| Infantile diarrhea disease, the second leading cause of death in children under five years old in the world, is a digestive tract disease caused by many pathogens and factors, characterized by increasing defecate frequency and the changes of traits of the defecate. The disease, one of the leading causes of death in children in developing countries, with complex etiology, is difficult to treat and easy to cause malnutrition, seriously impacting on the growth of infants and young children, even leading to death. So the WHO have classified the control of diarrhea as a global strategy. Viral diarrhea is the main type of infantile diarrhea. The main pathogenes caursing infantile diarrhea are human rotavirus (HRV), human norovirus (HNV), human astrovirus (HAstV), enterovirus (ETvirus), hepatovirus et al, and the most common pathogens are rotavirus, norovirus and astrovirus.This study is mainly focused on the molecular epidemiology infantile diarrhea virus of HRV, HNV and HAstV in Kunming, cloning part of HNV genome and HAstV genome by reverse transcription PCR (RT-PCR) amplification. In addition, A preliminary isolation has been carried on HAstV-1in HT-29cell, a human colon cancer cells.(1) Epidemiological study and viruses typing of HRV, HNV and HAstV in KunmingViral detection primers and typing primers were designed according to nucleotides sequence in highly conserved areas of HRV, HNV and HAstV. Stable detection system of HRV, HNV and HAstV were establish by optimizating the RT-PCR detection system and conditions.12HRV,12HAstV and13HNV, with30.8%,30.8%and33.3%were found in39infantile diarrhea feces.By RT-PCR and sequencing method, the virus genotype were that HNV were all GⅡ type, HAstV were HAstV-type1, and HRV had2cases? P[6],1case? P [9],1case? P [10],1case? P [8],1case of G2P [8],4cases of G2P [4],1case mixed infection of? P [4] and? P [8],1case of mixed infection of G2P [8] and G8P [8], G2type P [4] HRV with the highest prevalence33.3%, were the dominant strains in Kunming area.(2) Cloning viral genome cDNA of HNV and HAstV6pairs of viral genome amplification primers were designed according to the results of the alignment for each types of virus genomes of HNV and HAstV downloaded from NCBI GeneBank with DNAMAN software. The amplification results of HNV and HAstV genome by RT-PCR were as follows:3fragments of HNV genome with the length of3.6kb, total length of HAstV genome with6.8kb. The sequence was submitted to GenBank and received on accession number KC342249, named KM-1. By nucleotide sequences homology comparison, it was found to have high nucleotide homology with the same type strain SH-1(98.4%) and India (95.0%). With other type virus homology were G282.9%, G381.9%, G476.2%, G582%, G666.2%, G884.5%. KM-1amino acid homology comparison, found that KM-1have high homology with other types in protease and RNA-dependent RNA enzymes amino acid by ORFla and ORFlb coding, and KM-1ORF2coding viral capsid protein precursor amino acid have high homology with the same type, India strains (97.97%), Shanghai (SH-1) strains (98.35%), but have the lower homology with different types, between62.64%and75.79%. Recombination event analysis found that has3area of sequences recombinat, was1996-2229bp area,3608-4155bp area and3608-4155bp area, respectively.1996-2229region had the highest homology Russia strains (GU732187),3608-4155region had high homology with South Korea strains (JN887820), and5937-5981region probably from India strains (AF260508).(3) HAstV IsolationCells (40%-50%density of HT-29cells) infected with HAstVs from clinical samples, and infected cells were incubated in culture medium containing10ug of trypsin/ml and2%calf serum. The infected cells were harvested after3,6days, respectively. HAstV Virus directed by RT-PCR was indicated that successful isolated in HT-29cells. The HAstVs were used for Observed by electron microscopy after concentrated virus by PEG, HAstV was about40nm in diameter.This study aimed to investigate the epidemiological characteristics of HRV, HNV and HAstV in children with infantile diarrhea in2012-2013in Kunming. The above data will provide a theoretical basis for the further study of HRV, HNV and HAstV. |
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