Porcine circovirus-associated disease in weaner pigs in Western Australia.

OBJECTIVE: To report the occurrence and pathology of porcine circovirus type 2 (PCV2)-associated disease (PCVAD) of postweaning pigs in two Australian pig herds. METHODS: Mortality data from two commercial piggeries that experienced higher than normal postweaning illthrift and mortalities were examined. Gross and histopathological examinations were performed on the index cases, and at weekly intervals thereafter for a period of 10 weeks. Specimens were submitted to the laboratory for routine diagnostic testing and for exclusion of porcine reproductive and respiratory syndrome virus (PRRSV). The genomes of two strains of PCV2 isolated during testing were sequenced. RESULTS: Mortality rates in weaned, 5-12-week-old pigs spiked significantly during mid to late 2007. This increase in the mortalities was mainly attributed to salmonella-associated diarrhoea and illthrift. Salmonellosis was diagnosed in 73/110 cases inclusive of both piggeries. Many pigs also had chronic granulomatous lymphadenitis and diffuse histiocytic interstitial pneumonia consistent with PCVAD and associated with varying amounts of PCV2 antigen and inclusion bodies. All samples tested for PRRSV were negative. Sequence analysis of the PCV2 isolates showed strain differences between piggeries. CONCLUSION: This report describes the first outbreaks of PCVAD in growing pigs in Western Australia (WA) and describes lesions not previously seen in this laboratory. It also describes the first isolation of a PCV2 group 1 virus in WA associated with PCVAD. Although the outbreaks of PCVAD occurred with concurrent salmonellosis, the two diseases were unrelated. Neither of the outbreaks met the Australian case definition for the diagnosis of postweaning multisystemic wasting syndrome.

Thermal stability of porcine circovirus type 2 in cell culture.

International trade in pig meat has resulted in some countries placing restrictions on the importation of pig meat, with requirements for cooking of imported meat to destroy viral agents. This study investigated the in vitro resistance of an Australian strain of porcine circovirus type 2 (PCV2), the causative agent of post-weaning multisystemic wasting syndrome (PMWS), to heat treatment. The viability of the virus in cell cultures was determined by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) to detect viral transcripts, and immunohistochemistry (IHC) to visualize viral capsid antigen. PCV2 retained infectivity when heated at 75 degrees C for 15 min but was inactivated by heating at 80 degrees C and above for 15 min. The results provide important information on the thermal tolerance of PCV2, which can be taken into account in risk assessments for trade in pig meat and porcine-derived biological products.

Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR.

A nested multiplex PCR was developed as a rapid (<12h), sensitive test for the simultaneous identification of equine herpesviruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.

Genetic characterisation of Australian strains of porcine circovirus types 1 and 2.

OBJECTIVE: As post-weaning multi-systemic wasting syndrome (PMWS) has not been identified within Australia, to determine if the absence of disease was associated with genetic differences between the strains of porcine circovirus (PCV) present in Australia and those from countries in association with PMWS. DESIGN: Pig tissues were obtained from weaned pigs found dead or presenting with clinical signs of illthrift and also from neonatal pigs with congenital tremors and used as a source of virus DNA for sequence analysis. PROCEDURE: DNA was extracted from the tissues and PCV detected by polymerase chain reaction (PCR). PCR with PCV type-specific primers was used to amplify the entire genome from selected tissues. The genomes of three strains of PCV1 and seven strains of PCV2 from three Australian states were sequenced and subjected to phylogenetic analysis using standard procedures. RESULTS: The three Australian PCV1 strains had 98 to 99% nucleotide identity to strains in other countries and the seven Australian PCV2 strains had 94 to 99% identity to PCV2 strains in other countries where PMWS has occurred. Six of the seven Australian PCV2 strains were genetically similar to each other, while the seventh was more distantly related. There were no consistent differences in the predicted amino acid sequence of the Australian strains of PCV2 and strains associated with PMWS in other countries. CONCLUSION: There were no consistent differences between Australian strains of PCV and those that have been associated with PMWS in other countries and it appears likely that other factors are responsible for the absence of PMWS in Australia.

The detection of porcine circovirus in the Australian pig herd.

OBJECTIVE: To determine if porcine circovirus (PCV) type 1 (PCV1) or type 2 (PCV2) is present in the Australian pig herd, to conduct preliminary genetic characterisation of any viruses detected, and to determine if there is any obvious virological reason why post-weaning multisystemic wasting disease (PMWS), associated with PCV infection in other countries, has not been detected in Australia. DESIGN: Serum samples were collected from 14 randomly selected pig farms in Western Australia and used for detection of PCV antibody. Additional samples from one farm were obtained at 2-week intervals from pigs between 2 and 12 weeks of age to detect any age-associated variations in prevalence of infection. Veterinary practitioners from four Australian states submitted tissues of dead or unthrifty weaned pigs, and these were examined for evidence of PCV1 and PCV2 infection. PROCEDURE: Sera were tested for antibody to PCV using an indirect immunofluorescence assay (IFA). Tissues were tested for PCV1 and PCV2 genomic material using a multiplex PCR. RESULTS: PCV antibody was detected in approximately 30% of Western Australian pigs tested. PCV1 DNA was detected in tissue samples from Western Australia, South Australia and New South Wales and PCV2 DNA was detected in tissue samples from Western Australia, New South Wales and Queensland. Sequence analysis of the PCR products indicated the PCV1 and PCV2 present in Australia were very similar to strains in other countries where PMWS is endemic. CONCLUSION: Both PCV1 and PCV2 are present in Australia and the viruses present appear similar to those in countries with PMWS. The absence of PCV2-associated PMWS in Australia may be due to absence of essential secondary factors required for PCV2 to produce PMWS.

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle.

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Bali cattle (Bos javanicus), which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5'- and 3'-long terminal repeats (LTRs), 0.4kb of truncated gag and 1.1kb of 3'-env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences, including gag, pol, vif, tat and rev, but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped, disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2)x10(6)CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as well. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus.

Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity.

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.

The induction of a protective immunity against Jembrana disease in cattle by vaccination with inactivated tissue-derived virus antigens.

The ability to induce a protective immunity against Jembrana disease, an acute lentivirus disease of Bali cattle (Bos javanicus) present in Indonesia, was investigated. A protective immune response was induced in cattle by vaccination with virus-containing plasma and spleen tissue derived from acutely affected cattle. The virus was inactivated with Triton X-100 and emulsified in either incomplete Freund's adjuvant or a mineral oil adjuvant (MOA). The vaccination procedure suppressed the duration and severity of the disease but did not completely prevent the development of disease in animals challenged with 100 infectious doses of virus.

Genetic diversity of beak and feather disease virus detected in psittacine species in Australia.

The complete nucleotide (nt) sequence of eight isolates of beak and feather disease virus (BFDV) obtained from a range of psittacine species with psittacine beak and feather disease (PBFD) from throughout Australia were compared with the sequences of two BFDV isolates previously reported from Australia (BFDV-AUS) and America (BFDV-USA), respectively. All isolates had the same basic structure including the position of the open reading frames, the hairpin structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the three motifs of Rep protein encoded from ORF1 and involved in rolling circle replication, and the P-loop motif previously described, but the genome size of the eight isolates ranged from 1992 to 2018 nt. Overall nt identity of the isolates compared to BFDV-AUS ranged from 84 to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both coding and noncoding regions. The identity of the nt sequence of ORF2 compared to BFDV-AUS varied from 80 to 99%, while the identity of the deduced amino acid sequences varied from 73 to 99%. Phylogenetic analysis grouped the isolates into four clusters but there were no apparent regional differences or differences related to the psittacine species of origin. While seven ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the BFDV-AUS isolate described previously, only three of these ORFs were detected in all 10 BFDV isolates for which sequence data were available. The three ORFs were ORF1 that presumably encodes the Rep protein, ORF2 presumably the major capsid protein, and the ORF previously designated ORF5. The ORF5 was of two size classes in different isolates, 303 and 474 nt, and only the first 303 nt of the viruses with an ORF of 474 nt were common to the other isolates.

Novel bovine lentiviral vectors based on Jembrana disease virus.

BACKGROUND: 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. METHODS: Sequences from the JDV genome were amplified by splicing overlap extension polymerase chain reaction (PCR) for the construction of transfer vectors as well as a packaging construct. Co-transfection of these two plasmids into 293T cells with a third encoding a G glycoprotein of vesicular stomatitis virus produced pseudotyped, disabled, replication defective JDV vector particles. Viral titre was obtained by transducing the cells with the supernatant harvested from transfectants and determining the number of cells expressing the transgene. PCR and Southern blotting were used to detect the presence of potential replication-competent viruses as well as transgene integration. RESULTS: Bicistronic JDV vectors encoding the green fluorescent protein (GFP) and the neomycin phosphotransferase were harvested with a titre range of 0.4-1.2 x 10(6) colony forming units/ml from vector-producing cells and were further concentrated by ultracentrifugation to the high titre of approximately 10(7) CFU/ml. Vectors encoding GFP were shown to transduce and integrate efficiently into the chromosomes of a range of primary and transformed cells of different origins in different differentiation status, including growth-arrested cells, with an efficiency of 25-75%. Exhaustive testing with a marker gene transfer assay in combination with a reverse transcriptase assay and PCR amplification of samples of serially passaged, transduced cells showed that no detectable amount of replication competent lentivirus (RCL) was produced. CONCLUSIONS: We showed the feasibility of the development of gene transfer vectors based on a non-primate bovine lentivirus, which will provide the opportunity for examination of the efficacy and biosafety of lentiviral vector-mediated gene transfer in vivo in animal models. JDV-based vectors may be applicable and more readily acceptable than those from HIV for human gene therapy.

Lymphovascular invasion as a predictor of disease progression in prostate cancer.

The biologic heterogeneity of prostate cancer (PCa) is evident from the large discrepancy between incidence rates and disease progression and tumor-related deaths. One of the challenges in treating patients with PCa lies in developing nomograms to identify patients who might benefit from adjuvant therapies. Lymphovascular invasion (LVI) is among the variables in PCa recommended to be reported by the Cancer Committee of the College of American Pathologists (CAP), yet few studies have evaluated the prognostic significance and prevalence of LVI in PCa. In the present study, whole-mount specimens from 263 patients with pT3N0 PCa treated by radical prostatectomy by a single surgeon were evaluated for the presence, location, and number of foci of LVI. Foci of LVI were identified in 91 patients. In cases with LVI the number of foci ranged from 1 to 40 with the majority of patients having 1 or 2 foci. LVI was found to be a significant predictor of disease progression in univariate analysis (p <0.0001) and was significantly related to Gleason sum (p <0.001), extra prostatic extension (focal vs established; p = 0.033), and seminal vesicle involvement (p <0.001). Furthermore, in multivariate analysis, LVI was a significant independent predictor of disease progression as well (p = 0.0014). These findings support the CAP recommendations and provide merit for the inclusion of LVI in nomograms to predict disease recurrence in PCa.

Tissue tropism of avian reoviruses is genetically determined.

Two genome segments, M2 and S1, were preferentially selected in reassortants isolated in Vero cells. Analysis with monoclonal antibodies (MAbs) against RAM-1 strain showed that the 39-kDa protein encoded by the genome segment S1 contained epitopes involved in neutralisation of virus infectivity for both Vero and chicken kidney (CK) cells. The 39-kDa protein appeared to have two major epitopes that are attachment sites for cell receptors, one interacting only with CK cell receptors and the other with both CK and Vero cell receptors but principally Vero cell receptors. These results suggest that the strain RAM-1 may have developed an epitope for Vero cell receptors owing to mutation in the S1 genome segment, but still retained the epitope responsible for infection of CK cells.

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.

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.

Serological evidence of an Australian bovine lentivirus.

Recombinant 26 kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia. A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera. Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples. All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens. This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia.

Evidence for immunosuppression associated with Jembrana disease virus infection of cattle.

Jembrana disease virus (JDV) is a newly recognised bovine lentivirus causing an acute disease syndrome in Bali cattle (Bos javanicus) in Indonesia. We evaluated the effect of JDV infection on the antibody response to chicken ovalbumin (cOVA) and Brucella abortus Strain 19 in Bali cattle. In infected cattle the IgG and IgM response to cOVA was suppressed and delayed and the IgG response to B. abortus Strain 19 was delayed. The results indicate that the humoral immune response is suppressed and delayed in JDV infected cattle.

Association between the sigma C protein of avian reovirus and virus-induced fusion of cells.

Monoclonal antibodies (MAbs) against a 39 kDa (sigma C) protein of the avian reovirus RAM-1 strain inhibited virus-induced fusion of cells and the protein was expressed on the surface of infected cells. The fusion-inhibiting activity of the three MAbs reacting with the sigma C protein suggest two putative epitopes were involved: one epitope recognised by antibody 6H1 and involved in fusion of both Vero and CK cells and a second epitope recognised by antibody 1G1 involved in fusion of Vero cells but not CK cells. The activity of the MAb 6E2 was intermediate, suggesting it may have been located in an intermediate position between the two putative epitopes and inhibited fusion by steric hindrance.

Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus.

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem-loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.

Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

Detection of Jembrana disease virus in spleen, lymph nodes, bone marrow and other tissues by in situ hybridization of paraffin-embedded sections.

Jembrana disease virus (JDV) is a lentivirus that causes an acute, severe disease syndrome in infected Bali cattle in Indonesia. An in situ hybridization technique was developed that detected JDV genomic RNA in formalin-fixed paraffin-embedded tissue sections, using a digoxigenin-labelled riboprobe. Large numbers of JDV-infected cells were demonstrated in many tissue sections from experimentally infected animals early in the disease course, which was consistent with the extremely high circulating viraemia previously reported to occur during the febrile phase. The number of infected cells was consistently highest in sections of spleen, followed by many other tissues including lymph nodes, lungs, bone marrow, liver and kidney. Infected cells were also identified in the general circulation and within unusual intravascular lesions in lung sections. The relatively high level of infection found in bone marrow suggested that its involvement may be important in the disease pathogenesis, as it is with other lentiviruses.