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Objective: To investigate the prevalence of viral pathogen in children with severe pneumonia in Hunan., Method: Bronchoalveolar lavage fluid [BALF] were collected from 122 hospitalized children with severe pneumonia in People's Hospital of Hunan province from January 2011 to December 2011. Nested- or reverse transcription Polymerase chain reaction (PCR or RT-PCR) was used to screen Adenovirus (ADV), Human Bocavirus (HBoV), Parainfluenzaviruses1-4 (PIV1-4), Human Respiratory Syneytial virus (RSV), Influenza virus A (IFVA), Influenza virus B (IFVB), Human Rhinovirus(HRV), Human Metapneumovirus (HMPV), human coronaviruses NL63 and HKU1 (HCoV-NL63, HCoV- HKU1)., Results: Among the 122 bronchoalveolar lavage fluid, viral agents were detected in 60 samples(49.1%), among which ADV (40.98%) was the most common virus, followed by RSV (7.37%) and HBoV (7.37%). Two viruses were detected in 21 individual (35%) samples, of which 20 were dual positive for ADV (40%)., Conclusion: ADV is the most frequently detected viral etiology of severe pneumonia in children in Hunan during this year. And its Coinfection with other respiratory viruses was common.

Objective: To establish a rapid, sensitive and specific real-time PCR method for detection of Human Herpesvirus-6 (HHV-6)., Methods: According to the reference, a pair of primers and a probe were designed located in U65-66 gene and to set up the standards. We established a real-time RT-PCR method for detection of HHV-6, and to verify the specificity, sensitivity, reproducibility., Results: The correlation coefficient was 0.999, E = 97.9%, the coefficient of variation values of Ct were 0.61% and 3.13% in real-time PCR assay for inter and intra assay, respectively. The results of all viruses were negative except of HHV-6 for the assay. The quantitative detection limit of the assay was 3 x 10(0) copies/microl., Conclusion: The real-time PCR assay is highly specific, sensitive and reproducible, which can be used to quatitative detecting clinical samples.

Objective: To investigate the prevalence and clinical characterization of HCoV-NL63 (NL63) in children with acute respiratory tract infections (ARTIs) in Lanzhou with other respiratory viruses. The prevalence of HBoV1 in ALRTI was obviously city,China., Method: From November 2006 to October 2009,1169 nasopharyngeal aspirates (NPA) were collected from children under 14 years old with ARTIs. Samples were screened for NL63 using a reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Demography and clinical information were recorded., Result: NL63 was detected in 35 (2.99%) of the 1169 children. The peak of the positive rate were in August, September 2007, July, August 2008 (23.53%,17.65%, 50%, 33.33% separately). There are no NL63 positive samples was detected in December, 2007 to February 2009. 25 (25/35, 71.43%) were co-infected with other respiratory viruses, and human rhinovirus (HRV) were the most common additional respiratory virus. No significant differences of infective rate of NL63 was found between < or = 3 years age group and > 3 years age group. Bronchiolitis and pneumonia were the most frequent diagnoses in NL63 positive patients and the major symptoms were fever and cough in our study. Between the monoinfection group and the coinfection group of NL63-positive patients, no differences were found in symptoms and clinical diagnoses except symptoms of gastrointestinal., Conclusion: HCoV-NL63 is an important pathogen of acute respiratory tract infection in children in Lanzhou city. The peak of HCoV-NL63 infections was in summer. There were annual differences in the prevalence of HCoV-NL63. HCoV-NL63 infections existed a high rate of mixed infection, and mixed infection does not increase the severity of the disease.

Objective: The main objective of this study was to explore the prevalence and clinical characteristics of human coronavirus NL63 infection in hospitalized children with acute lower respiratory tract infection (ALRTI) in Changsha., Methods: Nasopharyngeal aspirates (NPA) samples were collected from 1185 hospitalized children with ALRTI at the People's Hospital of Hunan province, between September 2008 and October 2010. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to screen for coronavirus NL63, which is a 255 bp fragment of a part of N gene. All positive amplification products were confirmed by sequencing and compared with those in GenBank., Results: The overall frequency of coronavirus NL63 infection was 0.8%, 6 (60%) out of the coronavirus NL63 positive patients were detected in summer, 2 in autumn, 1 in spring and winter, respectively. The patients were from 2 months to two and a half years old. The clinical diagnosis was bronchopneumonia (60%), bronchiolitis (30%), and acute laryngotracheal bronchitis (10%). Four of the 10 cases had critical illness, 4 cases had underlying diseases, and 7 cases had mixed infection with other viruses. The homogeneity of coronavirus NL63 with those published in the GenBank at nucleotide levels was 97%-100%., Conclusion: Coronavirus NL63 infection exists in hospitalized children with acute lower respiratory tract infection in Changsha. Coronavirus NL63 infections are common in children under 3 years of age. There is significant difference in the infection rate between the boys and the girls: the boys had higher rate than the girls. The peak of prevalence of the coronavirus NL63 was in summer. A single genetic lineage of coronavirus NL63 was revealed in human subjects in Changsha. Coronavirus NL63 may also be one of the lower respiratory pathogen in China.

Objective: To understand the epidemiological characteristics of human metapneumovirus (hMPV) infection in children with acute lower respiratory tract infection (ALRTI) in Changsha area, China., Methods: A total of 2613 nasopharyngeal aspirate (NPA) specimens were collected from hospitalized children with ARTIs between September 2007 and February 2011 (a period of 3.5 years) in Changsha area, Hunan Province, China. Respiratory specimens were screened for hMPV M gene by reverse-transcription polymerase chain reaction (RT-PCR). All RT-PCR positive' amplification products were confirmed by sequencing or analyzed and compared with those in GenBank known hMPV reference strain., Results: hMPV was detected in 135 (5.2%) out of the 2613 children. The detected rate was significantly different between male and female (chi2 = 8.007, P = 0.003). The majority of the hMPV-positive patients (63.2%) were 0-1 year old infants. The majority of hMPV-positive patients were detected during spring season. Seasonal distribution showed that subtype A2b circulated predominantly in winter and spring, subtype B2 circulated predominantly in summer and spring. The most common genotype in 135 hMPV-positive patients were A and B in human subjects in Changsha. The predominant genotype of hMPV changed over the 3.5 year study period from genotype A2b to combined A2b and B2, and to B2. The 66 (48.9%) out of the 135 hMPV-positive patients were found to be co-infected with other respiratory viruses, and human bocavirus (HBoV) was the most common additional respiratory virus., Conclusion: These data suggest that some cases of acute lower respiratory infection in young children in Changsha area are related to hMPV and the majority of children infected with hMPV were less than 1 years old, the detected rate of boys was higher than that of girls. It circulated predominantly in spring. The subtype A2b genetic lineage and the subtype B1 genetic lineage of hMPV were revealed alternately in human subjects in Changsha area and the hMPV exhibited high co-infection rate with other respiratory viruses.

Objective: To study the clinical and molecular epidemiology characteristics of human Bocavirus 1-3 (HBoV1-3) in children for acute respiratory infection in Lanzhou area., Methods: Nasopharyngeal aspiration samples and throat swabs were collected from 524 children with ARTI at the First Hospital of Lanzhou University, Gansu Province, China, between December 2009 and November 2010. Nested PCR was employed to screening HBoV1-3, which amplified a 518-bp fragment of the partial NS1 gene. Furthermore, a standard reverse transcription-PCR was used to screen for other common respiratory viruses., Results: The overall frequency of HBoV was 8.2% (43/524), lining up behind human rhinovirus, RSV, parainfluenza virus 3. Thirty of the HBoV-postive children(69.8%) were co-infected with other respiratory viruses. The prevalence of HBoV1 in ALRTI was obviously higher than that in AURI. The 2 HBoV2 NS1 sequences shared 99% and 100% nucleotide sequence identity with HBoV2 strain CU47TH respectively. Two cases of HBoV2 postive children appears gastrointestinal symptoms. The one HBoV3 NS1 sequences shared 99% nucleotide sequence identity with HBoV3 isolate 46-BJ07., Conclusion: The HBoV3 was detected at the first time in lanzhou area. HBoV1-3 infection exists in children with acute respiratory tract infections in Lanzhou region, HBoV1 were dominant. The mixed infection rate was higher.

Objective: To investigate prevalence of Saffold virus (SAFV) in Changsha area of hospitalized children with respiratory tract infection, and to discuss whether this virus is related to respiratory tract infection of children., Methods: 643 nasopharyngeal aspirates samples were collected from hospitalized children with respiratory tract infection of the first affiliated hospital of Hunan nomal university during Nov. 2007 to Oct. 2008. Real-time fluorescent quanti-tative PCR(FQ-PCR) performed to screen the 5'UTR gene. And then analyze clinical data., Results: SAFV were detected in 67 patients (10.42%) out of the 643 children, it was not detected over 5 years of age. The virus were detected in 8 patients (25.81%) out of the 31 children with persistent pneumonia and chronic pneumonia, there was statistically significant., Conclusion: There existed SAFV infection in hospitalized children with lower respiratory infection in Changsha area; SAFV maybe related to disease onset with lower respiratory tract infection of children.

Objective: In order to understand the epidemiological and virologic characteristics of coronavirus HKU1 infection in hospitalized children with acute respiratory tract infection (ARTI) in Changsha., Methods: 1165 nasopharyngeal aspirates (NPA) specimens were collected from hospitalized children with ARTI between September 2007 and August 2008 in Changsha. Specimens were screened for pol gene of coronavirus HKU1 by polymerase chain reaction. All positive amplification products were confirmed by sequencing and compared with those in GenBank., Results: Coronavirus HKU1 were detected in 12 patients (1.03%) out of the 1165 children. The patients were from 8 days to 3 years. The most common clinical diagnosis was bronchopneumonia(83.33%). Similarity of coronavirus HKU1 with those published in the GenBank at nucleotide levels was 98.18% - 100%., Conclusion: Coronavirus HKU1 may be important pathogens in children with acute lower respiratory tract infection. Coronavirus HKU1 infections are common in children under 3 years old. There is no significant difference in the infectious rate between the boys and the girls. The peak of its prevalence is in spring and winter. A single genetic lineage of Coronavirus HKU1 was revealed in human subjects in Changsha.

Objective: To investigate the molecular epideiological and clinical feature of human metapneumovirus in children with acute respiratory tract infection in Nanjing city, China., Method: Nasopharyngeal aspirates and nasopharyngeal swab were taken from 642 outpatients or hospitalized pediatric patients with acute at the Children Hospital of Nanjing, Jiangsu Province, China, between August 2009 and July 2010. Respiratory speciments were tested for the M gene of hMPV by reverse-transcription polymerase chain reaction (RT-PCR). All RT-PCR positive products were sequenced and phlogenetic analysis was conducted., Result: hMPV was detected in 35 (5.5%) of the 642 children. Phylogenetic analysis revealed that 51.4% of the hMPV were B1, 31.4% were A2b. The peak of the positive rate was in April. The majority of the hMPV-positive patients(71.4%) were 0-1 years old. Of the 35 hMPV-positive patients, 15 (42.8%) were co-infected with other respiratory viruses, and human rhinovirus (HRV) were the most common additional respiratory virus. The most common clinical diagnosis was pneumonia (48.6%)., Conclusion: Human metapneumovirus is an important pathogen of acute respiratory tract infection in children in Nanjing city. The subtype B1 was the predominating lineage in 2009-2010 in Nanjing city. No significant differences were found for clinical characteristics between genotype A and genotype B human metapneumovirus infection in children in Nanjing.

Objective: To understand the genotypes of human metapneumovirus (hMPV) and the genetic character of hMPV attachment protein G sequence in Hunan, China., Methods: 232 nasopharyngeal aspirates (NPA) samples from hospitalized children with acute respiratory infections were collected from Hunan, China in 2005. HMPV was detected. The full length of G glycoprotein genes were amplified and sequenced. Bioinformatics soft-wares were employed to analyze the sequences., Results: 17/232 (7.3%) were showed hMPV positive. And co-infection rate with other viruses is 35%. The diagnoses of these hMPV positive cases are pneumonia, bronchiolitis and bronchopneumonia. Phylogenetic analysis for G genes from 13 hMPVs revealed the existence of four major subgroups: A1, A2, B1, B2 in Hunan, China in 2005. There are four types of sequence lengths of hMPV G glycoprotein, which are 711, 675, 660, 696nt. It is different in potential N-linked glycosylation sites and number of cysteine residues among these hMPVs of Hunan, China and Beijing, China. Also it is different from those in Japan and North America., Conclusion: The investigation of hMPV from Hunan, China in 2005 revealed the high speed of genetic variation and the marked character of geographic epidemic differences.

Objective: To investigate newly identified polyomavirus WUV and WUV and KIPyV are associated with acute respiratory infections in China, tests were developed to detect WUV and KIPyV gene fragments from nasopharyngeal aspirates collected from children with ARI fron Nov. 2006 to Oct. 2007., Methods: A total of 318 clinical samples were tested for WUV and KIPyV using PCR method. The positive products were sequenced and compared with those in GenBank., Results: 14 of the 318 Samples were positive (WUV was 2.2%, KIPyV was 2.2%). All of children who were positive for WUV or KIPyV had respiratory illness., Conclusion: Polyomavirus WU and KIPyV infection may be associated with upper and lower respiratory diseases.

Objective: To prepare human interferon-k (hIFN-kappa) and study its biological activities., Methods: Whole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities., Results: The purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity., Conclusion: Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.

Objective: To construct a novel recombinant rhIFN-epsilon155ser, and study its biological activities., Methods: The whole sequence of rhIFN-epsilon was artificially synthesized and some codons were altered according to the preferred codon using of E.coli. The sequence was cloned into plasmid vector pBV220 to express in E.coli DH5alpha. After purification and re-folding of rhIFN-epsilon155ser inclusion body, the final product was tested for its biological activities, including anti-viral, anti-proliferative and NK cell enhancing activities. At the same time, by using DNA microarray biochips, the gene expression patterns in the rhIFN-epsilon155ser and rhIFN-alpha2b treated cells were compared and analyzed., Results: The re-built rhIFN-epsilon155ser sequence was expressed in E.coli as a form of inclusion body. After purified and re-folded, the rhIFN-epsilon155ser protein reached a purity of above 95%. The rhIFN-epsilon155ser protein had a specific anti-viral activity of about 6 x 10(5) IU/mg in WISH/VSV system. Its anti-proliferative activity and NK cell enhancing activities in vitro seemed to be lower than that of rhIFN-alpha2b. Data obtained from microarray biochips indicated that there were 283 pieces increasing 2 folds and 1489 pieces decreasing 2 folds among totally 22,278 pieces of human genes were found in the rhIFN-epsilon155ser treated cells; more changes in gene expression pattern were detected in the rhIFN-alpha treated cells., Conclusion: A novel recombinant rhIFN-epsilon155ser was constructed, which belonged to type 1 interferon. The biological activities of rhIFN-epsilon155ser were compared with rhIFN-alpha2b. The changes of gene expression pattern in the interferon treated cells were detected, analyzed and discussed.