Analysis Of Virome In The Vagina Of Pregnant Women With Vaginitis And Identification Of A Novel Type Of HPV

A large number of different types of microbes exist in the surface and the cavity communicated with exterior of human body.Different parts of the body contain different microbial groups.Vaginal infection can cause further complications such as premature birth,puerperal infection and pelvic infection,increasing the risk of infections of pathogenic viruses,bacteria and funguses in women especially pregnant women.Women suffering from vaginitis will affect the quality of sperms during pregnancy,and even lead to infertility;During pregnancy,microbes are also easy to cause intrauterine infection,birth canal infection,and then infect the fetus,newborn,causing miscarriage,premature birth,congenital malformations,mental retardation and other serious consequences.At present,most of researches aim at the composition and distribution of bacterial community in vagina and its association with the related diseases such as vaginitis whereas less researches on the viral community in the vagina.As an important member of vaginal microflora,the species and distribution of the virus community in the vagina is of great significance to the health of women,especially pregnant women.To explore the complex composition of virus community in vigina and its relationship to health and disease of pregnant women,the composition and variability of viral community in the vagina microenvironment under specific physiological and pathological conditions can lay a theoretical foundation for the diagnosis and treatment of related diseases.Traditional bacterial and viral infections in pregnant women will not bring severe troubles to health because they may be treated with effective antibiotics,vaccines and antibodies for the purpose of treatment and prevention.However,new virus has yet to be discovered may be the chief culprit causing unknown intrauterine infection or birth canal infection inpregnant women and then infect the fetus,newborn,resulting in miscarriage,premature birth and other serious consequences.Digging the potential pathogenic new viruses in advance will be the first choice to conquer the emerging infectious diseases caused by new viruses.Viral Metagenomics is a new method of virus detection in recent years and has broken through the limitation of traditional virus detection methods which can only detect the known viruses.It takes all the viral nucleic acids in biological samples as the research object and is able to detect a variety of unknown new viruses quickly while identifying the existence of known viruses effectively.Therefore,in this research,we take pregnant women suffering vaginitis as the study object,to identify new viruses based on the analysis of the composition of virus community in pregnant women under vaginitis microenvironment using the method of Viral Metagenomic and then to explore the molecular origin and evolution of the new virus,and study the correlation between the new viruses and diseases,thus to provide theoretical basis and technical support for the prevention and clinical treatment of infections caused by new viruses in pregnant women.The main contents and results of this study are as follows:1.Analysis of the composition of virus community in pregnant women under vaginitis microenvironment:In this study,a total of 88 samples of vaginal secretions from pregnant women suffering from vaginitis were collected from Zhenjiang Maternal and Child Health Hospital to construct the next-generation sequencing libraries using Viral Metagenomics based on Miseq Illumina sequencing platform.Obtaining the original virus nucleic acid datas by deep sequencing of Miseq sequencing platform,we used analysis platform of bioinformatics based on viral metagenomic technology to analyze raw high-throughput sequencing datas to obtain all possible viral gene sequences in the libraries.Results indicated that: The gene sequences number of different viral family from large to small are Papillomaviridae 81.62%,Anelloviridae 13.36%,Unclassified viruses1.03%,HERV 0.85%,Parvoviridae 0.62%,Caliciviridae 0.51%,Phycodnaviridae 0.40%,Picornaviridae 0.23%,Mimiviridae 0.20%,Other viruses 1.18%.The composition of virus community of pregnant women under vaginitis microenvironment showed the following characteristics:(1)The virus community composition showed diversity of virus species.The eukaryotic virus community contains at least 30 virus families or groups.(2)Papillomavirus(HPV)constitutes the main body of the virus community in pregnant women under vaginitis microenvironment.We found that the HPV types were rich and the viral titer was at a relatively high level in the virus community in pregnant women under vaginitis microenvironment by further classification according species and types.The virus community includes high-risk types like HPV16,HPV52 and low-risk types like HPV6,HPV42.HPV71,the member of species Alphapapillomavirus 14,has the largest number of sequences,accounting for 38.64% of the total HPV sequences.(3)The virus community contains a variety of potential pathogenic viruses that can infect mammals such as Anelloviridae,Caliciviridae,Parvoviridae and Picornaviridae.2.Identity of a novel HPV and its genetic characteristics:By analyzing the virus community in pregnant women under vaginitis microenvironment and combining the phylogenetic analysis based on viral gene sequences,we found the library KYD08 contained 11 contigs with low amino acid homology with known HPV genomes in Gen Bank,suggesting it might be a novel HPV type.Based on the longest one of the 11 contigs,we designed a pair of nested detection primers to perform the PCR screening on 15 vaginal swabs in the library and found sample no.52 was positive.Based on the 11 HPV contigs in the metagenomic library,8 pairs of nested primers were designed to amplify the gap sequences between contigs by nested PCR and the whole genome sequence of the novel HPV was finally obtained.The HPV strain was named HPV-ZJ01,and the sequence of this virus strain was deposited in Gen Bank with Gen Bank no.KX082661.Genome sequence analysis indicated that the full-length genome of HPV-ZJ01 is 7,358 bp and contains six ORFs(E6,E7,E1,E2,L2,and L1)and a non-coding long control region(LCR).Phylogenetic analysis based on L1 protein and the whole genome of HPV-ZJ01 and other relative HPVs both indicated HPV-ZJ01 clusters with HPV161,HPV162 and HPV166(members of Gamma-PV genus,species Gamma-19).At the level of L1 gene,HPV-ZJ01 shares 71.1% nt sequence identities with HPV161 and according to the International Committee on Taxonomy of Viruses(ICTV),the PV types within a species share between 71% and 89%nucleotide identity within the complete L1 ORF.The above evolutionary analysis indicates that HPV-ZJ01 belongs to a new type within Gamma-19 species of Papillomaviridae.3.Preliminary study on the relationship between HPV and vaginitis and cervical cancer:In order to explore the correlation between the novel HPV and the vaginitis and cervical cancer,128 samples of cervical cancer in Zhenjiang area were collected,plus 88 vaginal samples of pregnant women suffering from vaginitis,nested PCR detection method based on HPV-ZJ01 sequence were used to perform PCR screening of this novel HPV on vaginal swab samples from pregnant women with vaginitis and cervical cancer samples from women.Results indicated that except the original sample 52 in the positive virus library,none of the other samples is positive.The results indicated that the new HPV strain was not associated with vaginitis and cervical cancer.The molecular epidemiological of this novel HPV on its pathogenicity needs a further study based on larger sample size.Conclusions:(1)the composition of viral community in pregnant women under vaginitis microenvironment was analyzed using the method of Viral Metagenomics.(2)The virus community in pregnant women under vaginitis microenvironment shows virus type diversity and contains various kinds of pathogenic viruses.(3)A novel HPV strain was identified in the virus community in pregnant women under vaginitis microenvironment and it belongs to a new type within species Gamma-19.(4)This new type of HPV may not be associated with vaginitis and cervical cancer.