Severe infectious diseases,i.e.antibody-dependent enhancement (ADE) resulted from successive infection with different serotypes of dengue virus.After its introduction into Brazil in 2015,Zika virus has spread rapidly to more than 60 countries and regions by the end of November 2016.Some south-east Asian countries including China have also reported cases of ZIKV infection.In recent studies,it was observed that sera cross-reactivity antibodies or such monoclonal antibodies have been elicited by two domains,ED1 and ED2,of envelope (E) protein on Zika or/and Degue virus,and ADE was easily induced by such antibodies.Dengue fever epidemic often occurred in Chinese coastal provinces each year.Then,it will be followed by Zika virus disease.Therefore,we must pay attention to and propose replying measurement for it.

Dengue is the most common vector borne viral disease of humans globally.Detection of viral RNA from suspected patient specimens is rapid,specific and confirmative in laboratory diagnosis of dengue infections during the acute phase.In this study,a multiplex nested reverse transcription PCR (RT-PCR) system was established for clinical specimens.While other nucleic acid amplification tests showed relatively low sensitivity,the multiplex nested RT PCR assay detected 4 cases among blood clots from 8 serologically confirmed dengue patients.These results suggested that blood clots of dengue patients could be used in laboratory diagnosis,and that the multiplex nested RT PCR assay,which simplified the detection procedure,could facilitate viral RNA detection of specimens in clinical laboratories.

Since several dengue viruses (DENV) have been isolated in Fujian Province during the past decade, sequencing and evolution analyses of viral envelope genes are helpful in determining their possible transmission origins. In this study, viral RNA was extracted from 12 DENV strains from Fujian between 20042010. Viral envelope genes were amplified, cloned into TA vectors and sequenced, and the sequence data were subsequently analyzed by bioinformatics software. Full-length E genes of DENV-1 or DENV-2 of 1 485 bp, and DENV- 3 of 1 479 bp were obtained. It was indicated, from BLAST analysis and phylogenetic trees, that DENV strains in Fujian Province during 20042010 shared the highest similarity with Southeast Asian strains, suggesting that DENV circulating in Fujian Province between 20042010 were probably imported from Southeast Asia. Hence, extensive monitoring on passengers from this region at the entry-ports should be strengthened.

For investigating the epidemiology and genetic characteristics of enterovirus 71 (EV71) in Fujian from 2010 to 2015,we analyzed the surveillance data of EV71 and sequenced VP1 genes of 72 EV71 strains randomly picked from the past 6 years.The overall infection rate was gradually down and one incidence peak (from May to July) was observed each year.Major infectious population were focused on Xiamen,Fuzhou,Nanping and Quanzhou,the ages ranged from one to three years old.Scattered children were the most infected ones.The proportion of EV71 in the severe case was higher than in the HMFD(χ2 =732.064 5,P＜0.000 1).EV71 circulated from 2010 to 2015 in Fujian Province was belonged to subgenotype C4a in consistent with vaccine strain (H07).Compared with the VP1 of vaccine strains,the divergence of complete VP1 nucleotide sequence was gradually expanding as time distance increased,but the sequence of amino acid was not found obvious difference.Variations in 4 key immune epitopes of amino acid had not appeared a regular pattern in year and not consistent with the trend of proportion of EV71 in HMFD.As a result,we considered the epidemiology characteristics of EV71 in Fujian was obvious,72 strains still belonged to C4a subgenotype and had no outstanding antigenic drift or mutation.Extensive epidemiology surveillance and genetic characteristic are needed for the application of EV71 vaccine.

To study the biological characteristics and mutations of influenza A(H1N1)pdm09 virus isolated from one case of pneumonia of unknown etiology (PUE),which would provide references for clinical treatment and disease control,the throat swab specimen from the PUE case was isolated in the Madin-Darby Canine Kidney (MDCK) cells,and then the antigenicity,pathogenicity and drug resistance of influenza A (H1N1) pdm09 virus were analyzed after sequencing.As a result,one influenza virus strain was isolated from the specimen and named as A/FujianGulou/SWL64/2016(H1N1).The similarities of nucleotide sequences and amino acids sequences compared with the vaccine strain A/California/07/2009 (H1N1) were 96.9％-98.9％ and 96.7％-99.5％,respectively.Eighteen amino acids had mutated in the HA and 4 mutations,K163Q,S185T,S203T and D222N,were involved in 3 different epitopes,which indicated that the antigenic drift had occurred in the influenza virus.The D222N mutation associated with receptor binding site made the virus infect lower respiratory tract more easily.The virus was still amantadine-resistance and oseltamivir-sensitive.In conclusion,the influenza A (H1N1) pdm09 virus in this study have occurred antigenic drift and has the molecular characterization of causing severe pneumonia,so further surveillance should be performed to prevent and control the influenza epidemic.

In order to investigate EV71 antibody levels among general population from Fujian Province after the 2008‐2009 HFMD epidemics ,390 sera‐specimens were collected from 390 participants in 2010 .EV71‐specific antibody was detected by neutralization test ,indicating 186 (47 .69% ) sera of 390 were EV71‐seropositive .Although the difference by gender was not statistically significant on positive rates and antibody titers ,significant differences were observed in positive rates and antibody titers among age groups .The positive rate was increasing with age ,while the 0 age‐group yielded the lowest positive rate of 16 .67% .Subsequently ,significant difference was detected among positive rates and antibody titers between age groups of 0 to 4 years‐old and 5‐years‐old ,with the positive rate of 25 .33% and 61 .67% ,respectively .Therefore ,the EV71 antibody levels among general population from Fujian Province after the 2008‐2009 HFMD epidemics was still in the low level ,especially the age groups of 0 to 4 years‐old .The epidemic of HFMD mainly caused by EV71 will still occur in the future ,and children under 5 years old are major susceptible population ,continuously intensive surveillance ,prevention and control are required .

Objective: To understand the epidemiological and virological features of influenza B viruses and the difference between the vaccine strains and epidemic strains, the antigenic and genetic characteristics on hemagglutinin (HA) gene of influenza B viruses circulating in Fujian during 2010-2015. Methods: The representative strains were selected randomly according to the lineage of influenza B viruses isolated from network laboratory in Fujian, 2010-2015. Viral RNA was extracted and gene fragments were amplified by reverse transcription polymerase chain reaction (RT-PCR ) and the PCR products were sequenced. The complete HA gene sequence was obtained and analyzed via bioinformatics. Results: Compared to the vaccine strains recommended by WHO, there were significant changes in genetic and antigenic characteristics on HA gene of B Yamagata lineage viruses from 2010 to 2015, especially in 2010, 2014 and 2015. There were major five amino acid residues substitutions (116, 150, 165, 196 and 202) involved in antigenic determinants, and the variable sites gradually increased as time on over. However, the variability of B Victoria lineage viruses on HA gene was less and there was no obvious trend over time. The results showed that the B Yamagata vaccine strains of 2010 and 2015 recommended by WHO had poor protective effect on influenza virus infection, while the B Victoria vaccine strain still play a satisfactory protective effect on humans in Fujian. Conclusions With time on, influenza B Yamagata lineage viruses had gradually mutated, causing a poorly match with vaccine strains in part of year, and emerging antigenic drift phenomenon. Strengthening further surveillance of mutations of B influenza virus remains essential to allow for early warning of influenza epidemic.

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:To establish a nucleic acid detection method for human parainfluenza virus (HPIV) based on reverse transcription recombinase-aid amplification (RT-RAA) combined with CRISPR/Cas12a.Methods:The type-specific primer pairs of RT-RAA and CRISPR RNA (crRNA) targeted on conserved sequence of nucleocapsid protein (N) gene of HPIV were designed. Fluorescence intensities from cleavage of fluorophore labeled probes mediated by Cas12a were measured for screening of crRNA and concentration optimization of crRNA, Cas12a as well as the probe. With the RNA transcribed in vitro and clinical specimen, the lower limit of detection and specificity of RT-RAA combined CRISPR/Cas12a detection were evaluated. Results:The crRNA specific to each type of HPIV 1-4, with strongest cleavage activity, were screened out. With the optimal concentration of crRNA, Cas12a as well as the probe, the lower limit of detection could reach 10 copies of target gene per reaction on fluorescence intensity measurement. No cross-reaction was found in clinical samples of eight other respiratory viruses detected by this method .Conclusions:The established HPIV1-4 fluorescence CRISPR nucleic acid detection method is rapid, specific, and does not require professional nucleic acid detection equipment.