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.

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.

Review: resolution issues in single-particle reconstruction.

Specific factors that affect the resolution of single-particle reconstructions are discussed. We present reconstructions of six particles (DNA-dependent protein kinase catalytic subunit, alphaB-crystallin, the ribonucleoprotein vault, hepatitis A virus, adenovirus type 2, and the adenovirus type 12/alpha(v)beta5 integrin complex), which have a variety of symmetries (asymmetric to 60-fold) and a wide range of molecular masses (470 kDa to 150 MDa). In the case of icosahedral viruses, we have found that applying a "soft" mask to remove regions of disordered density improves the resolution given by the Fourier shell correlation 0.5 criterion. This masking procedure is also useful during refinement to improve the quality of the reference model and thus aid in precise alignment of the particle images. For asymmetric particles, we note that image classification, although often a necessary step to generate a first reconstruction, can limit the achievable resolution. The diameter of the particle and the available computational power can also affect the resolution, as can structural variability within the particle.

Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles.

Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.

An epidemic of hepatitis A attributable to the ingestion of raw clams in Shanghai, China.

An epidemic of hepatitis A in 1988 in Shanghai had an overall attack rate of 4083/100,000 population (292,301 cases). The epidemic curve showed three peaks in January and February. A case-control study of 1208 matched pairs supported that clams were the vehicle for the virus (summary odds ratio, 9.47; P less than .001). Analysis of subsets who had eaten clams indicated that only 3.5% with hepatitis A had cooked their clams compared with 18.1% without hepatitis A, and those with the disease consumed more clams. A historical cohort study indicated that approximately 31.7% of the population had eaten clams one or more times between 9 December 1987 and 3 January 1988. The estimated attack rates in those who had and had not eaten clams were 11.93% and 0.52%, respectively (relative risk, 22.94; attributable risk, 11.41%). The three peaks in the consumption curve correlated with those in the epidemic curve. Hepatitis A virus was demonstrated in clams taken from the Shanghai markets and from the catching area.

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.

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.

[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.

Morphogenesis of hepatitis A virus: isolation and characterization of subviral particles.

The morphogenesis of hepatitis A virus (HAV) in BS-C-1 cells was examined by immunoblotting with antisera to capsid proteins and labeling of virus-specific proteins with L-[35S]methionine. Antiserum to VP2 detected two virus-specific proteins with apparent molecular masses of 30.6 and 30 kDa, representing VP0 and VP2, while antiserum to VP1 detected proteins with molecular masses of 33 and 40 kDa, representing VP1 and a virus-specific protein which we designated PX, respectively. Sedimentation of cell lysates revealed the presence of virions, procapsids, and pentamers, but particles analogous to the protomers of other picornaviruses were not detected. Although provirions and virions were not found as discrete species in our gradient system, it was evident that the rate of sedimentation was proportional to the relative amounts of VP0 and VP2 in particles, with slower-sedimenting particles (provirions) containing predominantly VP0 rather than VP2. Procapsids contained VP0 in addition to VP1 and VP3. Pentamers also contained VP0, but PX was present rather than VP1. These results suggest that PX is a precursor to VP1 and is most likely 1D2A. Primary cleavage of the viral polyprotein also occurs at the 2A-2B junction in cardioviruses and aphthoviruses, but assembly of pentamers containing 1D2A has not been reported for those viruses. The absence of detectable levels of protomers suggests a high efficiency of pentamer formation, which may be related to the high efficiency of viral RNA encapsidation for HAV (D.A. Anderson, B.C. Ross, and S.A. Locarnini, J. Virol. 62:4201-4206, 1988). The results of this study reveal further unusual aspects of the HAV replicative cycle which distinguish it from other picornaviruses and may contribute to its restricted replication in cell culture.

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 three-dimensional models for foot-and-mouth disease virus and hepatitis A virus.

Atomic models of foot-and-mouth disease virus and hepatitis A virus have been predicted using amino acid sequence alignments with the known structures of Mengo virus and human rhinovirus 14. The structural models are consistent with results of biochemical and immunological studies. The two viruses appear to have surface features exceedingly different than those of other picornaviruses. They also have large hydrophobic cavities within VP1 suggesting that it may be possible to inhibit their infectivity with suitably designed antiviral agents that block uncoating.

Immune electron microscopy in the detection of viruses other than enteroviruses on cell culture in untreated sewage.

Following the demonstration of enteroviruses in samples of untreated sewage by inoculation of cell cultures with an eluate fraction, immune electron microscopy (IEM) was employed in two stages to detect adenoviruses and the hepatitis A virus (HAV). 6/12 samples contained adenoviruses and 5/15 other samples contained viruses having characteristics of HAV. Cell culture can thus be usefully allied with IEM in the more precise determination of the potential health hazards of waste water.

Research of enterovirus, hepatitis A virus in a bathing area over a six month period and their salubrity impact.

Cell-culturable enterovirus and HAV levels in the effluent of a treatment plant were compared with those near the effluent outfall and in a neighbouring bathing area over a period of six months. Enteroviruses were found in all effluent samples, whereas only three contained HAV. No viruses were detected near the outfall nor in the bathing area. Despite the low impact of this scenario on human health, the view is expressed that the potential risk posed by the discharge of viruses from treatment plants must not be underestimated, since there is ample evidence that HAV can be epidemiologically transmitted via the consumption of shellfish.

An electron microscopical investigation of faecal small round viruses.

A retrospective study of small round featureless viruses (SRVs) initially identified by negative-staining electron microscopy of stool samples was performed. A variety of technique, including immunoelectron microscopy and caesium chloride gradient centrifugation, was applied in an attempt to classify further these viruses. Over a four-year period, 64 SRV-positive samples were reported (1.8% of the stool samples sent for electron microscopy and 6.2% of the total number of positive samples), of which 53 were available for further study. A significant degree of misclassification was found. Viruses previously identified as SRVs were shown to be astrovirus (n = 14), calicivirus (n = 2), and "Norwalk-like" virus (n = 1). The majority of the 36 remaining samples were identified as parvovirus-like (n = 27) (75%), 14 of which were associated with the presence of adenovirus particles. Enteroviruses (n = 3) and hepatitis A virus (n = 1) were infrequently detected. The remaining viruses (n = 5) could not be adequately classified. Parvovirus may be the predominant SRV associated with acute diarrhoeal disease in childhood.

A technology of preparing hepatitis A virus (HAV) from human stools.

Two-step differential centrifugation through 40% and 20% sucrose of 20% water extracts of HAV-positive stools yielded specific defined antigens for hepatitis A serological diagnosis by 3rd-generation methods. The antigens would be suitable also for other purposes. Products of comparable quality were obtained from HAV-positive stools yielding different amounts of virus. Optimal were incubation-period stools, where the loss in HAV yield was minimal; these preparations displayed the highest antigenic capacity and could be diluted up to 150 fold for serological reactions. Results obtained in extensive preliminary experiments, outlined in the Discussion, corroborated the advantages and efficiency of the proposed technology.

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus.

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.

[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.