- Volume 72, Issue 5, 1991

Spontaneous focus formation in the contact-inhibited C127 cell line, cl.2, harbouring multiple copies of a bovine papillomavirus type 1 deletion mutant, was associated with the evolution of further viral genomic deletions in addition to an amplification of the viral genome copy number. Three simple frameshift deletions of 308, 605 and 1291 bp, associated with separate transformation events, were mapped within the E1 open reading frame, implying a common mechanism of spontaneous transformation in this cell line. Furthermore, each transformed cell line also retained multiple copies of the intact E1 gene, suggesting that these novel deletion mutants might function by a dominant-negative mechanism to disrupt the normal control of viral DNA replication or viral transcription. These mutants had the potential to encode truncated E1 polypeptides with a common N-terminal region encoded by the 5′ end of E1, i.e. overlapping the previously described E1 modulator gene. A possible role for these mutants in diverting a lethal type of virus-cell interaction is discussed.

The nucleotide sequence of a region of DNA 30 kb from the right end of the orf virus genome has been determined. Examination of the sequences revealed an open reading frame encoding a 10K peptide with significant amino acid homology to the 14K ‘fusion’ protein reported in vaccinia virus. The orf virus sequence has a 31% identity with the vaccinia virus protein, but a higher level of homology of core predicted residues. The secondary structure of both proteins is also similar. The occurrence of the TAAAT sequence upstream from the initiation codon indicates that the sequence is likely to be transcribed late in infection.

Recombinant vaccinia viruses have been proposed as live virus vectors in man to generate both specific humoral and cellular immune responses. The generation of such responses following revaccination was followed in five normal volunteers. All five showed elevated (35- to 6500-fold) vaccina virus-specific IgG by 14 days post-vaccination, following uncomplicated vaccination by dermal scarification. Four of the five had elevated total serum immunoglobulins with increased spontaneous and pokeweed mitogen-induced immunoglobulin production. Major histocompatibility complex class I-restricted vaccinia virus-specific cytotoxic T lymphocytes (CTLs) were not detected either directly, from peripheral blood mononuclear cells, or following secondary in vitro stimulation, even by limiting dilution analysis. These studies would suggest that vaccination with recombinant vaccinia viruses may provide an insufficient stimulus to the generation of CTLs in vivo.

The role of immune responses to haemagglutinin-neuraminidase (HN) protein in protection against a Newcastle disease virus (NDV) infection was investigated using a recombinant vaccinia virus expressing HN (HN-RVV). Live HN-RVV replicated in chickens and completely protected them from lethal infection with virulent NDV. Inactivated HN-RVV also protected chickens when administered with adjuvant but not when administered without adjuvant. However, large amounts of the inactivated HN-RVV (100-fold excess) without adjuvant provided protection. Specific antibodies against the HN protein of NDV were detected in sera from survivors but not from dying birds. However, the kinetics of antibody responses in chickens inoculated with live HN-RVV and inactivated HN-RVV were considerably different. These results clearly confirm that immune response(s) solely to the HN protein of NDV can provide chickens with protection against NDV challenge, and show that the presence of antibodies to the HN protein correlates significantly with the protection from NDV infection at least in HN-immunized chickens.

Cultured human sensory neurons are directly susceptible to CVS rabies virus infection and produce virus yields of 105 p.f.u./ml; infection can persist for more than 20 days without any sign of c.p.e. The use of a compartmentalized two-chamber culture system, with access to either the cell soma or neuritic extensions, permitted the study of viral retrograde transport, which occurs at between 50 and 100 mm/day. Neurons of human origin were more susceptible to virus infection than rat neurons and the axonal transport of rabies virus was more efficient. Electron microscopy allowed virus transport and infection of human dorsal root ganglia neurons to be observed.

Twenty-one monoclonal antibodies (MAbs) directed against the NY-1 and the Osloss-c strains of bovine viral diarrhoea virus were produced and characterized by indirect immunofluorescence assay, radioimmuno-precipitation and neutralization tests. Fourteen MAbs directed against the NY-1 strain recognized the gp48 and showed a weak neutralizing activity in the presence of goat anti-mouse immunoglobulin serum.

Citrus tristeza virus (CTV) contains approximately 20000 bases of positive-sense ssRNA, encapsidated by a coat protein of approximately 25000 M r that has previously been reported to consist of at least two size variants, cp1 and cp2. In the present study, a cDNA library of the T36 isolate of CTV was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein. Five immuno-positive clones produced proteins in Escherichia coli that reacted with monoclonal as well as polyclonal antibodies to the CTV coat protein. Nucleotide sequence analysis of a region common to the five clones revealed the presence of a 669 nucleotide open reading frame flanked by numerous in-frame termination codons. The encoded protein has a predicted M r of 24909 and an amino acid composition consistent with that previously reported for the CTV coat protein. Comparison of the predicted amino acid sequence of the coat protein with the amino-terminal sequences of cp1 and cp2 indicated that these coat protein species arise from the same primary translation product, as a result of post-translational proteolysis at sites approximately 12 to 15 and 26 amino acids from the amino terminus respectively. These results are the first reported cloning and sequencing of a CTV gene and provide evidence that CTV may be translated using subgenomic RNA.