Identification Of The Whole Genome Analysis Of New HpeV Virus And The Intestinal Virome Of Children With Diarrhea In Some Areas Of Jiangsu Province

Diarrhea is a common type of intestinal disease that is common in the world,which brings a huge threat to human health.Diarrhea is easy to violate children and elderly groups,and acute diarrhea is a worldwide common disease,especially in underdeveloped countries,and is one of the main causes of infant mortality.Pathogenic microbial infections(including bacteria,viruses,fungi and parasites)are important causes of diarrhea,Which is the most important cause of viral infection,With the continuous development of economic strength,the continuous improvement of medical and health conditions,medical and health conditions continue to improve and drinking water and health conditions to improve,Human beings have developed a large number of effective antibiotics for the treatment of bacterial diarrhea,While viral infection caused by diarrhea has not been effectively controlled and concerned,resulting in the incidence of bacterial diarrhea gradually less than viral diarrhea,Pathogenic pathogens for viral diarrhea are still not clearly stated.Therefore,the present study is to understand and master the composition and prevalence of diarrhea virus and for the prevention and treatment of viral diarrhea is of great significance.At present,the traditional virus identification and detection methods are mainly for the known or variability of the virus,In the unknown or new virus caused by the new infectious disease pathogen confirmed on the larger limitations;In recent years,with the development of molecular biology technology and the need to tap new viruses,A large number of non-culture-dependent virus mining and identification technology came into being,Where in the viral metagenomic(Viral Metagenomics)gradually show its great advantages,which has become an unknown new virus identified an important means.The main purpose of this study is to use viral metagenomics to analyze the composition of viral communities in diarrheal children.At the same time,the evolution and genetic relationship of new diarrheal virus were studied.which is the basis for the prevention,diagnosis and treatment of acute diarrhea caused by new virus,which is of great practical significance for the rapid confirmation of pathogenscaused by new viruses in the future.The followings are the results of this study:1.The composition of intestinal virus population from children with diarrheaUsing non-culturally dependent virus biology research methods,collecting stool samples of diarrhea children,construction of a virus genome library for the Illumina Miseq platform,using the Miseq platform for high-throughput sequencing,After obtaining the original library nucleic acid data,a series of bioinformatics analysis based on viral nucleic acid information was performed,the visual community results in the output of the virus community.The results showed that the composition of the virus community in the diarrhea stool samples of children was as follows:Adenoviridae,Anelloviridae,Caliciviridae,Parvoviridae,Picornaviridae,Reoviridae,Astrovidae,Astroviruses,Astroviridae,Picobiriviridae,Podoviridae,Circoviridae,Dicistroviridae,Phycodnaviridae,Phytoxylidae,Poxviridae,Retroviridae,Tombusviridae,Baculovoridae,Herpesviridade,Microviridae,Iridoviridae,Papillomaviridae,Human Endogenous Retroviruses(HERV),Flaviviridae,Coronaviridae,Virgaviridae,and Undeclonal Viruses and Other virus.Virus community analysis suggested:(1)Children with enterovirus community composition Diarrhea-related viruses include: Caliciviridae,Astroviridae,parvoviridae,Picornaviridae,Picornaviridae,Reoviridae,Virus titer analysis results show that,These virus groups constitute the children 's diarrhea virus community constitute the main body of the virus community;(2)The results of virus titer analysis showed that the virus titer contained in DiaFeces05-DiaFeces09 virus library was higher and the virus type was more.While DiaFeces01-04 and DiaFeces10 virus titer is small;(3)in the previous cause of diarrhea caused by the main pathogens for the calicivirus class,although in this part of Jiangsu local children with diarrhea intestinal virus community composition of the cup virus is still more than half of the proportion of the library positive rate is high,the study of calicivirus is not the cause of diarrhea.2.Identification of a new human parechovirus(HPeV)in children with diarrhea(1)Analysis of genomic structure and genetic characteristics of novel HPeVBased on the bioinformatics analysis of the viral genome sequences in the viral community of diarrheal children,a new human parechovirus HPeV(named CH-ZXY1)was found in the stool samples of children with diarrhea,In the genetic relationship with the known virus has a greater degree of divergence,which may be a new type of HPeV strains.Thus,using the relevant informatics software such as the Assembly function in Geneious and the Genome Mapping method based on the viral genome,the whole genome sequence of the virus was obtained,The whole genome sequence was further confirmed by ordinary PCR.The results show that the CH-ZXY1 genome sequence is 7,213 nt,Based on the CH-ZXY1 whole genome sequence phylogenetic tree showed that CH-ZXY1 and HPeV5 CH-ZI1 aggregated to form a single evolutionary branch;Based on the whole genome sequence of CH-ZXY1 strain,the results of Blastn in GeneBank showed that the similarity of CH-ZXY1 to HPeV5 CH-ZJ1 was 84%.In this study,phylogenetic tree analysis was performed based on three major genes of HPeV virus(P1,P2 and P3).Based on the phylogenetic tree of three different gene regions,the CH-ZXY1 strain was closely related to HPeV5 CH-ZJ1,HPeV4 Fuk2005-123 and HPeV1 KVP6,respectively.And then BLASTn search based on the nucleotide sequence of P1,P2 and P3 regions in GeneBank,The results showed that CH-ZXY1 and P1,P2,P3 nucleotide sequence in GeneBank Blastn,the results showed CH-ZXY1 showed the highest sequence similairity to HPeV5 CH-ZJ1,HPeV4 Fuk2005-123,and HPeV1 KVP6 with similarity of 82.8 %,83%,and 91%,respectively.Based on the phylogenetic analysis and sequence analysis of CH-ZXY1 genome,the genetic relationship between different gene regions and other strains was different,suggesting that there may be genome homologous recombination.(2)HPeV virus recombination analysisBased on the whole genome sequence of the related strains of HPeV strain CH-ZXY1 and GenBank,the homologous recombination analysis of virus gene was carried out,The results showed that CH-ZXY1 could be a recombinant strain,which occurred between HPeV1 representative strain Br / 53/2006(HQ696575)and HPeV5 representative strain BNI-788st(EF051629).The phylogenetic tree analysis wasperformed based on the gene sequences at both ends of the gene homologous recombination breakpoint,the results in phylogenetic trees based on different sections showed that CH-ZXY1 was clustered with HPe V1BNI-788 st and HPe V5 Br / 53/2006,respectively,which confirmed the recombination event more accurately.The recombination results was further confirmed by PCR with primers covering the recombination breakpoints.(3)Prevalence of HPeV in Diarrhea Children in Zhenjiang AreaIn order to elucidate whether the HPeV strain is associated with diarrhea disease in children in Zhenjiang and the prevalence of HPeV in the diarrheal population of the region.The fecal samples of 110 children with diarrhea and 50 physical control children were collected from Zhenjiang area.Universal Degenerate Primers were used to detect the HPeV in children with diarrhea,the results showed that 15 cases of HPeV positive in fecal samples of diarrhea children,the total detection rate was13.6%.The amplified bands of the positive HPeV samples were cloned and sequenced,the results showed that the number of HPeV1 was 46.6%.Of the 50 normal control fecal specimens,5 copies of HPeV positive specimens,detection rate of 10%(5/50);Five positive HPeV samples were cloned and sequenced,the results showed that 3cases of HPeV1,and 2 cases of HPeV6,compared with the detection rate of diarrhea,there was no significant difference.In the diarrhea of stool samples also detected less popular HPeV6 and HPeV7,Based on the above results,the prevalence of HPeV1 in children with diarrhea in Jiangsu is the main epidemic type.Conclusion:(1)In this study,In this study,the viral metagenomic method was used to successfully analyze the composition of viral communities in diarrhea of parts of Jiangsu Provincethe(2)A new type of HPeV strain was isolated from the stool samples of diarrhea children and the whole genome sequence(3)Systematic evolution and recombination analysis showed that the strain was recombinant for the recombination events between different genotypes of HPeV(4)The epidemiological status of HPeV in diarrhea children in Zhenjiang area was elucidated.