Ian H. Holmes

Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis.

Abstract Electron microscopy of duodenal mucosa from nine children with acute non-bacterial gastroenteritis revealed virus particles in epithelial cells from six patients. The morphology of the virus particles was identical in each of the six children. The virus belonged to the orbivirus group. No virus particles were observed in duodenal mucosa obtained from three of these children after clinical recovery. This orbivirus is believed to have been an important cause of sporadic gastroenteritis in children in Melbourne during the 3 months of the survey.

DETECTION OF A NEW VIRUS BY ELECTRON MICROSCOPY OF FÆCAL EXTRACTS FROM CHILDREN WITH ACUTE GASTROENTERITIS

Abstract Electron microscopy of negatively stained faecal extracts revealed particles resembling orbiviruses in 11 of 14 children with acute non-bacterial gastroenteritis. No such particles were observed in faecal extracts from 9 control children. The method of differential centrifugation for extraction of faeces is simple, quick, and more sensitive than duodenol biopsy for detecting the virus. Combining results from duodenal mucosa and faeces, orbivirus particles were observed by electron microscopy in 81% of children with sporadic gastroenteritis in Melbourne.

Sequential partially overlapping gene arrangement in the tricistronic S1 genome segments of avian reovirus and Nelson Bay reovirus: Implications for translation initiation

ABSTRACT Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the ςC viral cell attachment protein initiates over 600 nucleotides distal from the 5′ end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3′-proximal ORF present on the NBV S1 genome segment also encodes a ςC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the ςC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and ςC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream ςC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal ςC start site.

Bunyaviruses and Bunyaviridae

A new family is described, the Bunyaviridae, which contains a single genus, Bunyavirus. The main characteristics of the family are as follows: single-stranded RNA, total molecular weight about 7 X 10(6) daltons, probably in three segments. Virions spherical, enveloped particles 90-100 nm in diameter. Envelope contains at least one virus-specified glycopeptide. Develop in the cytoplasm, mature by budding into smooth-surfaced vesicles in the Golgi region or nearby. Internal ribonucleoprotein composed of long strands 2-2.5 nm broad. There are at least 150 members, 87 serologically related bunyaviruses and other probable bunyaviruses.

IS LACTASE THE RECEPTOR AND UNCOATING ENZYME FOR INFANTILE ENTERITIS (ROTA) VIRUSES

Rotaviruses are now regarded as important causes of diarrhoea in man, cattle, pigs, mice, and possibly other animals. Characteristically, disease occurs in newborn and young animals, and infection seems limited to the differentiated gut epithelial cells. The major surface polypeptide of the calf scours rotavirus is glycosylated, and highly purified beta-galactosidase (lactase) interacts with the virus in vitro causing removal of the outer shell of the capsid (uncoating). It is suggested that lactase present in the brush border of the intestinal epithelial cell performs a similar function in vivo by acting as a combined receptor and uncoating enzyme for the rotavirus. This hypothesis is consistent with the observations that rotaviruses seem to infect only gut epithelial cells, and that infant animals, whose lactase concentrations are generally higher than those of adult animals, seem more susceptible to rotavirus infections. Implications of the hypothesis include possible new approaches to laboratory cultivation of rotaviruses, which should be more successful in cells selected for surface lactase activity, and the suggestion that the epidemiology of human rotavirus infections may be influenced by the fact that different ethnic groups have different lactase levels (and hence lactose intolerance) in adulthood.

Glycosylation, an important modifier of rotavirus antigenicity.

SummaryMutants of a non-glycosylated strain of SA 11 rotavirus (clone 28), were selected using a monoclonal antibody directed against the VP 7 protein. These mutants possessed an amino acid substitution at residue 238 of VP 7, whereas mutants of wild type SA 11 selected with the same antibody have previously been shown to contain a substitution at residue 211 (i.e., in the antigenic C region). In both cases the mutations produce new potential glycosylation sites, and these were found to be utilized. The mutations also lead to gross antigenic changes, and these were found to be reversible upon removal of the attached carbohydrate. The results suggest an important role for carbohydrate in influencing the exposure of antigenic determinants of the rotavirus serotype-specific protein, VP 7.

Is rubella an arbovirus? II. Ultrastructural morphology and development.

Abstract The morphology of rubella virus and its development in BHK21 and RK13 cells has been studied by electron microscopy. In thin sections, rubella virus has an electron-dense core 30 mμ in diameter, and a total diameter of about 60 mμ. The virus develops by budding through the cytoplasmic membrane or sometimes into cytoplasmic vacuoles. By negative staining, small spikes projecting 5–6 mμ from the viral envelope can be seen. The negatively stained particle tends to be somewhat deformed by drying, but after fixation in isotonic osmium fixative the particle appears spherical and 60–70 mμ in diameter. Both in morphology and development rubella closely resembles certain arboviruses, although there are size differences. It has here been compared directly with Semliki Forest virus and Bunyamwera virus, and the classification of all these viruses is discussed.

Nucleotide sequence of uk bovine rotavirus segment 4: Possible host restriction of VP3 genes

The bovine UK and simian SA11 rotaviruses are commonly used VP7-type reference strains. Since the surface protein VP3 is a significant neutralization antigen, it is important to fully characterize the VP3 types associated with current reference strains. Here we present the complete nucleotide and predicted amino acid sequence of VP3 from UK rotavirus (VP7 type 6) and compare it to the published sequences of SA114fm and RV-5. We also compare the deduced amino acid sequence covering the trypsin cleavage region of UK VP3 to 25 other available sequences. The UK protein is clearly different from that of bovine NCDV (another commonly used VP7 type 6 strain) and represents a second VP3 type associated with bovine rotaviruses. Our SA11 sequence differs from that determined by Lopez et al. [1985, Virology 144, 11-19; later referred to as SA114fM by Lopez et al. (1986, Virology 154, 224-227], their sequence being very similar to the published sequence of NCDV VP3. The significance of these results with regard to virus serotypes is discussed. Finally, in analyzing the nucleotide sequence surrounding the initiation codon, a potential hairpin-loop structure was identified which may be involved in translational regulation.

Characterisation of G serotype dependent non-antibody inhibitors of rotavirus in normal mouse serum

SummarySerotype specific (non-immunoglobulin) inhibitors of rotavirus have been identified in normal mouse serum obtained from BALB/c, CBA, and BL10 mice. Sialic acid was essential for the neutralising activity as sera treated with the neuraminidase from Vibrio cholerae failed to neutralise rotavirus. G serotypes 4, 5, 7, 8, 9, & 10 were unaffected by the inhibitor(s) while G serotypes 1, 2, 6 and two G3 strains were neutralised to significant titres. Assessment of neutralisation of reassortants suggested that VP7 is the virus protein involved in the interaction although it remains possible that VP7 is influencing VP4 binding. Analysis of the sera by Western blot followed by virus overlay confirmed that binding is dependent on the presence of sialic acid. The human strain tested, Wa, bound to two (glyco)proteins (50 & 80 kDa) while the bovine strains tested, NCDV and UK bound to one (55 kDa) and two (36 &55 kDa) proteins respectively. This indicates that while the bovine rotaviruses may bind to a common element, the human strain binds to clearly distinct proteins. We propose that these inhibitors interact with animal rotaviruses in a manner analogous to that by which they attach to target cells. The glycoprotein to which NCDV bound was purified and identified by N-terminal sequencing as murine alpha-1-anti-trypsin (MuAAT) and was confirmed to possess both neutralisation and anti-trypsin activity. Since MuAAT is known to possess only three N-linked glycans, identification and analysis of the actual virus-binding structure should now be possible.

The major surface glycoprotein of simian rotavirus (SA11) contains distinct epitopes.

The polypeptide specificities on monoclonal antibodies previously derived against the SA11 simian, NIC bovine, and Wa human strains of rotavirus were determined by radioimmunoprecipitation of infected cell lysates. All the monoclonal antibodies derived using NIC and Wa were found to be directed against the major component of the inner capsid, while most of the SA11 monoclones were directed against the major outer capsid glycoprotein. When several SA11 glycoprotein-specific monoclonal antibodies were used in competitive binding studies, four distinct epitopes, which correlated with the functional activities of the antibodies, were defined. One epitope appeared most critical for virus neutralization, another was involved to a lesser extent, and the remaining two epitopes seemed to have no role. A possible topographical arrangement of these epitopes is suggested.