Development And Application Of Metagenomics-based Detection Method For Porcine Viruses

With the substantial development of the pig industry in China,the scale and intensive level of breeding have been greatly developed.However,the current situation also poses great challenges to the pig industry.The situation of disease is becoming more and more complicated.Nowadays,mixed infections and continuous variation of pathogens,and emergence of new pathogens,all of these are serious threats to the pig industry of China.At present,the method of pathogens detection mainly include PCR,m PCR,q PCR,indirect immunofluorescence test,ELISA,colloidal gold test strip,etc.All of these methods are based on the known nucleic acid sequence of the pathogen.And the number of pathogens detected in a single test is limited.These methods lack the ability to cope with the complex situation and can not make a more comprehensive and accurate detection.This study focuses on the combination of viral metagenomics and next-generation sequencing technology to deal with the complex situation,to improve the capability for detecting,to enrich the knowledge of detecting,to provide ideas for the prevention and treatment.(1)A method for detection of porcine viruses was developed by means of viral metagenomics and high-throughput sequencingThe positive samples was a mixture of porcine circovirus type 2,porcine pseudorabiesvirus,porcine epidemic diarrhea virus and porcine reproductive and respiratory syndrome virus.First,the DNA and the RNA were extracted from the positive samples.Second,the nucleic acids concentration of DNA was increased by conducted multiple displacement amplification.After reverse transcription of RNA into c DNA,klenow polymerase was used to synthesize the double-stranded c DNA and completed the ends.Third,products of MDA and dsc DNA were amplified sequence-independent single primer amplification with random primers.The PCR products were purified and concentrated.Fouth,the qualified samples were utilized to construct sequencing library after repairing the end,added tail,connected adapter,and enrichment.Finally,the results were obtained by high-throughput sequencing and bioinformatic analysis.The results of library quality examination showed that the quality of the constructed library was good,which met the requirements of sequencing.And a total of 2.90 G clean data was obtained.The figure of base quality distribution showed that the accuracy of sequencing was above 99.90%.And PCV2,PRV,PEDV and PRRSV were detected in all the positive samples by bioinformatic analysis.(2)Etiological investigation of porcine reproductive failure and porcine diarrheaIn this study,10 cases of porcine reproductive failure were detected by using viralmetagenomics.A total of 2 914 036reads were annotated,which can classify into baculoviridae,phages,bunyaviridae,herpesviridae,retroviridae,parvoviridae,flaviviridae,circoviridae,anelloviridae,astroviridae.The main viruses detected in case 1was PRV,and in case 2 were PPV2,PPV6,PPV3,PBo V5 and JEV,and in case 3 were PPV6,PPV2,PCV2,PCV1 and TTSu V,and in case 4 were PPV2,PPV3,PPV6,PCV2and PCV1,and in case 5 was JEV,CSFV,PEDV and PRV,and in case 6 were TTSu V 1b and TTSu V1a,and in case 7 were JEV,and in case 8 were PCV2,PCV1,PEDV,PRV,JEV and PRRSV,and in case 9 were PPV2,PPV3,PPV6,PCV2,PCV1,JEV and TTSu V1b;in case 10 were PBo V5 and PAst V2.The detection rate of JEV was 50%(5/10).The detection rate of PPV2,PPV6,PCV2,PCV1 and TTSu V was 40%(4/10).The detection rate of PPV3 and PRV was 30%(3/10).The detection rate of PBo V5 and PEDV was 20%(2/10).The detection rate of CSFV,PRRSV and PAst V was 10%(1/10).Four cases of porcine diarrhea were detected by viral metagenomics.Using bioinformatics analysis,a total of 1 958 451 reads were obtained,which can classify into baculoviridae,phages,bunyaviridae,parvoviridae,circoviridae,coronaviridae,anelloviridae,flaviviridae.The main viruses detected in case 1 were PPV3,PPV6 and TTSu V1b and PEDV,in case 2 were PPV3,PPV6 and PEDV,in case 3 were PCV2,PEDV,PPV3,PPV6 and PPV2,in case 4 were PCV3 and PEDV.The detection rate of PPV3,PPV6 and PEDV was 75%(3/4).The detection rate of PPV3,PCV2,PCV3,JEV and TTSu V was 25%(1/4).Complete genome sequences of PCV2,PPV2,PPV3,PPV6 and PAst V2 were obtained by sequence splicing.Genetic analysis showed that these five viruses had higher homology with their corresponding viruses,and there were some changes in amino acid sites.(3)Development and application of porcine parvovirus type 2 3 and 6 multiplex PCR Through the previous research results,we found that the detection rate of PPV was high and there were mixed infection of multiple types of PPV.Many studies confirmed that the phenomenon was common on pig farms.At present,there is no method to detect porcine parvovirus type 2 3 and 6 at the same time.To understand the prevalence of PPV in pig farms,a multiple PCR assay was developed to identify PPVs.Specific primers for three genotypes of PPV were designed by using primer 5.0.A multiple PCR method,that there is capable of detecting PPV2 PPV3 and PPV6simultaneously,was established by optimizing the reaction temperature,and the number of reaction cycles,and the final concentration of the primers.The specificity and sensitivity of the method were evaluated by specificity test and sensitivity test.The limit of detection of PPV2 is 4.32×10~3copies/?l,PPV3 is 9.62×10~3copies/?l,and PPV6 is 4.25×10~3copies/?l,which are demonstrated that this method possess good sensitivity.There was no effective amplification of PCV2 PEDV PRRSV PRV HPS APP and JEV,suggesting that the method has good specificity.Detection results of 25 clinical samples showed that the detection rates of PPV2 PPV3,PPV6 were 12%(3/25),16%(4/25),16%(4/25)respectively.A total of 250 porcine reproductive failure-related samples from hubei province were collected to detect PPVs.The positive rate of PPV2 was 8.40%,PPV3 was 11.60%,and PPV6 was 9.60%respectively.PPVs mixed infections were prevalent in the time range of collected samples and the detection rate does not change with season.This study successfully established detection method for porcine viruses and provides a method for rapid detection for PPVs.