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 establish and optimize the in vitro enrichment method for human coronavirus NL63 reference strain (HCoV-NL63-NC_005831).Methods:HCoV-NL63 was cultured in rhesus monkey kidney cell line (LLC-MK2). Then at 135 000× g, the virus culture supernatant was centrifuged for 4 h, 6 h, 8 h, 10 h and 12 h to enrich the virus, and compared with the enrichment method of commercial PEG Precipitation Kit. Real time reverse transcription polymerase chain reaction (qRT-PCR), TCID 50 assay and plaque assay were used to quantitatively detect the viral nucleic acid and viral biological activity of the original virus solution and the enriched virus solution respectively. The virus morphology before and after enrichment was observed after negative staining under a transmission electron microscope. Results:After 100∶1 volume enrichment by ultracentrifugation and PEG precipitation, both of the concentration of virus nucleic acid and live virus were higher than that of the original virus solution ( P < 0.001), and before and after the enrichment of the two method, the normal negative staining morphology of virus particles can be observed under the electron microscope. After 4 h, 6 h, 8 h, 10 h and 12 h ultracentrifugation, the concentration of live virus was (7.35±1.62) times, (13.98±1.71) times, (36.36±10.41) times, (48.16±9.38) times, (48.16±9.38) times and (54.26±7.02) times that of the original virus solution, respectively; the concentration of live virus after PEG precipitation was (3.39±0.16) times that of the original virus solution, and the nucleic acid concentration and live virus concentration of concentrated virus solution obtained by ultracentrifugation were significantly higher than those obtained by PEG precipitation ( P < 0.05). After 8 h, 10 h or 12 h ultracentrifugation, the virus concentration was significantly higher than that of 4 h or 6 h ( P < 0.05). However, there was no significant difference in virus content between 8 h, 10 h and 12 h ( P >0.05). Conclusions:Compared with commercial PEG precipitation method, ultracentrifugation method can enrich HCoV-NL63 virus more effectively; when the relative centrifugal force is 135 000× g, the better enrichment effect in vitro can be obtained by selecting the super separation time of 8 h.

Objective:To construct 2019 novel coronavirus (2019-nCoV) 614D and 614G pseudovirus by HIV lentivirus packaging system and explore their biological specificity.Methods:The recombinant expression plasmids pCDNA3.1-614D and pCDNA3.1-614G were transiently cotransfected with psPAX2 and pLenti CMV Puro LUC into 293T cells respectively. After 72 hours, the supernatant was collected and ultracentrifuged with 20% sucrose cushion. The titer, morphology, protein expression and neutralizing activity of pseudovirus were determined.Results:S protein specific fluorescence was detected by indirect immunofluorescence test, Western blot analysis showed S protein was expressed, and the spike of pseudovirus was observed under transmission electron microscope. The titers of pseudovirus 614D and 614G were 1.12×10 4 and 2.52×10 4 TCID 50/ml, respectively. The pseudovirus 614D and 614G could be neutralized by S rabbit polyclonal antibody, indicating that the pseudovirus has high specificity. Conclusions:In this study, 2019-nCoV 614D and 614G pseudovirus was successfully constructed, which laid the foundation for the establishment of in vitro neutralizing antibody detection platform based on pseudovirus.

Objective:To evaluate the immunological efficacy of a novel DNA vaccine against West Nile virus (WNV) in a mouse model.Methods:A DNA vaccine VRC-prME expressing the precursor membrane (prM) and envelope protein (E) of WNV Xinjiang strain (XJ11129-3) was constructed and its ability to express virus-like particles was verified in vitro. C57BL/6 mice were immunized twice with VRC-prME via intramuscular injection combined with electroporation with an interval of four weeks. Enzyme-linked immunoassay (ELISA) was used to detect serum antibodies after immunization. WNV (NY99 strain) single-round infectious particles were used to detect neutralizing antibodies. Cellular immune responses were analyzed by enzyme-linked immunoblot assay (ELISPOT) and intracellular cytokine staining (ICS). Results:VRC-prME induced a strong Th1-biased antibody response in mice that could cross-neutralize the WNV (NY99 strain) single-round infectious particles two weeks after the boost immunization. Moreover, the vaccine also elicited antigen-specific multifunctional CD8 + T cell responses (IFN-γ, IL-2, TNF-α). Conclusions:The novel DNA vaccine prepared in this study, expressing the prME protein of WNV XJ11129-3 strain, could induce stronger humoral and cellular immune responses in mice, which was worthy of further research and development for the prevention of WNV infection in China.

Objective:To establish a quick and convenient method for detecting human adenoviruses (Human adenoviruses, HAdV) based on immunochromatographic assay (ICA).Methods:Two antibody clones, 3C11 and 7E6 were found to bind to all tested HAdVs and then subsequently processed into ICA. The specificity and sensitivity were evaluated using representative strains of the respiratory HAdV types, including HAdV-1, 2, 3, 4, 5, 6, 7, 10 and a gastroenteric type HAdV-41 together with the original throat swabs of 10 HAdV patients confirmed by nuclear acid testing (NAT).Results:The ICA exhibited high specificity to HAdVs and its detection limitation ranged from 0.16 to 10 3 half tissue culture infectious dose (TCID 50)/ml for different types of HAdVs. All clinic samples with successful virus isolation tested by this ICA showed positive result . Conclusions:The ICA developed in the present study will be suitable for HAdVs screening in clinic setting, especially for those of respiratory types.

Objective:To establish and optimize the isolation scheme of exosomes from herpes simplex virus type 1 (HSV-1) infected HEp-2 cells.Methods:The supernatant of HSV-1 infected HEp-2 cells were collected and pretreated with differential centrifugation and filtration, then purified by total exosome isolation (TEI) in combination with size-exclusion chromatography (SEC); and enriched the exosome with ultrafiltration. Western blotting, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were performed to characterize the purified exosomes.Results:The purified exosomes from HEp-2 cells with or without HSV-1 infection showed positive signals of exosome-specific markers (TSG101 and CD9), and the expression of virus protein gB, VP16 and ICP5 were detected in exosomes derived from HSV-1 infected cells. The purified exosomes from HEp-2 cells with or without HSV-1 infection showed a typical round-shaped morphology under TEM, with a mean diameter of 50~150 nm; meanwhile, no typical HSV-1 virus particles were detectable. The peak diameter of purified exosomes was 112.2 nm, furthermore, the peak concentration of exosomes from HEp-2 control cells was 3.4×10 9 particles/ml, while the peak concentration of exosomes from HSV-1 infected cells was 4.1×10 9 particles/ml. Conclusions:We accomplished an efficient and optimized approach for the isolation and purification of exosomes from HSV-1 infected cells.

Objective:To examine a survival prognostic model applicable for patients with intrahepatic cholangiocarcinoma (ICC) based on Bayesian network.Methods:The clinical and pathological data of ICC patients who underwent curative intent resection in ten Chinese hepatobiliary surgery centers from January 2010 to December 2018 were collected.A total of 516 patients were included in the study. There were 266 males and 250 females.The median age( M( Q R)) was 58(14) years.One hundred and sixteen cases (22.5%) with intrahepatic bile duct stones,and 143 cases (27.7%) with chronic viral hepatitis.The Kaplan-Meier method was used for survival analysis.The univariate and multivariate analysis were implemented respectively using the Log-rank test and Cox proportional hazard model.One-year survival prediction models based on tree augmented naive Bayesian (TAN) and na?ve Bayesian algorithm were established by Bayesialab software according to different variables,a nomogram model was also developed based on the independent predictors.The receiver operating characteristic curve and the area under curve (AUC) were used to evaluate the prediction effect of the models. Results:The overall median survival time was 25.0 months,and the 1-,3-and 5-year cumulative survival rates was 76.6%,37.9%,and 21.0%,respectively.Univariate analysis showed that gender,preoperative jaundice,pathological differentiation,vascular invasion,microvascular invasion,liver capsule invasion,T staging,N staging,margin,intrahepatic bile duct stones,carcinoembryonic antigen,and CA19-9 affected the prognosis(χ 2=5.858-54.974, all P<0.05).The Cox multivariate model showed that gender,pathological differentiation,liver capsule invasion, T stage,N stage,intrahepatic bile duct stones,and CA19-9 were the independent predictive factors(all P<0.05). The AUC of the TAN model based on all 19 clinicopathological factors was 74.5%,and the AUC of the TAN model based on the 12 prognostic factors derived from univariate analysis was 74.0%,the AUC of the na?ve Bayesian model based on 7 independent prognostic risk factors was 79.5%,the AUC and C-index of the nomogram survival prediction model based on 7 independent prognostic risk factors were 78.8% and 0.73,respectively. Conclusion:The Bayesian network model may provide a relatively accurate prognostic prediction for ICC patients after curative intent resection and performed superior to the nomogram model.

Objective:To examine a survival prognostic model applicable for patients with intrahepatic cholangiocarcinoma (ICC) based on Bayesian network.Methods:The clinical and pathological data of ICC patients who underwent curative intent resection in ten Chinese hepatobiliary surgery centers from January 2010 to December 2018 were collected.A total of 516 patients were included in the study. There were 266 males and 250 females.The median age( M( Q R)) was 58(14) years.One hundred and sixteen cases (22.5%) with intrahepatic bile duct stones,and 143 cases (27.7%) with chronic viral hepatitis.The Kaplan-Meier method was used for survival analysis.The univariate and multivariate analysis were implemented respectively using the Log-rank test and Cox proportional hazard model.One-year survival prediction models based on tree augmented naive Bayesian (TAN) and na?ve Bayesian algorithm were established by Bayesialab software according to different variables,a nomogram model was also developed based on the independent predictors.The receiver operating characteristic curve and the area under curve (AUC) were used to evaluate the prediction effect of the models. Results:The overall median survival time was 25.0 months,and the 1-,3-and 5-year cumulative survival rates was 76.6%,37.9%,and 21.0%,respectively.Univariate analysis showed that gender,preoperative jaundice,pathological differentiation,vascular invasion,microvascular invasion,liver capsule invasion,T staging,N staging,margin,intrahepatic bile duct stones,carcinoembryonic antigen,and CA19-9 affected the prognosis(χ 2=5.858-54.974, all P<0.05).The Cox multivariate model showed that gender,pathological differentiation,liver capsule invasion, T stage,N stage,intrahepatic bile duct stones,and CA19-9 were the independent predictive factors(all P<0.05). The AUC of the TAN model based on all 19 clinicopathological factors was 74.5%,and the AUC of the TAN model based on the 12 prognostic factors derived from univariate analysis was 74.0%,the AUC of the na?ve Bayesian model based on 7 independent prognostic risk factors was 79.5%,the AUC and C-index of the nomogram survival prediction model based on 7 independent prognostic risk factors were 78.8% and 0.73,respectively. Conclusion:The Bayesian network model may provide a relatively accurate prognostic prediction for ICC patients after curative intent resection and performed superior to the nomogram model.

Objective: To study the manifestations of digestive system of hospitalized patients with novel coronavirus pneumonia (NCP) in Wuhan, China, and to provide reference for disease control and treatment. Methods: The data of hospitalized patients with NCP in the Sino-French Branch of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology was retrospectively analyzed, which included general information, nucleic acid test, severity degree of disease, incubation period, initial symptoms and manifestations of digestive system. The general information, positive rate of nucleic acid detection, and manifestations of digestive system were compared between critical patients who required non-invasive or invasive assisted ventilation (critical group) and non-critical patients without assisted ventilation (non-critical group). Continuous corrected chi-square test and independent sample median test were performed for statistical analysis. Results: Among the 305 patients there were 146 males (47.9%) and 159 females (52.1%), median age 57 years old. Nucleic acid assay of nasopharynx swab or pharynx swab of 84.1% (228/271) patients were positive. Forty-six patients (15.1%) were in critical group and 259 patients (84.9%) were in non-critical group. The incubation period was one to fifteen days, and the median period was six days. The initial symptoms mainly were fever (81.1%, 163/201), cough (39.3%, 79/201), fatigue (54.7%, 110/201), and loss of appetite (50.2%, 101/201). In one to ten days after the disease onset, 79.1% (159/201) of patients developed gastrointestinal symptoms including nausea (29.4%, 59/201), vomiting (15.9%, 32/201), or abdominal pain (6.0%, 12/201). 49.5% (146/295) of patients had diarrhea, median time was 3.3 days, (3.3±1.6) times per day, and a duration of (4.1±2.5) days. Excluding possible drug-related diarrhea, the incidence of diarrhea still was 22.2%. Only 6.9% (4/58) of patients were found leukocytes or fecal occult blood positive in regular stool test. ALT, AST, or bilirubin increased in 39.1% (119/304) of patients at admission. Patients with ALT or AST ≥ 80 U/L only accounted for 7.9% (24/304) and 6.3% (19/304), respectively. About 2.0% (6/304) of patients also had increased bilirubin level, average level was (37.4 ± 21.1) μmol/L. The median age of critical group was older than that of non-critical group (65.5 years vs. 56 years), at admission the rates of abnormal liver function test abnormal and slightly increased AST (40~80 U/L) of critical group were both higher than those of non-critical group (67.4% (31/46) vs. 34.1% (88/258) and 47.8% (22/46) vs. 21.7% (56/228)), and the differences were statistically significant (x²=5.885, 18.154 and 15.723;all P <0.05). There were no statistically significant differences in the proportion of male (58.7% (27/46) vs. 45.9% (119/259)), the positive rate of nucleic acid detection (94.6% (35/37) vs. 82.5% (193/234)), the percentage of patients with gastrointestinal symptoms (85.0% (17/20) vs. 78.5% (142/181)), the rate of diarrhea (44.7% (17/38)vs. 50.2% (129/257)) and ratio of patients with abnormal bilirubin level (6.5% (3/46) vs. 1.2% (3/258)) (all P>0.05). Conclusions The manifestation of digestive system of hospitalized NCP patients in Wuhan is significant, the ratio of patients with diarrhea and abnormal aminotransferase level is high. And at admission the rate of patients with abnormal liver function rate of critical group is higher than that of non-critical group, which will provide reference for the prevention and treatment of NCP.