Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant swine diseases with near worldwide distribution. In the U.S., open reading frame 5 (ORF5) is commonly sequenced to investigate viral epidemiology. While glycoprotein 5 (GP5) is the major protein on the surface of the virion, the minor glycoproteins GP2a, GP3 and GP4 exhibit similar genetic diversity and are responsible for receptor binding and are important antigens for the immune response. Additionally, the non-structural protein 2 (nsp2) plays key role in viral pathogenicity and deletions in nsp2 have been associated with strains with increased virulence.
The goal of this project was to develop next generation (metagenomic) sequencing methodology to enable full PRRSV genome sequencing directly from clinical samples. The current standard GP5 sequencing for epidemiological investigations takes into account only <5% of the genome and advances in sequencing technology now make it cost-effective to determine a comprehensive picture of PRRSV genetics. Metagenomic sequencing of PRRSV-positive nasal swabs and oral fluids were able to detect PRRSV however read coverage was insufficient to determine complete genomes. In contrast, metagenomic sequencing of PRRSV-positive sera was successful in determining complete PRRSV genomes. Metagenomic sequencing was performed on a collection of 182 PRRSV-positive sera submitted to veterinary diagnostic laboratories. Complete PRRSV genomes were determined from 66 of these samples. Analysis of the viral structural proteins found 4-7 lineages currently circulating in the U.S. This study identified more diversity in the PRRSV structural proteins than previously recognized, possibly due to direct sequencing of clinical samples as opposed to sequencing viruses isolated in cell culture.
The goal of this project was to develop next generation (metagenomic) sequencing methodology to enable full PRRSV genome sequencing directly from clinical samples. The current standard GP5 sequencing for epidemiological investigations takes into account only <5% of the genome and advances in sequencing technology now make it cost-effective to determine a comprehensive picture of PRRSV genetics. Metagenomic sequencing of PRRSV-positive nasal swabs and oral fluids were able to detect PRRSV however read coverage was insufficient to determine complete genomes. In contrast, metagenomic sequencing of PRRSV-positive sera was successful in determining complete PRRSV genomes. Metagenomic sequencing was performed on a collection of 182 PRRSV-positive sera submitted to veterinary diagnostic laboratories. Complete PRRSV genomes were determined from 66 of these samples. Analysis of the viral structural proteins found 4-7 lineages currently circulating in the U.S. This study identified more diversity in the PRRSV structural proteins than previously recognized, possibly due to direct sequencing of clinical samples as opposed to sequencing viruses isolated in cell culture.
The added benefit to metagenomic sequencing of clinical samples is the ability to detect all viruses present in the sample in an unbiased manner. A large number of the serum samples contained porcine parvovirus 2 and porcine parvovirus 3, 4, and 5 were also detected. Importantly, we identified porcine parvovirus 6 (PPV6) forthe first time in the U.S., in 13.2% of the samples. A novel pestivirus, named atypical porcine pestivirus (APPV), was also identified in 2.1% of the samples. Genetic analysis of APPV found that is highly diverged from livestock pestiviruses bovine viral diarrhea, classical swine fever and border disease virus and is more closely related to a recently partially sequenced bat pestivirus. No information in available on the ability of PPV6 and APPV to cause disease.
The metagenomic sequencing methodology developed in this NPB grant should be of great value to swine producers and veterinarians. The Kansas State Veterinary Diagnostic Laboratory is now offering this test for $300 per sample. Given the ease of sample collection and relative low cost for metagenomic sequencing ($300 currently, prices expected to decrease), metagenomic sequencing will undoubtedly become more commonplace in veterinary diagnostics as producers and veterinarians are routinely paying ~$150 for GP5 sequencing alone. Determination of complete PRRSV genomes will greatly improve our understanding of PRRSV genetics, epidemiology and evolution and will enable more efficacious vaccine development and eradication efforts. The added ability to survey all other viruses present in a sample concurrently significantly improves our diagnostic testing capabilities and enables more comprehensive understanding of disease complexes which will lead to improved control measures.
