorly reacting with antibody.

[Spontaneous hepatitis A with a fatal outcome in rhesus monkeys]. / Spontannyi gepatit A so smertel'nym iskhodom u makak.

This is the first report of virologically verified spontaneous hepatitis A in M. rhesus monkeys with severe involvement of the liver leading to the death of the animals. In 21 out of 23 dead monkeys morphological lesions in the liver have been characterized as acute hepatitis. In 6 (26%) animals no other pathological processes were found. In 15 animals hepatitis was combined with other diseases (dysentery, parasitic infestations, coronavirus infection). Antigen of hepatitis A virus was detected by an enzyme immunoassay in the intestinal contents of 8 monkeys and in the livers of 3 of them. Immune electron microscopic studies detected in the intestines some virus particles morphologically and antigenically similar to human hepatitis A virus.