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Background & Aims: Hepatitis B virus (HBV) RNA in serum has recently been linked to efficacy and prognosis of chronic hepatitis B (CHB) treatment. This study explored the nature, origin, underlying mechanisms, and potential clinical significance of serum HBV RNA., Methods: The levels of HBV DNA and RNA were determined in the supernatant of induced HepAD38, HBV-expressing HepG2.2.15 cells and primary human hepatocytes (PHH), and in the serum of transgenic mice and CHB patients. NP-40 and proteinase K treatment, sucrose density gradient centrifugation, electron microscopy, northern blot, multiple identification PCRs and 5' rapid-amplification of cDNA ends were performed to identify the nature of serum HBV RNA., Results: Although significantly lower than HBV DNA levels, abundant HBV RNA was present in the serum of CHB patients. A series of experiments demonstrated that serum HBV RNA was pregenome RNA (pgRNA) and present in virus-like particles. HBV pgRNA virion levels increased after blocking the reverse transcription activity of HBV DNA polymerase, and decreased after blocking the encapsidation of pgRNA. Furthermore, the presence of HBV pgRNA virion was associated with risk of viral rebound after discontinuation of nucleot(s)ide analogues (NAs) therapy in CHB patients., Conclusions: Serum HBV RNA was confirmed to be pgRNA present in virus-like particles. HBV pgRNA virions were produced from encapsidated particles in which the pgRNA was non- or partially reverse transcribed. Clinically, HBV pgRNA virion might be a potential biomarker for monitoring safe discontinuation of NA-therapy., Lay Summary: HBV may have another virion form in which the nucleic acid is composed of RNA, not DNA. The level of HBV RNA virion in serum may be associated with risk of HBV viral rebound after withdrawal of treatment, and therefore, a potential predictive biomarker to monitor the safe discontinuation of nucleot(s)ide analogues-therapy., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The process by which eukaryotic viruses with segmented genomes select a complete set of genome segments for packaging into progeny virus particles is not understood. In this study a model based on the association of genome segments through specific RNA-RNA interactions driven by base pairing was formalized and tested in the Orbivirus genus of the Reoviridae family. A strategy combining screening of the genomic sequences for inter-segment complementarity with direct functional testing of inter-segment RNA-RNA interactions using reverse genetics is described in the type species of the Orbivirus genus, Bluetongue virus (BTV). Two examples, involving four of the ten BTV genomic segments, of specific inter-segment interaction motifs whose maintenance is essential for the generation of infectious virus, were identified. Equivalent inter-segment complementarities were found between the identified regions of the orthologous genome segments of all orbiviruses, including phylogenetically distant species. Specific interaction of the participating RNA segments was confirmed in vitro using electrophoretic mobility shift assays, with the interactions inhibited using oligonucleotides complementary to the interaction motif of one of the interacting partners, and also through mutagenesis of the motifs. In each example, the base pairing rather than the absolute sequence was critical to the formation of a functional inter-segment interaction, with mutations only being tolerated in rescued virus if compensating changes were made in the interacting partner to restore uninterrupted base pairing. The absolute sequence of the complementarity motifs varied between species, indicating that this newly identified phenomenon may contribute to the observed lack of reassortment between Orbivirus species.

The regulatory elements which control the processes of virus replication and gene expression in the Orbivirus genus are uncharacterized in terms of both their locations within genome segments and their specific functions. The reverse genetics system for the type species, Bluetongue virus, has been used in combination with RNA secondary structure prediction to identify and map the positions of cis-acting regions within genome segment 10. Through the simultaneous introduction of variability at multiple nucleotide positions in the rescue RNA population, the functional contribution of these positions was used to map regions containing cis-acting elements essential for virus viability. Nucleotides that were individually lethal when varied mapped within a region of predicted secondary structure involving base pairing between the 5' and 3' ends of the transcript. An extended region of predicted perfect base pairing located within the 3' untranslated region of the genome segment was also found to be required for virus viability. In contrast to the identification of individually lethal mutations, gross alteration of the composition of this predicted stem region was possible, providing the base-pairing potential between the two strands was maintained, identifying a structural feature predicted to be conserved throughout the Orbivirus genus. The approach of identifying cis-acting sequences through sequencing the recovered virus following the rescue of a degenerate RNA population is broadly applicable to viruses where reverse genetics is available.

The Hepatitis B virus X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). It has been suggested that the transcriptional activation of cyclin D1 by HBx is implicated in the development of HCC. However, numerous studies have shown that overexpression of cyclin D1 alone is not sufficient to drive oncogenic transformation. Herein, we investigated whether HBx can stabilize cyclin D1 and induce cyclin D1 protein nuclear accumulation, and thereby accelerate hepatocarcinogenesis. The effects of HBx on cyclin D1 stabilization were assessed in cell-based transfection, Western blot, immunoprecipitation, immunocytofluorescence staining, and flow-cytometric assays. The results demonstrated that ectopic expression of HBx in HCC cells could extend the half-life of cyclin D1 protein from 40-60 minutes to 80-110 minutes. HBx stabilized cyclin D1 primarily in the S phase of the cell cycle, in a manner dependent on the inactivation of GSK-3β, which was mediated by ERK activation. HBx also prompted the nuclear accumulation of cyclin D1, and cotransfection of the constitutively active mutant of GSK-3β along with HBx could reverse the nuclear accumulation and subsequent cell proliferation induced by HBx. Further, a positive correlation between HBx and nuclear cyclin D1 level was established in HCC specimens detected by an immunohistochemical assay. Taken together, our results indicated that HBx could stabilize and increase cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3β. This HBx-induced cyclin D1 upregulation might play an important role in HCC development and progression., (©2015 American Association for Cancer Research.)

A cohort based study has been undertaken to investigate the possible association of genetic polymorphisms in genes functionally related to follicular T helper (TfH) cells with non-responsiveness to hepatitis B virus (HBV) vaccination. A total of 24 single nucleotide polymorphisms (SNPs) in 6 TfH related genes (CXCR5, ICOS, CXCL13, IL-21, BCL6 and CD40L) were investigated in 20 non-responders and 45 responders to HBV vaccination. Genetic association analysis revealed that three SNPs (rs497916, rs3922, rs676925) in CXCR5 and one SNP (rs355687) in CXCL13 were associated with hepatitis B vaccine efficacy. In addition, significantly unbalanced distributions of two haplotypes, defined by three SNPs (rs497916, rs3922, rs676925) within CXCR5, were also seen between non-responders and responders. Furthermore, we demonstrated that the rs3922 "GG" genotype was associated with higher levels of CXCR5 than the "AG" and "AA" genotype in a group of healthy volunteers. A dual luciferase report assay was used to confirm that the "G" allele in rs3922 may lead to higher gene expression than the "A" allele, implicating that rs3922 might be a functional SNP affecting CXCR5 expression. These results indicated that polymorphism associated changes in CXCR5 expression in TfH cells may be associated with non-responsiveness to hepatitis B vaccination., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background & Aims: Although hepatitis B virus (HBV) integration into the human genome has been considered as one of the major causative factors to hepatocarcinogenesis, the underlying mechanism(s) was still elusive. Here we investigate the essential difference(s) of HBV integration between HCC tumor and adjacent non-tumor tissues and explore the factor(s) that determine the oncogenicity of HBV integration., Methods: 1115 HBV integration sites were collected from four recent studies. Functional annotation analysis of integration targeted host genes (ITGs) was performed using DAVID based on Gene Ontology and KEGG pathway databases. Array-based expression profiles, real-time qPCR and western blot were used to detect the expression of recurrent integration targeted genes (RTGs). The biological consequences of the overexpression of UBXN8 in 8 HCC cell lines were studied in vitro., Results: HBV is prone to integrate in genic regions (exons, introns, and promoters) and gene-dense regions. Functional annotation analysis reveals that, compared to those in adjacent non-tumor tissues, ITGs in HCC tumor tissues were significantly enriched in functional terms related to negative regulation of cell death, transcription regulation, development and differentiation, and cancer related pathways. 32% of the 75 RTGs identified in this analysis expressed abnormally in HCC tissues. UBXN8, one of the RTGs, was identified as a new tumor suppressor candidate which functions in a TP53 dependent manner., Conclusions: The oncogenicity of HBV integration was determined, to some extend by the function of HBV integration targeted host genes in HCC., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Aim: To determine the prognostic value of circulating indicators of cell death in acute-on-chronic liver failure (ACLF) patients with chronic hepatitis B virus (HBV) infection as the single etiology., Methods: Full length and caspase cleaved cytokeratin 18 (detected as M65 and M30 antigens) represent circulating indicators of necrosis and apoptosis. M65 and M30 were identified by enzyme-linked immunosorbent assay in 169 subjects including healthy controls (n = 33), patients with chronic hepatitis B (CHB, n = 55) and patients with ACLF (n = 81). According to the 3-mo survival period, ACLF patients were defined as having spontaneous recovery (n = 33) and non-spontaneous recovery which included deceased patients and those who required liver transplantation (n = 48)., Results: Both biomarker levels significantly increased gradually as liver disease progressed (for M65: P < 0.001 for all; for M30: control vs CHB, P = 0.072; others: P < 0.001 for all). In contrast, the M30/M65 ratio was significantly higher in controls compared with CHB patients (P = 0.010) or ACLF patients (P < 0.001). In addition, the area under receiver operating characteristic curve (AUC) analysis demonstrated that both biomarkers had diagnostic value (AUC ≥ 0.80) in identifying ACLF from CHB patients. Interestingly, it is worth noting that the M30/M65 ratio was significantly different between spontaneous and non-spontaneous recovery in ACLF patients (P = 0.032). The prognostic value of the M30/M65 ratio was compared with the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores at the 3-mo survival period, the AUC of the M30/M65 ratio was 0.66 with a sensitivity of 52.9% and the highest specificity of 92.6% (MELD:AUC = 0.71; sensitivity, 79.4%; specificity, 63.0%; Child-Pugh: AUC = 0.77; sensitivity, 61.8%; specificity, 88.9%)., Conclusion: M65 and M30 are strongly associated with liver disease severity. The M30/M65 ratio may be a potential prognostic marker for spontaneous recovery in patients with HBV-related ACLF.

Background: Recent research has suggested that the oncomir microRNA 155 (miR-155) is up-regulated in hepatocellular carcinoma (HCC). In this study, the authors investigated the tumorigenic mechanism of this oncomir in the development of HCC., Methods: Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to analyze the expressions of miR-155 and its potential target genes in paired tumor tissues and adjacent tumor-free tissues and in disease-free liver tissue samples. The in silico predicted target genes of miR-155 were assessed by dual-luciferase reporting assay, real-time RT-PCR, and Western blot analyses. U6 promoters that drive miR-155 precursor overexpression and miR-155 tough decoy knock-down constructs were used to study its affects on cell proliferation in vitro and on tumor formation in nude mice., Results: Quantitative RT-PCR demonstrated a gradual ascension of miR-155 expression in cirrhotic liver tissues and in HCC tumor tissues compared with low expression levels in normal liver tissues. Ectopic expression of miR-155 in HepG2 cells enhanced its tumorigenesis, whereas depletion of the endogenous miR-155 reversed these tumorigenic properties. Ectopic expression of sex-determining region Y box 6 (SOX6) was able to reverse the growth-promoting property of miR-155. Concordantly, the results demonstrated for the first time that SOX6 is a direct target of miR-155. Further analysis revealed that SOX6 reduced cell growth by up-regulating p21waf1/cip1 expression in a p53-dependent manner. In addition, a decline in p21waf1/cip1 expression caused by miR-155 could be reversed by SOX6 expression., Conclusions: The current data indicated that SOX6 is a novel target of miR-155 and that miR-155 enhances liver cell tumorigenesis at least in part through the novel miR-155/SOX6/p21waf1/cip1 axis. These findings suggest that miR-155 may be a potential target for HCC treatment., (Copyright © 2011 American Cancer Society.)

The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions, which, through site-directed mutagenesis of the N-linked glycosylation sites near the amino terminus of the protein, were shown to be dependent on its ability to become fully glycosylated. Real-time confocal microscopy was used to follow the appearance of similar cytoplasmic extrusions in virus-infected cells and revealed them to grow at a rate of ~2 microns/min to more than three cell diameters and to have a lifespan of 30-60 minutes. CellTracker dyes were used to label cell populations and facilitate the monitoring of the transfer of cytoplasm from virus-infected to surrounding uninfected cells through cytoplasmic extrusions that broke down to vesicles, seen both on the surface of and within uninfected cells in mixed cell populations. Staining of these tagged cell mixtures with monospecific antibody to NSP4 revealed the presence of the protein on uninfected cells, suggesting that the cytoplasmic extrusions formed by virus-infected cells facilitates the direct cell-cell spread of NSP4. This direct cell-cell transfer of infected cell material triggered by expression of glycosylated NSP4 in virus-infected cells may contribute to viral pathogenesis and facilitate host invasion by rotaviruses.

Expression of the rotavirus enterotoxin (NSP4) in transfected monkey kidney cells was found to result in a dramatic re-modeling of the microtubule (MT) network. This important centrosome organized cytoskeletal element was dissolved by expression of NSP4 and re-formed in a ring array at the periphery of the cell, similar to that seen following normal virus infection. Site directed mutagenesis of the N-linked glycosylation sites in NSP4 was employed to show that glycosylation of NSP4 was not required for it to promote changes in the MT network. This result together with experiments using conventional inhibitors indicated that NSP4's ability to cause elevation of intracellular calcium levels was also not necessary to effect the changes in the MT network. Use of the centrosome function inhibitor nocodazole demonstrated that NSP4 based remodeling of the MT network was dominant over the normal organizational role of the centrosome. Finally the remodeling of the MT network was shown not to be linked to cellular apoptosis or necrosis. The potential importance of this newly recognised role for NSP4 in the overall process of intracellular pathogenesis by rotaviruses is discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820-1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis.

The role of the rotavirus non-structural proteins NSP1 and NSP3 in regulating cellular and viral mRNA translation has been investigated by examining the effect of added recombinant NSP3 on protein translation in a T7-based in vitro coupled transcription-translation system. Addition of purified NSP3 to assays primed solely with cellular mRNA was found to have no effect on the translation efficiency of the mRNA. However, as expected, the addition of viral mRNA to such assays competitively inhibited the synthesis of cellular protein, and interestingly, this inhibition was enhanced by the addition of NSP3. Treatment of NSP3 with antisera raised against the purified protein abrogated its function, but only when used prior to mixing the protein with viral mRNA. Addition of partially purified NSP1 to the coupled system was able to alleviate the enhancement of the inhibition of cellular mRNA translation caused by NSP3. The role of NSP1 in this process appears to be to modulate the impact of the NSP3-based inhibition of cellular translation by binding to the 5' end of viral mRNAs.

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55(GAG)) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1-22) and central (aa 70-100) regions of Vif to be essential for its interaction with Pr55(GAG), but deletion of the carboxy-terminal (aa 158-192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55(GAG) showed that a 35-amino-acid region of the protein bridging the MA(p17)-CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp(21)) near the amino terminus of Vif showed it to be important for the interaction with Pr55(GAG). By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif-Pr55(GAG) interaction.

The 12kDa non-structural protein 6 (NSP6) is the least studied of the rotavirus proteins. In an attempt to further characterize this protein mono-specific antisera was generated using purified protein expressed in E. coli. Pulse/chase radio-labeling of virus infected cells was used to show that it is expressed at a steady but low rate throughout the virus replication cycle. In contrast to the other rotavirus non-structural proteins, NSP6 was found to have a high rate of turnover, being completely degraded within 2h of synthesis. NSP6 tagged with GFP was used to probe the intracellular distribution of the protein, perinuclear aggregates were observed in the cytoplasm of transfected cells. Following virus infection of these transfected cells the aggregates were seen to redistribute to the viroplasms. Consistent with its localization to the site of viral genome replication and packaging, NSP6 was found to be a sequence independent nucleic acid binding protein, with similar affinities for ssRNA and dsRNA.

A fluorescence-based assay is presented for measuring the cytoxicity of viral proteins added exogenously to cells. The assay is based on the use of two fluorescent dyes, calcein-AM and ethidium homodimer (EtD-1) to specifically stain living and dead cells respectively and employs fluorescence activated cells sorting (FACS) to achieve a rapid and accurate measurement of the cytotoxic capacity of a potential viral toxin. The assay has been developed using the group B homologue (ADRV-NSP4) of the NSP4 enterotoxin encoded by Group A rotaviruses but should be applicable to assaying any viral protein exhibiting cytotoxic activity.

The RNA segment (Gene 10) from a human group B rotavirus which encodes the homologue of the rotavirus enterotoxin (NSP4) has been cloned and sequenced. The gene is of the same length (751 nucleotides) as its better-characterized group A rotavirus counterpart but shows minimal homology (approximately 10%) to it at the primary sequence level. Despite this low level of sequence homology, secondary structure predictions for the group B protein (ADRV-NSP4) showed a close similarity of structural features with the group A protein. Full-length ADRV-NSP4 was expressed in Escherichia coli with an amino terminal 6xHis tag that was used to purify it to homogeneity. The cytotoxicity of the purified protein was examined in a rapid dye-uptake assay that assesses membrane permeability and was found to be comparable to its group A counterpart.

The protease (PR) and virus infectivity factor (vif) gene sequences of a cohort of HIV-1 infected patients showing evidence of developing protease inhibitor (PI) resistance whilst undergoing highly active antiretroviral therapy (HAART) have been determined. The PR sequences showed the presence of the classical mutations associated with resistance to PIs. The sequence of the Vif protein showed less variation in samples from PI treated patients than in specimens prepared from treatment-naive patients. In addition a number of amino acid positions within Vif showed highly significant preferences for a particular amino acid in the PI-treated cohort compared to the untreated control cohort.

The RNA segment encoding the guanylyltransferase (VP3) from 12 group A rotavirus isolates has been sequenced following RT-PCR and molecular cloning of the full-length amplicons produced. Alignment of the derived amino acid sequences including those of the four VP3 sequences available from GenBank revealed two levels of sequence divergence. Virus isolates from humans showed greater than 94% sequence identity, whereas those isolated from different mammalian species showed as low as 79% sequence identity. The exceptions were avian virus isolates, which diverged approximately 45% from those of mammalian origin, and the human virus isolates DS1 and 69M, which showed much closer (over 90%) identity to viruses of bovine origin, suggesting that these human isolates may have undergone recent reassortment events with a bovine virus. Analysis of the sequences for a putative enzymic active site has revealed that the KXTAMDXEXP and KXXGNNH motifs around amino acids 385 and 545, respectively, are conserved across both group A and C rotaviruses.

A region of genome from the NADL strain of BVDV corresponding to the coding sequence for the E2 glycoprotein has been molecularly cloned using RT-PCR. The viral cDNA sequence was used to construct vaccinia virus recombinants that expressed either the entire E2 coding sequence or fragments of it. These recombinants were used to immunise mice of three H-2 haplotypes to investigate their ability to elicit a neutralising antibody response against BVDV. Sera from mice immunised with the recombinant expressing full length E2 contained high levels of virus neutralising antibodies that in addition to giving neutralisation of the homologous NADL strain were also able to neutralise the Oregon C24V reference strain. These sera failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2 serotypes. These results were confirmed in gnotobiotic lambs. Expression of E2 fragments revealed the presence of at least two distinct neutralising epitopes, one of which was localised within the carboxy terminal 90 amino acids of the protein.

Age-specific patterns of rotavirus infection were investigated using a randomly selected and representative sample of sera from a suburban community of São Paulo, Brazil screened for class-specific antibodies to group A rotavirus. Age-serology of anti-rotavirus IgG showed primary infection predominant in young infants with a median age of around 18 months consistent with IgM serology suggesting highest rates of recent infection between ages 4 and 48 months. Anti-rotavirus serum IgA prevalence increased gradually with age. Paired samples from infants, collected 1 month apart, indicated high exposure rates with seroconversion occurring in several infants during the reported low transmission season. Between 5 and 10% of adults had elevated IgM levels indicative of recent infection and, potentially, of an important contribution adults may play to rotavirus transmission. Further understanding of the dynamics of rotavirus transmission within populations, at group and serotype level, would benefit the design and monitoring of future immunization programmes.

The cytotoxic T cell (CTL) response in C57/B16 (H-2b) mice to rotavirus has been analysed using a cognate set of vaccinia virus recombinants covering the 12 primary gene products of the UKtc strain of bovine rotavirus. The gene products of RNA segments 5 (VP5/NSP-1) and 8 (VP7) both elicited a classic CD8+ Class I MHC restricted CTL response. Using L cells transfected with specific Class I MHC loci as targets the VP5/NSP-1 response was found to be restricted at Db and the VP7 response at Kb. Vaccinia virus recombinants expressing VP7 genes from seven G serotypes were used to show that the CTL response to this antigen is completely cross-reactive. By contrast, using the same strategy the CTL response to VP5/NSP-1 was found to be virus strain specific. A vaccinia virus recombinant carrying RNA segment 5 from the deletion mutant P9D delta 5 was used to localize at least one CTL epitope in VP5/ NSP-1 to the first 150 amino acids of the protein. The expression of a number of fragments of VP7 in vaccinia virus recombinants was used to show that the CTL epitope (amino acids 31-40) previously identified through the use of synthetic peptides is virus serotype specific rather than cross-reactive.

In a survey of coronavirus and rotavirus-induced neonatal diarrhoea in 373 calves from 284 farms, nine (2.4%) of the animals were found to be infected with non-group A rotaviruses when their faeces were analysed in a commercial ELISA and by PAGE. Seven out of eight 1-4- day-old kids from a single farm were also infected with similar viruses. In a mini-PAGE, all the viruses displayed group B- and/or E-like 4223 electrophoretypes. In Northern hybridizations with cDNA chemiluminescent probes specific to rotavirus groups A (RF strain), B (adult diarrhoea rotavirus, ADRV) and C (Cowden strain) all the viruses belonged to group B.

Nucleotide sequencing of RNA segment 5 from seven strains of group A rotavirus has been carried out to investigate the extent of diversity and conservation, as well as possible selective pressures involved in driving the fixation of sequence changes in this gene. Analyses of the derived sequences revealed that sequence conservation could not be correlated either with rotavirus serotype or the species of origin of the virus strain. These sequences together with other published and unpublished sequences of this gene have raised the total number available for comparison to 17. Alignment of all the available sequences revealed that only 88 amino acid positions (17.6%) in the protein encoded by gene 5 (VP5) are absolutely conserved but that the metal-binding motif reported by others is conserved in all sequences. Despite the high degree of sequence divergence, alignment of secondary structure predictions for VP5 showed a high level of conservation, suggesting that constraints on sequence divergence may operate at the level of overall higher-order structure of the encoded protein.

The VP4, VP7, NS53 and VP6 genes of two porcine rotavirus variants which differ in their in vitro growth properties and pathogenicity have been cloned and sequenced. The VP4 genes show only 67.2% nucleic acid and 70.6% amino acid identity. The VP4 gene of one variant (4S) is closely related to that of the bovine UK rotavirus strain, whereas the VP4 gene of the other variant (4F) is only distantly related to known VP4 genes and is likely to represent a new P serotype. In contrast the NS53 (VP5), VP6 and VP7 genes of the 4F and 4S variants show greater than 99% nucleotide and amino acid identity, indicating that the two viruses are genetically related by a reassortment event. The implications for the role of VP4 in the determination of in vitro growth characteristics and pathogenicity are discussed.

We determined the capacity of vaccinia virus recombinants expressing individual rotavirus genes to induce virus-specific cytotoxic T lymphocytes (CTLs) in mice. Mice were orally inoculated with vaccinia virus recombinants containing genes which encode rotavirus outer capsid proteins vp4 or vp7, single-shelled virus proteins vp1, vp2, or vp6, or rotavirus nonstructural proteins NS53, NS35, NS28, or NS26/NS12. We found that (i) the greatest frequencies of virus-specific CTLs were induced by vaccinia virus recombinants expressing vp7, (ii) transport of vp7 beyond the endoplasmic reticulum was not necessary for induction of CTLs, (iii) recombinants expressing vp7 induced CTLs which reacted with different rotavirus serotypes, and (iv) CTLs were induced among both intestinal and nonintestinal lymphocytes after oral inoculation. These findings may be relevant to vaccine strategies which utilize vectors expressing individual rotavirus genes.

Three variants of group A rotavirus with large changes in their gene 5 structures have been analyzed at the molecular level. The first of these, P9 delta 5, was obtained during plaque purification undertaken as part of the biological cloning of a field isolate of virus. The gene 5 homolog in this isolate migrated just ahead of the normal segment 6 RNA, giving an estimated size of 1,300 bp. Molecular cloning and sequencing of this homolog revealed it to have a single 308-bp deletion in the center of the normal gene 5 sequence extending between nucleotides 460 and 768 of the normal gene sequence. This deletion caused a frameshift in the gene such that a stop codon was encountered 8 amino acids downstream of the deletion point, giving a predicted size for the protein product of this gene of 150 amino acids compared with the 490 amino acids of its normal-size counterpart. Attempts to detect this shortened protein in virus-infected cells were not successful, indicating that it was much less stable than the full-length protein and/or had suffered a large change in its antigenicity. The second two variants, brvA and brvE, were generated in an earlier study following the high-multiplicity passage of the UKtc strain of bovine rotavirus. Polyacrylamide gel electrophoresis analysis of these nondefective variants showed that brvA had a gene 5 homolog approximately equal in size to the normal RNA segment 2 (approximately 2,700 bp) and that brvE had a size of approximately 2,300 bp. Both variants showed changes in their gene 5 protein products, with brvA mimicking P9 delta 5 in failing to produce a detectable product whereas brvE produced a new virus-specific protein approximately 80 kDa in size. Full-length cDNA clones of the brvE gene 5 homolog were isolated, and analysis of their structure revealed a head-to-tail concatemerization of the normal gene 5 sequence with the first copy of the concatemer covering nucleotides 1 to 808 and the second covering nucleotides 92 to 1579, giving a total length of 2,296 bp. Sequencing across the junction region of the two copies of the gene showed that they were joined in frame to give a predicted combined open reading frame of 728 amino acids with the amino-terminal region consisting of amino acids 1 to 258 fused at the carboxy terminus to amino acids 21 to 490.(ABSTRACT TRUNCATED AT 400 WORDS)

The stimulation of reporter gene expression following co-transfection with the S4 gene of mammalian reoviruses was analysed. The sigma 3 protein of type 3 reovirus gave a five- to eightfold increase in expression of chloramphenicol acetyltransferase and beta-galactosidase but this was found to be dependent upon the nature of the promoter being used to drive reporter gene expression. The sigma 3 protein of reovirus type 1 failed to stimulate reporter gene expression under any of the conditions used. Hybrid constructs between the S4 genes of reoviruses type 1 and 3 were used to map the stimulation characteristic to the carboxy-terminal third of the gene. Analysis of the level of sigma 3 protein accumulation in transfected cells showed that the reovirus type 3 protein accumulated to a much higher level than that of reovirus type 1. Using the hybrid gene constructs this higher level of protein accumulation was shown to co-segregate with the ability to stimulate reporter gene expression.

Dot and Northern (RNA) blot hybridization assays were developed for the P typing of group A bovine rotaviruses (BRV) by using cDNA probes prepared from gene segment 4. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions (nucleotides 211 to 686) of BRV strain UK, IND, NCDV, and Cr VP4 cDNA by using specific oligonucleotide primers. The probes were P type specific (VP4) and exhibited little or no cross-reactivity with double-stranded RNA from heterologous rotavirus P types. Our studies indicate that at least three P types, as defined by polymerase chain reaction-derived VP4 gene probes from the UK, NCDV, and Cr strains, exist among the seven BRV isolates tested.

The aims of this experiment were: (1) to establish a reovirus type 3-induced murine model of biliary atresia/neonatal hepatitis that as far as possible corresponds to the human disease; (2) to demonstrate that the disease is histologically similar to the human disease, and to investigate the natural history of reovirus type 3 infection in this model; (3) to study the host-virus interrelationships at a molecular level; and (4) to develop sensitive assays that could be translated to the human disease. In this study we were unable to produce an exact model for extrahepatic biliary atresia (EHBA) in the laboratory mouse following a perinatal reovirus type 3 infection. However, the ability of reovirus type 3 to persist in the murine liver and the effects produced in the offspring of infected pregnant mice indicate that this preparation may provide the basis for the eventual development of the experimental model of EHBA.

Rotaviruses with genome rearrangements isolated from a chronically infected immunodeficient child (F. Hundley, M. McIntyre, B. Clark, G. Beards, D. Wood, I. Chrystie, and U. Desselberger, J. Virol 61:3365-3372, 1987) are the first recognized human isolates of serotype 10. This was shown by both a direct enzyme-linked immunosorbent assay and virus neutralization assays using serotype specific monoclonal antibodies. The serotype was confirmed by sequence analysis of the gene encoding VP7, which revealed a 96% amino acid homology to the bovine serotype 10 isolate B223.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.

Normal and rearranged RNA segments 10 of group A rotaviruses isolated from a chronically infected immunodeficient child were amplified by the polymerase chain reaction as full-length cDNA copies, and were subsequently cloned and sequenced. Compared with the nucleotide sequence of the normal RNA segment 10, the rearranged form contains a partial non-coding duplication at its 3' end and several point mutations. The normal RNA segment 10 was similar to that of bovine rotavirus.

Cytotoxic T lymphocytes (CTLs) generated in mice orally inoculated with rotaviruses lyse target cells infected with different rotavirus serotypes (cross-reactive CTLs). Using vaccinia virus recombinants expressing individual rotavirus proteins from two different rotavirus serotypes, we found that cross-reactive CTLs recognize target cells expressing outer capsid protein vp7 better than those expressing outer capsid protein vp4 or inner capsid protein vp6. These findings may be relevant to vaccine strategies which include immunization with reassortant rotaviruses or viral or bacterial vectors expressing individual rotavirus proteins. The region or regions of vp7 which are antigenically conserved among different rotavirus serotypes and recognized by cross-reactive CTLs remain to be determined.

We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.

Cytoplasmic polyhedrosis virus is an insect reovirus which is occluded in crystalline inclusion bodies that form in the mid-gut of certain insects. Inclusion bodies of cytoplasmic polyhedrosis virus from Bombyx mori were isolated and purified. These crystalline bodies, about 1-3 microns in linear size, were compacted in a capillary tube while immersed in buffer. X-ray diffraction photographs showed powder rings, extending to 8.2 A resolution, which could be indexed with a cell measuring a = b = 49.9 +/- 0.4 A, c = 41.5 +/- 0.4 A, alpha = beta = gamma = 90 degrees. The polyhedrin protein, which is the major component of the inclusion body, has a molecular weight of about 30,000 daltons and, hence, there are probably two molecules in the unit cell. Thus, the unit cell is monoclinic or possibly triclinic. A Patterson derived from the measured powder pattern intensities, assuming monoclinic symmetry, could be interpreted in terms of a molecule with two larger globes. Such a structure is roughly consistent with the breakdown of the polyhedrin into two larger fragments of molecular weight 17,000 and 14,000 when raising the pH to near 10. Under these conditions the inclusion bodies disintegrate, releasing virus and catalyzing the proteolysis of the polyhedrin.

The sequence of the gene encoding the major neutralization antigen (VP7) of the type member (bovine virus strain B223) of the proposed serotype 10 of rotavirus has been determined. This was done using a rapid new strategy involving direct sequencing by primer extension of cDNA generated from the relevant virus gene (gene 8) using a combined reverse transcription/polymerase chain reaction. The sequence obtained is 1062 bp in length and contains a single long open reading frame capable of encoding a protein of 326 amino acids. Comparison of the sequence to that of the corresponding gene of the other major virus serotype found in cattle, serotype 6, showed that it was approximately 25% divergent at the nucleotide level and 18% divergent at the amino acid level.

Two overlapping c-DNA clones, which hybridized in Northern blots to RNA segment 6 of the prototype strain (Cowden) of group C rotavirus, were selected from a c-DNA library in pBR322 and sequenced. The gene 6 sequence obtained was 1349 nucleotides and contained a single long open reading frame encoding a protein of 394 amino acids (total MW, 44,479) which is in line with the size of the major capsid protein VP6. Comparison of the group C sequence with that of the corresponding group A rotavirus gene revealed homology levels of 55 and 42% for nucleotides and amino acids, respectively. These values were surprisingly high in view of previous immunological and nucleic acid hybridization data which failed to show any cross-reaction between group A and non-group A rotaviruses. The epidemiological implications of these observations are discussed.

An assay protocol based on exploiting the polymerase chain reaction (PCR) for the detection of rotavirus in infected faeces is described. The assay is 100,000 times more sensitive than the standard electropherotype method that is widely used. It also gives a 5000-fold increase in sensitivity over the hybridisation based assay previously developed (Pedley and McCrae, 1984) and does not require the use of radioisotopes. The amplified product is a full length c-DNA copy of the gene encoding the major neutralisation antigen of the virus whose molecular cloning and sequence analysis will allow detailed information on the molecular basis of epidemiological variation to be rapidly collected.

A method is described that allows the rapid screening of field isolates of rotavirus for the detection of specific viral RNA segments. Cloned cDNA copies of viral genomic RNAs are employed for detection in the assay which makes use of the dot-hybridization technique of Thomas (1980). The assay developed was shown to be sufficiently sensitive and specific to allow the genetic composition of virions to be determined over a wide range of concentrations. The feasibility of using the method developed for both the analysis of genetic reassortants prepared in vitro and for screening field isolates to detect putative genome reassortment events is demonstrated.

The naturally non-polyadenylated mRNAs of reovirus were shown to have a half-life in excess of 3 days when injected into Xenopus laevis oocytes. Hybridization analysis and gel electrophoresis showed that all 10 mRNA species had a similar high stability, despite being translated at widely differing rates. We have confirmed previous findings indicating a role for the 5'-terminal cap structure in determining reovirus mRNA stability [Furuichi et al. (1977) Nature (Lond.) 266, 235-239]. The significance of these results in relation to a general role for poly(A) in messenger RNA function is discussed.

The use of T1 RNase fingerprinting of terminally labelled genomic double-stranded RNA species from various rotavirus isolates, to analyse the near terminal G-residue positions, has revealed an RNA species-specific fingerprint pattern covering approximately 40 nucleotides at the termini. These RNA species-specific terminal fingerprint patterns were found to be conserved in both rotavirus RNAs isolated from various animal species, and in isolates from a single animal species where gross divergence of internal RNA sequence for a particular RNA species was evident. This conservation of near terminal G-residue positions suggests that, internal to the short regions of absolute terminal sequence conservation that we have previously shown to be present on all rotavirus RNA species, there is a region of conserved sequence which is specific for a particular RNA species.

Bunyamwera virus-induced polypeptide synthesis in BSC-1 cell has been studied using polyacrylamide gel electrophoresis and autoradiography. Four virus-induced polypeptides were identified. Their molecular weights were 200 X 10(6) (L), 128 X 10(6) (G1), 31 X 10(6) (G2), and 23 X 10(6) (N). Pulse-chase experiments, short labeling experiments, and experiments using amino acid analogs failed to show evidence of polypeptides processing by proteolytic cleavage. Analysis of the kinetics of synthesis of these polypeptides showed that a clear division into early and late categories could be made, the onset of synthesis of polypeptide N and L rapidly reached a peak and then declined. Polypeptides G1 and G2 were made for several hours; their rate of synthesis then declined. All four polypeptides then continued to be made in relatively small amounts for many hours.

A sensitive and quantitative solution hybridization assay recently developed in this laboratory has been applied to the study of the regulation of viral gene expression in rotavirus-infected cells. Measurement of the cumulative level of viral plus-strand (mRNA) synthesis at hourly intervals throughout the growth cycle has provided evidence for both quantitative and qualitative regulation of transcription. Qualitative control was found only when cycloheximide was used to block protein synthesis in infected cells, when transcription of four of the viral genes (genes 5, 6, 7, and 9) was independent of protein synthesis. Quantitative regulation was demonstrated by the accumulation of mRNA to much higher levels for some of the viral genes (e.g., genes 2 and 7) relative to others (e.g., genes 4 and 6). In addition to quantitative control at the level of transcription, measurement of the relative molar amounts of the various viral proteins at 6.5 h postinfection showed that their levels did not directly reflect those of their encoding RNAs, indicating the existence of translational control of gene expression in the rotavirus system. Analysis of the levels of minus strand synthesized for each viral gene showed that they were not all accumulated to the same level. The significance of this result in the light of the presumed similarities in replication strategy to that of the mammalian reoviruses is discussed.