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.

Abstract: More than 300 mosquito-borne arboviruses have been found worldwide, among which Dengue virus, Chikungunya virus, Japanese encephalitis virus, West Nile virus, and Zika virus are circulating around the world, causing a huge public health burden and arousing widespread concern in the whole society. China's vast territory and complex geographical landscapes are suitable for the reproduction of various mosquito-borne arboviruses, so the species and geographical distribution of mosquito-borne arboviruses in China have attracted much attention. Since the 1980s, 38 mosquito-borne arboviruses belonging to nine families, including Flaviviridae, Bunyaviridae, Togaviridae, and Reoviridae, have been isolated and identified from more than 1 million mosquitoes and human and animal specimens collected across China by means of tissue cell culture and animal inoculation. The results of field and laboratory tests indicate the existence of various mosquito-borne arboviral diseases in China, including Japanese encephalitis, Dengue fever, West Nile encephalitis, and febrile diseases caused by Tahyna virus infection. This study summarizes the species and geographical distribution of mosquito-borne arboviruses, mosquito-borne arboviruses, and their vectors, and mosquito-borne arboviral infectious diseases in China, providing technical support for the control and prevention of mosquito-borne arbovirus and their corresponding diseases, endemic management, disease early warning and prediction. Joint prevention and control of arboviral diseases in Asia and even the world is also of long-term and practical significance.

Objective To investigate whether the characteristics of pathogens mediated by Wolbachia can interfere with Japanese encephalitis virus (JEV) replication and explore the regulatory role of Wolbachia on JEV replication transmitted by Culex mosquitoes. Methods Real-time fluorescence quantitative PCR (qPCR) and RNA fluorescence in situ hybridization (RNA-FISH) were used to detect Wolbachia density in Aa23 (naturally infected with Wolbachia) Aedes albopictus cell and negative control Aa23T Aedes albopictus cells (Wolbachia infection was removed by tetracycline treatment). The plaque assay was conducted to measure the viral titers and cytopathic effect (CPE) in Aa23T and Aa23 cells on days 1 to 8 after JEV (P3 strain) infection. Results qPCR and RNA-FISH results consistently showed that the symbiosis of Wolbachia was negative in Aa23T cell. In Aa23 cells, the copy number of the WSP gene of Wolbachia and the fluorescence signal intensity targeting Wolbachia 16S rDNA increased with cell growth time. In response to JEV infection, Wolbachia prolonged the CPE in viral infected Aa23 cells, which compared to infected Aa23T cells. The plaque assay result has obviously showed that JEV titer in Aa23 cells (106 PFU/mL) was significantly lower than that in Aa23T control cells (108 PFU/mL). Conclusions Wolbachia significantly delays CPE of JEV on cells and inhibits JEV replication in A. albopictus cells. To the best of our knowledge, this is the first report that Wolbachia strongly inhibits JEV infection in mosquito cells. It revealed the role of Wolbachia on inhibition of the viruses that transmitted by Culex mosquitoes. In particular, it provides important experimental data and theoretical basis for application of Wolbachia-based mosquito control technology in prevention and control of JEV.

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.

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.

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.

Objective:To evaluate the role of TANK-binding kinase-1 (TBK1) in renal fibrosis in mice with acute kidney injury (AKI) and relationship with endoplasmic reticulum stress.Methods:Twenty-four male wild-type C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group CON), group AKI, control plus TBK1 inhibitor group (group CON-GSK) and AKI plus TBK1 inhibitor group (group AKI-GSK). In group AKI and group AKI-GSK, folic acid 250 mg/kg was intraperitoneally injected to prepare AKI model.From the first day after folic acid injection, 1% dimethyl sulfoxide 20 ml/kg was intraperitoneally injected every other day in group AKI, and GSK8612 1.5 mg/kg was intraperitoneally injected every other day in group AKI-GSK, 7 times in total.In group CON and group CON-GSK, 1% dimethyl sulfoxide 20 ml/kg and GSK8612 1.5 mg/kg were intraperitoneally injected, respectively, every other day for 7 times in total.On the 14th day after injection of folic acid, the eyeball blood samples were taken to determine the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr), and the kidney tissues were also extracted, and the pathological results of renal tissue were observed by Sirius red staining, Masson staining and HE staining.The area of renal fibrosis was measured and the tubulointerstitial injury score was calculated.The expression of fibronectin, type I collagen and α-smooth muscle actin was detected by immunofluorescence.The expression of phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), activated transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), phosphorylated inositol-requiring kinase 1α (p-IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), apoptosis-regulating signal kinase 1 (ASK1), caspase-12 and phosphorylated c-Jun N-terminal protein kinase (p-JNK) was detected by Western blot. Results:Compared with group CON, the serum BUN and Cr concentrations, area of renal fibrosis and renal tubulointerstitial injury score were significantly increased, and the expression of fibronectin, type I collagen, α-smooth muscle actin, p-PERK, p-eIF2α, ATF4, CHOP, p-IRE1α, TRAF2, ASK1, caspase-12, and p-JNK was up-regulated in group AKI and group AKI-GSK ( P<0.05), and no significant change was found in the indexes mentioned above in group CON-GSK ( P>0.05). Compared with group AKI, the serum BUN and Cr concentrations, area of renal fibrosis, and tubulointerstitial injury score were significantly decreased, and the expression of fibronectin, type I collagen, α-smooth muscle actin, p-PERK, p-eIF2α, ATF4, CHOP, p-IRE1α, TRAF2, ASK1, caspase-12, and p-JNK was down-regulated in group AKI-GSK ( P<0.05). Conclusions:TBK1 is involved in the process of renal fibrosis in mice with AKI, and the mechanism may be related to the promotion of endoplasmic reticulum stress.

Objective:To disclose the molecular genetic differences of Culex flavivirus among mosquitoes in Gansu province in 2011 and 2019.Methods:Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to obtain the nucleotide sequences of Culex flavivirus genomes from mosquitoes in Gansu province in 2011 and 2019, and viral molecular biology and bioinformatics method were used to analyze the genetic differences of the viruses.Results:Nucleotide sequences of 10 strains of Culex flavivirus were obtained, including 8 strains (all from Culex pipiens pallens) obtained in 2011 and 2 strains ( from Culex tritaeniorhynchus and Anopheles sinensis) in 2019. Homology analysis of nucleotide and amino acid sequences of virus E gene showed that the nucleotide sequence similarity and amino acid similarity of viruses isolated from Gansu in 2019 and 2011 ranged from 98.3%-100% and 95.4%-97.3%, respectively. Phylogenetic analysis of Culex flavivirus E gene sequence showed that two strains of Culex flavivirus isolated in Gansu province in 2019 (GS1975 and GS1976) and eight strains of Culex flavivirus isolated in 2011 all belonged to group B of genotype 1 of Culex flavivirus. Further analysis found that GS1975 virus isolated in 2019 was in a common evolutionary cluster with viruses isolated from Liaoning (2010 and 2011) and Inner Mongolia (2018), while GS1976 virus isolated in 2019 formed a coevolutionary cluster with viruses isolated from Inner Mongolia (2018) and Gansu (2011). Conclusions:Although both Culex flaviviruses isolated in Gansu province in 2011 and 2019 are genotype 1 virus, the two viruses isolated in 2019 distributed in two different evolutionary clusters, suggesting that the local mosquito virus genome changes over time, therefore, long-term monitoring of molecular differences is needed to carry out.

Objective:To perform virological and molecular biological identification of the virus (SX1943) isolated from specimens of Culex tritaeniorhynchus in Shaanxi province in 2019. Methods:Mosquito ground fluid was inoculated into tissue culture cells, and isolates were made virological and molecular genetic analysis.Results:A virus isolate (SX1943) was obtained from a specimen of Culex tritaeniorhynchus collected in Shaanxi province in 2019, which developed significant cytopathic effect (CPE) on day 3 after inoculation into C6/36 cells, manifested as cell pyknosis, aggregation and shedding, and the isolate could be stably passaged. However, no significant CPE was observed after three consecutive passages in BHK-21 cells. Electron microscopy (negative staining) of C6/36 cells inoculated with SX1943 virus showed a large number of round virus particles, about 25 nm in diameter. The result of amplification and sequencing of the SX1943 viral genome showed that the total length of the viral genome sequence was 3 594 nt, encoding a total of three proteins, which were non-structural proteins (NS1 and NS2) and capsid proteins (VP), and the gene lengths of the three proteins were 2 376 nt, 1 098 nt and 1 136 nt, respectively. Phylogenetic analysis of the virus revealed that the SX1943 virus were in the same evolutionary clade as Culex pipiens pallens Densovirus (CppDNV) in the genus Brevidensovirus in the subfamily Densovirus, and the above result suggested that SX1943 virus was CppDNV. Conclusions:CppDNV was isolated from Culex tritaeniorhynchus in Shaanxi province.

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.