Canine parvovirus in asymptomatic feline carriers.

Canine parvovirus (CPV) and feline panleukopaenia virus (FPLV) are two closely related viruses, which are known to cause severe disease in younger unvaccinated animals. As well as causing disease in their respective hosts, CPV has recently acquired the feline host range, allowing it to infect both cats and dogs. As well as causing disease in dogs, there is evidence that under some circumstances CPV may also cause disease in cats. This study has investigated the prevalence of parvoviruses in the faeces of clinically healthy cats and dogs in two rescue shelters. Canine parvovirus was demonstrated in 32.5% (13/50) of faecal samples in a cross sectional study of 50 cats from a feline only shelter, and 33.9% (61/180) of faecal samples in a longitudinal study of 74 cats at a mixed canine and feline shelter. Virus was isolated in cell cultures of both canine and feline origin from all PCR-positive samples suggesting they contained viable, infectious virus. In contrast to the high CPV prevalence in cats, no FPLV was found, and none of 122 faecal samples from dogs, or 160 samples collected from the kennel environment, tested positive for parvovirus by PCR. Sequence analysis of major capsid VP2 gene from all positive samples, as well as the non-structural gene from 18 randomly selected positive samples, showed that all positive cats were shedding CPV2a or 2b, rather than FPLV. Longitudinally sampling in one shelter showed that all cats appeared to shed the same virus sequence type at each date they were positive (up to six weeks), despite a lack of clinical signs. Fifty percent of the sequences obtained here were shown to be similar to those recently obtained in a study of sick dogs in the UK (Clegg et al., 2011). These results suggest that in some circumstances, clinically normal cats may be able to shed CPV for prolonged periods of time, and raises the possibility that such cats may be important reservoirs for the maintenance of infection in both the cat and the dog population.

Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease.

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.

Canine parvovirus type 2 vaccine protects against virulent challenge with type 2c virus.

The ability of dogs vaccinated with a live attenuated CPV type 2 (Nobivac Intervet) vaccine to resist challenge with a current CPV2c isolate was investigated. Six SPF beagle dogs were given the minimum recommended course of vaccination, comprising a single inoculation of vaccine (Nobivac Lepto+Nobivac Pi) at 8-10 weeks of age followed 3 weeks later with a parvovirus vaccine in combination with distemper, adenovirus and parainfluenza virus (Nobivac DHPPi) and a repeat leptospirosis vaccine. Six control dogs were kept unvaccinated. All animals were challenged orally with a type 2c isolate of CPV and monitored for clinical signs, virus shedding, white blood cell fluctuations and serological responses. All vaccinated dogs were fully protected; showing no clinical signs nor shedding challenge virus in the faeces, in contrast to control animals, which displayed all the typical signs of infection with pathogenic CPV and shed challenge virus in the faeces.

Vaccination with inactivated virus but not viral DNA reduces virus load following challenge with a heterologous and virulent isolate of feline immunodeficiency virus.

It has been shown that cats can be protected against infection with the prototypic Petaluma strain of feline immunodeficiency virus (FIV(PET)) using vaccines based on either inactivated virus particles or replication-defective proviral DNA. However, the utility of such vaccines in the field is uncertain, given the absence of consistent protection against antigenically distinct strains and the concern that the Petaluma strain may be an unrepresentative, attenuated isolate. Since reduction of viral pathogenicity and dissemination may be useful outcomes of vaccination, even in the absence of complete protection, we tested whether either of these vaccine strategies ameliorates the early course of infection following challenge with heterologous and more virulent isolates. We now report that an inactivated virus vaccine, which generates high levels of virus neutralizing antibodies, confers reduced virus loads following challenge with two heterologous isolates, FIV(AM6) and FIV(GL8). This vaccine also prevented the marked early decline in CD4/CD8 ratio seen in FIV(GL8)-infected cats. In contrast, DNA vaccines based on either FIV(PET) or FIV(GL8), which induce cell-mediated responses but no detectable antiviral antibodies, protected a fraction of cats against infection with FIV(PET) but had no measurable effect on virus load when the infecting virus was FIV(GL8). These results indicate that the more virulent FIV(GL8) is intrinsically more resistant to vaccinal immunity than the FIV(PET) strain and that a broad spectrum of responses which includes virus neutralizing antibodies is a desirable goal for lentivirus vaccine development.

The receptor binding site of feline leukemia virus surface glycoprotein is distinct from the site involved in virus neutralization.

The external surface glycoprotein (SU) of feline leukemia virus (FeLV) contains sites which define the viral subgroup and induce virus-neutralizing antibodies. The subgroup phenotypic determinants have been located to a small variable region, VR1, towards the amino terminus of SU. The sites which function as neutralizing epitopes in vivo are unknown. Recombinant SU proteins were produced by using baculoviruses that contained sequences encoding the SUs of FeLV subgroup A (FeLV-A), FeLV-C, and two chimeric FeLVs (FeLV-215 and FeLV-VC) in which the VR1 domain of FeLV-A had been replaced by the corresponding regions of FeLV-C isolates. The recombinant glycoproteins, designated Bgp70-A, -C, -215, and -VC, respectively, were similar to their wild-type counterparts in several immunoblots and inhibited infection of susceptible cell lines in a subgroup-specific manner. Thus, Bgp70-A interfered with infection by FeLV-A, whereas Bgp70-C, -VC, and -215 did not. Conversely, Bgp70-C, -VC, and -215 blocked infection with FeLV-C, while Bgp70-A had no effect. These results indicate that the site on SU which binds to the FeLV cell surface receptor was preserved in the recombinant glycoproteins. It was also found that the recombinant proteins were able to bind naturally occurring neutralizing antibodies. Bgp70-A, -VC, and -215 interfered with the action of anti-FeLV-A neutralizing antibodies, whereas Bgp70-C did not. Furthermore, Bgp70-C interfered with the action of anti-FeLV-C neutralizing antibodies, while the other proteins did not. These results indicate that the neutralizing epitope(s) of FeLV SU lies outside the subgroup-determining VR1 domain.

A mutant of canine adenovirus type 2 with a duplication of the E1a region exhibits altered expression of early region 4.

The genomic DNA of a vaccine strain of canine adenovirus type 2 (Vaxitas; ICI Tasman) has been shown to contain two copies of the E1a region, the second being at the far right end of the genome. DNA sequence analysis of the right terminal 2.8 kbp of this vaccine strain showed that numerous point mutations have occurred in the second copy, which would preclude the synthesis of any functional products. However, expression vectors in which the E1a promoter from the right terminus were linked to the chloramphenicol acetyltransferase gene showed that the promoter was fully functional. Furthermore, the activity of the reiterated E1a promoter was considerably greater than that of the normal E4 promoter. This dramatic change in the regulation of E4 expression may be an important factor in determining the altered host cell specificity displayed by this vaccine strain virus.

Identification and nucleotide sequence of the early region 1 from canine adenovirus types 1 and 2.

The genome of canine adenovirus type 1 (CAV-1) has been cloned and restriction maps compiled. These maps are compared with those of canine adenovirus type 2 (CAV-2). The left ends of both genomes were further characterised by DNA sequence analysis. Several features of the DNA sequence and predicted polypeptide sequence are similar to those of the human adenoviruses. The level of homology observed across the E1 regions appears to be of the same order as the overall DNA similarity between CAV-1 and CAV-2 (75%). Transfection experiments using the presumptive E1a containing region of CAV-2 suggests that it encodes a transactivating function typical of the human adenovirus E1a genes.

Molecular cloning and restriction endonuclease mapping of two strains of canine adenovirus type 2.

The DNA of a field isolate and of a vaccine strain of canine adenovirus type 2 (CAV-2) were analysed by digestion with several restriction endonucleases. The PstI restriction fragments of the field isolate (CAV-2 Glasgow) and the vaccine strain were cloned into the plasmid pBR322. Physical maps of the two viral genomes were constructed by molecular hybridization of PstI, EcoRI, SmaI, BamHI and KpnI digests of the viral DNA with the cloned PstI fragments. The restriction profile of CAV-2 Glasgow was shown to be virtually identical to those of the two prototype CAV-2 strains, Toronto A26/61 and Manhattan. However, the restriction fragment pattern of the vaccine strain of CAV-2 showed characteristic alterations, in particular additional sequences at or near the genome termini.

Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping.

Fifty-four faecal specimens obtained from kennelled dogs with diarrhoeal disease were used to inoculate a range of cell types in tissue culture. Particles resembling adenovirus virions were seen in three specimens and 22 stools yielded an adenovirus upon culture. Viral DNA from each isolate was digested with the restriction endonucleases Bam H1 and Pst 1. Agarose gel electrophoresis revealed identical restriction patterns for all isolates. One isolate, 9228, was selected as a prototype and was compared with reference strains of canine adenovirus-2 (CAV-2) (Manhattan and Toronto A26/61) and CAV-1. Isolate 9228 was clearly distinct from CAV-1 but identical to both Manhattan and Toronto (A26/61) strains of CAV-2. However, restriction site polymorphism was observed in 9228 following digestion with Hpa II. Isolate 9228 was similarly compared with all commercially available vaccinal isolates of CAV-2 and was shown to be clearly distinct from these.

Construction and characterization of guaB-lacZ fusions in Escherichia coli K12.

GuaB-lacZ fusion strains of Escherichia coli K12 have been constructed using the Casadaban (1976) gene-fusion technique. The major modification to the procedure was the removal of the guanine requirement of Mu-induced guaA auxotrophs by introduction of a ColEl-gua+ plasmid prior to selection of the fusion. Conversion to prototrophy was necessary to overcome problems associated with repression of the gua operon by guanine added to selection media. Induction of the lysogenic fusion strains yielded plaque-forming lambda transducing phages that carried the lac genes and either the complete guaB gene and the gua promoter or only a distal portion of guaB.