Objective To expression prM/E gene of dengue vires type I in mammalia cells.Methods The full-length prM/E gene of dengue virus type Ⅰ strain GZ01/95 was amplified by RT-PCR,the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile,and three of the constructions were cloned into the poDNAS/FRT.Then they were transfected into 293T cells by lipofectamine respectively.The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA)as well as Western blot.Results In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA,the expressed producm for gene of dengue virus type Ⅰ were confirmed by IFA.The secreted expression products for gene of dengue virus type Ⅰ specific protein bands were confirmed by Western blot only existing in the cell supematants transfected with the modified recombinant plasmids DNA.Conclusion The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA.The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.

Objective Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. Methods The internal controls target gene were obtained by insertion of a 180bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. Results The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. Conclusion The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.

Objective To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coil to inhibit virus infection and induce the neutralizing antibody. Methods E Ⅲ protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21 (DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E Ⅲserum. Antibody titers were determined by neutralizing assay. Results The recombinant E Ⅲ proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2infection. All four type anti-E Ⅲ sera can neutralize DV2 but their efficacies are different. Conclusion E Ⅲproteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E Ⅲ proteins. Both E Ⅲ protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.

Objective To identify the epidemic characteristics and risk factors of an emerging infectious disease-severe fever with thrombocytopenia syndrome (SFTS) in Hubei province.Methods Active surveillance program on SFTS was set up in monitoring sites-hospitals,at the township level or above,in Suizhou,Huanggang and Wuhan from January to December,2010.Specific surveillance program on SFTS was launched across the province in hospitals above the county level.Cases that matched the definition of surveillance case were identified and reported to Centers for Disease Control and Prevention (CDCs).Cases were interviewed and their blood samples collected and detected using PCR and virus isolation.We also conducted serum antibody surveys among healthy population and livestock and surveillance on vector ticks in those high-epidemic areas.Results 188 cases that matched the definition of surveillance case and 21 deaths were reported in 11 cities,32 countries and 100 towns in 2010,with an incidence rate of 0.33/106.The fatality rate was 11.2％.Data showed that the patients were from hilly areas at the altitude elevated between 28-940 meters.The epidemic period was between April and December with the peak from May to September.The youngest case was an 11-year old,while the eldest was 81 with median age as 56-year old.95.3 ％ of the patients were farmers.All Patients did not have the history of traveling,two weeks before the onset of SFTS.93.6％ of the patients engaged in different kind of work which was associated with agriculture.52.8％ of the patients had been exposed to ticks.22.0％ of the patients had been bitten by ticks.Skin injury was found in 64.2％ of the patients.Samples from 129 cases (68.6％) were collected and detected,with 67.4％ of them (87 cases) showed positive by Real time-PCR for SFTS virus.An elevation in antibody titer by a factor of four or evidence of sero-conversion was observed in 11 patients; SFTS virus was isolated from 2 patients.The total antibody positive rates were 3.8％,55.0％ (6/11 ),36.7％ (2/3) and 80.0％ (4/5) respectively in healthy population,dogs,sheep and cows.Ticks from grass,cattle and sheep were detected positive by Real time-PCR.Conclusion Most cases of SFTS in Hubei were infected by SFTS virus,and cases of livestock were infected by SFTS virus.Ticks might serve as an important vector.Skin injury,exposure to tick bites seemed to be the risk factors.

Since December 2019, novel coronavirus pneumonia (NCP) has been reported in Wuhan, Hubei Province, and spreads rapidly to all through Hubei Province and even to the whole country. The virus is 2019 novel coronavirus (2019-nCoV), never been seen previously in human, but all the population is generally susceptible. The virus spreads through many ways and is highly infectious, which brings great difficulties to the prevention and control of NCP. Based on the needs of orthopedic trauma patients for emergency surgery and review of the latest NCP diagnosis and treatment strategy and the latest principles and principles of evidence-based medicine in traumatic orthopedics, the authors put forward this expert consensus to systematically standardize the clinical pathway and protective measures of emergency surgery for orthopedic trauma patients during prevention and control of NCP and provide reference for the emergency surgical treatment of orthopedic trauma patients in hospitals at all levels.