Objective To construct the recombinant rAAV2-hGAD65 vector and detect its function both in vitro and in vivo. Methods The cDNA of human glutamic acid decarboxylase 2 (hGAD65) gene, which was one of gamma-aminobutyric acid (GABA) synthetase, cloned by the method of RT-PCR, was subcloned into the adeno-associated virus (AAV) vector and formed the recombinant vector of AAV-hGAD65 (rAAV2-hGAD65). The recombinant vector was packaged by the AAV Helper-Free System and its titer was detected. The primary fibroblast, cultured from the rat lung, was transfected by the rAAV2-hGAD65. The expression of the hGAD65 in fibroblast was detected by immunohistochemical method and the level of GABA in the media was assayed by high performance liquid chromatograph (HPLC). In vivo, the hGAD65 was detected by immunohistochemical method in STN and the concentration of the GABA in the reticular part of substantia nigra (SNr) was assayed by HPLC after the rAAV2-hGAD65 was injected into the subthalamic nucleus (STN) by the stereotaxic method. Results The sequence of hGAD65 cDNA was in accordance with that in the Genebank. The amino acid sequence of hGAD65 had no mutation and the titer of rAAV2-hGAD65 was reached 4.5 ×10~(11) per milliliter. The efficiency of infection to the rat primary firoblasts was 80%, and the concentration of GABA in the media of fibroblasts was (45.66±6.07)nmol/L per liter. In vivo, hGAD65 could be detected in STN, and the concentrateion of the GABA in the SNr was increased from (5.66±1.07)nmol/g to (12.66±2.59)nmol/g.Conclusion The cDNA of hGAD65 was cloned by RT-PCR and the recombinant vector of rAAV2-hGAD65 was constructed. The AAV can infect the primary fibroblast in vitro and the hGAD65 can catalyse the glutamic acic to GABA. In vivo, the concentration of GABA in the SNr was heighten after the rAAV2-hGAD65 was injected into the STN.

Objective To discuss the prevalence of non-EV71,non-CA16 virus strains of hand,foot and mouth disease(HFMD) in Guangdong province between 2008 and 2009,and analyze the genetic evolution of these non-EV71,non-CA16 virus strains.Methods Isolated viruses from stool samples collected from outpatient and in-patient cases of HFMD between 2008 and 2009 by human rhabdomyosarcoma(RD) cell and HEp-2 cell,cultures that exhibited a characteristic enterovirus cytopathic effect were evaluated by RT-PCR.Those strains which identified non-EV71,non-CA16 were analyzed by VP1 sequencing and then were identified by BLAST program.A phylogenetic tree was constructed using the Neighor-Joinning method in the MEGA 4.0 software.Results Twenty-two virus strains of non-EV71,non-CA16 were obtained,and nine of the twenty-two virus strains in 2008 were classified into CA2,CA4,and CB3 by BLAST; thirteen of the twenty-two virus strains in 2009 were classified into EV80,Echo13,Echo30,CBS,Echo24,CA10,CA6,and poliovirus 1 by BLAST.The honology of all strains was low,and all the strains belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup.Conclusion Except for EV71 and CA16 was a major causative agent in prevail of HFMD in Guangdong province between 2008 and 2009,there also existed other subgroup Enterovirus.The other twenty-two strains respectively belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup,and none of those strains was predominant.Muti-species Enterovirus occurred concomitantly.

Objective To understand the genetic characteristics of Enterovirus 71 ( EVT1 ) circu-lating strains of Guangdong province in 2008. Methods We isolated an EV71 strain from the fatal case of the hand, foot and mouth disease during an epidemic of Guangdong in 2008. Its complete genome was se-quenced and analyzed comparatively. Results The results showed that the full length of EV71 GDFS-3 ge-nome( not including poly A tail ) is 7405 bp. No insertion or deletion is detected in the coding region. There are several insertions and deletions in 5'and 3'UTR. Phylogenetic analysis of GDFS-3 and reference strains showed GDFS-3 strain shares the highest nueleotide homology with TW984 strain(96.0% ) but low homology with SIN5865, MS and BrCr( about 81.0% ). GDFS-3 strain also shares the highest amino acid homology with TW984 strain(99.0% ). It clustered with reference strains of CA subgenotype in the phylogenetie tree. The nucleotide identity with CA reference strains is 91.0% -95.0%. Conclusion The phylogenetic analysis based on the entire genome demonstrates that GDFS-3 strain has the nearest genetic relationship with TW984 strains ( isolated in 2004). GDFS-3 may belong to the same subgenogroup ( CA ) with Taiwan predominant strains. Otherwise,Some mutations in 5'UTR of EV71 may play the very important role in heightened viru-lence.

Objective:To evaluate the immunogenicity of an experimental SARS-CoV inactivated vaccine.Methods:The virus suspension of F69 strain was inactivated with 0.4% formaldehyde and purified,then used as the immune antigen combined with Freund′s adjuvant.Eight adult New Zealand rabbits were immunized 4 times with this vaccine.12 sets of rabbit serum were sampled from the third day to 74th day after first immunization.Titers of specific IgG antibody and neutralizing antibody were determined by indirect ELISA and micro-cytopathic effect neutralizing test.Results:Rapid and potent humoral immune responses were induced by F69 inactivated vaccine in all eight immunized rabbits.Both specific IgG antibody and neutralizing antibody all peaked just with 2 vaccinations,with the maximum titer of 1∶81 920 and 1∶20 480 respectively about 6 weeks after first immunization.Across neutralizing reaction existed between F69 and Z2-Y3 strains.Conclusion:F69 inactivated vaccine owns strong immunogenicity.Similar antigenic epitopes exist between the F69 strain and Z2-Y3 strain,which ensured the cross neutralizing reaction.

Objective To study the epidemic and genetic characteristics of coxsackievirus A6 ( CVA6) strains isolated in Guangdong province.Methods Enterovirus strains positive for neither entero-virus A71 ( EV71) nor CVA16 were isolated from Guangdong province during 2008 to 2013 to screen CVA6 isolates by real-time PCR.The entire sequences of viral genes encoding VP1 of CVA6 positive samples were amplified and sequenced.The phylogenetic analysis was performed to analyze the full-length gene sequences encoding VP1 of CVA6 isolates and sequences downloaded from GenBank by using DNAStar6.0 and MEGA5.2 software packages.Results CVA6 strains accounted for 61.4%of the 1672 non-EV71 and non-CVA16 enterovirus strains isolated in Guangdong province during year 2008 to 2013.The positive rates were respectively 10.5%(4/38), 66.7%(34/51), 36.2% (81/224), 63.0% (182/289), 62.3% (325/522) and 73.0%(400/548) from 2008 to 2013 and the differences among different years were significant (χ2=133.79, P<0.01).The CVA6 isolates could be classified into four clusters in the phylogenetic tree, designated A, B, C and D (including D1, D2 and D3 subgenogroups) genogroups.The four clusters shared nucleotide diversity ranging from 15.5% to 23.1%.The CVA6 strains isolated in Guangdong province shared 88.7%-100.0% homologies in nucleotide and 95.7%-100.0% in amino acid.Subtype D2 strains circulated during 2008 to 2012 and subtype D3 strains circulated during 2009 to 2013.Conclusion CVA6 strains were the predominant enterovirus strains among non-EV71 and non-CVA16 enterovirus strains circula-ted in Guangdong province from year 2008 to 2013.The CVA6 isolates could be classified into A, B, C and D genogroups based on the sequence analysis of VP1 region.Subgroups D2 and D3 isolates were identified and the subgroup D3 isolates were the prevalent strains in Guangdong.

Objective To understand the enterovirus infection in family close contacts of patients with hand,foot and mouth disease (HFMD) and the contamination of environment.Methods Forty-one HFMD cases confirmed by laboratory from web-based surveillance system during July to August 2010 in Guangdong Province were selected.All members of the cases′ family were investigated by collecting their information on demography,habit of domestic hygiene and hygiene status in household.The stool samples of all members and the smear samples from the surface of family belongings from 16 families were collected and the enterovirus was detected by real time quantitative polymerase chain reaction.The data were analyzed by chi square teat and t test.ResultsForty-one HFMD cases′ families and 135 close contacts were included in this survey.The infection rate of the enterovirus was 39.2％ (53/135) in all close contacts.Of all the investigated families,the infectionrate was 58.5％ (24/41) in family with one or more close contacts and 9.8％ (4/41) in family with all close contacts.The differences of infection rates of enterovirus among the members of parents (32.5％,25/77),grandparents/aunts/ uncles (43.3％,13/30) and cousins (53.6％,15/28) didn′t show statistical significance (χ2 =4.07,P=0.131).The infection rate of enterovirus in close contacts from family with more than 5 members was higher than that from family with 4 or less members (OR=1.4,95％CI 1.1-1.9).Among 135 close contacts,27.4％ (37/135) were infected with the same types of entervirus as that of HFMD case in the family and 11.9％ (16/135) were infected with the different virus types.In 33 family belongings samples from 16 families,the positive rate of enterovirus detection was 6.1％ (2/33).Between 17 families with enterovirus testing negative and 23 families with enterovirus testing positive in close contacts,there were no statistical differences of the family hygiene status,hand-washing of babysitter,disinfection of tableware and drinking,sharing towels,airing bedding articles and toy cleaning (P＞0.05).ConclusionsThe infection rate of enterovirus in close contacts of HFMD cases is high and the enterovirus contamination exists in case family environment.Management of close contacts of HFMD cases and disinfection of the family environment are important in HFMD controls.

AIM: To study the pathological change in mouse organs immunitied by inactivated SARS-CoV vaccine. METHODS: Inactivated SARS-CoV vaccine was injected into BALB/c and C57BL/6 mice. Anti-SARS antibody was analyzed by ELISA. After 8 weeks, the immunitied mice were killed and those organs were analyzed by pathological methods. RESULTS: Anti-SARS antibody in mice was positive after 8 days. Only minimal injury was observed in a few lungs and livers, but the other organs were not. CONCLUSIONS: Inactivated SARS-CoV vaccine induced mice to create antibody, whereas they did not cause severe injury. This result will be valuable for vaccine into clinical research. [

ObjectiveTo investigate the morbidity rate, states of distribution and therapy of psychosis, and the influence of the patients with psychosis to society. MethodsClue exploration was used mainly, assisted with filling vacancy.ResultsTotal of the patients with psychosis was 4253,the time-spot rate of every psychosis was 4.59‰, the all-life rate was 6.24‰. ConclusionSome data of psychosis in Huicheng District have been obtained.

Objective:To prepare seven standard strains of 2019 novel coronavirus (2019-nCoV), including wild type (WT) strain, Beta variant, Delta variant, Omicron variants (BA.2, BA.5, BQ.1, XBB.1 branches), which could be used to apply for national standard strains.Methods:According to cytopathic effect (CPE), virus titer, whole-virus-genome-sequencing and detection of mycoplasma, the basic biological characteristics of clonal isolation were determined through plaque purification technology.Results:The CPE was mainly characterized by cell shrinkage and exfoliation in Vero cells after infection with seven 2019-nCoV clonal isolations (WT, Beta, Delta, Omicron, BA.2, BA.5, BQ.1 and XBB.1 branches). The result of mycoplasma detection were negative and titers of the clonal isolation from 2nd to 5th generations were stable at 10 5-10 8 TCID 50/ml; the electron microscope showed that the virions were all round or elliptical, with a diameter between 60 nm and 140 nm. The subtypes of 7 strains were identified by whole-virus-genome-sequencing and phylogenetic analysis, with genomic stability after the fifth successive generations of clonal isolation. Conclusions:The series clonal isolation of 2019-nCoV with typical CPE of coronavirus, clear morphological structure, good viral activity and stable genetic characteristics were prepared, and they could be used to apply for national standard strains.

The theory of "equal stress on bones and muscles" emphasizes that "the tendons bind to the bones, the bones are stretched, the bones are connected, and the bones are fractured. The relationship between bone and soft tissues are important, which is the law of Traditional Chinese Medicine in the treatment of orthopedic diseases. For patients with lumbar disc herniation, the percutaneous intervertebral foraminal technology remodels the disordered internal biological balance of the spine under pathological conditions. Among them, two common clinical minimally invasive approaches under endoscopy are paid attention to soft tissue protection, and active and appropriate functional exercises after surgery, which have become a typical manifestation of the theory of "equal stress on bones and muscles" in modern spinal orthopedic surgery.