Detection Of Virus In Pig Lung Tissue Samples By Metagenomic Next-generation Sequencing Technology And Application

In recent years,the scale of pig breeding in China has been expanding,and novel pathogens in the pig herd have emerged.However,the infectious diseases previously prevalent in pig populations have not been eliminated yet.Thus,these infectious diseases pose huge threats to the development of pig industry.Metagenomic next-generation sequencing(m NGS)technology is a newly emerging technology developed after Sanger sequencing to investigate virus communities.Also,this technology has been broadly exploited to detect unknown pathogens,which is regarded as an important tool of other biological applications.In this research,we intend to establish the technological approach based on m NGS for the detection of unknown pathogens in the lung specimens of piglets and sows.The following contents were included in this research:(1)In this study,the m NGS approach was used to analyze the DNA virus communities in the lung tissue samples from piglets and sows.Before sequencing,the samples were processed(the control is designated as the untreated group)to reduce host DNA contamination and improve the signal-to-noise ratio.The results showed that the host nucleic acid sequences in the untreated piglet samples accounted for 82.72% of the total reads,while the host nucleic acid sequences in the treated group only accounted for 0.08% of the total reads.Therefore,the type of pig-derived viral DNA and their reads in the treated specimens are more than those of the untreated group.Furthermore,the host nucleic acid sequences in the untreated group of sow samples accounted for 2.07% of the total number of reads,while the host nucleic acid sequence detected in the treated group accounted for 1.12% of the total number of reads.In addition,we have detected a variety of known or unknown pathogens,indicating that our treatments can effectively reduce residue of host nucleic acid,which further increase the detection rate of viruses.(2)In this research,SYBR Green I fluorescent quantitative PCR technology was used to confirm the data generated by the m NGS.By detecting the constructed DNA library,the qualitative and quantitative analysis of three DNA viruses: porcine circovirus type 2(PCV2),porcine circovirus type 3(PCV3),and porcine parvovirus(PPV),in the samples were conducted.The results showed that the piglet samples were PCV2 positive,and the sow samples were PCV2,PCV3 and PPV(except the processed group in sow sample)positive.Of note,15,146 PPV DNA contigs were read out in the sow samples(processed group)via m NGS,which indicates the results of the m NGS test were reliable and meet the requirements of clinical application.(3)Finally,m NGS method was also used to investigate the RNA virus communities in the lung tissue samples from piglets and sows.The results show that the reads number of the porcine reproductive and respiratory syndrome virus(PRRSV)was greatly increased in the processed samples.Simultaneously multiple other DNA viral genome sequences were also detected.These results indicated that the m NGS technology established in this study was suitable for the identification of various viral pathogens in pig samples.In summary,the m NGS approach used in this research can be developed a practical method for pathogen diagnosis,which will be a novel tool to monitor newly emerging porcine pathogens to promote the healthy development of the pig industry.