Objective To obtain the molecular epidemiology of human Parechovirus (HPeV)infections m children with central nervous system (CNS)-related disease and sepsis,as well as understand the pathogenic properties of HPeV infections by detecting HPeV in cerebrospinal fluid (CSF) and blood samples.Methods From January to December in the year of 2009,a total of 359enterovirus-negative specimens including 210 CSF and 149 blood samples were collected from 328children ＜14 years of age who were hospitalized for CNS-related disease and sepsis at Children's Hospital,Fudan University,Shanghai,China.HPeV was detected by nested reverse transcription polymerase chain reaction (RT-PCR),and then directly genotyped by sequencing nested RT-PCR product of VP3/VP1 region.Ninty-nine blood samples from healthy children were collected as controls during the same period.Results Twenty-seven children (8.2％) were HPeV positive in 328 children.HPeV infections were found in all age groups of children and the highest frequency was seen in children ＜3 months old (18.2％,12/66).HPeV was detected in several months,with the peak in December (18.8％,9/48).Of all the positive samples,20 were genotyped successfully and identified to be HPeV1.No HPeV infections were found in blood of healthy controls.ConclusionsHPeV is the pathogen of CNS infections and sepsis in children.HPeV screening should be enrolled in the routine virus testing in specimens obtained from children.HPeV1 is the prevalent type in children in the year of 2009 in Shanghai.

Objective To investigate the clinical and molecular epidemiologic features of human rhinovirus (HRV) in children with acute lower respiratory tract infection (ALRTI) in Shanghai by detecting HRV from bronchoalveolar lavage fluid (BALF) .Methods One hundred and thirty-five BALF samples were collected from children with ALRTI at Children′s Hospital of Fudan University from January to June in 2014 . The specimens were tested by one-step real-time reversible transcription (RT )-polymerase chain reaction (PCR) for HRV .Gene fragments of VP4/VP2 capsid protein of the HRV genome were amplified and sequenced .The phylogenetic trees of HRV strains were drawn .Chi-square test was used for statistical analysis .Result Among these 135 samples ,6(4 .4% ) were HRV positive , including 4 positive for HRV-A and 2 positive for unknown genotype .The age of children with HRV positive was 8 months to 8 years ,including 5 cases ≤ 3 years old .The nucleotide homology within 4 HRV-A samples varied 76 .5% -100 .0% .These 4 HRV-A sequences were respectively compared with reference strains of HRV-A ,HRV-B and HRV-C in GenBank .The nucleotide homology varied 73 .1% -91 .7% ,57 .8% -66 .0% and 59 .0% -65 .0% ,respectively .Among 135 children with ALRTI ,124 were diagnosed with ordinary pneumonia including 4 (3 .2% ,4/124) HRV positive and 11 were diagnosed with severe pneumonia (with respiratory failure and/or heart failure) including 2 HRV positive .All children with HRV presented with fever and cough .Most of them (5/6) were co-infected with other pathogens .In 5 children with HRV positive ,the peripheral blood leukocyte counts were less than 10 × 109/L , neutrophil percentages were less than 0 .50 ,C-reactive protein was less than 8 mg/L and procalcitonin was less than 0 .05 μg/L .All the children were clinically cured .Conclusions The detection of HRV in BALF samples indicates that HRV is one of the pathogens in children with ALRTI in Shanghai and HRV-A is more common .Gene fragments of VP4/VP2 capsid protein of HRV pathogenic strains display a high degree of genetic variability .

Objective To compare the molecular epidemic characteristics of human astrovirus (HAstV) between outpatient and hospitalized children with acute diarrhea,and to investigate the relationship between HAstY infection and diarrhea in children.Methods A total of 298 cases were randomly collected from hospitalized children from January 2008 to December 2010 in Children's Hospital of Fudan University,and 360 specimens were collected from outpatients with acute diarrhea from August 2010 to July 2011.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect rotavirus (RV),human calicivirus (HuCV),HAstV and human adenovirus (HAdV).H AstV genotype was determined by gene sequencing and phylogenetic analysis.Results Epidemiology of HAstV in hospitalized children was as follows:among the included 298 samples,HAstV was detected in 27.2％ (81/298) of the patients,compared with 33.9％ (42/124),33.8％ (25/74) and 14.0％ (14/100),respectively from 2008 to 2010.HAstV diarrhea occurred throughout the year and peaked in January,March,and April.95.1％ (77/81) of the infected children were 0-35 months old.All the episodes of HAstV were mixed with other diarrhea virus infection.Molecular epidemiology of HAstV in outpatient children with diarrhea was as follows:the overall incidence of HAstV was 1.9 ％ among the 360 cases (7/360).The seasonal distribution of HAstV's gastroenteritis showed a peak in November.All the outpatient children were 0-35 months old.Three cases were single infection with HAstV and the others were coinfection with RV (3 cases) or HAdV (1 case).All of the detected HAstV,either in inpatients or outpatients,belonged to HAstV-1.Conclusions The detection rate of HAstV in hospitalized children is significantly higher than that in outpatients.Most HAstV infections in hospitalized children are ascribed to nosocomial infections.Most episodes of HAstV infection were accompanied with other diarrhea viruses infection.HAstY single infection is seen in outpatient children while the detection rate is very low,implying that HAstV co-infection with other viruses plays a main role in diarrhea in most instances.

Objective To investigate the dynamic characteristic of molecular epidemiology of group A Rotavirus (RV) by analyzing viral genotypes,disease seasonality,and the patients' age distribution,so that to provide theoretical basis for preyention and control of RV diarrhea in children.MethodsA total of 380 RV antigen positive samples were selected from 5176 stool specimens collected from ＜5 year-old patients with acute diarrhea who were admitted to Children's Hospital of Fudan University during January 2006 to December 2008. Multiplex nested reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the RV genotypes.ResultsDuring 2006-2008,the incidence of RV related diarrhea peaked from October to December and about 96.8％ of all RV episodes occurred in patients younger than 3 years old,The predominant genotype was G3 which accounted for 58.4％ (222/380),G9 was an emerging genotype with the prevalence rate as high as 10.8％ (41/380).G1 and G2 types were rarely found during the three years.Infections with both G3 and G9 were the major mixed genotype G infection. Genotype P [8] was predominant with the prevalence rates of 64.6％ (53/82) and 46.8％ (58/124) in 2006 and 2008,respectively,whereasgenotype P[4] was predominant in 2007 (38,5％,67/174).P[6] and P[9] were found as minor types.The major mixed genotype P infection were genotype P[4] and P[8]. The proportion of undetermined genotype G and genotype P strains tended to increasing during 2006-2008.Genotype P [8]G3 was the major RV strain (20.5％) in Shanghai during 2006-2008 and the other prevalent genotypes included P[4]G3 and P[m]G3.Conclusion The infection of group A RV in Shanghai presents some new molecular epidemiology characteristics during 2006-2008,such as switch of predominant genotypes and diversification of prevalent genotypes.

Objective To clear, amplify and detect the activity of the recombinant adeno-assoeiated virus vector with adiponectin( rAAV2/1-Aerp30 ). Methods Recombinant plasmid pSNAV2.0-Acrp30 was obtained. The recombinant plasmid was then transfected into BHK21 cells using LipofectAMINETM 2000. The G418 resistant cells were obtained consequently. These cells were infected with HSVI-rc/△UL2 which has the function of packaging and copying recombinant AAV. After purification, the construction of recombinant rAAV2/1-Aerp30 was collected. Results The construction of recombinant pSNAV2.0-Acrp30 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequencing showed that the recombinant pSNAV2.0-Acrp30 was correct. The virus titer was about 1.0×1012 μg/ml. The purity f the recombinant AAV2/1 was fairly high using the SDS-PAGE method. Conclusion With this method, rAAV2/-Aerp30 with high virus titers and purity can be acquired successfully and it can meet the demands of the experimental study of Acrp30 gene therapy of GK rats.

Objective To study the clinical features of neonates with enteroviral infection.Method We collected all neonates with enteroviral infection confirmed pathologically in the Department of Neonatology in the Children's Hospital of Fudan University from March 2009 to July 2016 retrospectively.The clinical data of the patients (clinical manifestation,first laboratory examinations,imaging findings,treatment procedures and outcomes) were retrieved to summarize the clinical features of neonatal enterovirus infection and to analyze the factors affecting the prognosis of neonatal enterovirus infection.Result A total of 81 neonates with enterovirus infection were included in the study.Among them,55 were full-term infants and 26 were premature infants,and the birth weight was (3 029±728)g.The median age of hospital admission was 5 (1,16) d,and multiple systemic manifestations were common in the course of the disease.Among them,19.8％(16/81)died.The factors for death are preterm birth,decreased hemoglobin,platelet count or serum albumin levels,abnormal coagulation,elevated blood lactate levels and acidosis.A scoring system was established based on high-risk factors.A score of ≥ 4 could reliably predict adverse outcomes,with a specificity of 92.3％ and a sensitivity of 93.8％.Conclusion Enteroviral infection in neonates can be highlt critical multiple system involvement.Neonates enterovirus infections with high risk factors(premature delivery,hemoglobin/ platelet/ albumin hypoplasia,coagulation dysfunction,lactic acid increase and acidosis) had higher mortality rate.There is a need to identify these neonates for intensive care and better treatment is warranted to improve the prognosis of these patients early.

Objective:To investigate the epidemiological characteristics of classic human astrovirus (HAstV) among children under five years old with acute diarrhea, and to understand the role of HAstV in children acute diarrhea.Methods:A total of 1 010 fecal specimens were collected in 1 010 outpatients under five years old with acute diarrhea admitted to Children′s Hospital of Fudan University, Shanghai from January 2012 to December 2016. Reverse transcription polymerase chain reaction (PCR) or PCR was used for screening classic HAstV, group A rotavirus, norovirus and adenovirus. Genotypes of classic HAstV were determined by nucleotide sequencing and phylogenetic tree analysis.Results:The overall positive rate of classic HAstV was 2.7%(27/1 010). The detection rates of classic HAstV from 2012 to 2016 were 6.9%(10/144), 3.5%(5/144), 2.1%(3/144), 1.5%(4/265) and 1.6%(5/313), respectively. Almost 96.3%(26/27) of children infected with HAstV were 0 to 36 months of age. The prevalence of classic HAstV infections displayed a typical autumn/winter seasonality except in 2016. All the positive classic HAstV strains were genotyped as HAstV-1 with two lineages of HAstV-1a and HAstV-1b. Among them, the lineage of HAstV-1a was the predominant subtype (63.0%, 17/27). There were 77.8%(21/27) of the children with acute diarrhea only infected with classic HAstV, whereas for the remaining cases a variety of other enteric viruses were detected (three cases co-infected with HAstV and group A rotavirus, two cases co-infected with HAstV and adenovirus, and one case co-infected with HAstV, group A rotavirus and adenovirus).Conclusions:Children infected with HAstV are mainly less than 36 months of age. Although the genotype of classic HAstV detected in this study is single, but the lineages are in a state of dynamic change. Long-time and continuous monitor for the epidemiology of classic HAstV is needed to avoid outbreak of diarrhea in children.

OBJECTIVE：To systematically evaluate therapeutic efficacy of Gongliuqing capsules combined with mifepristone in the treatment of uterine leiomyoma ，in order to provide evidence-based reference for clinical medication. METHODS ：Retrieved from Cochrane Library ，PubMed，Embase，CJFD，VIP，CBM and Wanfang database ，randomized controlled trials （RCTs）about Gongliuqing capsules combined with mifepristone （trial group ）versus mifepristone alone （control group ）in the treatment of uterine leiomyoma were collected. After literature screening and data extraction ，the quality of included literatures was evaluated with modified Jadad scale. Meta-analysis was conducted by using Stata 14.0 software，and trial sequential analysis （TSA）was performed by using TSA 0.9 software. RESULTS ：A total of 12 RCTs were included ，involving 1 210 patients. The results of Meta- analysis showed that the total response rate of trial group [RR ＝1.12，95％CI（1.00，1.26），P＜0.05] was significantly higher than that of control group ；maximum uterine leiomyoma volume after treatment [SMD ＝－1.08，95％CI（－1.21，－0.95），P＜0.05]，uterine volume after treatment [SMD ＝－0.80，95％CI（－1.14，－0.45）， P＜0.05]，follicle stimulating hormone （FSH）level [SMD ＝ － 0.28，95％ CI（－ 0.45，－ 0.19），P＜0.05]，luteinizing hormone（LH）level [SMD ＝－0.44，95％CI（－0.52，－0.12）， 020-38076311。E-mail：867203217@qq.com P＜0.05]，E2 level [SMD ＝－2.69，95％CI（－3.08，－1.49）， P＜0.05] and progesterone （P）level [SMD ＝－1.27，95％CI（－1.69，－0.71），P＜0.05] of trial group were significantly lower or better than those of control group. Results of subgroup analysis showed that except for the level of FSH in 5 and 10 mg mifepristone groups （P＞0.05），maximum uterine leiomyoma volume after treatment ，uterine volume after treatment ，the levels of FSH，LH，E2 and P in trial group were significantly lower than control group. The results of TSA showed that there were definite evidences for total response rate of Gongliuqing capsules combined with mifepristone being better in the treatment of hysteromyoma. CONCLUSIONS ：Total response rate of Gongliuqing capsules combined with mifepristone in the treatment of hysteromyoma is better than mifepristone alone ，which can effectively decrease the volume of maximum uterine leiomyoma volume and uterine vilume ，and reduce the level of serum hormone.