Detection And Molecular Epidemiological Characteristics On Enteric Viruses In Children With Acute Diarrhea In Guangzhou,2015-2018

1.ObjectiveDiarrhea is a global problem that cannot be ignored.Of which,viruses occupy a large share.To understand the prevalence,epidemiological characteristics of viruses is of great significance for the prevention.2.Method2.1 Subjects,information and specimen collectionFecal samples of subjects were collected from a hospital in Guangzhou.A total of 774 fecal specimens of diarrhea patients under 5 years old,228 fecal specimens of diarrhea patients over 5 years old and 364 fecal specimens of non-diarrhea people under 5 years old were collected from January 2015 to January 2018.Demographic data and clinical data were collected at the same time.2.2 Virus detectionStool specimens were also screened for common gastroenteritis viruses,including HRV,HuCV,AstV,HBoV,AdV,HPeV,AiV and SaIV.2.3 Statistical analysis and phylogenetic analysisStatistical analysis was performed using SPSS20.0 software.BLAST,Bioedit and MEGA5.0 were used to conduct multiple sequence alignment and evolutionary analyse.2.4 Viral metagenomicsIllumina HiSeq was used for high-throughput sequencing.After quality control and filteration,the sequences was compared with NCBI MicroNR and MicroNT databases through BLAST,so as to obtain virus classification annotation and classification.Reads was mapped to virus genes to obtain functional abundance information of the virus genes.2.5 16S rDNA high-throughput sequencingBacterial 16S universal primer was used to amplify the V3-V4 region of the bacteria high variable area.Then build a library and use Illumina HiSeq for high-throughput sequencing.High quality sequences will be obtained through matching,quality control and filtering.Statistical analysis such as OTU clustering taxonomy,diversity index,community structure was conducted.3.Results3.1 Demographic characteristics of subjectsAccording to the diagnostic criteria for diarrhea,1,002 fecal specimens of patients with diarrhea were collected in this study,including 774 fecal specimens of diarrhea under 5 years old and 228 fecal specimens of diarrhea over 5 years old.The mean age and standard deviation of the diarrheal patients was 1.17 ± 0.96 years old and 31.50 ± 20.35 years old,respectively.A total of fecal specimens of 364 non-diarrhea control were collected in this study,all of which were under 5 years old,with an average age of 1.25 ± 1.56 years old.3.2 Molecular epidemiological characteristics of common enterovirus in the subjectsThe detection rate of common enterovirus in the diarrhea patients under 5 years old ranked were as follows:HRV(14.08%,109/774),NOV(10.98%,85/774),HBoV(5.17%,40/774),AdV(4.01%,31/774),savv(2.07%,16/774),AstV(2.58%,20/774),AiV(0.13%,1/774),SaIV(0.13%,1/774);The detection rate of common enterovirus in the non-diarrhea controls were as follows:HBoV(2.75%,10/364),NOV(2.47%,9/364),HRV(2.20%,8/364),AdV(1.10%,4/364),SaV(0.82%,3/364),AstV(0.55%,2/364);The detection rate of common enterovirus in the diarrhea patients over 5 years old were as follows:HRV(5.26%,12/228),NOV(8.33%,19/228),HBoV(2.19%,5/228),AdV(2.63%,6/228),SaV(2.63%,6/228),AstV(3.07%,7/228),AiV(0.44%,1/228).The detection rate of HPeV in the children under 5 years old with and without diarrhrea was 10.34%(80/774)and 2.75%(10/364),respectively.There was significant difference of the detection rate between the two groups(P<0.001).The nucleotide evolutionary tree based on VP3/VP1 90 hpev-positive strains.Homologous analysis of nucleotide sequences showed that 67 strains were hpevl,8 strains were hpev3,10 strains were hpev4,1 strain was hpev5 and 4 strains were HPeV14(4.44%),respectively.HPeV was not found in the patients with diarrhea over 5 years old.3.3 The results of viral metagenomicsAfter Illumina HiSeq sequencing,reads were annotated to viruses including 47 families,119 genera and 309 species in our study.The abundance of Caliciviridae and Adenoviridae was significantly higher in the childhood diarrhea group and childhood co-infected diarrhea group than other groups.Microviridae was obviously higher than in the adult diarrhea group than other groups.Myoviridae was dominant in the children group without diarrhea.3.4 The results of 16S rDNA high-throughput sequencingA total of 11 phylum,22 classes,37 orders,63 families,198 genera and 393 species was found in our research.The dominant phyla was Firmicutes and Proteobacteria in the childhood diarrhea group,with a decreased Bacteroidetes/Firmicutes ratio.4.Conclution(1)HRV and NOV are still the main viruses that cause diarrhea,but the high detection rate of HPeV in children with diarrhea should be paid attention to.Moreover,the relationship between AiV and SaⅣ and diarrhea should be further discussed.(2)The viral composition of children with diarrhea was different from that of adults with diarrhea and children without diarrhea.(3)The microbial flora of children with viral diarrhea presented imbalance,and the influence of diarrheal virus on the microbial community of the children was greater.