HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6.

The most common viral disease of cats worldwide is the infection with feline herpesvirus 1 (FeHV-1). This infection may be followed by Herpetic stromal keratitis (HSK), which is supposed to have an immunopathological basis. Experiments using herpes simplex viruses (HSV) in mouse models indicated that HSK may be treated by topical application of the interleukin 10 (IL-10) gene. The objective of this study was the construction of human herpes simplex virus type 1 (HSV-1)-based amplicon vectors expressing feline interleukin genes and delivery of these genes into cells of feline origin. HSV-1-based amplicon vectors encoding either the enhanced green fluorescent protein, the feline IL-6 or the feline IL-10 under control of the HSV-1 immediate-early 4/5 promotor were constructed, packaged into amplicon particles, transduced into feline cells, and tested for RNA synthesis and biological activity. Feline cells were successfully transduced by HSV-1-based amplicon particles and RNA specific for the transgene was detected already at 2h post transduction, with a maximum at 24h. The recombinant feline IL-10 was functionally active as demonstrated by the reduction of both IL-12 p40 and interferon-gamma-mRNA production in Pansorbin stimulated feline peripheral mononuclear cells. Similarly, the recombinant feline IL-6, which was secreted into the supernatant of transduced cells, was able to support the growth of the IL-6-dependent murine B cell hybridoma 7TD1. HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6. It will be of high interest to study the effects of these tools in vivo.

Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence.

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5' end (no 99-137), and two large, slightly overlapping ORFs, ORF1a (nt 297-12650) and ORF1b (nt 12605-20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.

Further analysis of the genome of porcine epidemic diarrhoea virus.

We report here the continued determination and analysis of the nucleotide sequence of both wild type (wt) and cell culture adapted (ca) porcine epidemic diarrhoea coronavirus (PEDV). These studies were undertaken with two objectives in mind: the identification of common and divergent features in the genomic sequences of wt and ca PEDV which can explain the differences in virulence of these isolates and the further exploration of the relationship of PEDV to other coronaviruses.

Sequence of ovine adenovirus homologs for 100K hexon assembly, 33K, pVIII, and fiber genes: early region E3 is not in the expected location.

Ovine adenovirus OAV287 was previously isolated from sheep in Western Australia. As a first step in characterizing the genome of this virus we have determined the sequence of its genome between map units 65 and 81. This region was expected to contain the nonessential E3 region which, in other adenoviruses, lies between the genes encoding the pVIII and fiber proteins, although its size and complexity varies. OAV287 genes coding for the hexon assembly, 33K, pVIII, and fiber proteins were identified by their homologies with human Ad2. These genes lie in the same relative positions in the OAV287 genome, but the intergenic region between the pVIII and the fiber genes is only 197 nucleotides and these appear to be incapable of coding for any protein. Thus, the ovine adenovirus E3 region is not present in the expected location. In addition, using cDNA synthesis, PCR amplification, and nucleotide sequencing we determined the location of splice junctions and transcription termination signals in mRNA species encoding these proteins. This showed that a family of variably spliced L4 RNAs is produced and that the region between the pVIII and the fiber genes contains several signals for RNA synthesis and processing. As the E3 region in human adenoviruses is nonessential for replication, in many instances it has been replaced with foreign DNA during the construction of recombinants. Because of this unexpected difference in the organization of the OAV287 genome further experimentation will be required to determine whether potential vaccine recombinants can be constructed for this adenovirus by making insertions into the pVIII/fiber intergenic region.

Characterisation of Australian ovine adenovirus isolates.

We have characterised two groups of adenoviruses isolated from sheep in Australia. Restriction endonuclease maps for enzymes BamHI, ClaI, SalI, SmaI and SphI have been determined for the genome of ovine adenoviruses related to bovine adenovirus serotype 7 (BAV 7) from sheep in Western Australia. Although previously serotyped as BAV 7 these isolates are different from bovine isolates of BAV 7 based on comparison with published restriction endonuclease profiles and maps of BAV 7 cattle isolates. Additional adenovirus isolates obtained from Victorian sheep have been serotyped as ovine adenovirus type 5 (OAV 5). On the basis of restriction endonuclease analysis these viruses are different from the sheep BAV 7 isolates. Following infection of sheep with ovine BAV 7 and OAV 5 isolates, virus was recovered from nasal and rectal swabs for several days. Antibodies detected by ELISA and serum neutralisation tests (SN) developed by 15 days after infection. Virus also spread from the infected sheep to an incontact control and one of ten sheep purchased for infection studies had SN antibodies to BAV 7 suggesting that BAV 7-like viruses naturally infect sheep in Victoria and Western Australia. With further development, these ovine adenoviruses may be suitable as vectors for the delivery of vaccine antigens to sheep and cattle.