| The site of Hongyuan county is located on the eastern Tibetan Plateau,with an average elevation of 3504 m,a relatively unique geographic location,and a relatively harsh environmental climate,which,together with the unique customs and customs of local residents,religious beliefs,lifestyles,and culture practices of animals,provides more opportunities for human and animal contact,and the Tibetan Plateau has long been characterized by an underdeveloped public health system,with viral diarrhea being the predominant local child To disease.Whereas zoonosis is a disease that can be naturally transmitted from vertebrates to humans or humans to animals,with most humans coming into contact with animals in some way,studies have shown that more than 60% of human pathogens originate from zoonotic infections.At present,the epidemiological investigation of diarrhea in children in Hongyuan county is not particularly sufficient,and the data of etiological studies are minimal.This study aims to use viral metagenomics to study diarrhoea viruses in diarrhoeal stools of local children,to understand diarrhoea associated virus species in diarrhoeal stools of children in Hongyuan County,to understand the possible zoonotic diarrhoea causing viruses in local children,and to further investigate their evolution and genetic relationships,to provide the basis for diagnosis and prevention of diarrhoea viruses in local people and animals.The achieved results are as follows:1.Viral metagenomics identifies virus species in diarrhea samples from children in Hongyuan CountyA total of 12 fecal samples from Hongyuan County pediatric patients with acute gastroenteritis from April 18th to November 4th,2019 were collected,which could be divided into two parts according to the sampling time,from May 24th to June 26th and from October 10th and November 4th,respectively,and the 6 samples in each part were composed into one pool sample,which respectively extracted total nucleic acid,reverse transcribed into double stranded c DNA,and built into a pool and C1 library,respectively,by Illumina novaseq sequencing.A total of 171368382 reads were obtained from library a,1149 RNA overlapping sequences(contigs)were assembled and 147342238 reads were obtained from library C1,and 1749 RNA was assembled and spliced Overlapping sequences,curated analysis of viral nucleotides in databases resulted in the identification of a total of seven viruses in two pools,of which five were identified in pool a: astrovirus,norovirus,reovirus,aichivirus,and adenovirus.The C1 library identified five species: astrovirus,coxsackievirus,sapovirus,mammalian orthoreovirus,and adenovirus.To validate the presence and distribution of the 7 viruses in pediatric diarrhea stool samples,RT-PCR and PCR assays were used to identify the 12 pediatric diarrhea stool samples used for library construction.The results showed that 7 viruses were detected as follows: astrovirus(66.67%),norovirus(8.33%),Sapporo virus(25%),Aichi virus(8.33%),coxsackievirus(8.33%),adenovirus(8.33%),and reovirus(0).From the results,the highest infection rate of astrovirus was observed,and some samples detected mixed infection with astrovirus and norovirus and Sapporo virus,reovirus failed to be detected,this study is the first to confirm the presence of the above 6 viruses in red won county children’s diarrhea samples.Astrovirus infection is the most common,and diarrhoea virus species are complex and diverse,with mixed infections,which provides reference data for the aetiological investigation of diarrhoea in children and the local prevention and control of diarrhoeal disease in Hongyuan county.2.Genome amplification and sequence analysis of astrovirusesTo further understand the genomic features of astrovirus,four pairs of primers were designed,and a full-length human astrovirus genome sequence(Hu / HY / 2019/ CHN)was obtained by segmental amplification,according to the gene fragment obtained by viral metagenomics and the astrovirus genome sequence in GenBank,and the sequence analysis found that the genome length was 6826 BP,with a GC content of 44.37%.Contains three open reading frames encoding 934 amino acids for orf1a:2803 BP,ORF1b: 1561 BP encoding 520 amino acids,and ORF2: 2364 BP encoding788 amino acids.A total of 81 nucleotide sequences were 42.6%-97.4% similar to all human derived astrovirus whole genomes available in GenBank.The corresponding ORFs from the upper human astrovirus type 1 strains that were homopolymeric as a large clade had nucleotide similarities of 84.1% - 97.5%,79.6% - 98%,79.1% -98.1%,respectively,whereas the amino acid similarities were 89.6% - 97.5%,79.8%- 98.4%,78.4 - 99.2%,respectively.The analysis showed the highest genome-wide nucleotide similarity(97.72%)with the Shanghai strain sh1 human astrovirus type 1(GenBank accession number: fj375756).Its subtype was further analyzed as human astrovirus type 1b.Alignment with the sh1 sequence revealed that orf1 a and ORF1 b shared 96 base mutations,30 amino acid mutations,ORF2 had 43 base mutations,and5 amino acid mutations.After prediction of the antigenic epitope,it was found that the amino acid mutation was not in the antigenic epitope position.No recombination events were found to have occurred after recombination analysis.In addition,although the Hu / HY / 2019 / CHN strain does not share a close genetic relationship with animal astrovirus strains,comparison with representative strains from various animals in GenBank(including chicken,duck,cat,pig,Greyhound,dog,dolphin,yak,cow,bat,sheep,rabbit)totaling 87 ORF2 amino acid sequence similarities revealed the highest similarity(74%)to cat.This study confirmed the presence and predominance of astrovirus 1 among childhood diarrhoea cases in Hongyuan County,and for the first time,we obtained a complete genome of human astrovirus 1 from Hongyuan County,and clarified its structural and evolutionary characteristics,which may provide valuable data for further research in pathogen detection and prevention.3.Genomic amplification and sequence analysis of norovirusesTo further characterize the human norovirus genome in Hongyuan County,11 primer pairs were designed based on the gene segments obtained by viral metagenomics techniques and the norovirus genome sequence available in GenBank,and a 7554 BP approximately full-length norovirus genome sequence(Hu / HY-1 / 19/ CHN)with a GC content of 49.85% was obtained by segmental amplification,which contains three open reading frames,ORF1(5100 BP)encoding 1699 amino acids,ORF2(1623 BP)encoding 541 amino acids and ORF3(807 BP)encoding 269 amino acids.The virus has structural features typical of noroviruses.Genomic blast analysis showed the highest similarity(98.73%)to GII.4 norovirus(GenBank accession number: mk934772).The phylogenetic tree constructed from the nucleotide sequence of the entire genome,the RdRp protein,the VP1 protein,the P2 domain,and the amino acid sequence of the VP2 protein,respectively,was found to cluster with all strains of the GII.4 syndney2012 cluster as a large branch,and the Hu / HY-1 / 19 /CHN strain was found to cluster with the GII.4 norovirus like cluster on the basis of the phylogenetic tree constructed from the amino acid sequences of the VP1 protein and P2 domain(GenBank accession no Transcript number: ky407116)were clustered into one branch and may have the same evolutionary relationship.The VP1 region amino acid sites were compared with the previously reported antigenic epitopes a(294,296,297,298,368,372),B(340,376),D(393,394,395),and E(407,412,413),B2(377,378)F(352,355,356,357)to compare and showed mutations at four amino acid sites,all occurring within the P2 domain,some of which were common to strains belonging to the sydney-2012 cluster of genes,amino acid change at position 297 from R to h,The amino acid at position 372 was changed from D to N,the amino acid at position 357 was changed from D to g,and only one site belonged to a separate mutation in the strains obtained in this study,as was the amino acid at position 393 changed from s to g.Because the P2 domain surface has a receptor binding function and contains strain specific determinants,binding sites,as well as neutralizing antibody binding sites,host immunity and virus receptor selection have collectively contributed to the evolution of the p region,resulting in the continuous generation of new subtypes.The biological significance of these mutations remains to be investigated.4.Genomic amplification and sequence analysis of Sapporo virusTo gain further insight into the genomic features of Sapporo virus,four pairs of primers were designed according to gene fragments obtained by viral metagenomic techniques and the Sapporo virus genomic sequence in GenBank,and a 7355 BP approximately full-length genomic sequence of Sapporo virus was amplified in segments with a GC content of 53.23%.With typical structural features of Sapporo virus,containing two open reading frames encoding 2266 amino acids for ORF1(6803 BP)and 168 amino acids for ORF2(504 BP),respectively.A total of 134 nucleotide sequences were 42.2% - 98.47% similar to all Sapporo virus whole genomes available in GenBank.Whole genome analysis revealed the highest similarity(98.47%)to Jiangsu giv.1 Sapporo virus tz-lib06-1(GenBank accession:mn161595).Alignment with the tz-lib06-1 strain sequence revealed the presence of mutations at 14 amino acid sites within ORF1,at only 1 position within VP1,and at 1amino acid site within ORF2.Phylogenetic trees constructed from the amino acid sequences of the VP1,VP2,and RdRp regions,respectively,showed that the giv.1Sapporo virus strains in this study did not show differences in phylogenetic clustering,but blast of the RdRp region sequences of the strains in this study for alignment and GenBank library for similarity revealed many GII.3 Sapporo virus sequences in the alignment and no recombination events.In this study,we confirmed the presence of Sapporo virus giv.1 in hongwon county and obtained the genome for the first time in Sichuan Province and enriched the whole genomic sequences of Sapporo virus in China to provide a reference for genetic evolution analysis of the Sapporo virus. |
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