Charaterisation pathogenicity and epidemiology studies on novel bocoviruses recovered from swine.

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Biological Sciences

Abstract

Recent research in our laboratories has identified 2 new bocaviruses in swine. We have isolated these 2 viruses, which are each antigenically different, from farms in Northern Ireland with clinical postweaning multisystemic wasting syndrome (PMWS) and preliminary partial molecular characterisation of these viruses has shown that they share genetic homology with bovine parvovirus-1 (BPV-1), canine minute virus (CMV) and human bocavirus (HBoV). To date, the disease-causing significance of these novel swine bocaviruses, as primary pathogens or perhaps as immunosuppresive triggers for other infectious agents, is undetermined. Investigation of the importance of these viruses in swine and humans as potential emerging pathogens is urgently required. It is possible that these newly identified swine bocaviruses could be pathogenic to swine and may also have a zoonotic potential. Whether swine bocaviruses have implications as a debilitating primary infection in pigs and/or humans or as a contributory infection, perhaps acting as an immunosuppressive infectious agent, merits urgent investigation. This genomic sequence data of the viruses generated to date will be expanded and used to produce tools for detection of swine bocaviruses in tissue samples and blood. Monoclonal and polyclonal antibodies will be produced to all 2 new swine bocaviruses to further study the biological characterisation, epidemiology and potential pathogenesis of swine bocaviruses in swine and other species. Experimental infections of swine will be carried out to determine sites of replication of these viruses and potential pathogenesis. Archived tissue samples from diseased and non-diseaed swine (including foetal material) in the UK will be examined and prospective field studies will be carried out. The possible 'contamination' of swine biologicals, including vaccines with swine bocavirus will also be investigated. On the basis of recent findings reported for other bocaviruses (including human bocavirus) we believe that our newly discovered swine bocaviruses may have the capacity to cause respiratory infections and/or reproductive problems and/or contribute to the severity of PMWS in swine. We also hypothesise that other existing porcine viral pathogens may have an increased severity due to the presence of these new viruses. Therefore, the hypothesis to be tested is that 'newly discovered swine bocaviruses will cause respiratory and other disease scenarios in pigs and that the virus isolates will exacerbate symptoms in pigs already suffering from other disorders. Additionally an experimental model of infection with swine bocavirus could prove useful in the study of human bocavirus infections. In this project hitherto-unavailable reagents, diagnostic tests and experimental models will be developed and applied to provide information relating to the characterisation, detection, epidemiology and pathogenicity of newly discovered bocaviruses of swine. Those newly discovered isolates will be fully characterised at nucleotide level and biologically using current techniques that have already been applied in our laboratory for the discovery of other novel viral pathogens.

Technical Summary

This project aims to fully characterise 2 newly isolated swine bocoviruses and provide information on the epidemiology and pathogenicity of these viruses, Complete genomic characterisation of the swine bocavirus (SwBo) isolates will be achieved using existing methodology and a modification of a random PCR method Protein profiling of SwBo will be carried out FRET-based probe assays for faeces, blood and tissue samples will be developed for the quantitative detection of swine bocaviruses nucleic acid, and validated. Probes for ISH will be developed. Polyclonal and monoclonal antibodies to the SwBos will be prepared. Clones will be selected that cross react with all 2 isolates and, if possible, SwBo individually specific clones will also be selected for characterisation/classification of the 2 viruses. Proactive and retrospective (archived samples) studies on farms will investigate the presence of SwBo antibodies and nucleic acid/virus. The material will also be assessed for the presence of other porcine viral infections to elucidate the possible interactions of SwBos with other viruses, related to disease expression. Sera from other species will be screened for SwBo antibodies and biologicals used in the swine industry will assayed by PCR for 'contamination' with SwBo. It is expected that a number of small pilot experiments in year 1 of this project in a CD/gnotobiotic model (3 to 4 pigs per experimental infection) will be necessary, probably with at least 2 of the 3 current isolates, with and without other viruses as a co-factor. In the light of the recent findings that a 28 year old human suffering from T-non-Hodgkin Lymphoma had developed severe atypical pneumonia associated with human bocavirus infection it would be of interest to experimentally inoculate immunocompromised swine with SwBo. Additional experimental infection studies will be carried out in year 2 and 3 of the project, but their nature will depend on results generated in year 1.
 
Description In recent years a number of small viruses with DNA genomes have been discovered in humans and animals. Some of these newly discovered viruses, (pig circovirus (PCVs)), were initially thought to be non-disease producing. However, following studies between 1997 and 2003, researchers at QUB discovered that infection of pigs with PCVs could cause severe disease and death and this was the cause of a new global epidemic of severe disease in pigs. In 2007 research by the same group identified a number of new bocavirus-like viruses in pigs in the UK. This represented the first report of these viruses in pigs and partial characterisation had shown that they were similar to the already characterised cattle parvovirus-1 (BPV-1), dog minute virus (CMV) and human bocavirus (HBoV). The disease significance of these bocaviruses was undetermined and merited investigation. On the basis of published research on bocavirus infections of other species, it was hypothesised that the new pig viruses (PBoVs) may have the capacity to cause respiratory infections and/or reproductive problems and/or contribute to underlying respiratory problems. This project was initiated to fully characterise these 2 new pig viruses, and to provide information on the spread, distribution and disease potential of these viruses.
In the initial work package of this project reagents and procedures were developed and validated for future use, including antibodies to different bocaviruses and techniques to use these antibodies for viral detection in tissue sections. Primers and procedures (real time polymerase chain reactions (RT-PCRs)) for the detection and quantification of porcine bocavirus nucleic acid (Northern Ireland and Swedish bocaviruses) and sequencing of the viral genomes were also developed. These reagents and procedures were used throughout the rest of the project to fully characterize the new cell culture grown porcine bocavirus isolates and bocaviruses recovered from samples by colleagues in Sweden.
The genomic sequences of the 2 viruses from Northern Ireland were elucidated as were the partial genomic sequence of a number of bocaviruses detected in tissues from Sweden and field investigations in the UK and Europe. The reagents and procedures were also used to determine the distribution of these newly discovered viruses in pigs in the UK and other countries in Europe, as well as cattle, humans and sheep. Surveys for specific antibodies in samples of blood from pigs were carried out and tests for the detection of the virus in tissue samples from pigs, humans, cattle and sheep were also completed. The viruses were widely detected in pigs but not in any other species tested. In the field studies carried out in pigs in France, a loose association between infection with bocaviruses and mild respiratory diseases was noted. However, extensive studies on tissue samples from a range of tissues from pigs with disease symptoms, including respiratory disease, abortions and gut disorders, failed to strongly associate bocavirus infections in the field with any obvious clinical condition.
Experimental infections of pigs with different bocaviruses, with and without co-infection with other pig viruses, were carried out. Infection with bocavirus alone failed to produce disease. However infection with bocavirus and PCV2 did produce clinical disease in one, and subclinical disease in six of 24 inoculates. However, further studies are required on the samples taken from these pigs and the relevant controls to confirm the significance of this observation. These results, combined with the results of the field studies outlined above, would suggest that the pig bocaviruses characterised and used in this project do not currently appear to have a major pathogenic role by themselves but may be important in combination with other infectious agents.
1. A major achievement of this project was the determination of the full coding sequence of PBoV3 and PBoV4. While partial sequence for both viruses have been submitted to GenBank, it is expected that the full CDS will be submitted now that it has been fully characterised.

2. The production of monoclonal antibodies against PBoV3 and PBoV4. To date, these reagents represent the only monoclonal antibodies against PBoVs currently in use worldwide.
3) The development of a range of molecular tools for the detection of these novel viruses. These assays afford researchers the opportunity to detect, sequence and characterise both PBoV3 and PBoV4 isolates and have the potential for the discovery of novel bocaviruses in other species.
Exploitation Route The findings listed above can be used by other research groups to advance the knowledge of the significance of bocavirus infections of swine.
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