G.E. Wilcox

Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus

A monoclonal antibody (mAb) that reacted specifically with a 16 kDa big liver and spleen disease virus (BLSV) protein was used to identify the protein in western immunoblots of infected liver extracts and enable partial amino acid sequence analysis of the protein. Based on this sequence, a degenerate primer was designed that was used in conjunction with random hexamers in a reverse transcriptase-POR (RT PCR), to amplify a 523 bp product from RNA extracted from homogenates of BLSV-infected livers. There was 62% nucleotide sequence identity between this sequence and the sequence of the helicase gene of human hepatitis E virus (HEV). POR primers designed from this 523 bp fragment were able to amplify a 490 bp product from livers of virus-infected chickens but not chickens from virus-free flocks.

Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae

The circular, single-stranded DNA genome of a novel circovirus of canaries, tentatively named canary circovirus (CaCV), was cloned and sequenced. Sequence analysis indicated that the genome was 1952 nucleotides (nt) in size and had the potential to encode three viral proteins, including the putative capsid and replication-associated (Rep) proteins. The CaCV genome shared greatest sequence similarity (58.3% nt identity) with the newly characterized columbid circovirus (CoCV) and was more distantly related to the two porcine circovirus strains, PCV1 and PCV2, beak and feather disease virus (BFDV) and a recently isolated goose circovirus (GCV) isolate (46.8-50.9% nt identity). In common with other members of the Circovirus genus, several nt structures and amino acid motifs thought to be implicated in virus replication were identified on the putative viral strand. Phylogenetic analysis of both the capsid and Rep protein-coding regions provided further evidence that CaCV is more closely related to CoCV and BFDV and more distantly related to GCV, PCV1 and PCV2.

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.

Characteristics of a retrovirus associated with Jembrana disease in Bali cattle

A virus causing Jembrana disease in Bali cattle (Bos javanicus) was demonstrated to have characteristics of a retrovirus. Reverse transcriptase activity was detected in virus purified by sucrose gradient centrifugation. Electron microscopic examination of tissue from the affected cattle indicated that the virus matured by C-type budding through the plasma membrane and into intracytoplasmic vacuoles of cells in lymphoid tissue, with the formation of circular enveloped virus particles ranging in diameter from 96 to 124 nm with an eccentric nucleoid. Western immunoblotting using sera from recovered animals demonstrated virus proteins of M(r) 100K, 45K, 42K, 33K, 26K, 16K and 14K. The 26K protein of Jembrana disease virus cross-reacted in Western blots with the 26K capsid protein of bovine immunodeficiency virus (BIV). The apparent morphogenesis, protein structure and antigenic relationship with BIV suggested the virus was a lentivirus.

A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae

ABSTRACT Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.

Studies of experimental Jembrana disease in Bali cattle. I. Transmission and persistence of the infectious agent in ruminants and pigs, and resistance of recovered cattle to re-infection.

The agent causing Jembrana disease, an enzootic disease of Bos javanicus (Bali cattle) occurring in Bali, Indonesia, was shown to occur at high titres in the blood of animals during the febrile period of the disease and to persist in cattle for 25 months after clinical recovery. During the febrile period of the disease, most of the infectious agent appeared to be associated with the plasma fraction of whole blood. There was a linear relationship between the number of organisms inoculated into susceptible Bali cattle and the incubation period, which varied from 4.5 to 12 days. Seventeen of 18 animals in which Jembrana disease had been experimentally induced up to 22 months previously did not develop clinical signs when re-challenged with the infectious agent. Ongole cattle (Bos indicus), Friesian cattle (Bos taurus), buffaloes (Bubalus bubalis) and pigs, but not sheep or goats, developed a mild febrile response, but no other overt clinical signs of the disease after inoculation with the Jembrana disease agent. Ongole and Friesian cattle, buffaloes, and sheep developed a persistent infection after inoculation; the infectious agent persisted in blood or spleen for at least 9 months in buffaloes and for shorter periods in the other species.

Novel bovine lentiviral vectors based on Jembrana disease virus

Safety is a concern that must be addressed prior to any clinical use of human immunodeficiency virus (HIV)‐based lentiviral vectors in human patients. Unfortunately, efforts to examine the biosafety of the vectors in preclinical animal models are hampered due to the lack of animal models for HIV infection. We have developed new lentiviral vectors based on the recently characterised Jembrana Disease Virus (JDV), which infects a specific species of cattle naturally in Bali, Indonesia.

Antibody response to Jembrana disease virus in Bali cattle.

An enzyme-linked immunosorbent assay (ELISA) and an agar gel immunodiffusion (AGID) test are described which detected antibody against Jembrana disease virus in infected Bali cattle. Both tests were specific and did not detect antibody in cattle from areas where clinical Jembrana disease has not been detected. The ELISA detected antibody in all infected cattle and had greater sensitivity than the AGID which detected antibody in less than 50% of infected cattle at any single time after infection. The antibody response to the virus was delayed; antibody was not detected by ELISA in a majority of infected cattle until 11 weeks after infection and a maximum antibody response was detected 23 to 33 weeks after infection. Antibody was still detectable 59 weeks after infection.

GENOMIC SEQUENCE ANALYSIS IDENTIFIES JEMBRANA DISEASE VIRUS AS A NEW BOVINE LENTIVIRUS

Jembrana disease virus, the cause of an acute, severe disease in Bali (Bos javanicus) cattle in Indonesia was recently identified as a retrovirus, and possibly a lentivirus. We have produced sequence data representing 598 bp of the pol gene, amplified by PCR from viral cDNA using broadly reactive universal primers for retroviruses and more specific genus-reactive primers for lentiviruses. When the sequence data were compared with that of known lentiviruses and other bovine retroviruses, the closest alignment was with bovine immunodeficiency-like lentivirus (BIV), showing 74% nucleotide sequence identity. This confirmed that JDV is a lentivirus and that it is distinguishable from BIV. The pathogenesis of Jembrana disease is most unusual for a lentivirus infection and differs markedly from that reported for BIV infection.

Canine Parvoviral Disease: Experimental Reproduction of the Enteric Form with a Parvovirus Isolated from a Case of Myocarditis

Five 7-week-old pups and four 4-week-old pups, all seronegative to canine parvovirus, were inoculated intravenously with 1000 haemagglutinating units of canine parvovirus originally isolated from the myocardium of a dog with naturally occurring myocarditis. After three days, pups in both litters became pyrexic, anorectic and depressed, with vomiting and diarrhoea. The 4-week-old pups were killed on day 4, and the 7-week-old pups died or were killed on day 5 post-inoculation. Histological examination showed degeneration and necrosis of intestinal crypt epithelial cells and villous atrophy. All pups had thymic atrophy caused by lymphoid depletion. Peyers patches, mesenteric lymph node and spleen also had lymphoid depletion. Lymphoid necrosis was present occasionally in these tissues. In the bone marrow, granulocytes and granulocyte and erythroid precursors were depleted. Amphophilic intranuclear inclusion bodies were abundant in crypt epithelial nuclei, less so in myocardial nuclei. Canine parvovirus was isolated from intestinal contents, thymus, spleen, mesenteric lymph node and liver in most pups, but not from kidney or myocardium.