Malcolm A. McCrae

Molecular characterization of rotaviruses with distinct group antigens.

Comparative serological and nucleic acid studies were carried out on rotavirus isolates, with and without the original group antigen. Indirect immunofluorescence allowed the atypical isolates to be divided into two groups each with its own distinct group antigen hence giving a total of at least three distinct rotavirus groups. Genome profile analysis of the atypical isolates again divided them into two groups which corresponded with those obtained by immunofluorescence and within which the overall pattern of genome segments was similar. A more rigorous examination of the degree of difference between the three virus groups was carried out using single-dimension terminal fingerprint analysis. This indicated that the viruses in the three groups did not carry any genome segments in common with each other. Therefore, experimental criteria have been established which allow the division of rotaviruses into three groups. Analysis of further atypical isolates, as they become available, will establish whether the division of rotaviruses into a number of separate groups, of the type seen in influenza virus, is justified.

Definition of two new groups of atypical rotaviruses.

Comparative antigenic and nuclei acid analyses were carried out on two new atypical rotavirus isolates coming respectively from chickens (D/132) and pigs (E/DC-9). Indirect immunofluorescence showed that each virus carried different group antigens which were also distinct from those of previously described rotavirus groups. By genome profile analysis each virus had a pattern of genomic RNAs clearly distinct from those of the other rotavirus groups. Comparative terminal fingerprinting of corresponding genome segments from the two viruses showed large differences between them, indicating that all of their genomic RNAs had significant differences in sequence both from each other and from the three previously defined rotavirus groups. On the basis of these results, extension of the number of rotavirus groups from three to five is proposed, with isolates D/132 and E/DC-9 being the type members of groups D and E respectively.

The molecular biology of rotaviruses II. Identification of the protein-coding assignments of calf rotavirus genome RNA species

Abstract The technique of in vitro translation of dimethyl sulfoxide denatured double-stranded (ds)RNA has been applied to the rotavirus system. Translation of an unfractionated mixture of genome RNA segments from calf rotavirus in a nuclease-treated rabbit reticulocyte system allowed the primary gene products of the viral genome to be defined. Polyacrylamide gel electrophoresis was used to resolve all 11 genome segments and then each was individually isolated and translated, to identify the primary gene product of each genome segment. RNA segments 1, 2, 3, 6, 7, and 11 were shown to code for structural proteins of the inner virus shell, segment 9 coded for the one completely nonstructural protein, and segments 4, 5, 8, and 10 coded for proteins or the precursors of proteins that were either completely or mainly outer shell structural polypeptides.

The application of polymerase chain reaction to the detection of rotaviruses in faeces.

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.

THE GENOMES OF ROTAVIRUSES ISOLATED FROM CHRONICALLY INFECTED IMMUNODEFICIENT CHILDREN

Abnormal RNA migration profiles were found in numerous rotavirus samples from two chronically infected children suffering from severe combined immunodeficiency. In both cases additional bands consisting of dsRNA were found migrating between RNA segments 1 and 7, and in one case RNA segment 11 was lost from the profile. Hybridization studies using segment-specific cloned cDNA probes indicated that some of the additional bands had sequence homologies with normal rotavirus dsRNA segments. In most cases these sequences were derived from genome segments of lower molecular weight by the formation of covalently linked concatemers.

Aberrant expression of microRNA 155 may accelerate cell proliferation by targeting sex-determining region Y box 6 in hepatocellular carcinoma.

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.

Sequence analysis of two porcine rotaviruses differing in growth in vitro and in pathogenicity : distinct VP4 sequences and conservation of NS53, VP6 and VP7 genes

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.

Re-evaluation of the Carcinogenic Significance of Hepatitis B Virus Integration in Hepatocarcinogenesis

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.

Molecular biology of rotaviruses. IX. Conservation and divergence in genome segment 5

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.

Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes

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.