A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA.

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Conformational changes in the hepatitis A virus capsid in response to acidic conditions.

Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.

A microcarrier cell culture system for large scale production of hepatitis A virus.

Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10(-2) contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.

Continuous production of hepatitis A virus in PLC/PRF/5 cell cultures: use of antigen for serology.

The strain CF53 of hepatitis A virus (HAV) previously adapted to growth in PLC/PRF/5 cells was grown in 175 cm2 flasks, at different passages. After infection, cells were incubated at 32 degrees C in RPMI 1640 medium supplemented with 2.5% foetal calf serum (FCS) for 6-12 months. HAV which was released continuously in the culture medium was harvested weekly. Hepatitis A virus antigen (HAAg) and infectious virus production was stable during each passage. The antigen titre, determined by radioimmunoassay, was about 50 for each passage whereas the infectious virus titre increased from 10(3.7) (passage 7) to 10(6.0) TCID50/ml (passage 13). Virus production was not influenced by the FCS concentration (0-2.5%) in the maintenance medium. The cell culture produced HAAg was used for detection of total anti-HAV antibodies, anti-HAV titration and IgM antibody capture assay and the results were identical to those obtained with commercial kits. HAAg produced by this practical and cheap method could easily replace primate derived antigen for the detection of anti-HAV antibodies.

Immunochemiluminescent focus assays for the quantitation of hepatitis A virus and rotavirus in cell cultures.

Two new immunological methods, the luminescent immunofocus assay (LIFA) and the luminescent immunofocus inhibition assay (LIF-IA), are described for the quantitation of cytopathic and non-cytopathic viruses propagated on cell culture monolayers. These methods use enhanced chemiluminescent detection to identify foci (luminescent immunofoci, LIF) of virus-infected cells. Viruses are propagated in susceptible cells under an agarose overlay, inactivated with ultraviolet irradiation, lifted onto nitrocellulose membranes, and probed with virus-specific monoclonal or polyclonal antibody followed by a second antibody conjugated to horseradish peroxidase. Membranes are then treated with a luminol-based detection reagent and exposed to light sensitive film for up to 10 min. The film is developed and foci appear as dark, discrete spots which are proportional to the dose of each virus. The LIFA detected both cytopathic and non-cytopathic hepatitis A viruses (HAV) and simian rotavirus. For the cytopathic HAV, the LIFA and plaque counts were comparable. The LIF-IA was developed for HAV using virus-specific antiserum which effectively attenuated LIF formation. The LIFA and LIF-IA may be completed 5 days faster than conventional radioimmunofocus assays for HAV and rotavirus and do not require the use of radiolabeled antibodies, offering safety advantages and making these techniques more adaptable for general use. Luminescent immunofocus assays should be useful for the detection and quantitation of virtually any cytopathic or non-cytopathic virus that can be propagated in monolayer cultures when virus-specific antiserum is available.

A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays.

Hepatitis A virus (HAV), a non-cytopathic picornavirus, has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridization. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.

An evaluation of immune electron microscopy as a method for the quantitation of virus particles.

A comparison is made between poliovirus titres obtained by immune electron microscopy and those obtained by plaque assay. The former method yields smaller values which cannot be explained by inefficient sedimentation of virus-antibody clumps or incomplete settling on the grid. Using this comparison and immune electron microscopy the theoretical titre of hepatitis A virus can be estimated.

Rapid concentration and detection of hepatitis A virus from lettuce and strawberries.

Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.

Propagation and assay of hepatitis A virus in vitro.

Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37 degrees C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.

Characterization of hepatitis A virus capsid proteins with antisera raised to recombinant antigens.

The capsid proteins of hepatitis A virus (HAV) were expressed as fusion proteins of beta-galactosidase in E. coli using the expression vector lambda gt11. Four fusion proteins were stably expressed and used to immunize rabbits to obtain mono-specific antisera. The antisera were unable to neutralize viral infectivity or react with HAV by radioimmunoassay. Three of the antisera were able to recognize HAV antigens in infected BS-C-1 cells by immunofluorescence and denatured capsid proteins by immunoblot analysis. The antisera were used to investigate the migration of the capsid proteins in gels by immunoblot analysis using standard SDS-PAGE conditions and in gels containing urea. The migration of VP1 and VP3 correlated with their molecular weights predicted from the nucleotide sequence and was consistent in either the presence or absence of urea. However, VP2 migrated with an apparent molecular weight significantly higher than the predicted value and, in gels containing urea, migrated as a doublet. It is proposed that the upper band of this doublet represents VP0, the proteolytic precursor of VP2 and VP4. The relative molecular mass (Mr) of VP4 was estimated to be less than 1 kDa, which is substantially lower than the 2.5 kDa predicted from the nucleotide sequence.

Effect of glutaraldehyde on the antigenicity and infectivity of hepatitis A virus.

The effect of glutaraldehyde on the antigenicity and infectivity of hepatitis A virus (HAV) was examined. The CF 53 strain, adapted to human hepatoma PLC/PRF/5 cells, was treated with glutaraldehyde using three different concentrations, 0.02, 0.10, and 0.50%, for various periods of time, 3, 10, and 30 min, respectively. After the virucidal assays, glutaraldehyde and HAV were separated by gel filtration, then the antigen (radioimmunoassay) titer and the infectivity titer were determined. The greatest antigen titer reduction was about 80% after 30 min using 0.10% glutaraldehyde and within only 3 min using 0.50% glutaraldehyde. Glutaraldehyde is an effective disinfectant against HAV: the infectious virus titer decreased by more than 3 log10 after 30 min using 0.10% glutaraldehyde and within only 3 min using 0.50% glutaraldehyde. Statistical studies showed that the decrease of antigen or infectious virus titer was affected by both glutaraldehyde concentration and exposure time.

Depuration of Mytilus galloprovincialis experimentally contaminated with hepatitis A virus.

Mussels (Mytilus galloprovincialis) were contaminated with known amounts of laboratory strains of hepatitis A virus and Poliovirus 1 and the effectiveness of a self-cleansing mechanism was studied using a pilot depuration system. Both viruses were rapidly bioaccumulated by mussels and the maximal concentration of about 10(4) TCID50/ml was reached within 1.5 hours. Depuration was carried out up to 24 h; infectivity titer decreased to 10(2) TCID50/ml and 10(3.2) TCID50/ml within 6 h in hepatitis A virus and Poliovirus 1 contaminated mussels, respectively, but only a very slight further decrease was obtained after 24 h. E. coli was used as a control; within 24 h the concentration decreased from 40 to 2 bacteria/ml of mussel (MPN). The elimination of bacteria is not a reliable parameter to control the effectiveness of viral depuration.

Cultivation of buffalo green monkey kidney cells persistently infected with hepatitis A virus.

Studies were carried out to determine the effect of prolongation of incubation periods, cocultivation with normal buffalo green monkey kidney (BGMK) cells and different concentrations of foetal calf serum (FCS) on the production of hepatitis A virus (HAV) by BGMK cell line persistently infected with HAV strain HM175. HAV could be detected from week 1 onwards. However, maintenance of cultures beyond this period was found to yield substantially higher quantities of virus. Cocultivation of persistently infected cells with normal BGMK cells also improved the antigen yields. Different concentrations of FCS did not show any effect on the amount of virus produced. The cell line was maintained up to 46 passages during which there was continuous production of HAV in the cells and release of small amounts of virus in the culture supernatants. Cell associated and cell free viral particles were found to be infectious. Supernatant derived virus was a highly suitable inoculum for infecting other susceptible cell lines. Persistently infected BGMK cell line appears to be a reliable and economical source to derive HAV in adequate amounts for diagnostic and research purposes.

Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus.

In Italy, the consumption of raw or slightly cooked mussels represents the most important risk factor for the transmission of hepatitis A virus (HAV). Although there exist effective methods for the bacterial depuration of contaminated mussels, these methods are poorly effective on enteric viruses. The objective of the present study was to evaluate the effectiveness of a closed-circuit depuration system that uses both ozone and UV light for disinfecting water and that allows salinity and temperature, important parameters for the metabolism of mussels (Mytilus galloprovincialis), to be maintained at constant levels. The results showed that this depuration method decreased the viral load (from 1.72 log 50% tissue culture infective dose [TCID50] ml(-1) to <1 log TCID50 ml(-1) within 24 h and from 3.82 log TCID50 ml(-1) to <1 log TCID50 ml(-1) within 48 h). However, in both cases, after 120 h of depuration, a residual amount of virus capable of replicating in cells was detected. These results show that depuration, even if performed with advanced systems, may not guarantee the absence of virus.

Fast growth of a Brazilian hepatitis A virus (HAF-203) in a primate cell line.

A hepatitis A virus (HAV, HAF-203) isolated in Brazil was submitted to 8 serial passages through fetal Rhesus kidney cells (FRhK-4). The kinetics of replication were monitored by enzyme immunoassay (EIA-HAVAg) and cDNA-RNA dot blot hybridization. The maximum level of RNA, which was observed 21 days post-infection (p.i.) during the 3rd passage, when HAVAg was still undetectable by EIA, served as a basis to establish subsequent passages every 21 days p.i. This schedule of passage resulted in a progressive reduction of time between culture infection and HAVAg and RNA production, together with an enhancement in antigen titer content of cell lysates. During the 7th passage, maximum HAVAg and RNA levels were detected at 7 days. Fourteen days after the 8th passage, clear morphological modifications appeared, suggesting a good adaptation of HAF-203 to FRhK-4 cells. Obtaining a fast-growing Brazilian HAV is very important for the development of vaccines.

Increased infectivity of hepatitis A virus cDNA clones with engineered 5'-terminal extra-cistronic sequences.

The genomic RNA of Hepatitis A virus (HAV), a picornavirus of the hepatovirus group, is a single-stranded molecule, ca. 7.5 kb in length of positive polarity. Translation of this uncapped RNA starts at the 10th (or 11th) AUG triplet (position 734-36), by a mechanism of internal initiation of translation. The long sequences extending between the uncapped 5'-end and the translation initiation site contain two (instead of just one) pyrimidine-rich tracts (PRTs) spanning nucleotides 94-140 and 711-724, respectively. The latter lies only 11 nucleotides upstream from the initiation site of translation, and the question arose as to whether the notoriously poor replication ability of HAV was a consequence of a down regulation of translation due to the too short "spacer" sequence intervening between the 3'-PRT and the initiation of the main open reading frame. To address this issue, a series of full-length HAV cDNA clones were constructed in which the "spacer" sequence (normally 11 nts) was brought to 45 nts. Following transfection of COS-1 cells with these constructs, the amount of HAV (+)-strand RNA was determined by dot hybridization using a strand-specific RNA probe. HAV cDNA clones carrying a 45-nt "spacer" increased two-fold the rate of (+)-strand viral RNA synthesis, suggesting that the poor translation ability of HAV RNA may be one of the mechanisms responsible for the lengthy replication cycle of HAV.

Growth of hepatitis A virus in murine cells.

In order to investigate the growth of hepatitis A virus (HAV) in murine cells, L929 cells of the established mouse cell line were transfected with the virion RNA or infected with the virions and examined for the formation of negative-strand RNA and the rise of the viral infectivity titer. In both the transfected and infected cells, the formation of negative-strand HAV RNA was assayed by the reverse transcription-polymerase chain reaction (RT-PCR). In the transfected cells, infectious HAV of an average titer of 10(1.8) TCID50/dish was obtained. The experiment with the virion infection was further extended by using other mouse cell lines, namely Balb/3T3 clone A31, NIH/3T3, and Swiss/3T3. Here, only NIH/3T3 cells were found capable to support the formation of negative-strand HAV RNA. Thus some murine cell lines are considered to have a complete cellular machinery for supporting the growth of HAV, though the efficiency of virus growth therein was considerably lower as compared to that in the susceptible primate cells.

Propagation of hepatitis A virus in a renal cell line JTC-12.P3 of cynomolgus monkey origin.

Human hepatitis A virus (HAV) derived from 10% HAV infected marmoset liver homogenate and faeces from acute hepatitis A was successfully propagated in vitro in a new cell line, JTC-12.P3. The cell line originated from the renal cortex of cynomolgus monkey which was adapted to growth in a serum free, protein free, chemically defined synthetic medium. Replication of the virus was followed by solid phase RIA, immunofluorescent staining, and immunoelectron microscopy. The propagation of HAV occurred over several passages, with the 1st and 2nd passages requiring at least 8 weeks each. However, with the increasing serial passage of virus, the period needed to detect it was shortened, suggesting the adaptation of HAV to the cells. The identity of the newly synthetized virus particles with HAV was established by immunoelectron microscopy and immunofluorescent blocking effect with human convalescent serum. The HAV propagated in JTC-12.P3 cells banded predominantly at a density of 1.32 g/cm3 in CsC1 gradient. The infected cells showed no specific signs of CPE. Ultrastructurally, clusters of virus particles 27 nm in diameter were observed mainly in the lysosomal vesicles and freely in crystalline array in the cytoplasm, too. Addition of 0.1% of various anti-HAV negative sera or of prostaglandin E1 to the culture medium caused accelerated propagation of HAV.

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.