| Objective:Yunnan Province is a border province in the southwest of China,bordering with Myanmar,Vietnam and Laos,with a vast territory and complex natural conditions.Banna virus(BAV),Chikungunya virus(CHIKV),Sindbis virus(SINV),Batai virus(BATV),and Dengue virus(DENV)have been isolated and identified in mosquitoes and/or human sera in Yunnan province.A case of Zika imported from Bangladesh was first reported in Yunnan in 2014.Tahyna virus(TAHV)has been isolated in Xinjiang,Qinghai and Inner Mongolia,but no cases have been reported in Yunnan Province and its neighboring Southeast Asian countries.In this study,for the first time,DENV NS1 negative serum samples of unexplained fever cases collected during the 2014-2015 dengue epidemic season in Ruili,Dehong Prefecture,were tested for the above seven viruses to find out whether there were infections of the above-mentioned 7 viruses and to provide scientific basis for the prevention and control of mosquito-borne diseases.Methods:The samples were 1738 DENV NS1 negative serum samples of unexplained fever cases in Ruili from 2014-2015,stored in a refrigerator at-80℃.Excel software was used to establish a database of study sample collection information and to analyze the data.The χ2 test of SPSS 20.0 software was used for the difference analysis of two sample rates,and P<0.05 was considered a statistically significant difference.Viral RNA was extracted using the virus RNA/DNA extraction kit from Xi’an Tianlong Technology Co.Primers and probes for the detection of seven viruses were synthesized with reference to the literature.After pre-experimental validation,triple-probe method real-time fluorescence RT-PCR was performed for DENV,CHIKV and ZIKV.The DENV-positive samples were then synthesized with DENV type 1-4 specific primers and probes according to "Dengue Diagnosis"WS216-2018,and DENV serotyping was performed by real-time RT-PCR.Some DENV-positive serum samples were selected for antigen detection by using domestic(Guangzhou Wanfu Biotechnology Co.,Ltd.)DENV NS1 antigen detection reagent.The detection of BAV,TAHV and BATV was completed by BAV single test,BAV and TAHV combined test,BATV and TAHV combined test and BATV single test.SYBR GREEN real-time RT-PCR and TaqMan real-time RT-PCR were used for the detection of SINV.DENV,CHIKV,ZIKV and BAV positive controls were from virus strains kept in our laboratory.Plasmid DNA standards containing the gene fragments of SINV,TAHV and BATV to be amplified were synthesized at The Beijing Genomics Institute(BGI)as positive controls for the detection of SINV,TAHV and BATV,respectively.The positive serum samples were inoculated into C6/36 Aedes albopictus cells for virus isolation and identification,and the positive sample sera or newly isolated strains were subjected to envelope(Envelope,E)gene and/or whole genome sequencing,and the sequencing results were analyzed for homology and evolution.Results:Sampling information:A total of 1738 serum samples of unexplained fever cases with negative DENV NS1 antigen test were collected in Ruili from 2014 to 2015,of which 657(37.80%)were collected in 2014,374(21.52%)were males and 283(16.28%)were females;1081(62.20%)were collected in 2015,556(31.99%)were males and 525(31.21%)were females.The youngest case was 2 months old and the oldest was 96 years old,with a median age of 24 years,with the highest percentage of young adults group(36.82%,640/1738).The number of samples collected from June to October was 1705(98.10%,1705/1738).There were 1250(71.92%)serum samples collected within one week of onset,of which 19(1.10%)were collected more than one week,and 469(26.99%)had an unknown onset date.Real-time RT-PCR:A total of 18 DENV positive(detection rate:1.036%)and 1 ZIKV positive samples(detection rate:0.058%)were detected.BAV,BATV,TAHV,CHIKV,SINV were not detected.One ZIKV-positive sample was collected in October 2015,and the patient was a local case.18 DENV-positive samples including 7 DENV-1 positive samples,including 6 local cases(all collected in June 2015)and 1 imported case from Myanmar(collected in October 2014);6 DENV-2 positive samples,of which 5 were local cases,collected in June(2 cases),September(1 cases)and October 2015(2 cases),and 1 case from Myanmar,collected in October 2015;Five cases were un-serotyped,all local cases,were collected in June(1 cases),August(2 cases)and September(2 cases)2015.The detection rate of DENV was 0.15%(1/657)in 2014 and 1.57%(17/1081)in 2015.There was a statistically significant difference in the DENV detection rate between the 2014 and 2015 samples by χ2 test(χ2=8.039,P=0.003).Antigen testing:Eight DENV-positive samples selected for antigen testing were negative.Virus isolation and identification:one strain of ZIKV was isolated and showed cytopathic effects(CPE)on day 3 after inoculation into C6/36 Aedes albopictus cells;four strains of DENV were isolated,two of which were DENV-1 and two of which were DENV-2.Only one of these four DENV strains(test no.15RL600)showed cell fusion on the fifth day of the third blind generation.The remaining three strains did not show typical CPE.Sequencing and evolutionary analysis:Sequencing yielded one ZIKV complete genome sequence(10804 bp),which belongs to the Asian genotype and is most closely related to the Myanmar strain imported into Yunnan in 2019(2019YNZIKV02);the same as the ZIKV strains imported into Yunnan from Bangladesh in 2014 and Myanmar in 2019,both with A188V mutation in the NS1 protein and no S139N and G18R mutations in the PrM and NS4B proteins.Sequencing yielded three DENV-1 E gene sequences(local cases),all of which belonged to genotype 1 and had the closest evolutionary relationship with the imported strains from Myanmar in 2015;sequencing yielded three DENV-2 E gene sequences.two of which were from local cases and one from Myanmar,all of which belonged to Asian Genotype and were most closely related to the 2015 imported DENV-2 from Myanmar.Conclusions:1.ZIKV was detected and isolated from local cases in Yunnan for the first time,and the complete genome sequence was sequenced,and the virus strain belonged to the Asian genotype,which was most recently related to the evolution of ZIKV imported from Myanmar to Yunnan in 2019.The newly isolated ZIKV had an A188V mutation in the NS1 protein,indicating high infectivity to Aedes aegypti,suggesting a risk of Zika virus disease outbreak in the border area between China and Myanmar.2.18 DENV-positive samples were detected from DENV NS 1-negative samples,suggesting that the diagnosis of cases in the field using only DENV antigen detection reagents would result in DF misses.3.No positive samples were detected for BAV,CHIKV,BATV,SINV and TAHV,but continued investigation and research is needed.4.There is an urgent need to raise clinicians’ awareness of Zika virus disease diagnosis and to conduct routine surveillance of Zika virus disease in the region.For suspected cases of dengue fever,the combination of antigen detection,DENV and ZIKV nucleic acid detection should be used to avoid missed detection. |
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