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.

Objective:To analyze the growth characteristics of different genotypes of Japanese encephalitis virus (JEV) in different cell lines, and to provide scientific basis for the selection of cell lines in the study of JEV.Methods:BHK-21, Vero, C6/36, PK-15, DF-1, N2a, SH-sy5y and MDCK cell lines were selected. The proliferation ability of genotype 1 (NX1889 strain), genotype 3 (P3 strain) and genotype 5 (XZ0934 strain) JEV in these cell lines was evaluated by plaque assay and RT-qPCR.Results:Significant cytopathogenic effects (CPE) were observed in BHK-21, Vero, C6/36, DF-1, N2a and PK-15 cell lines across all three JEV genotypes. However, no significant differences in CPE characteristics were observed within the same cell line. SH-sy5y and MDCK cell lines did not show significant CPE, but virus proliferation was detected in SH-sy5y cell line, while MDCK cell line were found to be insensitive to JEV. No significant difference was observed in the proliferation curves of G1, G3 and G5 JEV in BHK-21, Vero and SH-sy5y cell lines. In C6/36 and PK-15 cell lines, the titer of G1 JEV was higher than that of G3 and G5. In DF-1 cell line, G5 demonstrated a higher titer than the other two genotypes, whereas in N2a cell line, G5 showed a lower titer than the other two.Conclusions:There are differences in the proliferation of three different genotypes of JEV in different cell lines, which can provide reference for the study of JEV in different directions.

Objective　To establish a dual droplet digital PCR (ddPCR) assay for herpes simplex virus type I (HSV-1) and varicella-zoster virus (VZV). Methods The specific primers and probes were derived based on the conserved regions of HSV-1 and VZV genome. The primer-probe combinations were screened, and the annealing temperatures and primer-probe concentration ratios of the dual-droplet digital PCR reaction were optimized to establish a dual-droplet digital PCR reaction system for HSV-1 and VZV, which was tested for other viruses and validated for clinical samples. The sensitivity, specificity, and reproducibility of the established dual microtiter digital PCR method were analyzed. Results The optimal concentrations of primers and probes for the dual ddPCR detection method of HSV-I and VZV were determined to be 800 nmol/L and 250 nmol/L, respectively, with an optimal annealing temperature of 56 ℃. The correlation coefficient (R2) of the standard curve of the dual ddPCR assay was 0.99, showing a clear linear relationship. The method showed high sensitivity, with the lowest detection limit of herpes simplex virus type I being 2.97 copies/μL, and for VZV being 2.73 copies/μL. The repeatability was high with a small coefficient of variation and stable detection results; the specificity was excellent, and no cross-reaction was found with herpes simplex virus type Ⅱ, Epstein-Barr virus, Adenovirus, Coxsackievirus (CA6/CA10/CA16), Cytomegalovirus, Human Cytomegalovirus, Human enterovirus 71, Japanese Encephalitis virus, West Nile virus, Measles virus, Mumps virus, and human nucleic acids. Conclusions The dual droplet digital PCR assay for herpes simplex virus type I and varicella-zoster virus established in this experiment has strong sensitivity, specificity, and high repeatability, and can provide a solution for rapid quantitative detection of the two viruses in different scenarios.

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.

Chronic refractory wound (CRW) is one of the most challengeable issues in clinic due to complex pathogenesis, long course of disease and poor prognosis. Experts need to conduct systematic summary for the diagnosis and treatment of CRW due to complex pathogenesis and poor prognosis, and standard guidelines for the diagnosis and treatment of CRW should be created. The Guideline forthe diagnosis and treatment of chronic refractory wounds in orthopedic trauma patients ( version 2023) was created by the expert group organized by the Chinese Association of Orthopedic Surgeons, Chinese Orthopedic Association, Chinese Society of Traumatology, and Trauma Orthopedics and Multiple Traumatology Group of Emergency Resuscitation Committee of Chinese Medical Doctor Association after the clinical problems were chosen based on demand-driven principles and principles of evidence-based medicine. The guideline systematically elaborated CRW from aspects of the epidemiology, diagnosis, treatment, postoperative management, complication prevention and comorbidity management, and rehabilitation and health education, and 9 recommendations were finally proposed to provide a reliable clinical reference for the diagnosis and treatment of CRW.

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.

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 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.

Objective:To establish the best preventive measures for inadvertent perioperative hypothermia (IPH) in patients undergoing off-pump coronary artery bypass grafting (OPCABG) based on evidence, in order to reduce the incidence of IPH and reduce various complications in OPCABG patients.Methods:The evidence of preventive measures for IPH in OPCABG patients was systematically searched in major databases at home and abroad. The retrieval time limit was from the establishment of the database to August 31, 2019. Three researchers independently screened and evaluated the literature to obtain the best evidence for the prevention of IPH in patients with OPCABG. Using the cluster sampling method, 29 patients who underwent OPCABG in Zhejiang Provincial People's Hospital from January to April 2020 were selected as the control group, while 27 patients who underwent OPCABG from January to April 2021 were selected as the observation group. The differences in body temperature at admission, body temperature at 1 hour after anesthesia, body temperature after leaving the department, minimum intraoperative body temperature, drainage volume in the first 24 hours after surgery, length of stay in ICU and length of hospital stay after surgery were compared between the two groups.Results:A total of 17 papers were included, and 20 pieces of relevant evidence were obtained. After evaluation by experts, the best evidence suitable for the research environment was selected and applied in clinical practice. The body temperatures at admission of patients in the control group and the observation group were respectively (36.62±0.30) ℃ and (36.49±0.28) ℃, and the difference was not statistically significant ( t=2.85, P>0.05) . The body temperature at 1 h after anesthesia, the body temperature after leaving the department and the lowest body temperature during operation were (35.83±0.30) , (36.04±0.49) and (35.50±0.31) ℃ in the control group, and (36.43±0.38) , (36.62±0.27) and (36.21±0.28) ℃ in the observation group, respectively. The difference between the two groups were statistically significant ( t=37.65, 23.76, 58.13; P<0.01) . The incidence of IPH was 93.1% (27/29) in the control group and 11.1% (3/27) in the observation group, and the difference was statistically significant (χ 2=34.568, P<0.01) . The drainage volume in the first 24 h after operation in the control group was (260.0±70.3) ml and that in the observation group was (212.1±44.3) ml, and the difference between the two groups was statistically significant ( t=-3.025, P<0.01) . The length of ICU stay and hospital stay in the control group were respectively (49.0±13.4) h and (12.2±3.5) d, while those in the observation group were (39.8±13.8) h and (10.5±2.5) d, and the differences between the two groups were statistically significant ( t=-2.524, -2.035; P<0.05) . Conclusions:The best preventive measures for inadvertent perioperative hypothermia in patients with OPCABG provide scientific and rigorous procedures and specifications for the prevention of inadvertent perioperative hypothermia in this type of surgery, effectively reducing the incidence of inadvertent perioperative hypothermia and maintaining the intraoperative core temperature stability of patients, reduce postoperative bleeding and shorten the length of ICU stay and hospital stay, which is worthy of clinical promotion.