One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like HIN1, NS from classical swine H1NI, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.

To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection, the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2. In addition, the gene sequences coding hemagglutinin (HA), neuraminidase(NA) and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2, thus constructing the prokaryotic expression vectors pET32a-NS2-HA, pET32a-MS2-NA and pET32a-MS2-M. These recombinant plasmids were then transformed separately to E.coli BL21(DE3) with induction by IPTG. to express the virus-like particles. The virus-like particles observed under electron microscopy were identified by RT-PCR ,while their stability was confirmed by real-time RT-PCR. In this way, the virus-like particles were successively constructed and identified through PCR amplification, enzymolysis identification and sequencing analysis. These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm. Their stability was proved to be rather good. From these observations, it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.

Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P<0. 05) and 16. 9% (P>0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P<0. 05), and the levels of intracellular HBV DNA were respectively sup-pressed by 7. 2% (P>0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.

Objective To prepare and study the immunogenicity of hepatitis B virus surface anti-gen (HBsAg)-tetanus toxoid (TT) conjugate vaccine. Methods Tr was activated by cyangen bromide and reacted with adipic acid dihydrazide, then HBsAg-TT conjugate was prepared by carbediimide. Conjugate, HBsAg or hepatitis B vaccine was injected subcutaneously into mice. Anti-HBsAg and HBsAg-specific T cell response elicited by these immunogens were assayed. Results New HBsAg-TT conjugate elicited higher levels of anti-HBsAg and HBsAg positive conversion rates after the immunization than did HBsAg alone or hepatitis B vaccine. Conjugate induced mesdy antibodies of the IgG2a subclass, while HBsAg alone or hepa-titis B vaccine mainly elicited anti-HBsAg in the IgG1 subclass. The number of IFN-γand IL-2 secreting T cells induced by conjugate was also significantly higher than that did by HBsAg or hepatitis B vaccine. Con-clusion This study indicated new HBsAg-TT conjugate can induce both stronger humoral and TH1 type of cellular immune response.

Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.

Objective To evaluate the diagnostic value of detection of IgM antibodies to EV71-infection patients,and compared characterisation of RT-PCR,IgM capture ELISA and neutralization test.Methods Virus RNA,neutralization titer and IgM antibody in 115 EV71-infection patients were detected by EV71 real-time RT-PCR kit( EV71-PCR kit),neutralization test,and EV71 IgM-capture ELISA kit (EV71-IgM kit),respectively.Results Using EV71-IgM kit,the detection rate was 80.9％ (93/115,95％ CI:72.5-87.6) among the 115 EV71-infection patients,and was 2.6％ among the 228 healthy children.Simultaneously,sera collected after 1-2 day of disease onset showed an IgM positivity of 70.4％ (38/ 54).The positive rate of EV71-PCR among these patients was 82.6％ (95/115,95％ CI:74.4-89.0),so there was no statistically significant differences between it and EV71-IgM kit.In addition,the detection rate in EV71-infection patients could increase to 92.2％ by combined detection of EV71-PCR and EV71-IgM kit.Conclusion EV71-IgM kit was a rapid and valuable way for the early diagnosis of EV71 infection,and could significantly improve detection rate for EV71 infection by combining with EV71-PCR kit.

Objective To analyze the molecular characteristics and genetic origin of a novel avian influenza A H7N4 virus casuing a case of human infection in China. Methods Specimens were collected from the patient and chickens and ducks kept by the patient and neighbours and then detected by real-time quantitative PCR. The original specimens and virus isolates were analyzed by next-generation sequencing technology to obtain viral whole-genome sequences. Pairwise sequence alignments and phylogenetic analysis were performed by BLASTs,ClustalX and MEGA 6. 1 softwares. Results In January 2018, a human case infected with avian influenza A H7N4 virus was confirmed. Seven H7N4 viruses were isolated from speci-mens collected from chicken and ducks kept in the patient`s backyard. H7N4 virus was a novel reassortant vi-rus with all eight gene fragments derived from wild waterfowl in Eurasia. HA protein contained a single basic amino acid residue R in cleavage site, suggesting that H7N4 virus was low pathogenic. The receptor-binding sites of HA had QSG at 226-228 residues, which indicated that the virus retained avian-type receptor speci-ficity (SAα2-3Gal). Different from H7N4 viruses in avian, the virus isolated from the patient had substitu-tion at position 627 ( E→K) in PB2 protein, which might increase its adaptation in human host. Conclusion This study reported a case of human infection with a novel reassortant avian influenza A H7N4 virus, which revealed that the traditional backyard breeding models might facilitate cross-species transmission of avian in-fluenza viruses in southern China.

Objective: To investigate the pyroptosis induced by different enteroviruses in human neuroblastoma cells SH-SY5Y and the differences among them. Methods: SH-SY5Y cells were infected with nine strains of enterovirus respectively, including enterovirus A71 (EV-A71), Coxsackievirus A (CA), Coxsackievirus B (CB), Echovirus (Echo). The cellular morphology of infected and control groups were observed and activity of Caspase-1 of infected and control groups were detected by flow cytometry at 48 h post infection. Results: The activity of Caspase-1 induced by EV-A71 was higher than control (P<0.001), and the activity of Caspase-1 induced by EV-A71 isolated from severe case was significantly higher than that induced by EV-A71 isolated from common case (P<0.001). The activity of Caspase-1 induced by CA was at a lower level. The activity of Caspase-1 induced by CB and Echo were both significantly higher than that induced by EV-A71 and control (P<0.001). Cytopathic effects (CPE) were found to be related with the activity of Caspase-1. Conclusions EV-A71, CB and Echo all could induce pyroptosis mediated by Caspase-1 in SH-SY5Y cells, but the ability of CA to induce pyroptosis was at a lower level, which may provide evidences for further study on mechanism of neuropathy caused by enterovirus.

Objective: To establish a rapid and sensitive isothermal amplification assay for the detection of human Adenovirus. Methods: Primers and probe used for recombinase polymerase amplification(RPA)were designed based on the conserved region of the adenoviruses hexon gene. After optimizing the reaction temperature and times, the products of RPA were detected by capillary electrophoresis and lateral flow dipstick(LFD). Sensitivity and specicity of the assay were evaluated. The diagnostic value of the RPA-LFD assay was verified using clinical samples which were simultaneously tested by real time PCR assay. Results: The analytical sensitivity of RPA-LFD assay was 2 copies DNA molecules per reaction and no cross reaction with other pathogens was observed. Compared with real-time PCR assay, the sensitivity, and specificity of the present assay were all 100%. Conclusions The RPA-LFD assay developed in this study has the characteristics of high specificity, sensitivity, rapid and no requirement of expensive equipment which provided a new tool for rapid detection of human adenovirus.

Objective: To find out the characteristics and regularity trend of influenza activity according to the analyses of influenza surveillance data in Yangzhou from 2012 to 2017, and to provide scientific supports for predicting and controlling the pandemic outbreak of influenza effectively. Methods: The influenza samples were collected from Northern Jiangsu People′s Hospital, Yangzhou First People's Hospital and Gaoyou People’s Hospital, using fluorescent RT-PCR method to detect influenza virus nucleic acid and classifying influenza virus subtypes. Finally, the surveillance data from January, 2012 to December, 2017 of influenza like illness (ILI) cases of Yangzhou were analyzed. Results: Totally 18 083 throat swabs of ILI specimens were collected from 2012—2017 in Yangzhou, 1 983 samples were positive (10.97%), the difference in positive rates of adjacent years was statistically significant (χ²=167.93, P<0.001). In Yangzhou city, influenza virus kept activative in whole year. The influenza epidemic season was winter-spring and summer, accounting for 83.61% of all positive cases, with type A influenza prevalent throughout all year and type B influenza mainly prevalent in spring and winter, each subtype showed alternating prevalence. The influenza virus infection occurred in every age group. The highest positive rate was 16.33% in the age group of 10-19 years and the lowest was 8.52% in the age group of 20-29 years among all detected age groups. Conclusions From 2012 to 2017, the epidemic of influenza in Yangzhou was obviously seasonal and all the subtypes of influenza were in prevalent status alternately. The high-risk groups of Influenza were infants and teenagers who under the age of 19 years. So a long-term influenza surveillance is much needed for the early warning and forecasting the spread of influenza.