Author:(Huanyu HE)
1.Serological Investigation into the Infected Genotypes of Patients with Japanese Encephalitis in the Coastal Provinces of China
Zhang WEIJIA ; Zhao JIERONG ; Yin QIKAI ; Liu SHENGHUI ; Wang RUICHEN ; Fu SHIHONG ; Li FAN ; He YING ; Nie KAI ; Liang GUODONG ; Xu SONGTAO ; Yang GUANG ; Wang HUANYU
Biomedical and Environmental Sciences 2024;37(7):716-725
Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China. Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5. Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV. Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.
2.Expert consensus on the biosafety recommendation for arthropods of medical importance in field and laboratory
HE Changhua ; LUO Huanle ; YIN Feifei ; HAN Qian ; LIANG Lei ; SHI Yongxia ; YU Xuedong ; SUN Yi ; LIU Qiyong ; WANG Huanyu ; WANG Rong ; SHAN Chao ; DENG Fei ; YUAN Zhiming ; XIA Han
China Tropical Medicine 2024;24(2):119-
The emerging and re-emerging arthropod-borne infectious diseases pose a serious threat to global public health security. Field and laboratory studies of arthropods of medical importance are essential and critical for the prevention and control of arthropod-borne infectious diseases. Various institutions or universities in China have been conducting research in the field or laboratory study of arthropods of medical importance, but up to 2023, it is still lacking detailed biosafety guidelines or recommendations that can guide the related work for arthropods of medical importance. In order to proactively address potential biosafety issues in the field or laboratory activities related to arthropods of medical importance, improve the standardization of arthropod biosafety classification, operations, and protection, and ensure the safety of practitioners, an expert consensus on the biosafety recommendation of arthropods of medical importance in field and laboratory has been developed, aiming to guide the future work of arthropods and ensure the national biosafety and biosecurity of China.
3.Recombinant expression of Japanese encephalitis virus non-structural protein NS1 gene and its reaction with Flavivirus antigen and antibody
ZHANG Yijia ; YAO Xiaohui ; CAO Lei ; WANG Ruichen ; FU Shihong ; NIE Kai ; LI Fan ; YIN Qikai ; HE Ying ; WANG Huanyu ; XU Songtao ; MA Chaofeng ; LIANG Guodong
China Tropical Medicine 2023;23(12):1241-
Abstract： Objective To elucidate the antigenic antibody reaction of recombinant expression of non-structural protein 1 (NS1) of Japanese encephalitis (JE) virus with various mosquito-borne flaviviruses, including JE virus, and the antigenic antibody reaction of serum samples of patients infected with JE virus in acute stage. Methods In this study, Escherichia coli prokaryotic expression vector (pET) system was used to recombinant express Japanese encephalitis virus NS1 gene. Western Blot assay was performed to detect the antibody responses of the recombinantly expressed protein against a variety of mosquito-transmitted flaviviruses, including JE virus, as well as antigen-antibody reactions of serum from patients with acute JE virus infection. Results The NS1 gene expression product of JE virus (P3 strain) was in the form of an inclusion body, and the denatured and renatured expression product was displayed as a single band in the denatured gel (polyacrylamide gel electrophoresis, PAGE), with a molecular weight of about 45 000. The results of further antigen-antibody analysis showed that the antigen/antibody hybridization reaction of the expression product with polyclonal or monoclonal antibody of JE virus (mosquito isolates, encephalitis isolates) and serum samples of patients with acute JE virus infection could be completely consistent. The recombinant product showed negative antigen/antibody hybridization reactions with mosquito-transmitted flaviviruses, such as dengue virus and yellow fever virus polyclonal antibodies, but positive reactions with polyclonal antibodies to West Nile virus and Murray Valley encephalitis virus. Conclusions In this study, the recombinant expression of the NS1 protein of JE virus was successfully obtained, and the antigen/antibody reaction between the recombinant protein and samples of patients infected with mosquito-borne flavivirus and JE virus was analyzed. The study results provide important basic data for elucidating the antigen-antibody reaction between the NS1 protein of JE virus and mosquito-borne flavivirus. The recombinant expression protein obtained in this study provides an important material basis for further research on the function of JE virus NS1 protein.
4.Nam Dinh virus was detected and isolated in arbovirus investigation in Shanxi, China
Shenghui LIU ; Xiaodong TIAN ; Weijia ZHANG ; Hongmei ZHENG ; Junying ZHAO ; Chongxiao XU ; Yali ZHANG ; Shihong FU ; Kai NIE ; Fan LI ; Peifang DAI ; Qikai YIN ; Ying HE ; Jingxia CHENG ; Huanyu WANG
Chinese Journal of Experimental and Clinical Virology 2023;37(1):25-29
Objective:In this study, the collected mosquito samples were subjected to viral isolation to identify the species and branch characteristics of arboviruses in five regions of Shanxi Province.Methods:Eight arboviruses in mosquito samples collected from July to September 2020 were detected by real-time fluorescent quantitative PCR, and virus isolation was carried out through cell culture. Virus isolates were identified and analyzed by molecular biology and bioinformatics method.Results:We detected 1 batch of positive samples of Japanese encephalitis virus, 2 batches of positive samples of Culex flavivirus and 8 batches of positive samples of Nam Dinh virus among 121 batches of mosquito samples. Seven virus isolates were isolated, numbered: SX-YJ-Cxp-4、SX-YJ-Ars-2、SX-YJ-Cxp-1、SX-LY-Cxp-10、SX-GP-Ars-5、SX-GP-Cxp-2、SX-GP-Cxp-4, all of which were identified as Nam Dinh virus, and the whole genome sequencing was performed on one of them, and the result showed that Shanxi Nam Dinh virus isolate and Yunnan Nam Dinh virus isolate belonged to the same evolutionary branch.Conclusions:Nam Dinh virus was isolated and identified on the specimen from Shanxi province for the first time.
5.Laboratory identification and evaluation of national standard strains of Japanese encephalitis virus G1/G3/G5
Shenghui LIU ; Mengnan JIANG ; Weijia ZHANG ; Shihong FU ; Jingdong SONG ; Chongxiao XU ; Kai NIE ; Qikai YIN ; Ying HE ; Fan LI ; Songtao XU ; Guodong LIANG ; Qiang WEI ; Huanyu WANG
Chinese Journal of Experimental and Clinical Virology 2023;37(3):273-279
Objective:To determine the evaluation indexes of national standard strains of genotypes 1, 3 and 5 of Japanese encephalitis virus (JEV) and evaluate the national standard JEV strains.Methods:According to the national standard strain evaluation technical standards of pathogenic microbial bacteria (virus) species, based on the application of Japanese encephalitis virus research, and according to the morphological characteristics, biological characteristics, molecular biological characteristics and other research data to identify the characteristics of G1, G3 and G5 genotypes of JEV.Results:Spherical virus particles with a diameter of about 60 nm were visible under electron microscope of the three Japanese encephalitis virus strains. The cytopathic effect was mainly characterized by cell shrinkage and exfoliation in BHK-21 and Vero cell lines, cell fusion and exfoliation were shown after infection with C6/36 cell line; the virus titer was 10 5-10 7 PFU/ml, and the plaque size was different by genotype. The median lethal dose of intrabitoneal challenge in G1, G3 and G5 JEV in three weeks-old mice was 50.51 PFU, 6.98 PFU, and 8.13 PFU, and the median lethal dose of intracranial challenge in five weeks mice was 3 PFU, 0.3 PFU, 1.35 PFU. The whole genome length of G1, G3 and G5 JEV was 10 967 bp, 10 976 bp and 10 983 bp, respectively. Conclusions:Three genotypic national standard strains of JE V were identified and evaluated by electron microscopy, cell, animal and genome laboratory indexes, which provided reference for the identification and evaluation of other national standard strains of JEV.
6.Comparison of immune effects of varicella zoster virus gE protein combined with different adjuvants in mice
Jiehui WU ; Ruichen WANG ; Shihong FU ; Kai NIE ; Fan LI ; Qikai YIN ; Ying HE ; Guodong LIANG ; Huanyu WANG ; Hai LI ; Songtao XU
Chinese Journal of Experimental and Clinical Virology 2023;37(6):592-599
Objective:This study contrasts the immune efficacy of the varicella zoster virus glycoprotein E (VZV gE)using Al/CpG combined adjuvants and AS01 adjuvant in BALB/c mice.Methods:BALB/c mice were immunized at 0 and 21 days respectively, and serum antibodies were detected using enzyme-linked immunosorbent assay. Detection of neutralizing antibodies in mouse serum using varicella zoster virus; enzyme-linked immunosorbent spot assay was used to detect cellular immune response.Results:Following two intramuscular immunizations, mice in the experimental groups (Shingrix, gE+ Al/CpG, and gE+ AS01) demonstrated elevated neutralizing antibody titers and an augmented count of lymphocytes releasing IFN-γ and IL-4. The gE+ Al/CpG group displayed the highest neutralizing antibody titer (1943), yet the AS01-adjuvanted groups (Shingrix and gE+ AS01) showed increased lymphocyte counts secreting IFN-γ and IL-4 compared to the Al/CpG group (gE+ Al/CpG). In comparison to the AS01 adjuvant, Al/CpG adjuvants triggered a humoral immune response favoring Th2 in mice. The proportions of CD4 + T and CD8 + T cells were not significantly different among the experimental groups. Conclusions:Al/CpG adjuvant combined with gE protein resulted in high neutralizing antibody titers, while the intensity of the induced cellular immune response was inferior to that of AS01 adjuvant.
7.Establishment and preliminary application of RAA assay for varicella-zoster virus
Haoze LIU ; Ruichen WANG ; Weijia ZHANG ; Xiaohui YAO ; Shihong FU ; Kai NIE ; Fan LI ; Qikai YIN ; Ying HE ; Huanyu WANG ; Ruiping HU ; Songtao XU
Chinese Journal of Experimental and Clinical Virology 2023;37(6):631-636
Objective:To establish a rapid method for the detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA).Methods:The whole genome sequences of VZV were downloaded from the global shared database for comparison and analysis. Specific primers and probe were designed for the four conserved genes respectively and the optimal combination was selected. The optimal reaction system was selected through the concentration gradient of primers and probes, and a fluorescence RAA detection method was established. The sensitivity of the method was evaluated with VZV positive plasmid standard and clinical samples with gradient dilution, the repeatability of the method was evaluated with the lowest detectable limit concentration of positive plasmid standard, and the specificity of other viral nucleic acid method was evaluated. At the same time, this method and quantitative real-time PCR (qPCR) were used to detect clinical samples and the result were compared.Results:The optimal combination of primer pair F2/R2 and probe P2 targeting open reading frame (ORF) 28 gene was selected. Considering the cost factor, the optimal primer concentration was set at 500 nmol/L and the optimal probe concentration was 280 nmol/L. The minimum detection limit was 10 1 copies/μL, and the minimum clinical positive samples with a Ct value of 36.027 could be detected, and the result of repeated experiments were consistent. The method has no cross-reaction with other viral nucleic acids. The detection rate of clinical positive samples was 93.33%, which was almost identical to that of qPCR. Conclusions:This method is simple to operate with high sensitivity, strong specificity, low requirements for experimental conditions, visual detection result, and can detect VZV nucleic acid in samples within 20 minutes, which is a rapid VZV detection method that can be considered for clinical use for detection.
8.Analysis on the surveillance of infectious disease related public health bud-events in Shanghai from 2017 to 2020
Yinhao LU ; Yongchao HE ; Yi HE ; Huanyu WU ; Chunyan LUO ; Xiaoyan HUANG
Shanghai Journal of Preventive Medicine 2022;34(1):17-21
Objective To determine the epidemiological characteristics of infectious disease related public health bud-events in Shanghai and assess the effects of bud-event surveillance， so as to provide scientific evidence for improving the surveillance system. Methods Surveillance data of infectious disease related public health bud-events were collected from 16 districts of Shanghai from 2017 through 2020. Then the data were analyzed and compared with infectious disease related public health emergencies during the same period. Results A total of 6 376 infectious disease related public health bud-events were documented in Shanghai in 2017‒2020， which involved 29 792 cases. There were two seasonal peaks， April through June and November through December. Clustered events accounted for 38.85%， mainly caused by chickenpox （14.10%）， hand，foot and mouth disease （11.17%） and norovirus-associated infectious diarrhea （6.54%）. The 36.73% of the bud-events occurred in school settings， which involved 24 718 cases （accounting for 83.00% of all cases）. Median time duration between onset date of the first cases and report date of the events was 4 days， and median duration of the events was 14 days， demonstrating positive correlation. In addition， all the infectious disease related public health emergencies（
9.Establishment of TaqMan RT-PCR assay for Wuxiang virus
Danhe HU ; Xiaohui YAO ; Shihong FU ; Fan LI ; Tianmeng GU ; Junjie CHEN ; Ying HE ; Jiayu YIN ; Songtao XU ; Xiangdong LI ; Kai NIE ; Huanyu WANG ; Guodong LIANG
Chinese Journal of Experimental and Clinical Virology 2022;36(4):460-464
Objective:To establish a real-time fluorescent quantitative TaqMan reverse transcription polymerase chain reaction (Real-time RT-PCR) detection method for Wuxiang virus (WUXV).Methods:All gene sequences of WUXV were downloaded from GenBank, and multi-sequence alignment analysis was performed using Mega-X. Primers and probes designed for the highly conservative region of S-segment genes were selected to evaluate the specificity, sensitivity and stability of detection reactions.Results:The established method can specifically detect WUXV and does not cross-react with various arboviruses. The lowest detection limit was 1 pfu/ml. The inter-batch variation coefficients of repeated detection cycle threshold ( Ct) of the same sample were all less than 1.00%. TaqMan RT-PCR was used to detect 30 batches of sandflies nucleic acid samples, and 7 of them showed positive amplification curve of WUXV. Conclusions:TaqMan RT-PCR with high sensitivity, specificity and repeatability has been successfully established, which can be used to screen large quantities of samples of WUXV.
10.Establishment of TaqMan RT-qPCR assay for the detection Getah virus
Tianyuan WU ; Shihong FU ; Qikai YIN ; Jierong ZHAO ; Fan LI ; Ying HE ; Songtao XU ; Guodong LIANG ; Kai NIE ; Guang YANG ; Huanyu WANG
Chinese Journal of Experimental and Clinical Virology 2021;35(2):205-208
Objective:To establish a sensitive and specific real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) method for rapid detection of Getah virus (GETV).Methods:All the gene sequences of GETV were downloaded from GenBank database. Clustal X was used for sequence alignment, and specific primers and probes were designed according to highly conserved regions; we established a standard curve using the nucleic acid of GETV as a standard, and the sensitivity, specificity and stability of this method were evaluated respectively.Results:This method could specifically detect GETV and has no cross-reactivity with multiple arboviruses; the sensitivity was 1.0×10 pfu/ml, and the intra-assay and inter-assay coefficients of variation were less than 1%. One case was GETV positive in 196 batches of mosquitoes collected from Hunan province, Hebei province, Fujian province and Chongqing city.Conclusions:We established a TaqMan probe real-time quantitative RT-PCR with high sensitivity and specificity which can be used for screening.
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