- Volume 77, Issue 3, 1996

Specific pathogen free (SPF) domestic cats were inoculated with tissue homogenate obtained from a Chinese leopard (Panthera pardus japonensis) that had died in a North American zoo from a natural infection with canine distemper virus (CDV). The cats developed a transient cell-associated CDV viraemia along with pronounced lymphopenia but did not show any clinical symptoms. Plasma neutralizing-antibody titres against the homologous CDV (A92-27/4, isolated from the Chinese leopard) were consistently higher than against the CDV vaccine strain ‘Bussell’. The Chinese leopard CDV isolate showed in vitro biological properties reminiscent of virulent, wild-type CDV strains. Sequence analysis of the H gene of two large felid CDV isolates from the USA (A92-27/4 and A92-6) revealed up to 10% amino acid changes including up to four additional potential N-linked glycosylation sites in the extracytoplasmic domain as compared to CDV vaccine strains. Phylogenetic analysis was performed using the entire coding region of the H gene and a 388 bp fragment of the P gene of several morbillivirus species. Evidence was obtained that recent CDV isolates from different species in the United States (including isolates from large felids), Europe and Africa are significantly distinct from CDV vaccine strains. All wild-type CDV isolates analysed clustered according to geographical distribution rather than to host species origin. By sequence analysis a CDV epizootic among large felids in a Californian safari park was linked to a virus which most likely originated from feral non-felid carnivores.

Ross River virus (RRV) is the aetiological agent of epidemic polyarthritis (EPA) a predominantly rheumatic disease afflicting up to 5000 Australians annually. We show here for the first time that macrophages can be productively infected by RRV. Subneutralizing titres of anti-RRV IgG (but not IgM) also showed classical antibody-dependent enhancement (ADE) of RRV infection in macrophage and monocyte cell lines. No correlation between development of EPA and the preexistence of ADE titres was apparent, nor could sera raised against a related arbovirus, Barmah Forest, enhance RRV infection. Tumour necrosis factor-α, implicated in the immunopathogenesis of rheumatoid arthritis, was not secreted by RRV-infected monocytes or macrophages. Macrophage cell lines infected with RRV were, however, capable of producing virus for over 50 days. RRV-induced arthritis may therefore be due to the persistent productive infection of macrophages, perhaps established by a brief period of ADE early in infection.

Sequencing of the S1 genes of nine Australian strains of infectious bronchitis virus (IBV) identified two genotypically distinct groups of strains. The strains Vic S, V5/90, N1/62, N3/62, N9/74 and N2/75 comprised group I, sharing 80.7–98.3% identity in their deduced amino acid sequences. All group I strains were able to replicate in the trachea and kidney but only four strains, Vic S, N1/62, N9/74 and N2/75, were nephropathogenic, the latter three causing mortalities ranging from 32 to 96%. Group II contained strains N1/88, Q3/88 and V18/91 which only replicated in the trachea, inducing no mortalities. These viruses showed 72.3–92.8% amino acid identity to each other and only 53.8–61.7% identity to viruses of the first group. They were also distinct from the Massachusetts 41 and D1466 strains (47.5–55.7% amino acid identity). Thus N1/88, Q3/88 and V18/91 form a new group of viruses which are genotypically distinct from all previously characterized IBV strains. No definite correlations were established between the S1 amino acid sequences and the nephropathogenicity of strains.

Inhibitors of the human immunodeficiency virus (HIV) protease are a promising class of antiviral agents that dramatically reduce HIV replication both in culture and in infected patients. However, as for many other antiviral compounds, long-term efficacy of these agents is impeded by the emergence of virus variants with increased resistance to their inhibitory action, following selection of specific mutations in the protease coding sequence. We have studied HIV-1 variants that emerged at different stages of selection in the presence of the C2-symmetrical protease inhibitor ABT-77003. The selection of variants was a gradual process during which mutations accumulated at different sites in the protease, generating virus populations with increasing levels of resistance to the drug. The initially selected viruses had a low level of resistance as well as a markedly reduced replicative capacity. Further accumulation of mutations at secondary sites led to an improvement in both drug resistance and replication. In spite of their reduced infectivity, partially selected virus populations did not readily revert to wild-type when serially passaged in drug-free conditions. Instead, even in the absence of drug, secondary mutations identical to those selected in the presence of the inhibitor continued to emerge. These mutations improved both the intrinsic replicative capacity of the virus and its level of resistance to the inhibitor, suggesting that once committed to drug resistance, readaptation of the enzyme to its natural substrate leads to a reduction of its sensitivity to the inhibitor.

The vif gene (viral infectivity factor) of the human and simian immunodeficiency viruses (HIV and SIV) is present in almost all members of the lentivirus group of retroviruses. This gene is highly conserved among different HIV and SIV isolates and is therefore presumed to play an important role in pathogenesis. To analyse the role of Vif in SIV, three SIVmac mutants have been constructed by introducing site-specific mutations or deletions into vif of the pathogenic molecular clone SIVmac239. The effect of Vif on viral replication in T cells was examined by transfecting equal amounts of either vif-positive or vif-negative viral DNA into SupT1, CEM-SS and H9 cells. Reverse transcriptase assay of supernatants from transfected cultures revealed that both SupT1 and CEM-SS cell lines supported replication of all three vif mutants to a level comparable to the parental vif-positive virus, whereas vif mutants did not replicate in H9 cells. Our results demonstrate that the requirement for Vif in SIVmac replication is cell-type dependent and that sequences near both the N and C termini are required for its function. Vif-defective SIVmac239, produced in transfected SupT1 and CEM-SS cells, failed to infect primary T lymphocytes, whereas both vif-positive and vif-defective viruses established productive infection in CEMx174 cells. These findings in primary cells suggest that Vif plays an important role in viral replication in vivo.

Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological control of a persistent lentivirus infection and for evaluating the efficacy of various vaccine strategies. As an initial characterization of antibody responses associated with protective or enhancing immune responses elicited by experimental infections or vaccinations, we have utilized synthetic peptide ELISA to characterize the fine specificity of antibodies to linear determinants of the EIAV surface glycoprotein, gp90. The data indicated that serum antibodies associated with protective or enhancing immune responses differed quantitatively and qualitatively in their pattern of reactivity to gp90 peptides. Protective and enhancing EIAV vaccines could also be distinguished by their ability to evoke anamnestic antibody responses to gp90 peptides. These studies demonstrate for the first time definitive differences in the specificity of protective and enhancing antibody responses to EIAV and emphasize the importance of using native viral glycoprotein immunogens in lentivirus vaccines.

Moloney murine leukaemia virus (MoMLV) markedly accelerates thymic lymphoma development in mice carrying a transgene in which the human c-myc gene is linked to the CD2 locus control region. To investigate the mechanism of synergy and identify the genes which collaborate with myc in these clonal tumours, we analysed the sites of MoMLV insertion. Analysis of known viral integration loci revealed only a small number of insertions at bmi-1, pim-1 and ahi-1. Further cloning and hybridization analysis revealed a new common integration locus, designated til-1, which was occupied in 25 out of 77 lymphomas examined, with evidence of multiple clonal insertions in some cases. Mapping relative to established chromosomal markers in interspecific backcross mice located til-1 to mouse chromosome 17, distal to pim-1 and tic-1. These results suggest that the til-1 locus may harbour a novel myc-collaborating gene which acts as a target for activation in T cell lymphomas.

Many tumour cells are killed by the lytic replication of the autonomous parvoviruses H-1 and minute virus of mice (MVMp), whereas most untransformed cells (although they take up these viruses efficiently) are resistant, i.e. they do not produce infectious virus and are not lysed. Therefore, cells able to continuously produce large quantities of infectious virus have not yet been described. We have isolated such cells from the resistant cell line FR3T3 (Fisher rat fibroblast). These cells (called FR3T3C) produce infectious MVMp virions without being detectably lysed. Furthermore, a persistently infected population (R100FR3T3C) was generated by repetitive infection of FR3T3C cells with MVMp. Indeed, R100FR3T3C cells were successfully cultivated for two years and continuously produced infectious virus. Seventeen clones of R100FR3T3C cells isolated by limiting dilution produced infectious virions, indicating that in the R100FR3T3C cell population, virus production was not limited to a few cells. These cell lines may be useful for the production of MVMp and for the generation of a cell line for the packaging of recombinant viral genomes.

A short non-coding region (SNR) commonly exists between the E5 and L2 open reading frames of human papillomaviruses (HPVs). Except for the poly(A) signal for early gene transcripts, no biological functions have been discovered for the SNR. To test a possible promoter-like activity of the SNR, we carried out CAT reporter assays using constructs containing the SNRs from HPV-16, -18 and -33 linked to a promoterless CAT gene. We reproducibly observed enhanced expression of CAT gene by the SNRs. Co-expression of a transcriptional activator (LAP/NF-IL6) or deletion of the poly(A) signal augmented the promoter-like activity of the SNRs. RNase protection assays revealed a LAP-inducible CAT mRNA properly initiated from the HPV-16 SNR. These results may suggest that the SNR has a promoter activity that is regulated by keratinocyte differentiation.

The mechanism by which the E6 protein of human papillomavirus type 16 (HPV-16) transactivates heterologous virus promoters has not been established. In this study, the involvement of p53-mediated transcriptional repression in transactivation by the HPV-16 E6 protein was examined using several virus promoters. HPV-16 E6 transactivated the TATA box-containing simian virus 40 early promoter and the Rous sarcoma virus long terminal repeat in p53-containing cells but not in p53-deficient cells. In contrast, the adenovirus E2 promoter was transactivated both in p53-containing and p53-deficient cells. These results indicate that the transactivation activity of the HPV-16 E6 protein is mediated by p53-dependent and promoter-specific p53-independent pathways.

The major capsid protein L1 of human papillomaviruses (HPVs) has been identified as a promising candidate antigen for a prophylactic HPV vaccine. Since amino acid sequence heterogeneity has been demonstrated for the L1 genes within individual HPV types, nucleotide sequences for L1 were determined from six HPV-18 clinical isolates and the cervical carcinoma cell line SW756 and compared to the published HPV-18 prototype sequence. The sequences were almost identical between the clinical isolates and SW756 but differed markedly from the published prototype sequence. Resequencing the prototype HPV-18 revealed that these differences were due to sequencing artifacts of the prototype HPV-18 sequence archived in GenBank. Thus, the HPV-18 L1 genes seem to display a very high level of sequence conservation. The HPV-18 L1 gene derived from SW756 was expressed in Saccharomyces cerevisiae and self-assembly of the L1 protein into viruslike particles was demonstrated.

Haemorrhagic enteritis virus (HEV) is a member of a genetically ill-defined group within the genus Aviadenovirus which causes significant clinical disease in gallinaceous fowl. Using DNA obtained from a low virulence isolate of HEV passed in turkeys, we developed a genomic restriction map and estimated an apparent genomic length of 25.5 kb. No evidence for extensive DNA hybridization was found between the HEV genome and either the hexon or penton base genes of human adenovirus 2 (HAdV-2) and fowl adenovirus 10 (FAdV-10). The HEV penton base gene was identified by PCR using primers based on conserved adenoviral DNA sequences. The penton base gene was expressed in Escherichia coli as a fusion protein and detected by anti-HEV serum in both colony and denaturing gel immunoblots. DNA sequencing revealed a putative penton base ORF with a predicted amino acid sequence showing approximately 39.0%, 53.0% and 44.2% similarity with the penton base of HAdV-2, human adenovirus 40 (HAdV-40) and FAdV-10, respectively. The penton base gene was located at 43.3–48.6 m.u. on the HEV genome and had a remarkably low G+C content (33.8%). DNA sequencing also revealed ORFs for putative core proteins resembling pVII, p-mu and a partial ORF similar to pVI (hexon-associated protein) of HAdV-2 and HAdV-40. The results support the claim that HEV represents a distinct group of viruses within the genus Aviadenovirus.

The published sequence of the herpes simplex virus (HSV) type 2 strain HG52 neurovirulence gene RL1 indicated that, unlike the HSV-1 homologue, the gene contains a 154 bp intron. This intron contains six complete and one partial copy of a 19 bp repeat which encodes a stop codon; thus all three forward frames are blocked. By RT-PCR of poly(A)+ RNA, using primers on either side of the proposed intron, we have isolated a partial cDNA clone corresponding to the predicted spliced mRNA.

Previous studies have indicated that suberythemal ultraviolet B (UV-B) irradiation of C3H mice before primary infection with herpes simplex virus (HSV) type 1 does not result in increased morbidity or mortality, but a suppressed delayed type hypersensitivity (DH) to the virus can be demonstrated. Any effect of UV radiation on pathogenesis during secondary epidermal HSV infection has not been previously examined. Mice were immunized by subcutaneous injection of inactivated HSV and, 5 days later, one group was UV-B-irradiated. The next day all mice were challenged epidermally with HSV. Most of the mice (92%) in the irradiated group developed severe lesions, whilst 59% of the non-irradiated group had mild lesions and 30% no lesions. Infectious virus was not isolated from the adrenal glands after challenge in either group. In addition, the DH to the virus was not affected by the UV exposure. The numbers of lymphocytes and dendritic cells in the lymph nodes draining the site of epidermal infection were increased in the UV group compared with the non-irradiated group. Following challenge, the percentage of CD4+ and CD8+ lymphocytes in lymph nodes was unaltered but the MHC class II expression on dendritic cells in these lymph nodes was reduced by UV exposure. The lymphoproliferative response in vitro of lymph node cells revealed a suppressed response to HSV and to the mitogen concanavalin A in the irradiated group. Thus, UV irradiation prior to epidermal secondary infection with HSV led to more severe infections due, perhaps, to a modulation in local antigen presentation.

Equine herpesvirus type 1 (EHV-1) gene 71 encodes a heavily O-glycosylated 192 kDa protein with no identified herpesvirus homologue. Isolation of a deletion mutant in gene 71 (ED71) demonstrated that its protein product is not essential in vitro. To investigate the role of the gene 71 protein in the virus life cycle, ED71 has been characterized in vitro in terms of cellular adsorption, penetration, egress and transmission compared to wildtype and revertant virus. ED71 virions adsorbed to cells less efficiently than wild-type and revertant virus with a consequential effect on virus penetration; virus egress was significantly impaired and the timing of release was also delayed. The percentage of both full and empty capsids accumulating in the nuclei of ED71-infected cells was significantly higher than in wild-type virus-infected cells but the most notable differences were the low number of particles and the low ratio of enveloped to unenveloped capsids in the cytoplasm. The primary mode of transmission of the mutant virus is by direct cell-to-cell spread and the fact that a neutralizing antiserum did not reduce ED71 plaque size, supported the conclusion that deletion of gene 71 impairs the ability of virus to spread via release and readsorption to uninfected cells. Thus, deletion of EHV-1 gene 71 results in a defect in virus maturation and capsid envelopment. Progeny virus is consequently impaired in adsorption/penetration presumably due to the particles lacking the glycoprotein spikes predicted to be encoded by this gene and hence spreads by direct cell-to-cell contact.

The Epstein—Barr Virus (EBV) gene BZLF1 encodes the transcription factor EB1 (also known as Zta) which is essential for the switch from latency to the lytic cycle: EB1 expressed from a plasmid transfected into B cell lines carrying latent EBV episomes, induces a productive viral cycle. Furthermore, EB1-specific DNA-binding sequences (ZREs) have been found in the promoters of many EBV early genes, including the BZLF1 promoter PZ and the PR promoter. At promoter PR, bicistronic mRNAs are initiated which contain, from 5′ to 3′, the BRLF1 and the BZLF1 open reading frames (ORFs) encoding respectively the R and EB1 proteins. The current model for the activation of the EBV lytic cycle implies that downregulation of the PZ promoter activity is a key element for latency and that a limiting step in the activation of the productive cycle is the translation of EB1. Once made, EB1 autoactivates promoter PZ, activates the PR promoter at which an mRNA coding for the EBV transcription factor R is initiated and activates the EBV early genes and the ORIlyt, due to unrestricted accessibility of the EB1-responsive elements in the viral genome. We show here that EB1 expressed from a plasmid activated most if not all of the EBV early genes in the viral genome but not its own gene, BZLF1. Moreover, transfected EB1 induced the transcription of the bicistronic mRNAs from which R is efficiently translated but not EB1. Our results demonstrate that EB1 provided in trans, although competent to activate the productive cycle genes, was not sufficient to overcome the downregulation of the PZ promoter.

The reactivity of human cord blood sera was directed most frequently in Western blot assays to a protein with an apparent molecular mass of 85 kDa that belongs to the p85 complex, a family of antigenically related proteins identified previously in our laboratory with the aid of two MAbs. We show that the 85 kDa protein is phosphorylated. As antibodies present in the human sera were directed in part to proteins carrying cross-reactive epitopes between human herpesvirus 6 (HHV-6) and 7 (HHV-7), it is remarkable that reactivity to the 85 kDa phosphoprotein was maintained after preabsorption of the sera with HHV-6 antigen, but abolished after preabsorption with HHV-7 antigen. Therefore, the 85 kDa phosphoprotein may be considered a major determinant of the human immune response to HHV-7, discriminating HHV-6 from HHV-7 infection.