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.

Objective To explore the application effect of cross-team scenario simulation exercise in the training of fire emergency plan in operating room.Methods Totally 48 nurses,16 anesthetists and 16 workmates in operating room of Zhejiang Provincial People′s Hospital from January 2015 to March 2016 were selected and divided into eight groups. The training of fire emergency plan in operating room was carried out with the method of scenario simulation exercise. The system of exercise contained three phases. The first phase was the theoretical study;the second phase was a teaching of centralized operation exercise;the third phase was the exercise in groups according to the design of simulation scenarios. The score was evaluated according to the method of subjective comment with over 80 points as the qualified,and below 79 points as the unqualified. At the same time,the time of completing the process before and after the exercise was recorded.Results The mean score of exercise examination was (88.75±4.23) points among eight groups with six groups whose scores were more than 90 points. When the small fire in operating room could be put out within three minutes by themselves,the time from the occurrence of fire to the restart of surgery after training (428.8±19.1) s was less than that (605.0±17.9) s before training with a significant difference (P<0.05). When the big fire in operating room could not be put out within three minutes by themselves,the time of retreat after training (44.6±4.2) s was less than that (71.0±6.4) s before training with a significant difference (P<0.05).Conclusions The cross-team scenario simulation exercise can improve the mastery level of fire emergency plan in operating room of operating room members. It is worthy of popularization and application in clinic.

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.

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 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.

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 To investigate the effect and mechanism of suberoylanilide hydroxamic acid (SAHA) on the fear extinction in mice with chronic social defeat stress (SD). Methods Fifty-six male C57BL/6J mice aged 7-8 weeks were randomly divided into control group,social defeat group,control-SAHA group and social defeat-SAHA group to investigate the effect of SAHA and social defeat group,social defeat-AAV BDNF group and social defeat-AAV blank group to investigate the effect of BDNF. Fear extinction in mice was evaluated by fear conditioning test (FC). The levels of BDNF and HDAC2 in mice hippocampus were detected by Western blot (WB). The expression of BDNF-overexpressing virus in hippocampus of mice was detected by immunofluorescence assay. Results (1) Compared with control group,fear extinction in the social defeat group was significantly decreased (P<0. 05). Compared with control group, the level of HDAC2(0. 50±0. 02) in the social defeat group was significantly increased (P<0. 001),while the level of BDNF(0. 16 ± 0. 03) was significantly decreased (P<0. 001) in the social defeat group. ( 2) After using SAHA,fear extinction of mice significantly improved (P<0. 05). Compared with control group,the level of HDAC2 (0. 26±0. 02) in the control-SAHA group was significantly decreased(P<0. 001),and the level of BDNF (0. 40±0. 03) was significantly increased (P<0. 001). Compared with social defeat group,the level of HDAC2 (0. 39±0. 03) in the social defeat-SAHA group was significantly decreased (P<0. 001),and the lev-el of BDNF (0. 28±0. 01) was significantly increased (P<0. 001). (3)After injection BDNF-overexpressing virus,fear extinction was significantly improved(P<0. 05). Conclusion SAHA can enhance fear extinction in mice with chronic social defeat stress and its mechanism may be related to the up-regulation of BDNF ex-pression in hippocampus by inhibiting HDAC2 in hippocampal.

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:A highly sensitive and specific real-time quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for rapid and accurate detection of Tibet orbivirus (TIBOV).Methods:The TIBOV genomic sequences from GenBank were analyzed by Clustal X 2.1 and the specific primers and probe were designed in the conserved segment of VP4 gene. RNA standard was obtained from in vitro transcription and a TaqMan RT-PCR assay for TIBOV was established. The sensitivity, specificity and repeatability of this method were evaluated. Results:The assay showed a good amplification curve within the range of 1.0×10 2~8 copies/reaction template, the detection limit was 1.0×10 2 copies/reaction, the coefficients of variation of Ct values in repeat detections were all less than 1.5%. No cross-reaction was found in this assay. Variable mosquito samples were screened by this assay and the result showed TIBOV negative. The prepared TIBOV simulated positive samples were 100% detected. Conclusions:The assay developed in this study is specific and sensitive for detection of TIBOV and can be used for laboratory detection and routine surveillance.