HTS Based Screening Of Novel Zoogenic Virus And Virus Whole Genome Sequencing And Analysis

The emergency infectious diseases never stop appearing in nature,a large part of them are related to animals,such as Severe Acute Respiratory Syndrome,Middle East Respiratory Syndrome,avian influenza,Ebola haemorrhagic fever and other epidemics,which bring us extremely painful memories.The 2019 novel coronavirus which we are undergoing makes us realize that zoogenic viruses bring great loss to human health and economic life.The traditional high-throughput sequencing technology has played an important role in the detection and diagnosis of these major infectious diseases,but it is difficult to detect the samples with low titer and complex background.The proportion of host genome in the accounting of nucleic acid samples is overwhelming,resulting high tendency to cause amplification bias when building libraries,and the sequencing coverage of low-abundance virus is low or even unable to detect;match score of the novel virus genome and known sequences is low,and the sequence with low similarity may not be detected by the routine nucleic acid alignment,resulting in missed detection.For virus samples with low titer,it is often impossible to get the whole sequence through direct nucleic acid sequencing,and only a part of the sequence obtained from the first screening can not meet the needs of conventional whole genome amplification,while the novel viruses have low similarity with the reference sequences,hence,it is impossible to design primers based on the reference sequences.Considering the requirements of virus genome sequencing under these complex conditions,this paper improve the traditional high-throughput sequencing method,and explores a method for the whole genome sequencing of viruses with low titer or low homology with known sequences from different animal samples,which provides a new idea for the whole genome sequencing of viruses under complex conditions.The specific research contents are as follows:Detection of novel zoogenic virus.After high-throughput sequencing on Ion S5 sequencing platform,we used the new pathogen classification and analysis software independently developed by our laboratory to analyze the massive data obtained from sequencing: filtering the sequences to remove the low-quality data,homogenizing the sequencing coverage,comparing the sequences with all the viral protein libraries downloaded from NCBI,and blasting the virus-related sequences again,the relative sequences were compared with the viral nucleic acid sequence library to classify virus and identify similarity,the remaining nonmatched sequences were compared with the viral protein sequence library,significantly reducing the omission and discovering novel viruses.Using methods mentioned above,this paper detected a batch of serum samples from cattle and sheep raised by herdsmen in Tengchong City,Yunnan Province,different new hepaciviruses were detected among them,with the low nucleotide similarity with the known viruses.Respectively,they were detected for the first time in Yunnan Province,enriching the virus diversity in the border areas.Whole genome sequencing and bioinformatics analysis in complex situations.For the newly discovered hepatitis C viruses in the serum samples from cattles and sheep,the abundance of the new viruses in the nucleic acid samples containing the new virus was relatively low identified by fluorescence quantitative PCR,and the nucleic acid sequence was quite different from the known sequence,so it was difficult to determine the sequence by using the traditional PCR amplification after reverse transcription.In this paper,the reverse transcription method was improved,a known sequence was introduced into the terminal of c DNA using the new reverse transcriptase as a primer,PCR amplification was performed with another primer designed according to the obtained sequences.For viruses with low abundance,the number of amplification cycles was increased to obtain a sufficient amount of data.The data was analyzed with the sequence analysis platform of our laboratory,and the whole genome sequence was obtained using bioinformatics technology.With phylogenetic analysis of the new hepacivirus,we found that the virus found in bovine serum was a new subtype of Bovine Hepacivirus,and it was the only virus strain with the whole genome sequence detected in this genotype;the Hepacivirus found in sheep serum which was a new genotype was reported for the first time in China.This study promotes the understanding of virus diversity in border areas.Using the above methods,this paper also sequenced and analyzed a batch of samples from a pig farm in Qingyuan,Guangdong Province in August 2018.The samples were taken from the heart and brain tissue of the sick pig,and the nucleic acid was extracted through homogenization and filtration.The known 1375 bp length sequence was blasted with PRRSV strain HENZMD-9(login No.KU950374.1),and the highest similarity was 96%.HENZMD-9 was used as reference sequence to design amplification primers,PCR amplification was carried out with the c DNA obtained from reverse transcription with the new reverse transcriptase as the template.The library was analyzed by the high-throughput sequencing platform,the preliminary incomplete sequence was obtained by assembly,the primers for amplifying the whole genome sequence were designed with the obtained sequence as reference.The sequenced data were obtained,then the whole genome sequence of the virus was analyzed by bioinformatics and the phylogenetic tree was constructed.The detection of PRRSV confirmed the feasibility of the whole genome sequencing method for low titer virus.