Objective To screen the sensing elements for TNT detection in Escherichia coli genome.Methods A genome promoter library with cutting E.coli K-12 MG1655 genome was constructed.Bacterial luciferase luxCDABE was used as a reporter gene during promoter screening.We discovered TNT sensing elements through several rounds of screen-ing.Through analysis of sensitivity, specificity and timeliness, the promoter activity of the elements was evaluated,and the functional sequence of the elements was further confirmed.Results and Conclusion We successfully constructed an E.co-li K-12 MG1655 genome library , from which a TNT sensing element was discovered,which had a good performance in the analysis of sensitivity, specificity and timeliness.In this study, we reported that the topAp4 is a TNT sensing element for the first time.We also verified its excellent promoter activity.

Objective: To elucidate the epidemiological and etiological features of a local outbreak of dengue fever (DF) in Taijiang district in Fuzhou, Fujian province in 2017, and speculate possible viral source based on phylogenetic analysis. Methods: The clinical and demographic data of cases were collected through field investigation and the outbreak was characterized epidemiologically by descriptive method. The patient′s serum were collected and the adult mosquitoes were captured by anti-mosquito double-net method for the laboratorial test and viral isolation. The viral isolates were typed by real-time fluorescent RT-PCR and their full length of viral envelope (E) genes were amplified by RT-PCR and sequenced. The E gene sequences obtained in this study, together with the reference sequences, were used for the phylogenetic analysis. Results: A total of 13 cases of autochthonous DF were confirmed in the outbreak. All cases presented obvious clinical manifestations and clustered spatially and temporally. The Breteau Index (BI) of mosquito larva density was the highest in epidemic foci of Xingang street and was relatively low in surrounding areas. Four DENV-1 strains, three from patients and one from the captured adult Aedes albopictus, were isolated and identified by real-time RT-PCR. Full length E gene sequences of the four isolates were completely identical and phylogenetic analysis showed that the isolates were genetically closest to the strain (GenBank No. KT825033) from Vietnam in 2014, rather than the DENV-1 strains found in Fujian previously. Conclusions The DF outbreak occurred in Fuzhou in 2017 was caused by DENV-1 which was imported possibly from somewhere outside of Fujian province and subsequently led to local DF transmission in human via the mosquito Aedes albopictus.

Objective:To develop a triplex real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for dengue virus (DENV), yellow fever virus (YFV) and chikungunya virus (CHIKV), so as to achieve the rapid detection of these three viruses.Methods:The complete genome sequences of DENV(Ⅰ, Ⅱ, Ⅲ, Ⅳ), YFV and CHIKV were retrieved from Global Shared Database for comparative analysis, estimate its conservative region, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity was evaluated by other viral nucleic acids. The sensitivity was evaluated by in vitro transcribed RNAs of DENV, YFV and CHIKV. The repeatability of the method was evaluated by independent repeated experiments with different concentrations of viral nucleic acids. DENV detection method was validated with dengue patient serum. YFV and CHIKV detection methods were validated with simulated positive samples. The sera from healthy people were used for negative validation. Results:This method has no cross-reaction with other viral nucleic acids. The limit of detection (LOD) of DENV (Ⅰ、Ⅱ、Ⅲ、Ⅳ), YFV and CHIKV in vitro transcribed RNAs were less than 21.55 copies/PCR, 21.25 copies/PCR, 21.85 copies/PCR, 22.75 copies/PCR, 22 copies/PCR, 45.65 copies/PCR. The standard deviation of Ct values of each concentration was less than 0.5 and the coefficient of variation was less than 3%. The positive rate of clinical and simulated positive samples was 100%, and the negative rate of healthy serum was 100%. Conclusions:A triplex real-time fluorescent quantitative RT-PCR assay for DENV, YFV and CHIKV detection was established, and proved to be specific, sensitive and repetitive.

Objective:We attempted to investigate the variation of amino acids and molecular evolution trend of hemagglutinin(HA) genes of H9N2 avian influenza virus (AIV).Methods:In this study, we collected and aligned HA sequences of H9N2 AIV that submitted before April 2020 in the EpiFlu database. Then we analyzed the amino acid differences in the key sites of HA gene in bioinformatics method, including receptor binding sites, cleavage sites and glycosylation sites.Results:84.92% sequences have glutamine(Q) at the HA226 site in place of leucine(L) 84.75% sequences from avian and 72.97% sequences from mammals contained such HA-Q226 L mutation. 96.26% sequences still maintained the characteristics of low pathogenic AIV at the cleavage site of HA gene, except that 180 sequences from avian that two basic amino acids were inserted. Introducing one or two potential glycosylation sites were observed in 58.68% HA sequences, while loss of glycosylation site at HA210-212 in 66.44% sequences.Conclusions:The key protein receptor binding sites of the HA gene of H9N2 AIV showed obvious trends to binding human receptor. It revealed H9N2 AIV′s increasing adaptation to mammals. The cleavage sites and glycoylation sites of HA protein have a mutation tendency of characteristics of highly pathogenic AIV.

Objective:To isolate and identify the yellow fever virus (YFV) from the specimens of the imported yellow fever (YF) cases in Fujian province in 2016.Methods:Sixteen positive YFV nucleic acid samples including serum, urine and saliva were inoculated into C6/36 cells, respectively. The isolated strain was identified by YFV real-time RT-PCR. The complete gene sequence of this strain was obtained by high-throughput next-generation sequencing, and the phylogenetic tree was drawn.Results:Only one strain was isolated from the serum of one case three days after onset, and identified as a YFV strain by real-time RT-PCR. BLAST analysis showed that the complete gene sequence of this strain was identical to the strain CNYF01R/2016(KX268355) isolated from the first YF imported case in China in 2016. The phylogenetic tree showed that this strain belonged to the same phylogenetic branch as the epidemic strain Angola71 in Angola in 1971, and was significantly different from the 17D vaccine strain (X03700), indicating that the YFV strain isolated in this study belonged to the wild YFV strain of Angola genotype.Conclusions:An Angola genotype YFV strain was successfully isolated from samples of imported YF cases in Fujian Province in 2016.

Objective: To study the etiological characteristics of an imported Chikungunya fever (CHIK) epidemic in Fujian province in 2018. Methods: Serum samples collected at different days after the onset of the two CHIK cases were detected by real-time RT-PCR and ELISA. Structural protein E1 gene was amplified by RT-PCR and sequenced for nucleotide characteristics analysis and phylogenetic tree analysis. Results: RNA of Chikungunya virus (CHIKV) was detected in the 4 serum samples collected on the first 5 days of the disease, and the earliest IgM antibodies were detected in specimens on the 5th day of the disease, however, IgG antibodies were only detected in specimen on 10th day. Compared with the S27-African prototype strain, 12 mutant points were found in the amino acids of E1 genes in this study. The E1 genes of the two CHIK cases were exactly the same, and they were closest to the evolutionary relationship with the strain isolated in the Philippines in 2014. Their genotype was Asian genotype. Conclusions This epidemic was confirmed to have been imported from the Philippines after the infection with the Asian genotype CHIKV, which suggests that Fujian province should strengthen the monitoring of persons entering from the CHIK epidemic area, so as to prevent imported cases from causing local outbreaks.