Date Full Report Received04/27/2015
Date Abstract Report Received04/27/2015
InvestigationInstitution: ARS, Beltsville Area, USDA
Primary Investigator: Joan K. Lunney
Co-Investigators: James Reecy
Funded ByNational Pork Board
Despite extensive efforts to eliminate PRRS from US production facilities, it remains a key disease issue and poses a continued economic threat to the industry, particularly in pig dense areas. A major factor that complicates PRRS control is viral persistence. This project proposed to identify pigs which have persistent PRRSV infections. Persistence is a major epidemiological feature of PRRS virus. Pigs with persistent PRRSV infections, or carrier pigs, are a continuing threat to each production unit. Viral survival is maintained because a proportion of the herd have persistent virus which is shed occasionally (due to other diseases or stress). This shed virus then infects the remaining herd pigs which are naïve and thus susceptible. Transmission studies have verified that pigs can harbor the virus for >160 days, and likely longer. Closing a herd for 200 days was thought to be effective before new “clean pigs” could be reintroduced; some would argue for longer times.
Currently there is no good technology to accurately identify PRRSV carrier pigs, nor are there procedures to treat pigs to eliminate persistent virus from their tissues. This proposal determined the frequency of pigs with persistent PRRSV by quantitating viral RNA levels in tonsil as a surrogate measure of viral persistence. To perform this we took advantage of the repository of samples that were collected through the NPB funded PRRS Host Genetics Consortium (PHGC). Each PHGC pig, provided at weaning from current commercial breeding stocks, was infected with a virulent PRRSV isolate (NVSL 97-7985) and followed for 42 days post infection (dpi). Every pig that survived to 42 dpi had tonsil tissue archived. The PHGC database (www.animalgenome.org/lunney/index.php) has extensive data on each PHGC pig, including its pedigree, response to PRRSV infection (serum viral levels and weight gain data), and extensive genotypic information (60K SNP chip). As a results of efforts for the 3rd objective of this grant the PHGC database has been updated and expanded. It now has the capacity to archive data from more trials (up to PHGC25) and handle new data types such as next generation, deep sequencing gene expression data, as well as to save draft manuscripts and slide presentations.
For Objective 1 of this grant, PHGC trials 3 and 5 RNA was carefully extracted from tonsil of every pig that survived to 42 dpi. That RNA was then tested for viral RNA using a sensitive molecular assay. The resulting data clearly show that there is high variability in tonsil viral levels at 42 dpi with PRRSV isolate (NVSL 97-7985) in nursery pigs. Using this surrogate persistence measure, especially with sets of similarly PRRSV challenged pigs that have great variation in both serum and tonsil viral RNA levels, provided us with testable hypotheses to query for controls of persistence. Since sera from persistently infected pigs frequently are virus and antibody negative, these results and the PHGC data archive provided substantial means to affirm if there are any tissue or serological correlates of PRRSV persistence.
Our results affirmed that there is no correlation between tonsil viral RNA levels of pigs and 1) serum viral level at 42 dpi; 2) early serum viral levels (0-21 dpi); 3) weight gain changes (0-42dpi); or 4) immune gene expression in tonsil (limited survey). These results, while disappointing, were not unexpected given data accumulated from previous experiments.
These results set the stage for more detailed analyses. The wide variability of tonsil viral RNA levels opened up new avenues for querying factors that might be involved in tonsil virus persistence. Our new NPB grant (#14-223) will use sophisticated genome mapping techniques to determine whether there are genomic regions and host genes that regulate tonsil virus persistence. We will probe for mechanisms controlling tonsil PRRS viral levels using deep sequencing of tonsil RNA gene expression and statistical analyses. Comparing data from pigs with high versus low tonsil PRRS viral levels and using bioinformatic tools we hope to identify molecular pathways and genes involved in anti-persistence responses. With this knowledge, efforts can be planned to selectively breed for these pigs or, preferably, to identify means of stimulating these responses in pigs with high persistent PRRS viral RNA.