- Volume 74, Issue 10, 1993

Interferon gamma (IFNγ) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFNγ cannot be responsible for all cytokine effects during recovery from MCMV infection we have now tested the potential participation of tumour necrosis factor alpha (TNFα) in the antiviral defence. Neutralization of endogenous TNFα abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-deficient mice. These data suggest that the antiviral effect of the CD4 subset requires the presence of at least two cytokines, namely IFNγ and TNFα. Depletion of endogenous TNFα in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNFα also participates in CD8 T cell effector functions. Furthermore, endogenous cytokines were found to be required for survival after infection with lethal doses of MCMV, whereas immunotherapy with recombinant TNFα and IFNγ could not limit virus replication in vivo. The results suggest that, similar to IFNγ, TNFα is an integral part of the protective mechanisms involved in cytomegalovirus clearance.

The distribution of glycoprotein B (gB) among different human herpesvirus 6 (HHV-6) strains was analysed with a panel of three monoclonal antibodies (MAbs) derived from mice immunized with U1102-infected lymphocytes. MAb 2D9 reacted specifically by immunofluorescence and immunoprecipitation with the U1102 and GS isolates, and failed to react with Z29 and the variant B strains Hashimoto and SF. In addition, Z29, Hashimoto and SF gB had a lower M r than U1102 and GS gB. MAb 2D9 also failed to react with the exanthem subitum isolate CV, included in this study as an as yet poorly characterized isolate. Consistent with this result, CV failed to react with the variant A-specific MAb to gp82-105 and behaved as a variant B virus even with respect to the diagnostic HindIII endonuclease restriction cleavage site located in a fragment hybridizing to the pZVH14 probe. By contrast with MAb 2D9, MAbs 2B9 and 2D10 reacted with all of the isolates tested, strengthening the argument that they have common epitopes. Based on the antigenic and M r specificities of gB, the HHV-6 isolates tested were arranged into two non-overlapping clusters, which closely parallel the variant A and B strain groups, defined previously by several criteria, including restriction endonuclease polymorphism, antigenic variations, growth in in vitro cultures and sequence analyses.

The roles of topoisomerases I and II in Epstein—Barr virus (EBV) replication were investigated using Raji cells infected with EBV. The topoisomerase II inhibitor ellipticine inhibited the synthesis of EBV polypeptides at concentrations which did not affect total protein synthesis. Slot blot analysis of total cellular DNA showed that camptothecin and ellipticine inhibited replication of progeny EBV DNA in superinfected Raji cells at concentrations which did not inhibit synthesis of EBV early polypeptides prerequisite for EBV DNA replication. Analysis of the structure of EBV DNA termini demonstrated that both inhibitors affected the replicating EBV DNA. Gardella gel electrophoresis showed that both inhibitors affected the formation of the linear form of EBV DNA. However, restriction analysis of EBV DNA in superinfected Raji cells demonstrated that both inhibitors degraded neither endogenous nor exogenous EBV DNA. Cell viability was not affected by either inhibitor at the concentrations tested. These findings suggest that topoisomerase II is required for expression of the EBV genome and that both topoisomerases I and II are involved in replication of the EBV genome during the lytic phase of the life cycle. The effects of topoisomerase inhibitors on the circular form of EBV DNA during virus replication are discussed.

The goal of experiments reported here was to identify the genes that encode capsid proteins VP21 and VP24 of herpes simplex virus type 1 (HSV-1). Capsids were isolated from infected cells and the proteins were separated by SDS-PAGE. N-terminal amino acid sequence analysis of partial CNBr digestion products, and of intact VP21, showed that it is encoded within the UL26 open reading frame (ORF) of HSV-1 beginning with codon 248 and probably extending to the end of the ORF (codon 635). Similar analysis of digestion products confirmed that VP24 is specified by codons 1 to 247 at the 5′ end of the UL26 ORF. Each of the seven known capsid proteins has now been assigned to an ORF.

Bovine fetal palatine tissue infected with bovine papil-lomavirus type 4 (BPV-4) was implanted subcutaneously in athymic nude mice. The implants developed into cysts containing papillomas essentially the same as those in the natural host. In order to investigate the interaction of cocarcinogens with BPV-4 in cell transformation, the virus-infected implants were exposed in vivo to either the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the tumour initiator 7,12-dimethylbenz[a]-anthracene (DMBA). Papillomas were detected in a greater number of infected implants in the presence of either TPA or DMBA than in the absence of either of these chemicals indicating interaction between the virus and these two agents. Moreover, malignantly transformed cells arose with high frequency from infected implants that had been exposed to either chemical. In the presence of chemical and absence of virus or vice versa no neoplastic changes were seen histologically, indicating that cooperation between virus and cocarcinogen is required for transformation.

A phylogenetic tree based on the VP1 sequences of type O foot-and-mouth disease virus (FMDV) has been derived. Direct sequencing of PCR products has been used to obtain the VP1 gene sequences of new isolates. The tree exhibits four main lineages that largely correlate with the geographical origin of isolates. The analysis supports a close relationship between European O1 field isolates and vaccine strains, with the exception of O Thalheim Aus/81 and O Wuppertal Ger/82 which were probably of non-European origin. Analysis of nucleotide substitutions indicates that synonymous mutations play a major role in FMDV evolution.

Amino acid sequence comparisons between the capsid proteins of several human rhinovirus (HRV) serotypes identified residues potentially involved in the discrimination between the major and the minor group receptors. Amino acids conserved within minor group HRVs were substituted in a full-length cDNA clone of HRV2 for those found at equivalent positions in major group HRVs. Transfection of HeLa cells with RNAs transcribed from seven individual mutated cDNAs gave rise to only two viable viruses; growth characteristics and affinity for the minor group receptor of both were unchanged compared to wild-type. Similar mutations in HRV14 were previously shown to alter the affinity for its receptor; the contact sites between the minor group viruses and the respective receptor may therefore be different.

Analyses of the mRNA transcription processes of viruses in four genera (Bunyavirus, Hantavirus, Phlebovirus and Tospovirus) of the family Bunyaviridae have revealed a common mechanism of initiation using host-derived primers, known as cap-snatching. To provide similar information on the fifth genus in the family, the 5′ ends of Dugbe nairovirus S mRNA species were specifically cloned and sequenced. This revealed the presence of non-viral heterogeneous sequences, five to 16 nucleotides in length (average of 10 nucleotides) at the 5′ ends, confirming that cap-snatching to prime mRNA synthesis is a familial characteristic of the Bunyaviridae. Inspection of the sequences in the primers on nairovirus, bunyavirus and phlebovirus mRNAs suggests that in some cases polymerase slippage occurs shortly after initiation, resulting in a partial reiteration of the 5′-terminal nucleotides of the viral RNA.

Two new cell lines developed from chinook salmon with plasmacytoid leukaemia have been found to be producing a virus. The virus has been identified as a retrovirus based on: type of c.p.e. induced in culture; morphology and density of the particle; presence of Mn2+-dependent, poly(rA)-directed reverse transcriptase activity which was associated with a density of 116 to 118 g/ml in sucrose; electrophoretic pattern of the polypeptides from purified virions; elevated [3H]UTP labelling of RNA in the cell cultures occurring at a density of 116 to 118 g/ml in sucrose. This report describes the first isolation of a retrovirus from a salmonid cell line.

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

The nucleotide sequences of the coat protein genes and 3′ non-translated regions (3′-NTRs) of three isolates of bean common mosaic virus (NL1, NL3 and NY15) and one isolate of blackeye cowpea mosaic virus (W) were determined. Comparison of these sequences revealed that the coat proteins of NL1, NY15 and W were identical in size (287 amino acids) and exhibited an overall sequence similarity (94 to 97%), and 84 to 98% in their N-terminal regions. Furthermore, their 3′-NTRs were very similar in length [253 to 256 nucleotides (nt)] and sequence (93 to 96% similarity). In contrast, the coat protein of NL3 had only 261 amino acids and showed 87 to 89% similarity with NL1, NY15 and W whereas its N-terminal region revealed only 46 to 61% similarity. The 3′-NTR of NL3 also displayed appreciable differences, both in length (240 nt) and sequence (56 to 63% similarity). These results, in combination with earlier serological findings, justify the conclusion that NL1, NY15 and W should be considered strains of the same virus, i.e. bean common mosaic virus, and that NL3 is a strain of a different potyvirus for which the name ‘bean black root virus’ is proposed.

The genomic RNA of the potato virus X (PVX) strain HB, isolated in Bolivia and able to overcome all known resistance genes, has been cloned and sequenced. The PVX HB RNA sequence is 6432 nucleotides long and contains, similarly to the RNAs of other PVX strains, five open reading frames encoding proteins of M rs 165.1K, 24.5K, 12.4K, 7.6K and 25.1K (coat protein), respectively. Multiple amino acid sequence alignments of the coat proteins of four PVX strains identified eight amino acid residues unique for PVX HB . Structural prediction comparisons of the coat proteins of PVX HB and of the other strains suggest a general structural similarity. However, two of the eight amino acid residues unique for strain HB gave rise to a change in the predicted coat protein structure, suggesting a possible involvement in the resistance-breaking activity of PVX HB .