Objective: To understand the epidemiological characteristics of Human coronavirus (HCoV), the patterns of emergence and circulation, and the genotype distribution of human coronavirus OC43 (HCoV-OC43) from November, 2009 to April, 2016 in Shanghai. Methods: A total of 6 059 respiratory specimens, including pharyngeal swab, sputum, nasopharyngeal aspirates and alveolar lavage fluid, as well as relative clinical data were collected from patients with acute respiratory infections from seven sentinel hospitals during November, 2009 to April, 2016 in Shanghai. Respiratory specimens were tested by RT-PCR with HCoV-conserved primers and subsequently genotyped by DNA sequencing. Using specific primers to amplify and sequence full-length Spike (S), RNA-dependent RNA polymerase (RDRP) and nucleocapsid (N) gene from HCoV-OC43 positive samples. Further genotype and phylogenetic analysis of HCoV-OC43 were performed by conducting phylogenetic trees. Results: Among 6 059 patients, the total frequency of HCoV was 63 (1.04%), in which HCoV-OC43 was the most frequently detected species with 34 positive samples, followed by human coronavirus 229E (HCoV-229E) and human coronavirus HKU1 (HCoV-HKU1) with 18 and 10 positive sample respectively. However, other HCoV like human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV), were not been detected, which illustrated that HCoV-OC43 was the dominant subtype. The full-length of S, RDRP and N gene were obtained from 29 HCoV-OC43 positive samples. According to the sequence-analysis, 27 of which was genotype D, 2 of which was genotype B and others genotype, including genotype E, F and G, were not detected. The result indicated that the genotype D may be the dominant genotype. Further analysis of S protein that help HCoV-OC43 to entry host cell and stimulate the host immune system to produce neutralizing antibody found that two important functional domains in S protein, N-terminal domain (NTD) and receptor-binding domain (RBD) contained more amino acid substitution and positive selection sites, accompanied with amino acid insertion/deletion. 13 positive selection sites were all located in the NTD or RBD, 10 of which were located in the NTD and 3 in the RBD. Conclusion Human coronavirus OC43 was the major circulation human coronaviurs in Shanghai from 2009 to 2016, in which genotype D was the dominant genotype. NTD and RBD regions of the S protein were hypervariable region during HCoV-OC43 evolution, and had amino acid substitutions as well as amino acid insertion/deletion.

Objective: To analyze the changes in the expression of hypoxia inducible factor-1α (HIF-1α) and inflammatory cytokines and to investigate the role of HIF-1α in regulating the production of inflammatory cytokines during influenza A (H1N1) virus infection. Methods: BALB/c mice were injected with H1N1 virus to establish the mouse model of H1N1 virus infection. Fifteen BALB/c mice were randomly divided into three groups: control group, H1N1 virus group and H1N1 virus+ HIF-1α inhibitor group. Inflammatory cytokines (IL-6, TNF-α, IL-1β and IL-10) in samples of serum and lung tissues were detected by Luminex and ELISA. Levels of HIF-1α in serum and lung tissue samples were detected by Western blot and ELISA, respectively. Results: Compared with the control group, the levels of inflammatory cytokines in serum (IL-6, TNF-α, IL-1β and IL-10) and lung tissues (IL-6 and TNF-α) and the expression of HIF-1α in serum and lung tissues in the H1N1 virus group were significantly increased. The levels of HIF-1α, IL-6, TNF-α IL-1β and IL-10 in lung tissues in H1N1 virus+ HIF-1α inhibitor group were significantly lower than those of the H1N1 virus group. Conclusion During H1N1 virus infection, the levels of inflammatory cytokines and HIF-1α were significantly increased. The production of inflammatory cytokines was significantly reduced after inhibiting HIF-1α expression, suggesting that HIF-1α might promote the production of inflammatory cytokines.

Objective: To analyze the drug resistance of clinical isolates of Candida tropicalis in patients with infectious diseases, and preliminarily study their molecular characteristics. Methods: 95 strains of Candida tropicalis were isolated from the fungal culture specimens of 87 patients with infectious diseases in Shanghai Public Health Clinical Center from 2012 to 2015. Meanwhile, basic clinical data of patients were collected. The drug resistance of the strains to fungal drugs was analyzed by ATB FUNGUS 3 drug sensitivity test strips. All strains were classified by Multilocus sequence typing(MLST). Then, homology analysis was conducted by MEGA 5.2 software, and the evolutionary tree was mapped by using UPGMA method. Results: Patients distribution of strains was rendered as following: 31 strains from TB patients, 21 strains from HIV/AIDS patients, 19 strains from patients with liver disease, and 24 strains from rare cause infection or fever patients. The drug resistance rate to five antifungal drugs commonly used in clinical (amphotericin B, 5-fluorine cytosine, fluconazole, itraconazole, voriconazole) were 2.11% (2 strains), 0, 26.32% (25 strains), 26.32% (25 strains), and 26.32% (25 strains) respectively. Among the 25 azole-resistant strains: 14 strains were from rare cause infection or fever patients, 8 strains were from HIV/AIDS patients, and 3 strains were from tuberculosis patients. In MLST, 72 sequence types (ST types) were produced, 70 of which were new types. Evolutionary tree analysis showed that 95 strains of clinical strains distribute as three large clusters. 24 azole resistant strains (96.0%) were located in CLUSER Ⅰ. Conclusion The isolated Candida tropicalis were mainly resistant to azole drugs. MLST typing indicates that they was closely related to their genetic background.

Objective: To establish fluorescence quantitative polymerase chain reaction (PCR) to detect integrated HIV DNA in peripheral blood mononuclear cells. Methods: A total of 30 HIV-seropositve individuals were enrolled in this study, including 10 subjects with a detection limit of 20 copies/ml of plasma, 10 patients with drug resistance and 10 patients with no history of antiretroviral therapy (ART). Cultivated ACH2 cells carried a single copy of the integrated HIV genome. We have built pMD19T-CD3 plasmid and calculated the copy number. We used oligonucleotides ULF1 specific for the long terminal repeats (LTR) regions and two oliligonucleotides specific for human Alu sequences to pre-amplified the integrated HIV DNA. Samples and serial dilutions of ACH2 cells were all pre-amplified, the products of which were used for the second round fluorescence amplifications. The Lambda T primers, UR2 primers and HIV Taqman probes were used for second round amplifications in integrated HIV DNA assay. The CD3IN5 primers, CD3IN3 primers and CD3 Taqman probes were used for CD3 quantification. Results: Serial 5-fold dilutions of the plasmid were used as standards for CD3 gene quantifications. The equation of the linear regression was y=-2.731x+ 43.01(R²=0.953). The cellular input was quantified by number of human genome equivalents (CD3 gene, 2 copies/cell). The copies of ACH2 cells was 4.271×10^4, which was the cellular input of the ACH2 cells. Serial 5-fold dilutions of ACH2 cells were used to generate a standard curve for the integrated HIV DNA assays. The equation of the linear regression was y=-3.146x+ 39.11 (R²=0.968). The ratio between the number of copies of the integrated HIV DNA form and the number of cells (CD3 copies) was calculated to obtain the frequency of cells. Based on the test between different groups, each group had no difference (P> 0.05). Conclusions This method presented advantage in detection of the lower copies of virus. It hinted that the current antiretroviral therapy can not effectively attack against latent viral reservoirs. It also provided a basis for new therapeutic intervention.

Background:Acute gastroenteritis is the second largest public health problem in the world. Rotavirus(RV)is one of the pathogens of acute gastroenteritis in adults,researches focusing on RV infection may provide the basis for prevention and control of the disease. Aims:To determine the epidemiological characteristics of RV infection in adults with acute gastroenteritis in Shanghai,China. Methods:From Jun. 2010 to Dec. 2013,stool samples and clinical data in adults with acute gastroenteritis in a designated hospital in Shanghai Changning District were collected. ELISA and PCR were used to detect RV infection and its genotypes. Results:A total of 1 554 eligible stool samples from acute gastroenteritis patients were recruited,of them 691 were males and 863 were females,the mean age was(46. 19 ± 15. 59)years old. RV was detected in 189 patients with a detection rate of 12. 2% ,163(10. 5% )were categorized as group A RV and 26(1. 7% ) were group B/ C RV;the most common genotypes in group A RV were G9(30. 1% )and G1(25. 2% ). Watery stool and vomiting were more prevalent in RV-positive patients than in RV-negative patients(P < 0. 05). The detection rates in years 2010,2011,2012 and 2013 were 12. 2% ,14. 9% ,6. 8% and 16. 3% ,respectively. When analyzed by age group,the detection rate was significantly lower in 18-39 years group than those in 40-59,60-79,and ≥80 years groups(8. 7% vs. 14. 8% ,14. 2% ,and 17. 1% ,P < 0. 05). The peak of epidemic was from Nov. to next Feb. Conclusions:RV infection in adults with acute gastroenteritis is more popular in middle aged and elderly people and shows a winter seasonality in Shanghai,China. The most common genotypes of group A RV are G9 and G1.

Objective To compare and analyze the differences and characteristics of three strain mouse models in-fected by influenza virus aerosol inhalation, and provide the reference for choosing the appropriate infection model in the re-search of pathogenesis of influenza and the development of vaccines and drugs.Method C57BL/6, BALB/c and ICR mice were infected with A/Puerto Rico/8/34 (H1N1) virus strain by aerosol inhalation.The symptoms and body weight of mice were observed every day.At 3, 7, 14 days after infection, the mice were sacrificed.The lungs of mice were weighed, then virus assay and pathological observation were carried out.Results The three strains of mice were infected.The sur-vival rate in the C57BL/6 mice was lower than those in the BALB/c and ICR mice.The lung index and viral load of C57BL/6 mice were significantly higher than those of ICR mice ( P<0.05) at 3 days after infection.The pathological changes of C57BL/6 mice were also more obvious than other two strains.Compared with other two mouse strains, the weight recovery of BALB/c mice was the slowest.The survival rate in BALB/c mice was higher than that of C57BL/6 mice and lower than that of ICR mice.The lung index and viral load were not significantly different among the three strains of in-fected mice.The pathological changes among the three strains of infected mice were similar, but the degrees of pathological changes in the BALB/c mice were milder than in the C57BL/6 mice and worse than in the ICR mice.Compared with other two mouse strains, the process of disease is similar, but the body weight, mortality, lung index, viral load, and the micro-scopic pathological changes were lighter in the ICR mice than in the other two strain mice.Conclusions The three strain mouse models can be established by influenza virus aerosol inhalation, but showing different characteristics.Appropriate strain mice can be chosen to build model according to different research purpose in the experiment.

Objective To investigate the epidemiology of adults acute viral gastroenteritis in Shanghai Changning district. Methods All of 1 554 stool specimens of adults acute gastroenteritis in Shanghai Changning district from June 2010 to December 2013, and the enzyme-linked immunosorbent assay and multiple polymerase chain reaction was used to detecte different viruses. Results In all of 1 554 cases, the average age was (46.19 ± 15.59) years. Among them, 691 persons were male, 863 persons were female. Virus infection was detected in 407 cases, and the detection rate was 26.19%. Among them, 395 cases (97.05%) were single virus infection, and 12 cases (2.95%) were mixed infection. The peak of epidemic was from every November to next February. The incidence of watery diarrhea, vomiting and fever in virus positive group was significantly higher than that in virus negative group:95.09%(387/407) vs. 88.14%(1 011/1 147), 31.20%(127/407) vs. 18.83%(216/1 147), and 11.06%(45/407) vs. 7.59%(87/1147), P<0.01 or<0.05. Conclusions Rotavirus infection is common in adults with acute viral gastroenteritis. Patients with positive virus infection had a higher incidence of watery diarrhea, vomiting and fever. The peak of epidemic is winter.

OBJECTIVETo analyze virulence genes and molecular characteristics of Vibrio parahaemolyticus isolated from sporadic cases with diarrhea in tow sentinel hospitals of Shanghai, 2010-2012.

METHODSA total of 2 729 stool samples were collected from two surveillance sentinel hospitals in Shanghai 2010-2012. Vibrio parahaemolyticus strains were isolated and identified from diarrhea out patients using TCBS agar plates and biochemical reactions. Thermostable direct hemolysingene (tdh), thermostable-related hemolysin gene (trh), hemolysin gene (tlh) were detected by multiplex PCR method. Isolates were analyzed by PFGE and MLST. The PFGE profiles were analyzed using BioNumerics software.

RESULTSA total of 30 clinical Vibrio parahaemolyticus strains isolated from 2 729 stool samples. The anually Vibrio parahaemolyticus isolation rate during 2010 to 2012 were 1.1%(11/973), 1.0%(11/1 120) and 1.3%(8/636) respectively. The PCR positive rates of virulence genes tlh, tdh and trh were 100%, 97% and 0 respectively. The Vibrio parahaemolyticus strains were divided into 13 PFGE types (P1-P13)and 3 ST types (ST-189, ST-799, ST-3). Among 13 PFGE types, P4 was the main PFGE type, accounting for 30%(9/30). P9, P10 were accounting for 12% (4/30) respectively, P1, P2, P12, P13 were accounting for 7%(2/30) respectively, the others types were 3%(1/30) respectively. MLST analysis results showed there are three ST types, ST3 was 84%(25/30), ST189 and ST799 were accounting for 13% (4/30) and 3% (1/30) respectively.

CONCLUSIONThe infection rate of Vibrio parahaemolyticus was not very high from 2010-2012 in Shanghai, all strains were positive for tlh and negative for trh. ST3 was the major type of Vibrio parahaemolyticus.

China ; Diarrhea ; Genotype ; Hemolysin Proteins ; Hospitals ; Humans ; Multilocus Sequence Typing ; Outpatients ; Polymerase Chain Reaction ; Sentinel Surveillance ; Vibrio Infections ; Vibrio parahaemolyticus ; Virulence

OBJECTIVETo determine the function of twin-arginine translocation system (Tat) and gene cluster in Vibrio strains and to analyze the homology of tat gene cluster among different Vibrio spp. strains based on N16961 and tatABC mutant strains N169-dtat.

METHODSDifferent serotypes of biotype strains of Vibrio spp. were selected to detect the transcription of 4 genes of Tat transport system and upstream ubi aarF gene and downstream cyt551 gene by the total RNA reverse transcription and homologicity of the gene cluster by sequencing analysis.

RESULTSOur results showed that the 4 genes of tat cluster (tatA, tatB, tatC, and tatE) were intragenic and co-transcribed. We found that ubi aarF gene could be co-transcribed with tatA, tatB, but not with tatC. The electron transport chain and energy metabolism-related genes, cytochrome C551 peroxidase gene, and 4 genes located at upstream of tatABC operon were not transcribed with tatABC. Although the co-transcription between ubi aarF and tatAB was blocked in N169-dtat strain, they were still transcribed separately. Homologous analysis of genes of tat cluster in different types of Vibrio cholerae showed that tat gene cluster was a very conservative.

CONCLUSIONThe ubi and aarF gene might be co-transcribed with genes of tat cluster in Vibrio cholerae, which and the close relationship showed that they might play a key function in Vibrio cholerae.

Arginine ; Bacterial Proteins ; Cytochrome c Group ; Membrane Transport Proteins ; Vibrio cholerae

Objective To understand the epidemiologic feature of human parainfluenza virus type 3 (HPIV-3) in Shanghai,and to provide scientific evidence for formulating prevention and control measures in the future.Methods A total of 164 nasopharyngeal aspirates samples taken from children with acute respiratory infection (ARI) were collected from Xinhua Hospital Affiliated to Shanghai Jiaotong University and sent to Shanghai Public Health Clinical Center from June 2009 to June 2010.Samples were detected for HPIV-3 by reversed transcription-polymerase chain reaction (RT-PCR).Full-length hemagglutininneuraminidase (HN) gene (1 719 bp) of five positive samples were sequenced for phylogenetic analysis.Comparison between two groups was evaluated by the precise chi-square test (two sided).Results Of 164 samples,70 samples were infected with parainfluenza virus,and HPIV-3 was detected positive in 23 samples with the positive rate of 32.86％.HPIV-3 infections were most common in spring and summer,and most of infections were mainly found in 13-36 month-old infants.Five Shanghai isolates and 36 reference sequences from different countries and areas were divided by HN gene-based phylogenetic tree into three clusters (A,B and C).Five Shanghai isolates and five Beijing isolates belonged to C3a group.The homologies of nucleotide and amino acid sequences between five Shanghai isolates and five Beijing isolates were 99.0％-99.5％ and 99.7％-100.0％,respectively.Conclusions HPIV-3 accounts for a high proportion in children with ARI in Shanghai.C3a group may be the main lineage of HPIV-3,which suggests that HPIV-3 may be of regionally correlation.