- Volume 79, Issue 7, 1998

The ability of a bovine herpesvirus-1 (BHV-1) recombinant expressing the G protein of bovine respiratory syncytial virus (BRSV) to protect against BRSV infection was examined in calves. A synthetic G gene was inserted behind the gE promoter of BHV- 1 to give a gE-negative, BHV-1/G recombinant. Gnotobiotic calves, vaccinated intranasally and intratracheally with BHV-1/Gwere challenged 6 weeks later with the Snook strain of BRSV. As controls, calves were vaccinated with a gE-negative mutant of BHV-1 which contains a frame-shift (BHV-1/gEfs). Whereas infection with BHV-1/gEfs induced only mild clinical signs, infection with BHV-1/G resulted in more severe clinical disease and higher titres of BHV-1/G were isolated from the lungs when compared with BHV-1/gEfs. Thus, expression of the G protein of BRSV increased the virulence of BHV-1 for calves. Vaccination with BHV-1/G induced BRSV- specific antibody in serum and respiratory secretions. However, only one calf developed low levels of BRSV complement-dependent neutralizing antibody. Although BHV-1/G primed calves for BRSV- specific lymphocyte proliferative responses, there was no evidence for priming of BRSV-specific cytotoxic T cells. After challenge with BRSV, there was a significant reduction in nasopharyngeal excretion of BRSV in BHV-1/G-vaccinated calves compared with controls and BRSV was isolated from the lung of only one of five vaccinated calves compared with all four control animals. In addition, the extent of gross pneumonic lesions 7 days after BRSV challenge was significantly reduced in calves vaccinated with BHV- 1/G compared with controls given BHV-1/gEfs.

Plaque area is a measure of the degree of cytopathic effect and a predictor of neurovirulence for tissue culture adapted morbilliviruses. In the present work, the cellular stress response was shown to be a determinant of the expression of distinct measles virus large plaque phenotypes in Vero cells. The emergence of these large plaque phenotypes was associated with increased mean viral transcriptional activity and expression of the viral fusion glycoprotein, but not upregulation of the virus receptor CD46.

The nucleoprotein (N) gene of Hendra virus (HeV), an unclassified member of subfamily Paramyxovirinae in the family Paramyxoviridae previously known as equine morbillivirus, was cloned and sequenced. The majority of the deduced amino acid sequence was further confirmed by direct sequencing of peptide fragments of the N protein derived from purified virions. The 3′ untranslated sequence of the HeV N gene mRNA was 568 nt and was much longer than that observed in other Paramyxovirinae. The N protein was 532 amino acids in length with a molecular mass of 58·5 kDa. Although the HeV N protein had a slightly higher amino acid sequence identity to those of the genus Morbillivirus than to those of other Paramyxovirinae genera, the level of identity was much lower than that observed within the morbilliviruses. Our results indicated that HeV could not confidently be classified as a member of the genus Morbillivirus, Paramyxovirus or Rubulavirus and suggest that the virus be classified in a new genus within the Paramyxovirinae.

A monoclonal antibody, LMBH6, was derived from mice which had been sequentially immunized with bromelain-cleaved haemagglutinin (BHA) from influenza virus A/Aichi/2/68, A/Victoria/3/75 and A/Philippines/2/82 (all H3N2). LMBH6 recognizes the haemagglutinin (HA) of all H3N2 influenza A strains tested, which were isolated between 1968 and 1989. HA in the low-pH-induced conformation is not recognized, and cleavage of the HA0 precursor to HA1 and HA2 is needed to obtain efficient binding. Compared to other monoclonal antibodies, binding of LMBH6 to virus and to virus-infected cells is weak, while binding to BHA is comparable. Electron microscopy demonstrates binding to the membrane proximal end of the stem structure. The antibody shows no haemagglutination-inhibition activity, but inhibits polykaryon formation and the low-pH- induced conformational change of BHA. However, LMBH6 cannot prevent infection of MDCK cells but slows the growth of virus when included in a plaque assay overlay.

Human immunodeficiency virus type 2 (HIV-2) strains that infect cells in the absence of cellular CD4 emerge spontaneously in vitro after culture in CD4 T-cell lines. The HIV-2ROD/B strain can use the CXCR4 chemokine receptor for efficient entry into CD4 cells. Here we have shown that the rat homologue of CXCR4, in the absence of CD4, failed to mediate CD4-independent entry by ROD/B. Furthermore, using rat-human chimeric CXCR4 receptors we have demonstrated that the second extracellular loop (E2) of human CXCR4 is critical for HIV-2 infection of CD4 cells. E2 is also important for HIV-1 infection of CD4 cells. Our results therefore indicate that the role of E2 in HIV entry is conserved for HIV-1 and HIV-2 and for infection in the presence or absence of CD4.

Deletion of the simian immunodeficiency virus (SIV) nef gene leads to an attenuated virus phenotype in vivo. We have previously shown that these viruses induce a potent cellular immune response in macaques. To extend these studies, we established virus-specific short-term T-cell lines from four rhesus macaques infected with a nef deletion mutant of SIV. These T-cell lines proliferated upon restimulation with whole SIV or SIV gp140 antigen in vitro. The proliferating cells were characterized as CD4 helper T-cells (TH) and their antigen recognition was MHC class II DR-restricted. After antigenic stimulation, they transcribed mRNA for various TH1- and TH2-like cytokines. Using these SIV-specific cell lines, a variety of helper T-cell epitopes in the SIV Env protein were determined with overlapping peptides. TH epitopes were identified throughout the whole SIV Env including both constant and variable regions. Although the recognition of TH epitopes was heterogeneous among different animals, five more broadly reactive T-cell epitopes were identified. As expected, recognition was associated with the MHC class II DRB background of the animals. This is the first report on helper T-cell epitopes in SIV-infected monkeys. Such studies should be of considerable significance for AIDS/ vaccine research.

Simian immunodeficiency viruses (SIVs) have been shown to infect many Old World African primate species. Thus far, no work has been published on southern African primates. In this study we investigated the genetic diversity between SIV strains from Kenyan and South African vervets (Cercopithecus aethiops pygerythrus). We amplified and sequenced a 1113 bp region of the env gene. Phylogenetic analysis of these sequences showed that all strains clustered with members of the vervet subgroup of SIVagm. The SIVs from South African vervets differed by 7% from each other and by 8–14% from the Kenyan SIV strains, while the Kenyan SIV strains differed by 10–21% from SIVagm of other east African vervets. We also isolated and sequenced, for the first time, a SIV strain from a healthy chacma baboon (Papio ursinus), caught in South Africa. Phylogenetic analysis of the env region showed the virus to be closely related to the South African vervet SIV strains, while analysis of its pol region confirmed the virus to be a SIVagm variant.

Prunus necrotic ringspot virus (PNRSV) occurs as numerous strains or isolates that vary widely in their pathogenic, biophysical and serological properties. Prior attempts to distinguish pathotypes based upon physical properties have not been successful; our approach was to examine the molecular properties that may distinguish these isolates. The nucleic acid sequence was determined from 1·65 kbp RT-PCR products derived from RNA 3 of seven distinct isolates of PNRSV that differ sero-logically and in pathology on sweet cherry. Sequence comparisons of ORF 3a (putative movement protein) and ORF 3b (coat protein) revealed single nucleotide and amino acid differences with strong correlations to serology and symptom types (pathotypes). Sequence differences between serotypes and pathotypes were also reflected in the overall phylogenetic relationships between the isolates.

Purified virions of bamboo mosaic potexvirus-V isolate (BaMV-V) were found to contain three major RNA species, the 6·4 kb genomic RNA and two RNAs of 2·0 and 1·0 kb, in addition to associated satellite RNA (0·85 kb). Results of Northern blot hybridization, primer extension analysis and cDNA sequencing showed that the packaged 2·0 and 10 kb RNAs of BaMV-V were subgenomic RNAs. In contrast, in the BaMV-O isolate, only genomic RNA was packaged and encapsidated subgenomic RNAs were not detectable. The transcription initiation sites for the 2·0 and 1·0 kb subgenomic RNAs of BaMV-V were located 11 and 16 nt upstream of the initiation codon of open reading frames (ORFs) 2 and 5, respectively. The 2·0 and 1·0 kb subgenomic RNAs functioned as messengers for the ORF2 protein and capsid protein, respectively. Packaging of the 1·0 kb subgenomic RNAs resulted in the formation of rodshaped particles about 70 nm in length. Our results indicate that BaMV isolates have evolved distinctly for packaging of subgenomic RNAs.