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Objective: To investigate the infection status and epidemiological characteristics of influenza virus and respiratory syncytial virus (RSV) in influenza-like illness (ILI) of children ( ≤ 14 years) in Wuhan area from 2008 to 2012., Methods: A total of 2854 cases of ILI patients ( ≤ 14 years) in a hospital of Wuhan were recruited in the study from July 2008 to June 2012. The sample of pharyngeal swab was collected from each patient, to extract the virus nucleic acids. Real-time fluorescent quantitation reverse transcription PCR (RT-PCR) method was applied to detect the subtypes of influenza virus and RSV, and then analyzed the time and age characteristics., Results: Out of the 2854 cases, 758 (26.6%) were positive for influenza virus,including 547 (19.2%) influenza A virus positive samples and 211 (7.4%) influenza B virus positive samples. Usually, there were two peaks present in the annual curve of influenza virus, namely summer peak and winter/spring peak. The positive rate of influenza virus in 6-14 years old children (48.0%, 275/573) was significantly higher than that in 3-5 years old children (26.6%, 213/801) and that under 3 years old children (18.3%, 270/1480). The difference showed statistical significance (χ(2) = 187.432, P < 0.01). A total of 219 (7.7%) cases were positive for RSV,including 108 RSV-A positive samples and 112 RSV-B positive samples (1 co-infection). The epidemic of RSV showed an obvious seasonal pattern with peaks in autumn,winter and spring,which accounted for 96.8% (212/219) of all the cases; however, the annual incidence of RSV fluctuated greatly. The predominant subtype shifted every 2 years. RSV-B predominated during September 2008 and May 2009, December 2009 and March 2010, accounting for 76.6% (36/47) and 96.9% (62/64) respectively. RSV-A predominated during November 2010 and March 2011, September 2011 and April 2012, accounting for 92.5% (37/40) and 100.0% (48/48) respectively. With the increase of the age, the positive rate of RSV-A and RSV-B decreased gradually (RSV-A: χ(2) = 36.223, P < 0.01; RSV-B: χ(2) = 36.281, P < 0.01). The positive rates of RSV-A in children < 1,1,2,3,4,5-9 and 10-14 years old were 7.0% (26/373), 5.9% (39/662), 4.0% (18/445), 3.2% (13/406), 1.3% (3/236), 1.4% (7/517) and 0.9% (2/215) respectively; while, the positive rates of RSV-B in each age group were 6.4% (24/373), 6.0% (40/662), 4.5% (20/445), 4.4% (18/406), 1.3% (3/236), 1.0% (5/517) and 0.9% (2/215) respectively. The children aged 0-3 years old were more susceptible for RSV infection,accounting for 90.0% (197/219) of the total positive samples. During the outbreak of influenza A H1N1 in November 2009, the positive rate of RSW was 3.0% (3/100), lower than that in the same month of 2008, 2010 and 2011,which were separately 18.2% (6/33), 10.8% (10/93) and 10.0% (4/40). The difference showed statistical significance (χ(2) = 8.450, P < 0.05). During the outbreak of influenza A (H1N1) in January 2011,the positive rate of RSV was 5.7% (3/53), lower than those in the same month of 2009, 2010 and 2012, which was separately 21.7% (5/23), 28.6% (22/77) and 16.0% (8/50). The difference showed statistical significance (χ(2) = 11.233,P < 0.05). During the period of less influenza happened in September 2011, the RSV positive rate was 25.0% (10/40), higher than those in the same month of 2008, 2009 and 2010, which was separately 11.4% (4/35), 1.7% (2/118) and 0.0% (0/109). The difference showed statistical significance (χ(2) = 32.521, P < 0.01)., Conclusion: Both influenza virus and RSV were important etiological agents of ILI of children in Wuhan. The characteristics of seasonal and age distributions of the two viruses were notably different; meanwhile, a certain inhibitional effect of influenza virus on RSV could be observed.

Objective: To survey the distribution of influenza A subtypes in external environment and investigate the infectious status of highly pathogenic avian influenza (H5N1) in poultry-exposed population in Wuhan., Methods: Seventy-eight external environmental samples (water, cage surface and fecal samples) were collected from 3 habitats of wild migratory birds and 5 urban live-poultry markets in 2010. In 13 avian influenza monitoring points, 249 serum samples were collected from people living around habitats of wild migratory birds or working in live poultry markets. Real-time RT-PCR method was adopted to detect influenza A virus from external environmental samples; and multiple RT-PCR method and specific H3, H5, H7 and H9 primers were then applied to analyze the subtypes of the positive samples. The levels of H5N1 antibody in poultry-exposed population were tested by horse hemagglutination inhibition test and two avian influenza inactivated antigens: A/Hubei/1/10 and A/Anhui/1/05., Results: Of the 50 external environmental samples collected from live poultry markets, 17 samples were determined to be influenza A virus positive (positive rate 34.0%), including specific subtypes as follows: 4 samples of H5 single-positive subtype, 3 samples of H9 single-positive subtype, 4 samples of H3 and H5 mixed-positive subtype, 2 samples of H3 and H9 mixed-positive subtype, 2 samples of H5 and H9 mixed-positive subtype, 2 samples of H3, H5 and H9 mixed-positive subtype, but no H7 positive subtype was found. The 28 external environmental samples collected from habitats of wild migratory birds were all influenza A virus negative. Considering different types of external environmental samples, the influenza A virus positive rates in water, cage surface and fecal samples were 37.5% (6/16), 16.7% (5/30) and 18.8% (6/32), respectively. There were total 100 samples of serum whose A/Hubei/1/10 antigen inhibiting titers ≥ 40, accounting for 40.2%; while 36 samples of serum (14.5%) whose A/Anhui/1/05 antigen inhibiting titers ≥ 40 were found. The difference had statistical significance (χ(2) = 41.433, P < 0.05). Among the 249 serum samples collected from poultry-exposed population, 5 samples were H5N1 antibody positive against A/Hubei/1/10 antigen (inhibition titer ≥ 160), which came from 4 different live poultry markets, however, no positive serum sample against A/Anhui/1/05 antigen was found., Conclusion: Multiple subtypes of avian influenza virus simultaneously prevailed in Wuhan urban poultry markets. Moreover, results from the distribution of avian influenza virus in external environment were consistent with the level of H5N1 antibody in poultry-exposed population.

Objective: To analyze the molecular characteristics of the newly isolated two Japanese encephalitis virus strains (JEV) in Wuhan., Methods: The mosquitoes were collected in Wuhan from April to October in 2009. The envelope (E) protein gene of JEV was detected using RT-PCR and sequenced. Sequence comparisons and phylogenetic analysis were conducted using DNAstar and MegAlign., Results: Two Japanese encephalitis virus (JEV) strains (WHJX09-9, WHJX09-10) were isolated from Culex tritaeniorhynchus among 16 mosquito pools and identified as genotype I. The result showed that the homology of the two strains was 98. 9% in nucleotides and 100% in deduced amines. The comparison between the new genotype 1 JEV strains and live attenuated vaccine strain SA14-14-2 in E gene showed that the homology of nucleotide sequence was 87.4% and 87.9%, the homology of amino acid was 96.9% (total 15 amino acid were different) in E gene. The mutation sites of amino acid distributed among three different coding domain, but no antigen binding site and neurotoxin-involved site of amino acid were changed., Conclusion: Wuhan had appeared a new genotype of JEV which was different from the former strain isolated in Wuhan, the new JEV strains still had neurotoxicity but had high homology with the vaccine strains adopted in Wuhan. The vaccine could still be adopted to prevent Japanese encephalitis if steps were take to eradicate mosquitos at the same time. laboratory surveillance were also an important task to build an early-warning mechanism against JEV.

Background: From April to July in 2009 and 2010, unexplained severe hemorrhagic fever-like illnesses occurred in farmers from the Huaiyangshan mountains range., Methods: Clinical specimens (blood, urine, feces, and throat swabs) from suspected patients were obtained and stored. Mosquitoes and ticks in affected regions were collected. Virus was isolated from 2 patients and characterized by whole genome sequencing. Virus detection in additional patients and arthropods was done by virus-specific reverse transcription (RT) PCR. Clinical and epidemiological data of RT-PCR confirmed patients were analyzed., Results: An unknown virus was isolated from blood of two patients and from Haemaphysalis ticks collected from dogs. Whole genome sequence analysis identified the virus as a novel member of the family Bunyaviridae, most closely related to the viruses of the genus Phlebovirus within which it forms a separate lineage. Subsequently, infection was confirmed by RT-PCR in 33 of 58 suspected patients. The illness in these patients was characterized by fever, severe malaise, nausea, vomiting, and diarrhea. Prominent laboratory findings included low white cell- and platelet counts, coagulation disturbances, and elevation of liver enzymes. Hemorrhagic complications were observed in 3 cases, 5 (15%) patients died., Conclusions: A novel tick-borne Bunyavirus causing life-threatening hemorrhagic fever in humans has emerged in the Huaiyangshan mountain areas of China. Further studies are needed to determine the epidemiology, geographic distribution and vertebrate animal ecology of this virus.

Objective: To study the prevalence of hepatitis C virus (HCV) infection and characteristics on molecular biology related to HCV among patients who were enrolled in a Methadone maintenance clinic in Wuhan., Methods: Serum samples from 332 injection drug users (IDUs) were obtained and anti-HCV IgG was detected by enzyme linked immunosorbrent assay(ELISA), together with 86 anti-HCV positive specimens genotyped. A reverse transcriptase-polymerase chain reaction (RT-nPCR) assay using conserved primers deduced from the core-envelopel (C-E1) region of the HCV genome was employed to amplify a 474 bp fragment. Phylogenetic analysis of the C-E1 sequences was conducted by direct sequencing of the RT-nPCR products and alignment with determined by nucleotide sequencing followed by composition of a phylogenetic tree., Results: There were 313 cases (94.3%) appeared positive anti-HCV IgG in the 332 patients from a Methadone maintenance clinic in Wuhan. It was demonstrated that there were four different subtypes of HCV in that clinic in Wuhan, including 6a--71 cases (82.5%), 3b--7 cases (8.2%), 1a--5 cases (5.8%) and 1b--3 cases (3.5%)., Conclusion: Infection of 6a genotype HCV was predominant in patients from the Methadone maintenance clinic in Wuhan, followed by HCV 3b, 1a and 1b.

Heroin addicts are a high-risk group for hepatitis C virus (HCV) infection and the development of chronic HCV disease. We thus examined whether morphine, the active metabolite of heroin, has the ability to inhibit intrahepatic interferon (IFN)- alpha expression, facilitating HCV replication in human hepatocytes. Morphine inhibited intrahepatic IFN- alpha expression, which was associated with an increase in HCV replication in hepatocytes. Moreover, morphine compromised the anti-HCV effect of recombinant IFN- alpha . Investigation of the mechanism responsible for the morphine action revealed that morphine inhibited expression of IFN regulatory factor 5 in the hepatocytes. In addition, morphine suppressed the expression of p38, an important signal-transducing molecule involved in IFN- alpha -mediated anti-HCV activity. These findings indicate that morphine plays a cofactor role in facilitating HCV persistence in human hepatocytes.

Objective: To study influenza epidemic and analyze antigenic and genetic characterization of the predominant strains in Wuhan area in 2003., Methods: Epidemiological data and specimens from influenza patients were collected from surveillance sites weekly. Viruses were isolated from the specimens. Three H3 isolates were chosen to do antigenic analysis by hemagglutination inhibition (HI) test and their HA1 region was sequenced., Results: Totally 58 influenza viruses were isolated from 418 specimens, 57 of them were identified as H3 subtype and 1 of them was B subtype; both monthly positive rate and numbers of influenza like illness had two peaks of winter and summer, the highest peak appeared in July. The 3 new H3 isolates were antigenically different from vaccine strain A/Panama/2007/99, 14 amino acid changes have been found in HA1 domain of these 3 strains compared with A/Panama/2007/99, phylogenetic analysis also confirmed the difference in HA1 domain., Conclusions: Influenza epidemic had two peaks in Wuhan area in 2003. The activity of H3 virus was strengthened remarkably. And they are antigenically and genetically different from the vaccine strain.