Search

Bovine turbinate (BTu) and lamb testis (LT) cell lines persistently infected with bovine viral diarrhoea virus (BVDV) arose as a result of a single change of medium containing commercial foetal calf serum. Infected cells comprise 30% and 50% respectively, of the total cell population, determined by immunohistochemical staining. The ratio of positive cells has remained unchanged during successive passages. Characterization of the persistently-infected BTu cells (BTuI) showed that only full length viral RNA was detected by northern blot hybridisation, indicating that DI particles were not involved. Secreted and intracellular virus from these cells was fully infectious for fresh BTu and LT cells. The BTuI cell line was fully permissive for a cytopathic BVDV isolate and a bovine herpesvirus, but non-permissive for two non-cytopathic BVDV isolates. Attempts to induce the permissive state in the BVDV-negative cells of the BTuI culture by treatment with actinomycin D and 5′-aza-cytidine failed. These cells provide a convenient model to study aspects of BVDV pathogenesis and replication.

BACKGROUND: The epidemiology and disease association for the GB virus type C (GBV-C) or hepatitis G virus (HGV) are poorly understood. STUDY DESIGN AND METHODS: This study describes the exposure rates to GBV- C/HGV in diverse Australian population groups by testing for current infection and evidence of past infection with a reverse transcriptase polymerase chain reaction and an anti-E2 enzyme-linked immunosorbent assay, respectively. Subjects included volunteer blood donors, hepatitis C antibody (anti-HCV)-positive donors, children, hemodialysis patients, pregnant women attending a prenatal clinic, injecting drug users (IVDUs), and adult hemophiliacs. RESULTS: Combined GBV-C RNA and E2 antibody prevalence was 6.5 percent (6/93) in children, 13.3 percent (75/565) in blood donors, 14 percent (14/99) in pregnant women, 22.5 percent (18/80) in hemodialysis patients, 80 percent (56/70) in anti- HCV-positive donors, 88.6 percent (31/35) in IVDUs, and 85.7 percent (54/63) in adult hemophiliacs. Children had the lowest antibody rate, 1.1 percent, whereas the rate was 10.8 percent for blood donors and rose to 45.7 percent for IVDUs, 57.1 percent for anti-HCV-positive donors, and 74.6 percent for hemophiliacs. In contrast, current infection rates were comparable for children, blood donors, and pregnant women (5.4, 2.6, and 6%, respectively), rising to 11.1 percent for hemophiliacs, 24.3 percent for anti-HCV-positive donors, and 48.6 percent for IVDUs. Ten of 12 blood donors had persistent viremia, while 2 had recent infections, 1 with apparent resolution. CONCLUSION: Exposure to GBV-C can commence at an early age, although ongoing exposure may also occur among adults with no apparent risk factors. GBV- C RNA positivity was not associated with abnormal plasma alanine aminotransferase levels among blood donors.

The genomic sequence of an Australian isolate of hepatitis C virus (HCV) was determined from overlapping cDNA clones obtained from a small amount (1.2 ml) of serum from a single individual with hepatitis C. The isolate (HCV-A) comprises 9 379 nucleotides (nt) including 324 nt of a 5′ untranslated region (5′UTR), a single long open reading frame of 9 033 nt encoding a polyprotein of 3 010 amino acids (aa), and 22 nt of a 3′ untranslated region (3′UTR). Sequence analysis of a 251 nt region within the 5′UTR and a 222 nt region within NS5B showed the genotype of HCV-A to be subtype 1b. A striking difference in the amino acid sequence of the hypervariable region 1 (HVR-1), and not in the surrounding sequence, was seen in cDNA clones synthesised from serum taken 52 weeks after the initial sample, indicating a significant population diversity of HCV genomes.

To permit accurate identification of the 5' end of the HCV genome, we used RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) where an RNA molecule of known sequence (transcribed in vitro from a cDNA template) was ligated to RNA purified from a hepatitis C virus (HCV)-infected liver sample. After ligation, the product was amplified by nested polymerase chain reaction (PCR) and amplicons that were equal to or greater in size than those predicted from the recognized 5' terminus of the HCV genome were cloned into pBluescriptKS. Twelve clones were sequenced, including three that were identical and contained an additional eight nucleotides, namely CCCCCCCA. Thus, the HCV 5' untranslated region (UTR) is now recognized as comprising 349 nucleotides, although it is possible to speculate that these additional nucleotides are part of a second, as yet undetected, stem-loop at the extreme 5' terminus of the genome.

The replication of bovine viral diarrhoea virus (BVDV) RNA is considered to involve replicative intermediates (RI) and replicative forms (RF) of virus RNA. These RNA species were radiolabelled metabolically by 3H-uridine in BVDV-infected cells and separated by sucrose gradient centrifugation. RNA in the fractions was then digested with RNase A in high salt (2 × SSC) and the RNase-resistance was determined. Using the Baltimore formula, this led to the calculation that the RI of BVDV contained 6–7 nascent strands on the template. This suggests that the structure of the BVDV RI is similar to that of poliovirus and dengue 2 virus.

A quantitative assay of hepatitis B virus (HBV) binding to hepatocyte plasma membranes was adapted to show that leucocyte plasma membranes bind serum-derived HBV saturably, and that this binding is inhibited using synthetic peptides representative of the large envelope protein of HBV. Using a panel of ligand-blocking monoclonal antibodies (mAb) to opsonin receptors, it was shown that the three classes of Fc gamma R and CR3 are not major receptors for HBV on leucocytes or hepatocytes. It was also shown that HBV does not utilize the receptor for IgA, Fc alpha R, for attachment to leucocytes, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. Evidence is presented that the receptor for HBV on leucocytes may differ from the hepatocyte receptor(s), based on synthetic peptide inhibition assays of HBV binding. Furthermore, it was observed that glycosaminoglycans influence the HBV-liver and leucocyte interactions, providing evidence that HBV attachment may be a multi-stage process.

COS7 cells and other cell lines expressing the SV40 large T-antigen, have been of great benefit in transfection studies using transient expression vectors which contain the SV40 origin of replication, as these cells allow plasmid replication to a high copy number. As no T-antigen expressing cell line derived from a well characterised hepatocyte-like continuous cell line currently exists, the establishment of such a cell line for studies which require expression of hepatocyte-specific factors would be extremely useful. A HepG2-derived stable cell line (THT1) was therefore developed which demonstrates a high level of transfection efficiency whilst retaining hepatocyte-like features, such as the production of hepatitis B virus. The THT1 cell line displayed chromosomal integration of the SV40 T-antigen gene and nuclear expression of the antigen. The cell line also maintained the general morphological features of its parent cell line, and showed an increased rate of cell growth.

The structural proteins, core, E1 and a C-terminal truncated E2 (C-E1-E2p) as well as the 5'UTR linked to the core gene (5'UTR-C) of the hepatitis C virus were expressed in Sf9 cells. Expression of the C-E1-E2p polyprotein from a single vector resulted in expression of multiple forms suggesting that the cleavage of the polyprotein in the insect cells is incomplete. The structural proteins were expressed as insoluble forms and were solubilized by freeze/thaw cycles or detergent treatment. Analysis of the co-expressed (5'UTR-C and C-E1-E2p) proteins through a 20-60% sucrose gradient revealed that the core protein migrated rapidly into the gradient, in common with the sedimentation profile for core protein expressed from the core gene only. Neither E1 nor E2 proteins were involved in the core-core protein interaction. Expression of the 5'UTR-C in insect cells resulted in a high level of mRNA transcription but no translation of core protein, presumably due to a failure to initiate HCV translation by an internal ribosome entry mechanism, since the mRNA transcript was translated in vitro, suggesting that the 5'UTR can influence HCV tropism.

Transcription and replication of hepatitis delta virus (HDV) RNA are performed by the cellular enzyme RNA polymerase II (Pol II). As DNA is the normal template for Pol II, the enzyme must undergo template switching. The mechanism for this is unknown, but since HDV RNA can form a rod-like molecule by extensive intramolecular base pairing, it has been suggested that a double-stranded region(s) of HDV RNA serves as a recognition site for Pol II. A bidirectional promoter has been identified previously on HDV cDNA (T. B. Macnaughton, M. R. Beard, M. Chao, E. J. Gowans, and M. M. C. Lai, Virology 196:629-636, 1993). In the present study, genomic RNA corresponding to this region was able to direct the synthesis of antigenomic RNA in a nuclear extract transcription reaction, whereas genomic RNA species containing a mutation that resulted in a replication-defective phenotype were unable to do so. Thus, this region, the location of which is defined as nucleotides 1608 to 1669 on the basis of a highly conserved structure, represents a RNA-RNA promoter. Computer analysis of the RNA secondary structure predicted that the promoter contains two bulge regions in a stem-loop structure which encompasses a GC-rich motif. This predicted model was confirmed by enzyme digestion and primer extension analysis. The promoter is located at one end of the rod and has some homology with the simian virus 40 late promoter. A number of other mutations were introduced within this region, and expression plasmids were constructed to examine the effects of mutations in the promoter on HDV replication. Disruption of the overall secondary structure, particularly the bulge regions, totally inhibited HDV RNA replication.

A reverse transcription-PCR assay which successfully amplified hepatitis C virus RNA from poorly stored archival sera was optimized. Maximum sensitivity was achieved with Moloney murine leukemia virus RNase H- reverse transcriptase and by a single round of PCR amplification of a short (112-bp) fragment of the 5' untranslated region of the viral genome.

Aims: In order to examine more carefully the natural history of hepatitis C virus infection and to determine a role for anti-core in the discrimination of indeterminate samples, a solid phase ELISA to detect antibody of the immunoglobulin G class to the hepatitis C virus core antigen was developed using purified protein expressed in Escherichia coli from a major portion of the core antigen coding region. Methods/Results: In a study which examined 651 samples submitted for routine testing by a commercial ELISA (Ortho), only 11 samples showed discrepant results; of these, 10 were Ortho ELISA positive, anti-core negative and one was Ortho ELISA negative anticore positive. Supplemental tests showed that 510 of these samples were anti-HCV negative by RIBA and the reciprocal 5 were negative for anti-C22 but positive for anti-C100 and anti-C33. The Ortho ELISA negative, anti-core positive sample was weakly positive for anti-C22. The anti-core ELISA was then used to examine 67 indeterminate samples from the blood bank; 1111 samples which were HCV-RNA positive were anti-core positive and 756 samples which were HCV-RNA negative were anti-core positive. The anti-core titre was then examined in two groups of indeterminate samples; group 1, polymerase chain reaction-positive, anti-core positive and group 2, polymerase chain reaction-negative, anti-core positive. The geometric mean anti-core titres in these groups were 1×10 −3.6 and 1×10 −2.3 , respectively. Thus in this group of indeterminate samples, all samples (except one) with an anti-core titre ≥ 1200 were polymerase chain reaction-positive, confirming a close correlation between anti-core levels and hepatitis C viraemia. Anti-core was detected with equal efficiency in patients infected with genotypes which differed to that used to express the recombinant core antigen.

Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA,they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.

Hepatitis C virus (HCV) antigen expression was examined by immunohistochemical staining in liver tissue taken at biopsy from 8 anti-HCV positive patients. Frozen liver sections were stained by indirect immunofluorescence for capsid, E2/NS1, NS3, NS4 and NS5 using polyclonal antibodies raised to synthetic peptides from these regions. The antigens E2 and NS3 were localised in scattered hepatocytes and also in cells within and around areas of inflammation. A weaker signal was observed for NS4 and NS5 and no signal was seen for capsid antigen. No staining was seen in liver tissue from 9 individuals, including 3 hepatitis B virus-positive and 2 hepatitis delta virus/positive patients, who were negative for serological markers of HCV. The specificity of the staining reaction was also confirmed by the lack of staining in HCV-positive liver samples, after the antisera was pre-adsorbed against the specific peptide. Collectively, the data suggests that HCV may not only be hepatotropic but also lymphotropic, and this may be an important factor in the pathogenesis of HCV infection.

SUMMARY The prevalence of HCV antibodies was determined by a second-generation ELISA and a four-antigen rccombinant immunoblot assay in nine intravenous immunoglobulin (IVIG) preparations commercially available in Italy. In addition, the clinical safety of six of them was ascertained by polymerase chain reaction (PCR) of HCV RNA and a prospective study in 14 patients with immunodeficiency disorders. Results indicated that all IVIG preparations were anti-HCV-positive. However, there were substantial variations in their anti-HCV antibody titres. The preparations retained IgG subelass reactivilies to HCV-associated structural (C223) and non-structural (C33c, C100-3) proteins. Our sensitive and specific PCR assay was unable to detect HCV RNA in the six preparations tested. Clinical surveillance of IVIG-trcated patients prospectivcly evaluated over a mean period of 8·3 months failed to delect clinical and/or biochemical evidence of hepatitis.

The risks of acquisition of hepatitis C infection, the histological spectrum of liver disease, and the presence of viraemia were investigated in anti-hepatitis C virus (HCV) antibody positive blood donors. All 357 (0.64%) blood donors to the South Australian Red Cross Transfusion Service found to have anti-HCV antibody during the first seven months of testing in 1990 were assessed, and 70 (19.6%) were found to have elevated alanine transaminase levels. These subjects were referred for participation in the study; 31 presented for enrolment. Sixteen (52%) of the 31 patients had previously used intravenous drugs, four (13%) had been transfused, two (6%) had a history of occupational exposure to blood, and three (10%) had tattoos and ear-piercing as possible risk factors for acquisition of hepatitis C. There was no history of parenteral exposure in six (16%). None of these donors had clinical evidence of liver disease, but in all 24 of the 31 who had a liver biopsy there was histological evidence of significant liver damage. Twelve had evidence of chronic active hepatitis. All 24 subjects biopsied were viraemic as judged by the presence of HCV RNA in serum. (Aust NZ J Med 1993; 23: 176–180.)

Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is ill-defined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridization with 125I-labelled riboprobes, representing approximately 35% of the HCV genome. Only a few positive cells were identified in the HCV-infected liver samples, but not in a normal liver sample. Hybridization with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages.

A trimer of hepatitis delta virus (HDV) cDNA in a retrovirus expression vector was transfected into subclone of the PLC/PRF/5 human hepatoma cell line, and a stable cell line (H1 delta 9) was clonally selected that supported the synthesis of both genomic and antigenomic sense HDV RNA. The H1 delta 9 cell line also expressed hepatitis delta antigen (HDAg) in cell nuclei in three distinct morphological patterns, including patterns typically seen in HDV-infected livers. HDAg expression was restricted to the smaller (p24) of the two HDAg-associated polypeptides in early passages of the H1 delta 9 cell line, but continuous passage of the cells resulted in increasing of expression of the larger (p27) HDAg-specific polypeptide. Passage of the H1 delta 9 cells also led to sustained expression of monomeric HDV RNA and a reduction in the levels of dimeric- and trimeric-HDV RNA. This was accompanied by an attenuation of virus-related cytotoxicity which was a feature of early cell passage numbers. HDV RNA replication in these cells was resistant to actinomycin D suggesting that replication was not dependent on continued expression from the transfected HDV cDNA and thus was likely to be self-sustaining.

A radioimmunoassay was developed to detect duck hepatitis B virus surface antigen and antibody; viraemia (DHBV DNA or DHBsAg) was detected in all ducks inoculated within 3 weeks post-hatch, and persistent infection developed in 93% of birds in this group. In contrast, only 80% and 60% of ducks inoculated 4- and 6-weeks post-hatch respectively developed viraemia, and approximately 70% of the viraemic ducks became carriers. Markers of viraemia were undetected in ducks inoculated 8 weeks post-hatch and in uninfected controls. A typical anti-DHBs seroconversion developed subsequently in 2 of 4 birds that showed transient viraemia, and antibody also developed in 3 of 7 ducks inoculated 4-8 weeks post-hatch that showed no viraemia. However, gene amplification by the polymerase chain reaction demonstrated DHBV DNA in ducks from the latter group suggesting that the antibody did not result from passive vaccination. Thus, increased resistance to infection develops with increasing age that may be related to several factors including host immunity. This model may help elucidate similar age-related features of human hepatitis B virus infections.

The gene encoding the hepatitis delta virus (HDV) structural antigen (HD Ag) was inserted into a Rous sarcoma virus expression vector and the recombinant plasmid used to direct the synthesis of recombinant HD Ag (rHD Ag) in a continuous hepatoma cell line. A competitive radioimmunoassay for serum antibody to HDV using rHD Ag was developed and was found to be equally suitable for diagnostic purposes to a radioimmunoassay using infected liver-derived HD Ag. Similarly, rHD Ag was shown to be serologically equivalent to liver-derived HD Ag within the limit of the blocking titrations performed. The rHD Ag-positive cell line was also used in an indirect immunofluorescence assay to detect anti-HD. Similar titres of anti-HD were detected by both radioimmunoassay and immunofluorescence and identical samples were positive for anti-HD by either assay. In a sample of prison inmates with high prevalence of both HBV and HDV, anti-HD was confined almost exclusively to those with persistent HBV infection and not to those in whom HBV infection had cleared. The availability of rHD Ag will permit wider development of diagnostic anti-HD assays, and the use of two such assays is presented in this study.

A chemically synthesized peptide representing the C-terminal subunit (p13-C) of the p13 protein of GB virus B (GBV-B), the most closely related virus to hepatitis C virus (HCV) showed ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to potassium ions than to chloride ions. Amantadine but not hexamethylene amiloride (HMA) inhibited the ion channel function of p13-C in the lipid membranes. However, neither agent was able to inhibit the replication and secretion of GBV-B from virus-infected cultured marmoset hepatocytes, which were harvested from a marmoset that was infected in vivo or inhibit replication after in vitro infection of naive hepatocytes. These data suggest that the GBV-B ion channel, contrary to the data derived from the lipid membranes, is either resistant to amantadine or that virus replication and secretion are independent of ion channel function. As the p7 protein of HCV also has ion channel activity that is apparently resistant to amantadine in vivo, the former possibility is most likely. Ion channels are likely to have an important role in the life cycle of many viruses and compounds that block these channels may prove to be useful antiviral agents.

Hepatitis delta virus (HDV) is a defective human pathogen which is dependent on hepatitis B virus (HBV) for replication and expression. The full extent of the helper function is still unknown, but HBV provides a hepatitis B surface antigen envelope that surrounds teh HDV-specific internal components of the HDV virion. These components are a circular single-stranded RNA genome of approximately 1.7kb and hepatitis delta antigen (HDAg). The genome has five potential genes, but a protein product (HDAg) has only been identified from one. HDAg is found in the liver and serum of infected individuals as two related polypeptides of 24- and 27-kDa. The genome also shares a number of similarities with the viroids, highly pathogenic agents of plants including (1) the ability for RNA self-cleavage in vitro ; (2) genome replication by the rolling circle mechanism; (3) nuclear replication by host cell RNA polymerase II. A number of studies have shown that HDV RNA replication is dependent on HDAg; however recent evidence suggests that the role of HDAg is to transport HDV RNA to the nucleus but it does not form part of the transcriptional complex per se . Virus replication is generally associated with cell death, both in vivo and in vitro , that may be a result of the expression of HDAg p24 which appears to inhibit host cell RNA synthesis.

Hepatitis B virus (HBV) core antigen (HBcAg) detected by the peroxidase-antiperoxidase technique was present in the nucleus and cytoplasm of some infected hepatocytes but only in the cytoplasm of other hepatocytes. When cells expressing HBcAg were examined by in situ hybridization for the presence of HBV DNA, the intracellular level of cytoplasmic HBV replicative intermediate DNA correlated with the level of cytoplasmic HBcAg, but not with the presence or absence of nuclear HBcAg. This suggests that nuclear HBcAg may not be directly involved in hepadnavirus replication.

To better assess the extent of the tissue tropism of mammalian hepadnaviruses, 10 tissues from each of six woodchucks were examined for the presence and state of woodchuck hepatitis virus (WHV) nucleic acids 15 months after experimental WHV infection. The tissues examined were peripheral blood lymphocytes, lymph node, spleen, bone marrow, thymus, pancreas, kidney, ovary, testis, and liver. Tissue samples from three chronically infected animals and three animals with serologic patterns of recovery (serum: WHsAg − , anti-WHc + , anti-WHs + , WHV DNA − ) from acute WHV infection were analyzed in parallel by in situ hybridization and Southern and Northern blot techniques. WHV nucleic acids were detected in several individual tissues from each animal examined, although not all tissues in every animal contained WHV. Substantial differences were observed among the various tissues and animals with respect to the frequency, level, and intratissue distribution of WHV nucleic acids, as well as the presence of different viral genomic forms. Active WHV DNA replication was present only in the liver and spleen of the chronically infected animals. No evidence of ongoing WHV DNA replication was found in any of the tissues from the recovered animals. WHV DNA was homogeneously distributed among all hepatocytes in the livers of the chronic carriers. By contrast, WHV DNA in all the extrahepatic tissues, and in the livers of the recovered animals, was detected only in scattered foci of cells.

This study examined the detection of cellular poly(A) sequences in mouse liver sections by in situ hybridization using a 3H-labelled poly(dT) probe. Parameters examined included possible losses of target poly(A) sequences from sectioned cells, access of probe to target sequences, section thickness, hybridization conditions, autoradiographic efficiency, specific activity of probes and specificity of reaction. An improved protocol was devised that resulted in good preservation of histological detail in sectioned tissue blocks, and a calculated hybridization efficiency of 50%-100%. With the use of probes of defined sequence, the protocol should allow detection of unique mRNA sequences within single cells with an estimated sensitivity of 6-12 unique mRNA molecules per sectioned cell.

Plasmid pHBV 114 DNA, which contains 73% of the genome of hepatitis B virus (HBV), was radiolabelled with tritium to 1-2 X 10(8) dpm/microgram by nick translation and used as a radioactive probe to detect HBV DNA present in sections of infected liver tissue by in situ hybridisation followed by autoradiography. Factors affecting the sensitivity of the reaction were examined, including different methods of fixation, hybridisation time, temperature, and buffers. The specificity of the reaction for detecting viral DNA was carefully established by the use of unrelated DNA probes, pretreatment of sections with DNAase, and comparing the stability of the binding of DNA probe at different temperatures, with the melting curve of double-stranded DNA in solution. In the one liver studied in detail, cells containing large amounts of viral DNA were distributed in foci corresponding to areas containing morphologically damaged hepatocytes. This observation suggested a relationship between active viral replication and cell damage. Viral DNA was found mainly in the cytoplasm, although a minority of nuclei in these foci were also positive.

Thirty-five 1-day-old Pekin-Aylesbury ducks were inoculated intravenously or intraperitoneally with duck hepatitis B virus, and the time-course of infection was examined by Southern-blot, dot-blot, and in situ hybridization and by immunohistochemistry. Randomly scattered single infected hepatocytes were first seen on days 1 and 2 after inoculation and by day 3 occurred as single cells, pairs, and groups of 5–10 adjoining cells. From day 4 after inoculation all hepatocytes were positive for duck hepatitis B surface antigen and deoxyribonucleic acid. Duck hepatitis B virus deoxyribonucleic acid levels in liver extracts and serum increased logarithmically from days 2 to 3 to a plateau by days 4 to 5 after inoculation. Infected and control birds showed no significant differences during the first 7 days in terms of liver histology, hepatocyte morphology, or mitotic activity. It was concluded that (a) virus gains access to randomly distributed hepatocytes without first replicating in other cell types, and then begins disseminating to adjacent cells following anatomic boundaries; (b) markers of infection in liver and serum show reproducible kinetics, thus making this in vivo system amenable to further quantitative study; and (c) hepatocytes in this system are highly permissive to virus replication without the development of significant cytopathology.

Hitherto undescribed tubular and crystalline structures were detected by negative contrast electron microscopy in purified preparations of Newcastle disease virus. It is suggested that these are viral in origin and are composed of aggregates of viral glycoprotein.

A monoclonal antibody to human interferon-alpha (IFN-alpha) was produced using affinity-purified IFN-alpha, that reacted with recombinant human IFN-alpha 2, but not with IFN-alpha 1, IFN-alpha M1 or IFN-beta. Indirect immunofluorescence using this monoclonal (designated 6C3) and anti-IFN-alpha polyclonal antibodies identified cells expressing IFN-alpha. After Sendai virus induction of normal human buffy-coat cells the proportion of monocytes and lymphocytes expressing IFN-alpha rose progressively from 0% to 50% and 34% respectively, preceding peak IFN-alpha titres in the culture supernatants. Around 80-90% of polymorphs were IFN-alpha-positive using both antisera, with or without IFN induction, although very little IFN bioactivity was released to the supernatant of polymorph cultures after IFN induction. Sections of hepatitis B virus infected human liver tissue showed foci of IFN-alpha-positive infiltrating mononuclear cells and (to a lesser extent) fibroblasts in patients who had active cirrhosis and evidence of virus replication. These findings suggest that polymorphs constitutively express IFN-alpha 2 related antigenic activity, whose biological activity is at present unknown; and demonstrates the identification of IFN-alpha-expressing cells in sections of tissue undergoing natural virus infection.

Liver sections from five patients with persistent hepatitis B virus (HBV) infection and active cirrhosis were shown to contain intracellular HBV DNA by in situ hybridization using cloned 3H-labelled HBV DNA probes. Two classes of infected cells, with different distributions throughout the liver, were distinguished: (i) cells containing a low copy number of double-stranded HBV nucleotide sequences, confined to the cell nucleus and thought to represent HBV DNA, and (ii) cells containing large amounts (estimated to be greater than 10 or 15 genome copies per cell) of HBV DNA, much of it in a single-stranded form and largely confined to the cell cytoplasm; these single-stranded regions represented widely separated regions of the HBV genome, in contrast to the structure of the DNA in mature virions. It is likely that these latter cells may be supporting viral DNA synthesis. Cells with large amounts of cytoplasmic HBV DNA invariably contained hepatitis B surface antigen (HBsAg) and in addition contained either no detectable hepatitis B core antigen (HBcAg), or cytoplasmic HBcAg or nuclear HBcAg in that order of frequency. Cytoplasmic HBcAg was highly predictive of the presence of large amounts of cytoplasmic HBV DNA in the same cell, while either nuclear HBcAg, or cytoplasmic HBsAg, were often seen both in cells with and without such levels of DNA. These patterns, relating HBV DNA and antigen content in naturally occurring asynchronous infection in a heterogeneous cell population, should provide a background to further studies of the virus replication cycle with a defined experimental system, when such a system becomes available.

Seventy, 1-day-old ducklings inoculated intraperitoneally with duck hepatitis B virus and 30 controls have been studied over a 2-year period. Infection with duck hepatitis B virus occurred in all inoculated ducks, although this was not associated with clinical morbidity. Duck hepatitis B virus DNA was first detected in liver on Day 3, in pancreatic acinar cells on Day 4, serum on Day 6, splenic red and white pulp on Day 7 and in the renal glomurulus on Day 14, using a combination of dot, Southern blot and in situ hybridization techniques. Peak levels of circulating virus, as determined by DNA polymerase levels, occurred 1 to 4 weeks postinoculation. Mild degrees of portal inflammation were seen in sections of liver tissue in both infected and control ducks. However, moderately severe inflammatory changes were present in 8 of 22 infected birds compared with 0 of 18 controls (p less than 0.025). Appearance of this inflammatory infiltrate 6 weeks postinoculation coincided with a decrease in levels of duck hepatitis B virus DNA in hepatocytes and within the pancreatic acinar cells. At the same time, duck hepatitis B virus DNA became increasingly localized to the splenic germinal centers, and viral DNA was first detected in pancreatic islet cells. No histological changes accompanied the extra-hepatic tissue infection. The sequence and significance of duck hepatitis B virus infection in liver and extra-hepatic tissues is discussed in relation to the pathogenesis of hepatitis B virus infection in man.

Tissues from a 10-week-old Pekin duck, experimentally infected at 1 day of age with duck hepatitis B virus (DHBV), were examined for the presence of replicative levels of DHBV DNA by in situ and Southern blot hybridization. Hepatocytes, pancreatic lymphoid follicle, exocrine, and endocrine cells, and splenic mononuclear cells all contained DHBV DNA localized predominantly to the cytoplasm of infected cells. Duck hepatitis B surface antigen distribution in the same tissues correlated well with the presence of DHBV DNA in many of these cells. In hepatocytes and pancreatic islet cells, 60% of DHBV DNA was present as single-stranded DNA, indicating the likelihood of ongoing virus replication in these cell types and providing further evidence that hepadnavirus DNA replication occurs largely within the cell cytoplasm. In contrast, DHBV in mononuclear cells within splenic germinal centers was wholly double-stranded, suggesting that limited, if any, DHBV DNA replication was occurring in this cell type. These data provide further information about the pathogenesis and cell-specific sites of DHBV infection.

We obtained two lines of evidence that monolayer cultures of primary woodchuck hepatocytes support replication of the genome of human hepatitis delta virus (HDV). (i) From a Northern (RNA blot) analysis of the HDV-related RNA in infected cultures, both genomic and antigenomic 1.7-kilobase RNA species were detected at 11 days after infection. The ratio of genomic RNA to antigenomic RNA was 2:1 to 10:1, comparable to that previously reported in studies of experimentally infected chimpanzees and woodchucks. (ii) Replication in culture was also demonstrated by in situ hybridization with a strand-specific probe. Such studies showed that only a small fraction of the cultured cells supported replication and that in such cells the relative and absolute levels of the HDV RNAs were comparable to those in liver cells infected in vivo. Furthermore, as with the in vivo studies, the HDV RNAs were predominantly localized to the nucleus. In summary, we demonstrated that cultured cells supported both the early events of HDV adsorption and penetration and the intermediate events of genome replication.

The dengue virus membrane (M) protein is a key component of the mature virion. Here, we characterised the cellular behaviour of M using a recombinant protein construct to understand its inherent properties. Using confocal microscopy, we showed that M and its intracellular precursor, prM, localised to the endoplasmic reticulum. M protein was also detected on the cell surface and secreted, suggesting that M can enter the secretory pathway. In addition, cross-linking studies showed that M can form dimers and tetramers. These findings suggest that M behaves as a secretory protein analogous to the major envelope protein E.

Ultrastructural examination of a line of MDBK cells persistently infected with Newcastle disease virus (MDBKpi cells) revealed the presence of cytoplasmic aggregates of both smooth and granular nucleocapsids. Only granular nucleocapsids aligned under modified areas of plasma membrane and were incorporated into virus particles. On the grounds of morphogenesis, there was no apparent explanation for the persistent, not-cytocidal nature of the infection. Both nuclear and cytoplasmic aggregates of smooth nucleocapsids were present in MDBKpi cells which had been held without subculture for between 40 and 130 days (aged MDBKpi cells). Modified areas of plasma membrane with associated alignment of nucleocapsids were not present in aged MDBKpi cells, and neither budding nor released virus particles were observed, indicating a block in virus maturation. It is suggested that the granular material coating granular nucleocapsids allows them to interact with modified areas of plasma membrane, thereby inducing virus budding. A deficiency of this material, as apparently occurs in aged MDBKpi cells, would therefore cause a block in virus maturation. The nature of this granular material is discussed, and we suggest that it consists of M protein.

Liver sections from 18 patients positive for hepatitis B surface antigen (HBsAg), and from 12 negative patients, were examined for the presence of hepatitis B virus (HBV) DNA using an in situ hybridization assay that would identify only those hepatocytes containing more than 10 to 15 HBV genome equivalents per cell. Such cells are likely to be undergoing active viral replication, rather than latent infection. The findings were correlated with results of tissue immunofluorescence for HBV antigens and the presence of serum hepatitis B e antigen (HBeAg), together with histologic assessment of each liver. HBV DNA detected in the above assay was predominantly cytoplasmic; it was associated with the presence of hepatitis B core antigen (HBcAg) in hepatocytes and HBeAg in serum, and to a lesser extent with cirrhosis and immunosuppression, but not with the presence of HBsAg in hepatocytes, nor with histological evidence of disease activity judged by the presence of piece-meal necrosis and lobular and portal tract inflammation. These findings support the view that liver HBcAg and serum HBeAg are markers of virus replication, and demonstrate that active liver disease in HBsAg-positive patients may occur with or without such markers of replication. It is proposed that alternative mechanisms for hepatocyte injury may apply in different chronic HBV patients, one related to virus replication and one dependent on immunological factors.

Summary The neuraminidase content of purified virus particles of three velogenic strains of Newcastle disease virus was compared with that of three lentogenic strains. Velogenic strains possessed approximately three times as much enzyme as lentogenic strains.

To the casual observer,in situ hybridization (ISH) suggests a method to detect and localize nucleic acid sequences in cell preparations, tissue sections or chromosome spreads. This paper is an attempt to demonstrate that ISH is very much more versatile than is immediately apparent; a number of possible alternatives in different steps in the procedure have been defined and specific methods recommended that are known to be successful. The text is limited to the detection of viral nucleic acids in tissue sections and cells, although many of the concepts may be applied more generally. Methods and practical details have been reduced to a minimum as they are covered in the accompanying paper by Dr. F. Negro et al. (see page 177 of this issue) and in references. A comprehensive review of ISH procedures has been compiled recently12.

Examination of a naturally infected human liver and experimentally infected chimpanzee and woodchuck livers by in situ hybridization showed that hepatitis delta virus (HDV) RNA was restricted to hepatocytes. Genomic RNA was 20–30 times more abundant than antigenomic RNA and was predominantly single-stranded while antigenomic RNA was predominantly double-stranded. In acute delta hepatitis, viral RNA was a more reliable marker of virus infection in single cells than hepatitis delta antigen (HDAg) while in chronic hepatitis both markers were usually present in the same cell. In all cases, viral antigen and RNA were localized predominantly to the nuclei of infected cells. Thus, replication of HDV RNA is closely associated with HDAg expression at the cellular and intracellular level and it is likely that this new class of defective animal RNA viruses replicates in the nucleus of the infected cells.

Cells expressing alpha 2-interferon were identified by indirect immunofluorescence using both a polyclonal and a monoclonal anti-alpha-interferon antibody reagent. In hepatitis B or delta virus infection, focal clusters of alpha-interferon-positive infiltrating mononuclear cells and (to a lesser extent) fibroblasts were regularly seen in liver sections from patients who had chronic active hepatitis and cirrhosis and evidence of virus replication, but in a minority of patients with chronic persistent hepatitis B and not in nonvirally infected livers. This report provides evidence for local alpha-interferon production near the site of virus replication in hepatitis B infection, identifies mononuclear cells and fibroblasts (but not hepatocytes) as the main cell types producing interferon in this infection and suggests that locally produced alpha-interferon may be a natural regulator of virus replication in HBsAg-positive chronic active hepatitis. Furthermore, serological characterization of the interferon species produced locally may predict which particular interferon species could be of the greatest therapeutic benefit in specific disease states or individual patients.

The correlation between hepatitis B e antigen (HBeAg) and hepatitis B virus (HBV) DNA was studied in a number of hepatitis B surface antigen (HBsAg)-positive serum samples which had been submitted for routine hepatitis screening. In patients with either acute or persistent HBV infection, a good overall correlation between HBeAg and HBV DNA was noted, although in persistently infected patients with chronic liver disease, 28% of samples with hepatitis B e antibody (anti-HBe) were also positive for HBV DNA. Around 3% of the samples that were positive for HBV DNA were negative for HBeAg and anti-HBe while, conversely, 19% of HBeAg-positive sera were negative for HBV DNA. These results indicate that the presence or absence of HBeAg/anti-HBe may not necessarily reflect that of serum HBV DNA, particularly in persistent infection; this provides further support for the replacement of HBeAg/anti-HBe assays by detection of HBV DNA in the routine assessment of possible infectivity and chronic liver disease in HBsAg-positive patients.

Cytoplasmic and cell membrane associated hepatitis B core antigen (HBcAg) were found to be more widespread within infected liver using indirect immunofluorescence on frozen sections than with the widely used direct immunofluorescence method. Fixation of frozen sections with carbon tetrachloride improved tissue histology without reducing the sensitivity of antigen detection. In tissue blocks fixed with formalin or ethanol-acetic acid, detection of HBcAg was reduced in comparison with frozen sections, and many cells containing low concentrations of (usually cytoplasmic and membranous) HBcAg could not be identified even using indirect immunofluorescence or peroxidase-antiperoxidase reactions. In contrast, intracellular hepatitis B surface antigen (HBsAg) was well detected in fixed sections, but membrane associated HBsAg was not detectable after fixation.

A nested polymerase chain reaction was used to assess viraemia in blood transfusion recipients with no serological evidence of hepatitis C virus (HCV) infection (naive recipients) and in recipients with prior or existing HCV infection (infected recipients), who were transfused with HCV-positive blood. In 10 hepatitis cases in naive recipients, defined as primary infection, nine showed clinical hepatitis, and one was sub-clinical; the time between transfusion and elevation of alanine aminotransferase (ALT) levels was 15-60 days (37.9 +/- 13.9). All 10 naive recipients showed abnormal ALT, and 10/10 and 7/10 were persistently positive for anti-HCV and HCV-RNA, respectively, for more than 1 year. Similarly, in five cases in previously infected recipients, defined as re-infection, 4/5 showed clinical hepatitis, the time to elevation of ALT was 30-46 days (34.8 +/- 6.4), and 5/5 and 3/5 were persistently positive for anti-HCV and HCV-RNA, respectively, for more than 1 year. All five infected recipients showed abnormal ALT. In conclusion, there was no significant difference (P = 0.05) in the frequency of the markers of infection resulting from primary or re-infection with HCV, suggesting that primary infection fails to induce a protective immune response.