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： 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 evaluate the performance of an artificial intelligent (AI)-based automated digital cell morphology analyzer (hereinafter referred as AI morphology analyzer) in detecting peripheral white blood cells (WBCs).Methods:A multi-center study. 1. A total of 3010 venous blood samples were collected from 11 tertiary hospitals nationwide, and 14 types of WBCs were analyzed with the AI morphology analyzers. The pre-classification results were compared with the post-classification results reviewed by senior morphological experts in evaluate the accuracy, sensitivity, specificity, and agreement of the AI morphology analyzers on the WBC pre-classification. 2. 400 blood samples (no less than 50% of the samples with abnormal WBCs after pre-classification and manual review) were selected from 3 010 samples, and the morphologists conducted manual microscopic examinations to differentiate different types of WBCs. The correlation between the post-classification and the manual microscopic examination results was analyzed. 3. Blood samples of patients diagnosed with lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative neoplasms were selected from the 3 010 blood samples. The performance of the AI morphology analyzers in these five hematological malignancies was evaluated by comparing the pre-classification and post-classification results. Cohen′s kappa test was used to analyze the consistency of WBC pre-classification and expert audit results, and Passing-Bablock regression analysis was used for comparison test, and accuracy, sensitivity, specificity, and agreement were calculated according to the formula.Results:1. AI morphology analyzers can pre-classify 14 types of WBCs and nucleated red blood cells. Compared with the post-classification results reviewed by senior morphological experts, the pre-classification accuracy of total WBCs reached 97.97%, of which the pre-classification accuracies of normal WBCs and abnormal WBCs were more than 96% and 87%, respectively. 2. The post-classification results reviewed by senior morphological experts correlated well with the manual differential results for all types of WBCs and nucleated red blood cells (neutrophils, lymphocytes, monocytes, eosinophils, basophils, immature granulocytes, blast cells, nucleated erythrocytes and malignant cells r>0.90 respectively, reactive lymphocytes r=0.85). With reference, the positive smear of abnormal cell types defined by The International Consensus Group for Hematology, the AI morphology analyzer has the similar screening ability for abnormal WBC samples as the manual microscopic examination. 3. For the blood samples with malignant hematologic diseases, the AI morphology analyzers showed accuracies higher than 84% on blast cells pre-classification, and the sensitivities were higher than 94%. In acute myeloid leukemia, the sensitivity of abnormal promyelocytes pre-classification exceeded 95%. Conclusion:The AI morphology analyzer showed high pre-classification accuracies and sensitivities on all types of leukocytes in peripheral blood when comparing with the post-classification results reviewed by experts. The post-classification results also showed a good correlation with the manual differential results. The AI morphology analyzer provides an efficient adjunctive white blood cell detection method for screening malignant hematological diseases.

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 explore the buffering effect of positive childhood experiences (PCEs) on mental health risks among adolescents before and after COVID-19 epidemic. Methods: In October 2019 (before the outbreak of COVID-19), 1 322 students from grades 4 to 9 were recruited from primary and secondary schools in two counties of Chizhou city, Anhui Province. A questionnaire survey was conducted to collect general demographic information, PCEs, depressive symptoms, anxiety symptoms, self harm behavior, suicidal ideation. Follow up survey was conducted after school re opening (May 2020). Mental health status before and after the COVID-19 epidemic was compared among students with different PCEs by multiple logistic regression analyses. Results: The detection rates of depressive symptoms, anxiety symptoms, self harm behavior and suicidal ideation (22.6%, 16.0%, 40.0%, 29.9%) of the respondents after school re opening were significantly higher compared that before the epidemic (16.5%, 13.5%, 31.1 %, 22.6%). There were no significant differences in the detection rates of depressive symptoms, anxiety symptoms,self harm behavior and suicidal ideation between high PCEs group before and after the epidemic ( Z =-0.05,0.27,0.84,1.84, P >0.05). Multivariate Logistic regression analysis showed that the risk of depressive symptoms and self harm behavior in the low PCEs group after school re opening was 1.39 times higher than that before the epidemic (95% CI= 1.05 -1.84, P <0.05). The risk of non suicidal self injury behavior in the low PCEs group after school re opening was 1.31 times higher than that before the epidemic (95% CI= 1.05 -1.62, P <0.05). There were no significant differences in mental health detection rates in high PCEs group before and after the epidemic ( P >0.05). Conclusion During the time of COVID-19 epidemic, PCEs is associated with lower rates of depressive symptoms, anxiety symptoms, self harm behavior and suicide ideation in adolescents. The findings suggest that more support and help should be given to adolescents from the perspectives of family, school and peers, so as to reduce the adverse effects of public health emergencies on adolescents mental health.

Objective: To investigate the epidemiological characteristics of premature eruption of permanent molars and its aasociation with body mass index (BMI), to provide a reference for childhood oral health promotion. Methods: A total of 861 children aged 9 to 12 years from two primary schools in Bengbu City were selected by cluster sampling method. Parental questionnaire was administered to collect socio demographic information. The eruption of second permanent molars were examined. Data was analyzed by multivariate Logistic regression model and margins command. Results: The detection rate of premature eruption of second permanent molars was 26.5%(228), 27.5% in boys and 24.9% in girls( χ 2=0.73, P =0.39). Early detection rate of second permanent molars (39.0%) was significantly higher in obese group than normal weight group (21.5%)( χ 2=21.85, P <0.01). Logistic regression analysis showed that obesity was positively correlated with the risk of premature eruption of second permanent molars( OR= 3.55 , 95%CI=2.14-5.87, P <0.01). Overweight was not associated with higher risk of premature eruption of second permanent molars( OR=1.64, 95%CI=0.95-2.81, P =0.07). Being female was associated with higher risk of premature eruption of second permanent molars compared to age matched peers( OR=2.19, 95%CI=1.42-3.39, P <0.01). Conclusion Childhood obesity is associated with higher risk for premature eruption of second permanent molars. Girls are more likely to have second permanent molar erupted in advance compared to age matched boys.

BACKGROUND: Gestational diabetes mellitus (GDM) brings health issues for both mothers and offspring, and GDM prevention is as important as GDM management. It was shown that a history of GDM was significantly associated with a higher maternal risk for GDM recurrence. The incidence of GDM recurrence was unclear because of the incidence of second-child was low before 2016 in China. We aim to investigate the prevalence of GDM recurrence and its associated high-risk factors which may be useful for the prediction of GDM recurrence in China. METHODS: A retrospective study was conducted which enrolled participants who underwent regular prenatal examination and delivered twice in the same hospital of 18 research centers. All participants were enrolled from January 2018 to October 2018, where they delivered the second baby during this period. A total of 6204 women were enrolled in this study, and 1002 women with a history of GDM were analyzed further. All participants enrolled in the study had an oral glucose tolerance test (OGTT) result at 24 to 28 weeks and were diagnosed as GDM in the first pregnancy according to the OGTT value (when any one of the following values is met or exceeded to the 75-g OGTT: 0 h [fasting], ≥5.10 mmol/L; 1 h, ≥10.00 mmol/L; and 2 h, ≥8.50 mmol/L). The prevalence of GDM recurrence and development of type 2 diabetes mellitus were calculated, and its related risk factors were analyzed. RESULTS: In 6204 participants, there are 1002 women (1002/6204,16.15%) with a history of GDM and 5202 women (5202/6204, 83.85%) without a history of GDM. There are significant differences in age (32.43 ± 4.03 years vs. 33.00 ± 3.34 years vs. 32.19 ± 3.37 years, P  < 0.001), pregnancy interval (4.06 ± 1.44 years vs. 3.52 ± 1.43 years vs. 3.38 ± 1.35 years, P  = 0.004), prepregnancy body mass index (BMI) (27.40 ± 4.62 kg/m2vs. 23.50 ± 3.52 kg/m2vs. 22.55 ± 3.47 kg/m2, P < 0.001), history of delivered macrosomia (22.7% vs. 11.0% vs. 6.2%, P < 0.001) among the development of diabetes mellitus (DM), recurrence of GDM, and normal women. Moreover, it seems so important in the degree of abnormal glucose metabolism in the first pregnancy to the recurrence of GDM and the development of DM. There are significant differences in OGTT levels of the first pregnancy such as area under the curve of OGTT value (18.31 ± 1.90 mmol/L vs. 16.27 ± 1.93 mmol/L vs. 15.55 ± 1.92 mmol/L, P < 0.001), OGTT fasting value (5.43 ± 0.48 mmol/L vs. 5.16 ± 0.49 mmol/L vs. 5.02 ± 0.47 mmol/L, P < 0.001), OGTT 1-hour value (10.93 ± 1.34 mmol/L vs. 9.69 ± 1.53 mmol/L vs. 9.15 ± 1.58 mmol/L, P < 0.001), OGTT 2-hour value (9.30 ± 1.66 mmol/L vs. 8.01 ± 1.32 mmol/L vs. 7.79 ± 1.38 mmol/L, P < 0.001), incidence of impaired fasting glucose (IFG) (fasting plasma glucose ≥5.6 mmol/L) (31.3% vs. 14.6% vs. 8.8%, P < 0.001), and incidence of two or more abnormal OGTT values (68.8% vs. 39.7% vs. 23.9%, P < 0.001) among the three groups. Using multivariate analysis, the factors, such as age (1.07 [1.02-1.12], P = 0.006), prepregnancy BMI (1.07 [1.02, 1.12], P  = 0.003), and area under the curve of OGTT in the first pregnancy (1.14 [1.02, 1.26], P  = 0.02), have an effect on maternal GDM recurrence; the factors, such as age (1.28 [1.01-1.61], P  = 0.04), pre-pregnancy BMI (1.26 [1.04, 1.53], P = 0.02), and area under the curve of OGTT in the first pregnancy (1.65 [1.04, 2.62], P = 0.03), have an effect on maternal DM developed further. CONCLUSIONS The history of GDM was significantly associated with a higher maternal risk for GDM recurrence during follow-up after the first pregnancy. The associated risk factors for GDM recurrence or development of DM include age, high pre-pregnancy BMI, history of delivered macrosomia, the OGTT level in the first pregnancy, such as the high area under the curve of OGTT, IFG, and two or more abnormal OGTT values. To prevent GDM recurrence, women with a history of GDM should do the preconception counseling before preparing next pregnancy.
Adult ; Blood Glucose/metabolism* ; China/epidemiology* ; Diabetes Mellitus, Type 2/epidemiology* ; Diabetes, Gestational ; Female ; Fetal Macrosomia ; Glucose Intolerance ; Humans ; Male ; Pregnancy ; Retrospective Studies