Flow cytometric analysis of lymphocyte subset kinetics in Bali cattle experimentally infected with Jembrana disease virus.

Jembrana disease virus (JDV) is an unusual bovine lentivirus that causes an acute and sometimes fatal disease after a short incubation period in Bali cattle (Bos javanicus). The pathological changes occur primarily in lymphoid tissues, which feature proliferating lymphoblastoid-like cells predominantly throughout parafollicular (T-cell) areas, and atrophy of follicles (B-cell) areas. Five Bali cattle were experimentally infected with JDV and all developed typical clinical signs of Jembrana disease characterised by a transient febrile response, enlargement of superficial lymph nodes and a significant leukopenia. Flow cytometric analysis of PBMC during the acute (febrile) disease phase showed that the reduced number of lymphocytes was due to a significant decrease in both the proportion and absolute numbers of CD4(+) T cells, but not CD8(+) T-cells or CD21(+) B-cells. At the end of the febrile phase, total numbers of both CD8(+) T-cells and CD21(+) B-cells increased significantly, while CD4(+) T-cell numbers remained below normal values, resulting in a significantly reduced CD4(+):CD8(+) ratio. We speculate that the persistent depletion of CD4(+) T cells following JDV infection, through lack of CD4(+) T cell help to B cells, may explain the lack of production of JDV-specific antibodies for several weeks after recovery despite an increase in CD21(+) B cell numbers. Further, our previous data showing that IgG(+) plasma cells are targets for JDV infection, correlated with our current data demonstrating an increase in CD8(+) T cell numbers, supports the suggestion that anti-viral cytotoxic T cell or other cell-mediated immune responses may be critical in the recovery process, although this remains to be formally demonstrated for JDV.

Serologic prevalence of MPV1 in mouse strains in a commercial laboratory mouse colony determined by using VP1 antigen.

A mouse parvovirus (designated MPV1f) was identified in a commercial laboratory mouse colony in Australia. The infection had not been detected by using an rNS1 parvovirus ELISA antigen even though the virus was genetically similar to other MPV1 variants reported previously. A recombinant biotinylated protein based on a truncated VP1 protein of the MPV1 strain was produced and used as antigen for ELISA and Western immunoblots to detect virus infection and determine the seroprevalence of infection in a colony of approximately 45,000 mice. Antibody-positive mice were detected in 8 of 11 rooms sampled, indicating that infection was widespread in the facility. Antibody was detected in 16.2% of 1161 sera obtained from 20 strains of mice. Seroprevalence varied among mouse strains, suggesting genetic variation in the susceptibility of mice to MPV1 or in their antibody response to infection, as has been reported previously in experimentally infected mice. Seroprevalence was high in some inbred strains, including DBA/2JArc and the random-bred strains Hsd:NIH and Arc:Arc(s). Antibody was not detected inC57BL/6J strains, and BALB/c strains showed low seroprevalence of MPV1f.

Strain- and age-associated variation in viral persistence and antibody response to mouse parvovirus 1 in experimentally infected mice.

The effect of mouse strain and age at infection on viral replication and concurrent antibody response to mouse parvovirus 1 (isolate MPV1f) was evaluated for 305 d after inoculation in 4 strains of mice. The results confirmed previous reports that mouse strain and age at infection are significant factors in viral persistence and antibody development and detection. Randombred Arc:Arc(s) mice originally bred from CD1 stock inoculated as juveniles (4 wk) or adults (8 wk) developed persistent viral infection for 152 d after inoculation and an antibody response that persisted for 295 d. Mice of C57BL/6J background inoculated as juveniles had detectable viral DNA in large intestinal content and tissues for 24 d after inoculation and an antibody response that persisted for 288 d. However, viral DNA was not detected in tissues of C57BL/6J mice inoculated as adults, although an antibody was detected for 111 d after inoculation; these results suggest probable viral replication in adult C57BL/6J mice but at levels below the limits of detection. BALB/cArc mice inoculated as juveniles or adults had detectable virus DNA in tissues for 108 to 242 d after inoculation, but no antibody was detected. Similarly, BALB/c-Foxn1(nu)/Arc mice had detectable levels of viral DNA in tissues for 98 to 131 d but no measurable antibody. The difficulty of detecting antibody in mice with a BALB/c background indicates they are unsuitable for routine surveillance of MPV1f infection.

Prior bovine immunodeficiency virus infection does not inhibit subsequent superinfection by the acutely pathogenic Jembrana disease virus.

In cattle the interaction between the two genetically and antigenically related bovine lentiviruses, the acutely pathogenic Jembrana disease virus (JDV) and the non-pathogenic Bovine immunodeficiency virus (BIV) has not been reported although both JDV and a BIV-like virus have been reported in the Bali cattle (Bos javanicus) population in Indonesia. The outcome of infection of Bali cattle with the R29 strain of BIV prior to superinfection 42 days later with JDV(TAB/87) was determined. All BIV-inoculated cattle were successfully infected and developed an antibody response to the TM and CA proteins. BIV infection did not prevent subsequent infection with JDV or ameliorate the clinical signs of Jembrana disease in the infected cattle. It did, however, modify the dynamics of the JDV infection with an earlier onset and end of the acute disease process, and a reduction in the duration of viremia that exceeded 10(6) genome copies/ml of plasma.

Bovine immunodeficiency virus produces a transient viraemic phase soon after infection in Bos javanicus.

Infection of Bali cattle (Bos javanicus) in Indonesia with a non-pathogenic bovine lentivirus similar to Bovine immunodeficiency virus (BIV) is suspected but efforts to detect the virus have been unsuccessful. To define the kinetics of BIV infection in Bali cattle, 13 were infected with the R-29 strain of BIV and monitored for 60 days. No clinical effects were detected. Proviral DNA was detected in peripheral blood mononuclear cells from 4 to 60 days with peak titres 20 days post-infection (dpi). There was a transient viraemia from 4 to 14 dpi with a maximum titre of 1x10(4)genome copies/ml plasma. An antibody response to the transmembrane (TM) glycoprotein commenced 12 dpi but an antibody response to the capsid (CA) protein was detected in one animal only and not until 34 dpi. The results indicated that detection of BIV in infected Bali cattle would have a greater chance of success soon after infection and prior to the onset of a CA antibody response.

In vivo infection of IgG-containing cells by Jembrana disease virus during acute infection.

Jembrana disease virus (JDV) is an unusual bovine lentivirus which causes a non-follicular proliferation of lymphocytes, a transient immunosuppression and a delayed humoral response in infected Bali cattle in Indonesia. A double-immunofluorescent labeling method was developed to identify the subset of mononuclear cells in which the viral capsid protein could be detected. Viral antigen was present in pleomorphic centroblast-like cells which were identified as IgG-containing cells, including plasma cells, in lymphoid tissues. There was no evidence of infection of CD3(+) T-cells or MAC387(+) monocytes in tissues but large vacuolated cells with a macrophage-like morphology in the lung were found to contain viral antigen although they could not be shown conclusively to be infected. The tropism of JDV for mature IgG-containing cells may be relevant to understanding the pathogenesis of Jembrana disease, the delayed antibody responses and the genetic composition of this atypical lentivirus.

Comparison of immunoassay and real-time PCR methods for the detection of Jembrana disease virus infection in Bali cattle.

A sensitive diagnostic assay for the detection of infections with the bovine lentivirus Jembrana disease virus (JDV) is required in Indonesia to control the spread of Jembrana disease. Immunoassays are used routinely but are compromised by cross-reactive epitopes in the capsid (CA) protein of JDV and the genetically related bovine immunodeficiency virus (BIV). JDV gag-specific primers were tested in a real-time PCR assay to detect proviral DNA in peripheral blood mononuclear cells from 165 cattle from the Tabanan district of Bali. JDV-specific amplicons were detected in 9% of the cattle and only 33% of the real-time PCR positive cattle were seropositive. The delayed seroconversion that occurs after infection with JDV could explain the low concordance between these assays but other factors may be responsible. BIV proviral DNA was not detected in any of the PBMC DNA samples. A high concordance value of 98.6% was found between the JDV plasma-derived antigen Western blot and the JDV p26-his recombinant protein ELISA. Only 21% of the seropositive cattle had detectable levels of proviral DNA suggesting that the proviral load in recovered cattle is low. A combination of real-time PCR and JDV p26-his ELISA is recommended for the detection of infection with JDV in Indonesia.

Vaccination reduces the viral load and the risk of transmission of Jembrana disease virus in Bali cattle.

The efficacy of a tissue-derived vaccine, which is currently used in Indonesia to control the spread of Jembrana disease in Bali cattle, was determined by quantifying the viral load in plasma following experimental infection with Jembrana disease virus. Virus transmission is most likely to occur during the acute phase of infection when viral titers are greater than 10(6) genomes/ml. Vaccinated cattle were found to have a 96% reduction in viral load above this threshold compared to control cattle. This would reduce the chance of virus transmission as the number of days above the threshold in the vaccinated cattle was reduced by 33%. Viral loads at the onset and resolution of fever were significantly lower in the vaccinated cattle and immune function was maintained with the development of antibody responses to Env proteins within 10-24 days post challenge. There was, however, no significant reduction in the duration of the febrile period in vaccinated animals. The duration and severity of clinical parameters were found to be variable within each group of cattle but the quantification of viral load revealed the benefits of vaccinating to reduce the risk of virus transmission as well as to ameliorate disease.

Analysis of Jembrana disease virus replication dynamics in vivo reveals strain variation and atypical responses to infection.

Jembrana disease virus (JDV) is an acute lentiviral infection of Bali cattle in Indonesia. Data generated during a series of cattle infection experiments was examined and significant differences were identified in the mean plasma viral load on the first and second days of the febrile response in cattle infected with JDV(TAB/87) compared to those infected with JDV(PUL/01). The peak and total viral loads >or=10(6) genome copies/ml during the acute stage of the disease were significantly higher in JDV(TAB/87) infected cattle. JDV(PUL/01) infected cattle developed peak rectal temperatures earlier than the JDV(TAB/87) cattle but there were no differences in the duration of the febrile responses observed for the 2 groups of animals. The plasma viremia was above 10(6) genome copies/ml for almost 3 days longer in JDV(TAB/87) compared to JDV(PUL/01) infected cattle. Atypical responses to infection occurred in approximately 15% of experimentally infected animals, characterized by reduced viral loads, lower or absent febrile responses and absence of p26-specific antibody responses. Most of these cattle developed normal Tm-specific antibody responses between 4-12 weeks post-infection.

Analysis of Jembrana disease virus mRNA transcripts produced during acute infection demonstrates a complex transcription pattern.

Jembrana disease virus (JDV) is an unusual bovine lentivirus that causes an acute disease syndrome with a 20% case fatality rate after a short incubation period in Bos javanicus (Bali cattle) in Indonesia. Analysis of tat mRNA transcription patterns has identified up to six differently spliced transcripts indicating that, in common with other lentiviruses, JDV uses a complex splicing pattern. RT-PCR analysis of mRNA transcripts produced during the acute phase of infection with JDV(TAB/87) revealed at least 12 differently spliced transcripts involving 9 different splice sites. A single unspliced gag/pol transcript, singly spliced vif and tmx specific transcripts and alternatively spliced env, tat and rev transcripts were identified. A 67 nucleotide putative non-coding exon was identified that shared the same splice acceptor (SA) as vif and was incorporated into alternative transcripts of tat, rev and env.

Sequence analysis of mRNA transcripts encoding Jembrana disease virus Tat-1 in vivo.

Jembrana disease virus (JDV) is a lentivirus which replicates to very high titres in vivo and its Tat-1 protein has been shown to be a potent transactivator in vitro. Analysis of tat mRNA transcripts produced early in infection studies identified four predominant species which were generated by multiple splicing events. The use of a splice donor downstream of tat-1 was common indicating that a Tat-1 protein of 97 amino acids is expressed during the acute phase of Jembrana disease. The presence of an in-frame stop codon between tat-1 and tat-2 was identified in transcripts from three different strains of JDV confirming that expression of a single exon Tat-1 correlates with high viral titres in vivo. Sequence conservation in the regions of tat-1 that are critical for RNA binding and transcription activation in three different virus strains was high and the tat-2 sequences were completely conserved.

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.

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 ( approximately 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.

Sequence analysis of Jembrana disease virus strains reveals a genetically stable lentivirus.

Jembrana disease virus (JDV) is a lentivirus associated with an acute disease syndrome with a 20% case fatality rate in Bos javanicus (Bali cattle) in Indonesia, occurring after a short incubation period and with no recurrence of the disease after recovery. Partial regions of gag and pol and the entire env were examined for sequence variation in DNA samples from cases of Jembrana disease obtained from Bali, Sumatra and South Kalimantan in Indonesian Borneo. A high level of nucleotide conservation (97-100%) was observed in gag sequences from samples taken in Bali and Sumatra, indicating that the source of JDV in Sumatra was most likely to have originated from Bali. The pol sequences and, unexpectedly, the env sequences from Bali samples were also well conserved with low nucleotide (96-99%) and amino acid substitutions (95-99%). However, the sample from South Kalimantan (JDV(KAL/01)) contained more divergent sequences, particularly in env (88% identity). Phylogenetic analysis revealed that the JDV(KAL/01)env sequences clustered with the sequence from the Pulukan sample (Bali) from 2001. JDV appears to be remarkably stable genetically and has undergone minor genetic changes over a period of nearly 20 years in Bali despite becoming endemic in the cattle population of the island.

TaqMan real-time reverse transcription-PCR and JDVp26 antigen capture enzyme-linked immunosorbent assay to quantify Jembrana disease virus load during the acute phase of in vivo infection.

Jembrana disease virus (JDV) is an acutely pathogenic lentivirus that affects Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute phase of the disease process. We report for the first time two techniques that enable quantification of the virus and the use of these techniques to quantify the virus load during the acute phase of the disease process. A one-step JDV gag [corrected] TaqMan real-time reverse transcription-PCR (RT-PCR) assay was developed for the detection and quantification of JDV RNA in plasma. The limit of detection was 9.8 x 10(2) JDV viral RNA copies over 35 cycles, equivalent to 4.2 x 10(4) JDV genome copies/ml, and a peak virus load of 1.6 x 10(12) during the acute febrile period. An antigen capture enzyme-linked immunosorbent assay (ELISA) was also developed to quantify the levels of JDV capsid (JDVp26) over a linear range of 10 to 200 ng/ml. Viral RNA and JDVp26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (R = 0.860 and r(2) = 0.740) was observed between the two techniques within the range of their detection limits. The relatively insensitive capture ELISA provides an economical and feasible method for monitoring of virus in the absence of more sensitive techniques.

Recombinant Jembrana disease virus gag proteins identify several different antigenic domains but do not facilitate serological differentiation of JDV and nonpathogenic bovine lentiviruses.

In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and nucleocapsid proteins were produced from JDV gag and the expressed proteins were tested by Western blot using JDV and BIV hyperimmune sera. JDV matrix and truncated capsid proteins were recognised by both JDV and BIV hyperimmune sera indicating that there were multiple cross-reactive epitopes present in JDV gag. At least three epitopic regions were identified in these constructs, including the major homology region, by monoclonal antibody binding studies. JDV nucleocapsid recombinant protein was not recognised by either JDV or BIV hyperimmune sera and none of the recombinant gag proteins were able to differentiate between JDV positive sera from Jembrana disease endemic and Jembrana disease-free areas. Additionally, a 40 amino acid recombinant subunit protein encompassing the region recently found to contain an epitope unique to BIV [Zheng, L., Zhang, S., Wood, C., Kapil, S., Wilcox, G.E., Loughin, T.A., Minocha, H.C., 2001. Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity. Clin. Diagn. Lab. Immunol. 8, 283-287] was tested but was not recognised by either JDV positive sera from Jembrana disease-endemic or Jembrana disease-free areas.

Molecular detection of a virus, Pilchard herpesvirus, associated with epizootics in Australasian pilchards Sardinops sagax neopilchardus.

In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen over their entire geographical range on the Australian coastline. A virus with typical herpesvirus morphology was identified as the causative agent, although the source of the virus remains unknown. At the time of the mortality events, the only available diagnostic test available for the detection of the virus was electron microscopy and hence, development of a rapid diagnostic test for detection and identification of the virus was required. Initial sequence data for Pilchard herpesvirus (PHV) was acquired by comparing the highly conserved region of the ORF 62 gene of other piscine herpesviruses (Ictalurid herpesvirus 1 [Channel catfish virus, CCV] and Salmonid herpesvirus 2 [Oncorhynchus masou virus, SaHV2, OMV]), and designing primers that successfully amplified a fragment of PHV. Here we describe the use of polymerase chain reaction (PCR) technology to detect PHV in infected tissues. Sequence analysis of amplified fragment resulting from this PCR is different from all known herpesviruses and can therefore distinguish PHV from all known viruses.

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.