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Objective:To perform adenovirus detection and genetic evolutionary analysis on specimens from a fever outbreak in Kunming city.Methods:Pharyngeal swabs from typical febrile patients were collected and tested for nucleic acids of 30 common respiratory pathogens using TaqMan Array Card technology. The full-length sequences of three important genes of adenovirus, Penton base, Hexon and Fiber, were amplified, sequenced and typed using Nanopore high-throughput sequencing. A phylogenetic tree was constructed. Molecular variations and genetic evolution of the three genes were analyzed.Results:Five specimens were collected and all of them tested positive for adenovirus and Haemophilus influenzae. The sequences of the full-length coding regions of the Penton base, Hexon and Fiber genes were obtained by Nanopore sequencing. The homology of the three gene sequences in the five specimens was 100.0%, 99.9%-100.0% and 100.0% in nucleotide sequences, and 100.0% in amino acid sequences. The three genes in the specimens had the highest homology with those of the reference strain of human adenovirus type 3 (HAdV3, accession number: AY599834) in nucleotide sequences, which was 98.6%, 98.7% and 98.9%, respectively. Results of the phylogenetic analysis of the three genes were basically consistent. These Kunming strains were clustered into an independent clade with the reference HAdV3 strain and had a distant relationship with the strains isolated in foreign countries and Taiwan, China in the early years. They were closely related to the domestic and foreign strains in recent years and highly homologous to the 2019 Japanese strain (accession: LC703523) and the Guangzhou strain (accession: MZ540961). Compared with the reference strain, these Kunming strains had five amino acid variations in Penton base, 10 in Hexon and 11 in Fiber. Conclusions:All of the adenovirus strains isolated in this outbreak belong to P3H3F3 type based on the full-length sequences of Penton base, Hexon and Fiber genes. They share high homology with the domestic and foreign HAdV3 strains, including the reference strain. Compared with the reference strain, several amino acid mutations are identified in these Kunming strains, and most of them are in the high variability region or functional regions. M7L in the Hexon protein is an unique amino acid mutation site of Kunming strains.
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Objective@#To investigate the molecular epidemiological characteristics of Human Calicivirus (HuCV) infection among children less than 5 years in Kunming city, Yunnan province, it might be provide effective evident for prevention and control the diarrhea related with HuCV infection.@*Methods@#Four sentinel hospitals were recruited in the study from Kunming city, Yunnan province, 850 diarrhea cases and 170 non-diarrhea subject were recruited in this study from 2014 to 2015. RT-PCR was performed to screen HuCV infection, and gene sequencing was used to ensure positive infection subtypes and genotypes.@*Results@#The positive rate of HuCV was higher in children with diarrhea than in non-diarrhea children (11.5%, 98/85; 4.7%, 8/170, χ2=7.083, P=0.008), and the positive rate of Norovirus (NoV) GII was higher in non-diarrhea children were (11.1%, 94/85; 4.7%, 8/170, χ2=6.353, P=0.012). There was no significant difference in the positive rate of NoV GI (0.1%, 1/850; 0.0%, 0/170, P=0.833) and Sappovirus (0.4%, 3/850; 0.0%, 0/170, P=0.578) in diarrhea children and non-diarrhea children. GII.P4 (10%, n=102) was the most important genotype of NoV GII detected in diarrhea and non-diarrhea individuals. Despite no significant difference in Norovirus GII infection between different age groups (χ2=0.038, P=0.846) and sex(χ2=0.620, P=0.733), infection rate of NoV GII varied with season (χ2=9.867, P=0.020), having close relationship with diarrhea in autumn (15.6%), primarily caused by GII.4 and GII.12 genotype (χ2=8.881, P=0.031; χ2=7.917, P=0.039).@*Conclusions@#NoV GII diarrhea had higher epidemic rate, which was caused by multiple genotypes, GII.P4 was a dominant genotype, and was a major pathogenic agent of diarrhea in infants f in Kunming city.
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Non-O1/O139 group of Vibrio cholerae can cause human acute diarrhea disease,while compared with the O1 and O139 groups;it often ignore the risk of the disease for human being.Therefore,we analyzed the molecular characteristics of 31 V.cholerae isolated from Yunnan Province.We used the agar disc diffusion method (K-B) to carry out the antibiotic sensitivity test;polymerase chain reaction (PCR) amplification for the detection of virulence gene;at the same time,all of strains were performed for pulse field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).The drug sensitivity test showed that 67.74％ strains were resistant to rifampin,29.03％ resistant to nalidixic acid and cotrimoxazole,all of the isolates were sensitive to gentamicin and ciprofloxacin;PCR results showed that all strains had the ompW gene,87.10％ strains had hly gene,25.81％ strains had rstREl tor,16.13％ strains had rstRClassical and tcpAEl tor,while CT rfbO1 and rfbO139 gene were negative;PFGE results showed that 31 strains had a trend of discrete height,the same PFGE identity pattern was not nearly found;for the analysis of MLST,we found the one new alleles of gyrB,four new alleles of mdh gene,six new alleles of metE gene,two new alleles of pntA,three new alleles of purM and four new alleles of pyrC gene.After permutation and combination,we found 17 new ST types for V.cholerae(ST273-ST289).Non-O1/O139 group V.cholerae showed a high degree of diversity,while the non-O1/O139 group of V.cholerae in Yunnan Province has a certain geographical features,which enriched the existing molecular typing system of V.cholerae.