Investigation Of Epidemic Keratoconjunctivitis Of Tibet Breskout In 2016 And Study On Its Pathogenic Characteristics

Objective: From April 2016 to November 2016,epidemics of epidemic keratoconjunctivitis(EKC)broke out in Gongbo'gyamda County and Namling County of Tibet,and reported cases were up to 197 ones.The research firstly defined the pathogen for two times of EKC epidemics in Tibet in 2016,identified its type,and then analyzed the features and recombination for primary structural genes of human adenovirus type 8(HAdV-8)-the pathogen for this epidemics,to provide theoretical reference for the prevention of diseases caused by HAdV-8.Method:1.By the distributions of time,spot and population,epidemiological analysis of two EKC epidemics in Tibet was made2.Pathogen identification:53 portions of conjunctival swab for the two time of EKC epidemics in Tibet in 2016 were collected,including 41 samples for the first one and 12 samples for the second one.The real-time PCR method was adopted for detection with the universal primer of human adenovirus and universal primer of enterovirus to define the pathogen of the epidemics.3.Type identification of adenovirus:The real–time PCR positive sample of adenovirus amplified loop2 fragment for hypervariable region 7 of hexon.The sequence of loop2 fragment was determined to use BLAST method and bioinformatics method for preliminary genetyping for adenovirus of the epidemics pathogen.4.Virus isolation:The virus was isolated by hep-2 cell.All the samples of adenovirus which were identified as positive in real-time PCR were blind-passed to three generations.5.Feature analysis of genetic characteristic:The penton gene,hexon gene and fiber gene of HAd V are susceptible to recombination and are mostly recombined within the genus.The isolated EKC epidemic virus strains from Tibet were selected for the full-length sequencing of the major structural gene pentan gene,hexon gene and fiber genes.Based on these three major proteins,a phylogenetic tree was constructed using 4 strains isolated from Tibet and 10 representative strains selected from the world and 30 strains belonging to the HAdV-D for phylogenetic analysis.Result:1.A total of 50 samples from two outbreaks of EKC in Tibet showed PCR positive for adenovirus.By amplifying Loop2 in the hexon gene and sequencing analysis,the results of BLAST analysis and phylogenetic tree analysis showed that two outbreaks of EKC in Tibet were preliminary.It was identified as the same pathogen HAdV-8.2.The results of phylogenetic analysis of the full-length sequences of the 12 strains of hexan,pentan,and fiber genes successfully isolated were determined.The pathogen of this outbreak was HAdV-8.The penton,hexan and fiber genes of the 12 isolated strains had 100% nucleotide homology,confirming that the two EKC epidemics were all caused by the same strain of HAdV-8.HAdV-8 strains in Tibet have high nucleotide homology with HAd V-8 strains in other parts of the world,and the Penton,hexon,and fiber gene sequences of HAdV-8 are highly conserved.Conclusion:1.This EKC epidemics was caused by HAdV-8,and the pathogens in two times of epidemics were the same,being continuous epidemic of HAdV-8 in Tibet2.Since HAdV-8 was isolated and identified for the first time in 1955,outbreak or spreading and epidemic of HAdV-8 have been reported in a great many countries and regions,while before this epidemics in Tibet,there was no formal report on this virus in Chinese Mainland.This two times of EKC epidemics in Tibet is the firstly reported EKC outbreak caused by HAd V-8 in China,so findings of this research can provide important scientific data for prevention and control of adenovirus epidemics in China,and is favorable for prevention and control of EKC.3.The adenovirus and EKC surveillance systems in China are incomplete,which is part of the reason for the continuous outbreak.Currently it is urgently needed to establish a perfect monitoring network to respond to the outbreak of epidemics.