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Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories.

ABSTRACT: The value of next-generation sequencing (NGS)-based applications for testing purposes in human medicine is widely recognized. Although NGS-based metagenomic screening may be of interest in veterinary medicine, in particular for intensively farmed livestock species such as pigs, there is a lack of protocols tailored to veterinary requirements, likely because of the high diversity of species and samples. Therefore, we developed an NGS-based protocol for use in veterinary virology and present here different applications in porcine medicine. To develop the protocol, each step of sample preparation was optimized using porcine samples spiked with various RNA and DNA viruses. The resulting protocol was tested with clinical samples previously confirmed to be positive for specific viruses by a diagnostic laboratory. Additionally, we validated the protocol in an NGS viral metagenomics ring trial and tested the protocol on viral multiplex reference material (NIBSC, U.K.). We applied our ViroScreen protocol successfully for 1) virus identification, 2) virus characterization, and 3) herd screening. We identified torque teno sus virus and atypical porcine pestivirus in a neurologic case, determined the full-length genome sequence of swine influenza A virus in field samples, and screened pigs using pen floor fecal samples and chewing rope liquid.

SUBMITTER: Kubacki J

PROVIDER: S-EPMC7953084 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories.

Kubacki Jakub J Fraefel Cornel C Bachofen Claudia C

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20201224 2

The value of next-generation sequencing (NGS)-based applications for testing purposes in human medicine is widely recognized. Although NGS-based metagenomic screening may be of interest in veterinary medicine, in particular for intensively farmed livestock species such as pigs, there is a lack of protocols tailored to veterinary requirements, likely because of the high diversity of species and samples. Therefore, we developed an NGS-based protocol for use in veterinary virology and present here ...[more]

PMID: 33357110

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Project description:IntroductionCurrent clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains.AimTo leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods.Materials and methodsTen blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq.ResultsTwo common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits.ConclusionThe common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being from Singapore.

Project description:We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.

Dataset Information

Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories.

Publications

Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories.

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