Study Of The Molecular Epidemiology Of Rotavirus And The Origin, Evolution And Pathogenic Characteristics Of The Predominant Strain In Kunming

Rotavirus(RV)is the most important pathogen causing non-bacterial diarrhea in human infants and other mammals,children under age of five infected by RV at least once,almost 60%of inpatient children with acute diarrhea around the world were caused by RV infection.Of Chinese children,the infection and hospitalization rates were 28%-65%and 30-50%respectively,which resulted in a series of social burdens and economic losses.As a bordering province,Yunnan has the undeveloped economy and the poorer medical conditions,resulting in the widespread of infectious diseases including AIDS,viral hepatitis and dengue fever,which became a serious threat to public health.Due to special geographic location and suit environment,the pathogens of those infectious diseases were introduced from Southeast Asian countries and presented a co-circulation of various genotypes or subtypes in this region.Thus,Yunnan was considered the hub of pathogen’s transmission from neighbored countries into China,and then to inland provinces.In view of the diversity and widespread epidemic of RV,we believe that it is very necessary to establish a RV infection and genetic diversity monitoring network which is centered in Kunming and covered other regions of Yunnan.This study was carried out to investigate the overall endemic of RV,for further understanding its genetic diversity,to analyze the origin,evolution and epidemiological characteristics of the dominant epidemic strain.Furthermore,the dominant RV strain was isolated and cultured to investigate the pathogenic characteristics of RV.This study can provide the scientific basis for RV prevention and control,and lay the primarily foundation for the RV monitoring network in Yunnan province.From February 2015 to January 2016,samples and information were collected from outpatient and inpatient diarrhea children in two representative pediatric hospitals of Kunming,the first People’s Hospital of Yunnan province and Kunming Children’s Hospital.The overall prevalence of RV was screened using the antigen detection method.It was revealed that the positive rate of RV among 16311 diarrhic samples was 33.1%,children under age 2 were more susceptible with an infection rate of 87.4%.The occurrence of RV diarrhea was seasonal;RV infection rate was peaked in autumn and winter.It means the cold and dry is suitable for RV transmission.Moreover,120 samples were randomly selected from RV positive samples for virus RNA extraction,genome amplification,sequencing and analysis to determine its genotype.As a result,the most prevalent RV genotype in Kunming was G9P[8](85/107,79.4%),followed by G2P[4](15/107,14.0%),4 G9P[4](4/107,3.7%)strains were sporadic found.Notably,the once major prevalent GIP[8]and G3P[8]in China were not dominant in this region with the infection rates of 0.9%(1/107),respectively.However,G9P[8]has became the predominant RV genotype in this region.Homologous analysis showed that the source of G9 was diversified and closely linked to strains from other parts of China and neighboring countries.The amino acids variations of Chinese G9 were circulated in recent 5 years were found in comparing to Japanese,South Korean and Indian strains.In addition,several new variations were found in Kunming G9 strains,some of them were located in the antigen epitopes.The epidemiological curve of G9 was dynamic,has an upward trend in the current time.It may replace Gl,G2,G3 and other genotypes circulating in the surrounding countries in the near future.In order to further investigate the molecular characteristics of the major epidemic viruses,the whole genome of 12 dominant epidemic strains was amplified and the phylogenetic trees of different gene segments were constructed respectively,evolutionary analysis and variation comparison of VP7 were performed because this gene has the low homologous and high variation in 11 gene segments.Based on the constructed 11 segment genes phylogenetic trees,11 of the 12 strains showed a typical Wa-like constellations,one strain(km15118)possessed a complete Wa-like genomic backbone and recombined a DS-1-like genome segment 8(NSP2),all of them have high homology with human G9 RV.Evolution analysis showed that the average evolutionary rate for the G9 was estimated to be 1.63×10-3(1.1704-2.1068)substitutions/site/year,TMRCA analysis estimated the G9 strain was emerged around 1928,12 Kunming G9 strains belonged to lineage Ⅲ and lineage Ⅵ,emerged at 1988 and 1984 respectively,those two lineages containing human and porcine RV strains and were the most prevalent G9 subtypes around the world,they all branched from lineage I in the phylogenetic tree.To further understood the pathogenic characteristics of G9P[8]RV,one strain km15119 was isolated and cultured with MA104 cell,infected 4 days old balb/c mice with 3×105PFU could induce diarrhea,the diarrhea was self-limiting,duration for 5 to 7 days and the peak time is the third day,the diarrhea improved after 7 days but the sustainable detoxification could lasted to the tenth day.Irn addition to the intestinal,km15119 could be spread to the renal in infected mice,located in the renal proximal tubule epithelial cells,morphological showed the renal proximal tubule were swelling.No shedding and necrosis were found in the small intestine of the infected mice,the villi cells represented a wide range of vacuolar degeneration,microstructure and organelle lesions were the mainly changes observed by electron microscopy.In summary,the prevalence of RV was seasonal in Kunming,its infection rate was 33.1%among diarrhea children and children under 2 were most susceptible.G9P[8]is the predominant RV genotype in Kunming area,having a high evolutionary rate and the tendency of increasing transmission.The mouse model showed that the G9P[8]infection was mild and the disease was self-limiting,the pathological mainly presented as microstructure and organelles lesion in intestinal villi cells.Based on the study of the molecular epidemiology of RV and the molecular and pathogenic characteristics of the predominant strain,this study illustrated the prevalence of RV in Kunming,identified the origin,evolution and the prevalence feature of the predominantly G9P[8]RV,its pathogenicity were then discussed on the mouse model.The obtained results provide the scientific basis for the RV prevention,controlling and treatment of G9P[8]RV.