- Volume 75, Issue 5, 1994

Comparison of the matrix (M) protein gene sequences of various vaccine-associated mumps virus isolates reveals that in contrast to the SH gene the M gene is highly conserved and its sequence is probably not affected during virus attenuation.

Mouse infection with lactate dehydrogenase-elevating virus (LDV) leads to lifelong viraemia, despite the production of neutralizing antiviral antibodies. To test whether viral persistence correlated with the development of resistance to these antibodies, we compared the neutralization of viral particles derived from acutely and chronically infected animals, using polyclonal and monoclonal anti-LDV antibodies. Whereas virus isolated during acute infection was efficiently neutralized, titres of LDV from chronically infected mice were only slightly reduced by antiviral antibodies. In addition, LDV from animals acutely infected with such poorly neutralizable virus from chronically infected mice was resistant to anti-LDV antibodies like their parental viral particles. These results suggest that LDV variants capable of escaping neutralization by antiviral antibodies can emerge in chronically infected animals.

The carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (α-galactosyl) is abundantly expressed on cells of non-primate mammals, prosimians and New World monkeys, where it is synthesized by the enzyme α1,3-galactosyltransferase (α1,3GT). Old World monkeys, apes and humans lack α1,3GT and hence do not synthesize α-galactosyl epitopes. Instead, these species produce a natural antibody, anti-Gal, which interacts specifically with α-galactosyl epitopes and which constitutes up to 1% of circulating immunoglobulins in humans. We have used eastern equine encephalitis (EEE) virus as a model to examine the differential expression of α-galactosyl epitopes on the glycoproteins of virus propagated in cells that either produce or lack α1,3GT. As predicted, virus propagated in Vero cells (derived from the African green monkey, an Old World monkey) did not express α-galactosyl epitopes. In contrast, virus propagated in mouse 3T3 cells (EEE3T3) expressed approximately 80 α-galactosyl epitopes per virion on both the E1 and the E2 envelope glycoproteins. Thus, expression of the α-galactosyl epitope on virions paralleled that on host cells. The binding of anti-Gal antibody to these epitopes on EEE3T3 virions partially neutralized virus infectivity, raising the possibility that anti-Gal production in hosts may influence the initial infectious stage of viruses expressing α-galactosyl epitopes.

The non-structural glycoprotein NS1 of dengue virus type 2 contains sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 synthesized in infected cells is glycosylated at both locations. We have now examined the dimerization and secretion of NS1 lacking one or both of these sites by transient expression of mutagenized cDNA inserted into a simian virus 40-based vector. Immunoblotting and radioimmunoprecipitation were used to detect NS1 associated with transfected cells and in the extracellular medium. Elimination of one or both glycosylation sites did not abolish dimerization and secretion of NS1. However, NS1 lacking Asn-207 showed reduced dimer stability and secretion. Treatment of secreted NS1 with endoglycosidase H demonstrated that complex glycans were attached at Asn-130 and high-mannose glycans at Asn-207.

Flow cytometry and two-colour immunofluorescence were used to detect cytoplasmic bovine viral diarrhoea virus (BYDY) antigen in leukocytes from viraemic cattle. Monoclonal antibody to the p80 protein of BVDV, a non-structural viral antigen, was used to identify the subpopulations of leukocytes in which viral protein synthesis had occurred. Viral antigen was detected in 23 % of peripheral blood mononuclear cells. Monocytes were found to have the highest frequency of infection (35 %). A higher proportion of CD2+ T cells (23 %) were infected, compared with B cells (11 %) or WC1+ γδ T cells (11 %). No significant differences in percentages of different leukocyte subpopulations in blood were detected in persistently viraemic animals compared with controls.

The complete sequence of the spike (S) gene of the Brl/87 isolate of porcine epidemic diarrhoea virus (PEDV) was determined from cDNA clones. The predicted polypeptide was 1383 amino acids long, contained 29 potential N-linked glycosylation sites and showed structural features similar to those of the coronavirus spike protein. The PEDV S protein, like that of the members of the transmissible gastroenteritis virus (TGEV)-related subset, lacks a proteolytic site to yield cleaved amino and carboxy subunits S1 and S2. Viral polypeptide species of the expected M r, i.e. 170K/190K, were observed in PEDV-infected cells. Sequence comparison confirmed that, within the subset, PEDV was most closely related to the human respiratory coronavirus HCV 229E. However, PEDV S protein has an additional 250 residue N-terminal domain which is absent from HCV 229E and porcine respiratory coronavirus, the respiratory variant of TGEV. Alignment of the S1 regions revealed a second domain of about 90 residues with increased sequence divergence which might possibly express virus-specific determinants.

Both positive [( + )] and negative [(−)] sense versions of two satellite RNA (satRNA) genes from cucumber mosaic virus (CMV), the necrogenic I17N and the non- necrogenic R, have been introduced into the genome of tobacco plants. On infection with satRNA-free CMV, satRNA was amplified in plants expressing each of the four genes. All four genes confer protection against CMV infection. However, co-inoculation of plants with viral RNA and CMV satRNA transcripts synthesized in vitro showed that (−) sense transcripts were less active than the corresponding (+) sense transcripts. This is the first report that (−) sense CMV satRNA transcripts can serve as a template for satRNA replication.