Objective To investigate the genetic variation and molecular evolution of human bo-cavirus 1 (HBoV1) strains isolated during 2009 to 2014 in Hangzhou, China.Methods Throat swab sam-ples were collected from children with acute respiratory tract infections in the Children′s Hospital Affiliated to the Zhejiang University School of Medicine from 2009 to 2014.Real-time PCR was performed for the detec-tion of HBoV1 strains.Fifteen HBoV1 strains with high virus load were screened out for the amplification and sequencing of complete genomes.The complete genomes were submitted to GenBank for further analysis with bioinformatics software.Results A total of 48 nucleotide mutations were detected in the complete genomes of 15 HBoV1 strains, resulting in 11 amino acid mutations with 5 of them located in the active region of phospholipase A2 ( PLA2) .The 15 HBoV1 isolates along with 16 HBoV1 strains in GenBank were classified into three clusters as indicated by the phylogenetic analysis based on their complete coding sequences.All of the 15 strains were belonged to clusterⅠ, the representative strain of which was the Sweden prototype strain ST2.The phylogenetic trees constructed using genes encoding the capsid proteins VP1 and VP2 were highly similar to those based on the complete coding sequences.The estimated mean evolutionary rate of HBoV1 with regard to the complete coding sequence was 3.03×10-4(95%HPD, 2.14×10-4-3.92×10-4 ) substitu-tions per site per year.With regard to each gene, the NS1 gene was considered to the most conserved gene while the NP1 gene showed the highest substitution rate.The dN/dS ratios (ω) of the four genes were all less than 1, indicating that all of them were under negative selection.Moreover, the VP2 gene was under the strongest negative selection, while the NP1 gene was under the weakest negative selection.Conclusion All of the HBoV1 isolates circulating in Hangzhou province during 2009 to 2014 were belonged to ST2 genotype with a relatively high mutation in the area of PLA2.Despite the complete genome was conservative, its evo-lutionary rate was high.Among the four genes, the NP1 gene showed the highest substitution rate.All of the four genes were under negative selection, of which the VP2 gene was under the strongest negative selection.

Objective To detect adenovirus from children with acute upper/lower respiratory tract infections and to investigate the genetic evolution of virus .Methods A total of 1 178 clinical specimens were collected from the Children ’ s Hospital , Zhejiang University School of Medicine during March 2011 and February 2013, including 513 throat swabs from children with acute upper respiratory tract infection and 665 nasopharyngeal aspirates from children with acute lower respiratory tract infections .Besides, 9 specimens in an outbreak of adenovirus infection during 2011 and 2014 were also collected .Adenovirus was identified by real-time fluorescent polymerase chain reaction (RT-qPCR).The hypervariable region (HVR)-7 region of hexon gene in positive samples was amplified and sequenced for typing and phylogenetic analysis .Other respiratory viruses were also detected with RT-qPCR in adenovirus positive samples .Clinical characteristics of adenovirus infection were analyzed in children with lower respiratory tract infections .Chi-square test and Fisher exact probability were used for data analysis .Results Among 1 178 samples from sporadic cases , 104 samples (8.83%) were adenovirus positive .The rates of adenovirus infection in upper respiratory tract infection group and lower respiratory tract infection group were 13.65%(70/513) and 5.11%(34/665), respectively (χ2 =26.193, P<0.05).Compared with that in lower respiratory tract infections , positive rates of adenovirus were higher in upper respiratory tract infections in children aged 0-1 year and >3 years (χ2 =6.575 and 7.334, P<0.05 or <0.01).Adenovirus infection might occur throughout the year and peaked in spring and summer .Among 54 adenovirus-positive samples from 104 sporadic cases , adenovirus types 1, 2, 3, 4, 7 and 31 were identified in 4, 6, 26, 2, 15 cases and 1 case, respectively.While among 9 adenovirus-positive samples from outbreak cases , type 4 and type 3 were identified in 1 and 8 cases, respectively .The HVR-7 region of hexon gene was highly homologous in the same type , and the sequence alignment indicated that the sequence of HVR-7 might have regional differences .Nine out of 70 children (12.86%) were co-infected with other virus in upper respiratory tract infection group , while the rate of co-infection in lower respiratory tract infection group was much higher [58.82%(20/34), χ2 =24.045, P<0.05 ].There was no significant difference in clinical manifestations between children infected with adenovirus only and those with co-infection in lower respiratory tract infection group (P>0.05), but two children with co-infection died.Conclusions Adenovirus infection is more common in upper respiratory tract infection .Adenovirus type 3 and type 7 are the most prevalent serotypes in sporadic cases , while type 3 is the most prevalent serotype in outbreak cases .

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We investigated the genetic diversity and evolution of the M gene of human influenza A viruses in Hangzhou (Zhejiang province, China) from 2009 to 2013, including subtypes of A(H1N1) pdm09 strains and seasonal A(H3N2) strains. Subtypes of analyzed viruses were identified by cell culture and real-time reverse transcription-polymerase chain reaction, followed by cloning, sequencing and phylogenetic analyses of the M gene. Assessment of 5675 throat swabs revealed a positive rate for the influenza virus of 20.46%, and 827 cases were diagnosed as. infections due to influenza A viruses. Seventy-six influenza-A strains were selected randomly from nine stages during six phases of a virus epidemic. Sequences of the M gene showed high homology among six epidemics with identities of amino-acid sequences of 98.98-100%. All strains contained the adamantine-resistant mutation S31N in its M2 protein. Two of the A(H1N1)pdm09 strains had double mutants of V27A/S31N or V271/S31N. One of the seasonal A(H3N2) viruses had another form of double-mutant R45H/S31N. Evolutionary rate of the M gene was much lower than that of the HA gene and NA gene. Compared with A(H3N2) strains, higher positive pressure on the M1 and M2 proteins of A(H1N1) pdm09 viruses was observed. Separate analyses of M1 and M2 proteins revealed very different selection pressures. Knowledge of the genetic diversity and evolution of the M gene of human influenza-A viruses will be valuable for the control and prevention of diseases.
Amino Acid Substitution ; China ; epidemiology ; Evolution, Molecular ; Genetic Variation ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Selection, Genetic ; Viral Matrix Proteins ; genetics ; Viral Proteins ; chemistry ; genetics

Objective To investigate the epidemiological characters of human metapneumovirus (hMPV) infection in children with influenza-like illness (ILI).Methods A total of 1 164 throat swabs were collected from children with ILI symptoms in Children's Hospital,Zhejiang University School of Medicine from January 2011 to December 2012.hMPV was detected by using nucleic acid assay,the fusion (F) protein gene of hMPV was amplified by RT-PCR,gene sequencing was performed and the sequences were compared with those in GenBank.Positive rates of hMPV in different age groups were compared with Chi-square test.Results Among 1 164 samples,hMPV was positive in 73 (6.27％) samples.hMPV infection was the most popular in ＞2-4 y age group (33/220,15.0％),and the positive rates of hMPV in different age groups were of statistical significance (x2 =40.69,P ＜ 0.05).hMPV infection occurred throughout the year,but it was most common in winter and spring.The highest incidence of hMPV infection was observed in December 2012 (12/51,25.53％).Among 24 samples of hMPV,14 were with genotype B1,2 were with genotype B2,and 8 were with genotype A2.The most common genotype was B1 in 2011 (10/12),and A2 in 2012 (8/12).Homology between nucleotide sequences of the 24 samples of hMPV were 81.6％ to 100.0％.Conclusions hMPV infection exists in children with ILI in Hangzhou,and the epidemic seasons are winter and spring.hMPV infection is more likely to be found in children aged 2 to 4 years old,and different genotypes may predominated alternately.

Objective To explore the establishment of skin photoaging model and the protective effects of nitroxide tempol on skin in guinea pig. Methods The guinea pig skin photoageing model was established by using solar-simulaten radiation (SSR). Dermal structure was observed with hematoxylin-eosin staining. The structure and expression of elastic fiber were analyzed by Weigert's staining. The uhrastructure of dermal fibroblasts and elastic fiber were observed by electron microscopy. Tempol was used before each exposure at the concentration of 5 mg/ml or 0. 5 mg/ml, and the protective effects of tempol on skin were assessed. Results After seventeen weeks' exposure, there was typical "solar elastosis" damage in the upper dermis. Mature elastic fibers were severely degraded and there was large amount of elastotic material accumulated in the upper dermis. Dermal fibroblasts appeared metabolically hyperactive and mitochondria in the cells were damaged. Some cells even broke up. Tempol at the concentration of 5 mg/ml or 0. 5 mg/ml could prevent photodamage of the photoageing model in the dermis, and Tempol at the concentration of 5 mg/ml had stronger protective effects. Conclusions Guinea pig can be applied as an useful animal model of skin photoageing. Antioxidant tempol has photoprotective effects on photodamage of the photoageing model in guinea pig and can be used as an anti-photoageing agent.

Objective:To analyze the epidemiological and etiological characteristics of dengue fever in Hangzhou in 2018.Methods:RT-PCR was used to detect the nucleic acids and analyze the serotypes of dengue viruses (DENV) in serum samples collected from dengue fever cases. Phylogenetic trees based on the E gene sequences of DENV isolated from the serum samples were then constructed and analyzed. Epidemiological characteristics of these dengue fever cases were analyzed. Results:A total of 80 cases of dengue fever were detected in Hangzhou in 2018 with 55 imported cases and 25 indigenous cases (24 caused by DENV-1 and one by DENV-3). These indigenous cases mainly occurred during late July to early October with people above 50 years old accounting for 68%. Phylogenetic analysis showed that DENV-1 strains isolated from the indigenous cases in Yuhang, Jianggan-Shangcheng and Qiantang districts all belonged to genotype Ⅰ, and were respectively closely related to the strains from Indonesia in 2015, Myanmar in 2017, Ningbo in 2018 and Hangzhou imported cases from Thailand in 2018. The indigenous DENV-3 strain belonged to genotype Ⅲ, and shared 99.5% homology with the Singapore strain in 2013.Conclusions:Imported cases accounted for a large fraction of the dengue fever cases in Hangzhou, which brought a high risk to indigenous outbreak. Due to multiple imported cases, the current epidemic presented a characteristic of multiple small-scale outbreaks.

Objective To study the epidemiological characteristics and genetic evolution of human bocavirus ( HBoV ) infection in hospitalized children with severe acute respiratory infection ( SARI ) in Hangzhou.Methods A total of 1388 clinical specimens were collected from children with SARI admitted in Affiliated Children' s Hospital, Zhejiang University School of Medicine from January 2011 to December 2014.HBoV1-4 and other respiratory pathogens were identified by fluorescent real -time polymerase chain reaction (fRT-PCR).The VP1 gene in HBoV1 positive samples was amplified and sequenced for genetic analysis with Clustal X and MEGA 6.0.Chi-square test and Fisher exact probability were used to analyze the data.Results Eighty five HBoV positive samples were detected from 1388 samples (6.12％), among which 83 (97.65％) were HBoV1 positive and 2 (2.35％) were HBoV2 positive.The positive rates of HBoV in males and females were 6.54％and 5.35％(χ2 =0.780, P>0.05).The posititve detection rate of HBoV in all age groups was statistically significant (χ2 =47.446,P <0.01).The detection rate in children aged 6 months-1 year was highest (12.84％), in children aged >3 years was lowest (1.64％), in children aged ≤6 months and aged 1-3 years was 3.04％ and 3.33％, respectively.The detection rate of HBoV in summer was the highest (14.97％), followed by that in autumn (7.14％), spring (3.19％) and winter (1.97％) (χ2 =58.807, P<0.01).The detection rates of HBoV in 2011 to 2014 were 7.39％, 7.31％, 5.58％ and 4.72％ (χ2 =3.447, P >0.05 ).The co-infection rate with other respiratory pathogens was 62.35％.The main pathogens were human rhinovirus (33.96％), parainfluenza virus (28.30％) and respiratory syncytial virus (20.75％).The incidence of anhelation and wheezing in HBoV positive group was higher than that in HBoV negative group (χ2 =15.161 and 13.914, P <0.01). Sequence analysis of VP 1 gene showed that 44 isolates belonged to the same branch ( clade 1 ) as Swedish strain ST2, and 2 isolates HZ12-S32 and HZ12-S199 belonged to a separated branch.Conclusion HBoV is an important causative agent of hospitalized children with SARI in Hangzhou area and has high co -infection with other respiratory pathogens.Most of the strains belong to the same clade as the Swedish strain ST 2, and two strains of HZ12-S32 and HZ12-S199 are identified in a separated clade.

Objective:To investigate the characteristics of clinical bacteria and H3N2 influenza A virus coinfection and variations in the hemagglutinin (HA) cleavage site of H3N2 among influenza-like cases.Methods:A total of 12 250 samples were collected from influenza-like cases for real-time PCR detection of H3N2 influenza virus from January 2013 to December 2018. To analyze the characteristics of co-infection, some H3N2-positive samples were selected and analyzed by real-time PCR to detect Staphylococcus aureus, Streptococcus pneumoniae and haemophilus influenzae type B. HA genes of H3N2 isolates were amplified by RT-PCR and sequenced. A phylogenetic tree was constructed based on HA gene sequences. Amino acid variations in cleavage sites were analyzed. Results:H3N2 influenza viruses had been detected every year since 2013, causing 44.69% influenza-positive cases. There were 295 randomly selected H3N2-positive samples, of which 29.2% had clinical bacterial infection. The HA cleavage sites of 210 H3N2 isolates were sequenced and 68 had variations, including 63 carrying K342R (PEKQTR to PERQTR) single-amino acid site variation. The co-infection rate was 31.25% (45/144) in unmutated samples and 23.53% (16/68) in mutated samples (χ 2=1.34, P>0.05). The H3N2 influenza viruses circulating in Hangzhou mainly belonged to two evolutionary clusters of 3c.3a and 3c.2a, and the viruses with K342R mutation at the cleavage site belonged to the evolutionary cluster of 3c.3a. Conclusions:H3N2 influenza virus played an important role in the epidemic of influenza virus in Hangzhou. There were some bacterial co-infections in influenza-positive cases. Cleavage site variations showed regional epidemic characteristics, but had no significant correlation with bacterial co-infection.

Objective: To determine the epidemic characteristics of respiratory viruses, mycoplasma pneumonia(MP) and chlamydia pneumoniae(CP) in outpatients and hospitalized children with acute respiratory tract infections(ARI), to lay a foundation for the prevention and control of ARI. Methods: From 2011 to 2013, children with ARI, including outpatients and inpatients, were involved in this study. One nasopharyngeal aspirate or throat swab specimen was collected from each patient.Real time PCRs were performed to detect common respiratory tract viruses, MP and CP. Results: At least one pathogen was identified in each of 610 out of 908 patients and the overall positive rate was 67.2%. The positive rate in inpatient(76.7%)was higher than that in outpatient(43.0%) (χ²=94.79, P<0.001). Simultaneous detection of two or more viruses was found in 206 cases.Co-infection was more frequent in inpatients than in outpatients(29.0% VS 6.6%, P<0.001). Significant differences of the detection rate were observed in RSV, PIV, HRV, Flu, human bocavirus (hBoV), adenovirus (AdV), saffold virus(SAFV), MP and CP between the inpatient and outpatient group. Respiratory syncytial virus(RSV)(34.5%) was the most prevalent virus detected among hospitalized children, followed by MP(15.0%)and human rhinovirus(HRV)(14.6%). Whereas adenovirus(AdV) (15.2%)was the most frequently identified virus in the outpatient group, followed by influenza virus(Flu)(11.7%))and PIV(7.8%). Except for RSV and Flu, co-infection of the other pathogens was more frequent than its mono-infection in inpatients. Significant differences of the AdV co-infection rate were observed between the inpatient and outpatient group(χ²=18.90, P<0.001). Compared with mono-infection, co-infection has no significant effect on the clinical presentation. Conclusions The detection rate of respiratory pathogens was higher in inpatients than in outpatients with ARI, and co-infections were more popular in children hospitalized, it may show that co-infection had some correlation with disease severity.