The 1.23 kb DNA fragment encoding the early protein EP0 of pseudorabies virus (PRV) Ea strain was amplified by PCR technique and cloned into pBluescriptII sk+.Three sequencing plasmids containing the partial fragment of the EP0 gene were constructed and the sequences were obtained by Sanger's sequencing technique. Compared with PRV InFh strain, there were multipile site-mutations and a deleted-mutation in the EP0 gene of PRV strain Ea，and the diversity of amino acid residues also existed.Then, the EP0 gene was inserted into an expression vector, pET-28a, fused into the downstream of the 6ΧHis-Tag in frame, to yield the expression plasmid pETEP0. After induction by IPTG, a high expression of fusion protein was obtained, SDS-PAGE analysis and Western blotting showed that the fusion protein was 62kD and the protein was specific to antisera against PRV Ea strain. This indicated that the EP0 gene be expressed in BL21(DE3) and the expression products have immuno-genicity.

ObjectiveTo evaluate the diagnostic performance of CTCA in predicting myocardial perfusion defects through comparative analysis between MPI defects and severity of coronary stenosis on CTCA.MethodsFour hundred and seventy-eight patients who underwent CTCA and 99Tcm-MIBI MPI in the same period were analyzed retrospectively.According to the severity of coronary stenosis judged by visual evaluation of the vessel diameter,the patients were divided into five groups:no stenosis,mild stenosis,moderate stenosis,severe stenosis and total occlusion.MPI results were classified as negative or positive for perfusion defects,and the prevalence of perfusion defects in every group was calculated per-patient and per-vessel basis.The cut-off of stenotic severity for predicting myocardial perfusion defects was designated as 50％ or 75％,with MPI as standard reference.True positive,true negative,false positive and false negative statistics were thus determined separately on patient and vessel basis.The diagnostic performance for CTCA were calculated and compared.Pearson Chi-square and its partition tests or Fisher exact test were used to compare ordinal variables.ResultsFifty-eight patients showed myocardial perfusion defects.Either by patientbased or vessel-based analysis,the prevalence of myocardial perfusion defects showed an increasing trend with greater coronary artery stenosis in each group,and there were statistical differences among them (x2 =116.62 and 483.83,both P ＜ 0.05).On patient-based analysis,sensitivity ( SN),specificity ( SP),positive predictive value( PPV),negative predictive value(NPV) and accuracy (AC) for CTCA predicting myocardial perfusion defects were 62.1 ％ ( 36/58 ) and 34.5％ ( 20/58 ) (x2 =8.84,P ＜ 0.05 ),84.5％(355/420) and 97.1％ (408/420) (x2 =40.16,P ＜0.05),35.6％ (30/101) and 62.5％ (20/32) (x2 =7.19,P＜0.05),94.2％ (355/377) and 91.5％ (408/446) (x2 =2.18,P ＞0.05),81.8％ (391/478)and 89.5 ％ (428/478) (x2 =11.66,P ＜ 0.05 ) when the cutoff was set to 50％ and 75％,respectively.On vessel-based analysis,the SN,SP,PPV,NPV and AC for CTCA predicting myocardial perfusion defects were 58.8％ (40/68) and 30.9％ (21/68) (x2 =10.73,P ＜ 0.05),95.9％ (1768/1844) and 99.0％ (1826/1844) (x2 =36.72,P ＜ 0.05 ),34.5％ (40/116) and 53.8％ (21/39) (x2 =4.59,P ＜0.05 ),98.4％ (1768/1796) and 97.5％ ( 1826/1873 ) (x2 =4.14,P ＜ 0.05 ),94.6％ ( 1808/1912 ) and 96.6％ ( 1847/1912 ) (x2 =10.31,P ＜ 0.05 ),respectively.ConclusionsThe prevalence of myocardial perfusion defects correlates positively with the severity of coronary stenosis seen on CTCA.CTCA may predict perfusion defects with high SP and NPV.However,the PPV of CTCA in predicting myocardial perfusion defects is poor when the stenosis cut-off is set at 50％.It is significantly improved when the cutoff value is set at 75 ％.

Objective: To evaluate the accuracy of automated classification of ICD-O-3 morphology code from pathology reports by text-mining and support vector machine ( SVM ) , in order to provide basis for automated tumor coding in Chinese. Methods: The tumor report cards of Zhejiang residents from 2017 to 2019 were collected from Chronic Disease Surveillance Information Management System of Zhejiang Province. According to ICD-O-3, the keywords of the pathology reports were extracted, and SVM was used for automatic classification. The classification results were compared with those of 16 professionals with more than two years of experience in tumor coding, and the accuracy rate, recall rate and F-score were calculated for effect evaluation. Results: Totally 83 082 cases from 2017 to 2019 were included and were categorized into 17 morphological classifications, with 52 877 ( 63.65% ) cases of adenocarcinoma, squamous carcinoma and transitional cell carcinoma. A total of 1 090 keywords were enrolled into main corpus. The total F-score, accuracy rate and recall rate are 85.69, 77.20% and 96.27%, respectively. Conclusion Text-mining combined with SVM can improve the efficiency of ICD-O-3 morphology coding; however, the accuracy needs to be further improved.

Objective To evaluate the association between total fluid intake and the time of urination per day and the risk of bladder cancer. Methods A population-based case-control study was conducted in urban Shanghai, China, during January 1996 to December 1998. The study included 608 incident cases of bladder cancer and 607 age- and sex-matched controls. Unconditional logistic regression models were used to estimate the odds ratios(ORs)and their corresponding 95%confidence intervals(95%CIs)for bladder cancer associated with frequency of urination, after adjusted for age, gender, smoking status, history of occupation with high risk, history of bladder infections, body mass index and other confounding factors. The level of statistical significance was set at 0.05(two-sided). Results No significant trend was observed for the association between total fluid intake, time of nighttime urination and the risk of bladder cancer. Increasing time of urination during daytime was associated with decreased risk of bladder cancer(P for trend=0.014). ORs(95%CIs)for subjects who voided 4 times, 5 times and 6 or more times per day[0.72(0.49-1.05),0.60(0.41-0.87)and 0.62(0.43-0.90), respectively], when compared with those with less than 4times per day after adjustment of confounding factors. Data showed that smokers and nonsmokers who voided at least 6 times per day had the ORs of 0.72(95%CI: 0.45-1.15)and 0.46(95%CI:0.25-0.87)when compared to their counterparts who voided 3 times or less per day during the daytime. Subjects who urinated at least 6 times per day and consumed more than 1500 ml of total fluid per day experienced a significant 57% reduction in risk compared to subjects who urinated 3 times or less and consumed less than 750 ml of total daily fluid intake. Conclusion Increased urination frequency and total fluid intake, especially among those who never smoked might be associated with a reduced risk of bladder cancer.

OBJECTIVETo obtain isolated human metapneumovirus (HMPV) strains from clinical specimens collected from infants and children in Beijing and to promote the investigation on this important respiratory pathogen.

METHODClinical specimens including throat swabs from outpatients and nasopharyngeal aspirates from hospitalized children were collected from infants and children visited the affiliated children's hospital for acute respiratory infections during May 2008 to April 2009. HMPV positive specimens identified by RT-PCR and/or direct immunofluorescent assay with monoclonal antibody against HMPV were inoculated to LLC-MK(2) cells and incubated at 37°C and 33°C, respectively. The replication of the virus in the cells was detected by direct immunofluorescent assay followed by RT-PCR. The genotypes of the isolated virus strains were identified by RT-PCR.

RESULTOut of 1092 clinical specimens, 81 were HMPV positive by RT-PCR, the positive rate was 7.4% (81/1092). Among these positive specimens, 33 were inoculated to LLC-MK(2) cells and the replication of HMPV was revealed by antigen detection and RT-PCR from 5 out of these 33 inoculates. These isolated viruses could be passed in LLC-MK(2) cells and were not cross-reacted with other common respiratory viruses, such as ADV, RSV and Parainfluenza viruses 1/2/3 by monoclonal antibodies against these viruses in direct immunofluorescent assay. The HMPV was more likely to be isolated from fresh specimens within 24 hours after the collection of specimens which were not frozen. Four of the 5 isolated strains were identified as genotype A and 1 as genotype B. Unlike other respiratory viruses, these isolated HMPV did not show specific CPE in cell culture and the replication of the virus was identified by antigen detection and RT-PCR.

CONCLUSIONHMPV of both genotypes were isolated from infants and children with acute respiratory infections in Beijing which will accelerate the investigation of this important virus.

Acute Disease ; Child ; China ; Genes, Viral ; genetics ; Genotype ; Humans ; Infant ; Metapneumovirus ; isolation & purification ; Respiratory Tract Infections ; virology

Be subjected to study the application in the hypersaline organic wastewater treatment,A moder-ately halophilic bacterium YS-1 was isolated from the salty water of a Ludaokou salina of Weihai in China.The polyphasic taxonomy study was processed through identifying its morphological features by AFM,researched the features of culture and measured physiologic-biochemical index,analyzed 16S rDNA se-quence homology Phylogenetic analysis based on 16S rDNA gene sequences showed that the closest relative of the strain was Halomonas sp.(AB167061).Experimented of intensifying hypersaline organic wastewater treatmented processing in the SBR demonstrate a significant result.The wastewater salt content of initial wastewater was 12% and initial CODcr was 1494 mg/L.After 72 h,the removal efficiency of CODcr achieved 90.0% and after 120 h,it achieved 98.1%.The experiments also showed the hypersaline organic wastewater treatment and presented the feasibility of acclimating of halophilic bacteria and running of proc-essing system.

Objective To analyze the characteristics and trend of incidence and mortality of thyroid cancer in Zhejiang Province from 2007 to 2011.Methods Data from cancer registry and death registry in Zhejiang province were used to calculate the crude incidence and mortality,age -specific incidence and mortality,China - and World -standardized incidence and mortality of thyroid cancer.Trend Chi -square test was used to analyze the trend of incidence and mortality. Results From 2007 to 2011,the reported incidence rate of thyroid cancer in Zhejiang Province was 8.37 /100,000 (China -and World -standardized incidence were 5.28 /100,000 and 6.14 /100,000 respectively).The mortality rate was 0.34 /100,000 (China -and World -standardized mortality were 0.17 /100,000 and 0.24 /100,000 respectively). The incidence and mortality were both significantly higher in females and urban residents than in males and rural residents (both P <0.01).With age increased,the mortality increased.However,the incidence increased at the beginning and then declined with a peak age of 30 -59.From 2007 to 2011,the incidence of thyroid cancer increased rapidly with a speed of 29.95% per year while the mortality did not show the similar trend.Conclusion The incidence of thyroid cancer in Zhejiang Province is growing rapidly and the relative risk factors should be taken into consideration in future researches.

To study the molecular epidemiological characteristics of norovirus gastroenteritis outbreaks in Guangdong, we collected fecal and anal swabs specimens from 24 outbreaks of acute gastroenteritis from 2005 to 2008 to detect norovirus. Specimens were detected by RT-PCR and then sequenced. The descriptive data were also collected. According to our research, 19 of 24 outbreaks of gastroenteritis were positive for norovirus. The occurrence time was from October to next February mainly. The strains in 2005 belonged to G II-3 genotype and all outbreaks occurred in kindergarten and school. But from autumn of 2006, the outbreaks were all caused by G II-4/2006b variant and occurred in universities and community. The number of outbreaks in 2007 increased greatly and covered all over province. The nucleotide sequences of Guangdong strains in some sites showed high regional identity. Our results showed that with the shift of genotype from G II-3 to G II-4, occurrence of norovirus outbreaks increased greatly. The outbreaks of norovirus caused by G II-4/2006b variant spreaded widely and the involved population covered children and adult, indicating the strong invasiveness of this variant.
Adolescent ; Adult ; Base Sequence ; Child ; China ; epidemiology ; DNA-Directed RNA Polymerases ; genetics ; Disease Outbreaks ; Gastroenteritis ; epidemiology ; Humans ; Molecular Sequence Data ; Norovirus ; classification ; enzymology ; genetics ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

BACKGROUNDHuman metapneumovirus (hMPV) was discovered by scientists in the Netherlands as a novel respiratory virus in 2001 and had been found in children with acute respiratory tract infections (ARTI) in China. The objective of this study was to determine the importance of hMPV infection in children in Beijing and the genotypes of the circulating virus by the surveillance during a four-consecutive-year period.

METHODSClinical specimens collected from children with ARTI from January 2006 to December 2009 were tested for hMPV by RT-PCR using primers targeting the matrix (M) gene, followed by genotyping of hMPV directly from positive samples by diplex PCR with primers for glycoprotein (G) genes. Sequence analysis was used for genotyping of those un-typable samples. Common respiratory viruses in these clinical specimens were tested by virus isolation and antigen detection, in addition to hMPV detection.

RESULTSOf 4730 tested specimens, 191 (4.0%) were positive for hMPV and 62.8% of 191 were identified as genotype A. The positive rate of hMPV from hospitalized patients was higher than that from outpatients each year. Most of hMPV positive children were under five years old. The peak of hMPV activity mostly occurred in late spring and overlapped with or followed that of respiratory syncytial virus (RSV) and followed by parainfluenza virus 3. Of hMPV infected cases, 68.6% were lower respiratory tract infection, among which 79.4% were hospitalized, and upper respiratory tract infection was diagnosed for 31.4% of hMPV infected children. The 9.4% of hMPV positive samples were found to co-exist with other respiratory viruses.

CONCLUSIONShMPV was an important pathogen for ARTI in pediatric patients, especially those under five years old. Both genotypes A and B circulated simultaneously in Beijing.

Adolescent ; China ; Female ; Genotype ; Humans ; Male ; Metapneumovirus ; genetics ; pathogenicity ; Respiratory Tract Infections ; virology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo identify variations in hemagglutinin genes from influenza viruses (H3N2) isolated from infants and young children with acute respiratory infection (ARI) between March, 2004 and April 2005.

METHODSRNAs from influenza A virus strains (subtype H3) isolated from specimens collected from ARI children were extracted followed by amplification for HA1 fragments from hemagglutinin (HA) genes by RT-PCR. The sequences of the fragments were defined by direct sequencing for the PCR products or the target inserts after the PCR fragments were cloned into the TA-cloning vector pBS-T and analyzed by bioinformatic software.

RESULTSFragments of 987 bps of HA1 (encoding 329 amino acids) from a total of 32 strains of influenza A virus (subtype H3) isolated from the 2004 season and 1 from the 2003 season were amplified and the sequences were compared with vaccine reference strains recommended by WHO which were used in recent years. There were several consistent amino acid variations which involved in both antigenic epitopes A and B and receptor binding site (RBS) for isolated strains in the 2004 influenza season compared with the vaccine strains used during the recent years and the virus strains isolated in March 2004, indicated the antigenic drift of the viruses isolated in 2004 influenza season may lead to variant viruses.

CONCLUSIONThe variations of the HA genes from influenza virus (subtype H3) strains in the 2004-2005 influenza season were confirmed by sequence analysis for the HA1 regions of the hemagglutinin genes, which indicate that the antigenic drift would have been caused by the diversification of the genes and the efficacy of the recently used vaccines should be kept under close watch.

Antigenic Variation ; Child ; China ; epidemiology ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; epidemiology ; immunology ; virology ; RNA, Viral ; genetics ; Sequence Analysis, RNA