Prevalence of human metapneumovirus in children with acute lower respiratory infection in Changsha, China.

Human metapneumovirus (hMPV) causes acute respiratory infections in children. The prevalence and clinical characteristics of hMPV were determined in nasopharyngeal aspirates of children in Changsha, China. Reverse transcription-polymerase chain reaction (RT-PCR) or PCR was employed to screen for both hMPV and other common respiratory viruses in 1,165 nasopharyngeal aspirate specimens collected from children with lower respiratory tract infections from September 2007 to August 2008. All PCR products were sequenced, and demographic and clinical data were collected from all patients. Seventy-six of 1,165 (6.5%) specimens were positive for hMPV, of which 85.5% (65/76) occurred in the winter and spring seasons. The hMPV coinfection rate was 57.9% (44/76), and human bocavirus was the most common virus detected in conjunction with hMPV. Phylogenetic analysis revealed that 94.7% of the hMPV detected were of subgroup A2, 5.3% were subgroup B2, and none belonged to either the A1 or B1 subgroups. No significant differences were found in terms of the frequency of diagnosis and clinical signs between either the co- and mono-infection groups, or between patients with and without underlying diseases. It was concluded that hMPV is an important viral pathogen in pediatric patients with lower respiratory tract infections in Changsha. Only hMPV genotypes A2 and B2 were co-circulating in this locality; human bocavirus was the most common coinfecting virus, and coinfection did not affect disease severity.

Prevalence of adenovirus in children with acute respiratory tract infection in Lanzhou, China.

BACKGROUND: Human adenovirus (HAdV) is an important agent causing respiratory tract infection in children. Information on the epidemiological and clinical features of HAdV is limited in children with acute respiratory tract infections (ARTIs) in China, especially those of a novel genotype, Ad55. METHODS: In total, 1169 nasopharyngeal aspirates were collected from children younger than 14 years with ARTIs between November 2006 and November 2009. The polymerase chain reaction (PCR) was used to screen HAdVs. All PCR-positive products were sequenced. RESULTS: 74 of 1169 (6.33%) specimens were positive for HAdVs. Among positive cases, AdV3 (58/74) was detected most frequently, followed by AdV11 (10/74), AdV2 (2/74), AdV7 (2/69), AdV6 (1/74), and AdV1 (1/74). AdV55 was found in one case. The incidence of HAdV infection peaked in children aged 3-7 years. The most common clinical diagnosis was upper respiratory infection, and the most common syndrome was fever and cough.The comparison of HAdV and RSV group revealed that Children infected with group AdV were significant older than children infected with group RSV, had more fever but less frequently wheezing, and cough, crackles, and cyanosis, The duration of hospitalization between the AdV group and RSV group was not significant, but a greater frequency of LRTIs was observed in RSV group. CONCLUSIONS: HAdV is an important viral agent in children with ARTIs in Lanzhou City, China. Multiple HAdV serotypes co-circulated with Ad3, which was predominant in this 3-year study. The novel AdV55 genotype was found in one case. No fixed seasonal rhythm could be identified.

[Viral etiology of 1165 hospitalized children with acute lower respiratory tract infection].

OBJECTIVE: To explore the viral etiology of acute low respiratory tract infection (ALRTI) among hospitalized children in Changsha of Hunan Province of China. METHODS: Nasopharyngeal aspirates were collected from 1165 hospitalized children with ALRTI in Changsha from September 2007 to August 2008. Respiratory syncytin virus (RSV), human rhinovirus (HRV), influenza virus A (IFVA), influenza virus B (IFVB), parainfluenza 1-3 (PIV 1-3), human metapneumovirus (hMPV), human coronaviruses NL63 (HCoV-NL63), and human coronaviruses HKU1 (HCoV-HKU1) were detected by reverse transcription polymerase chain reaction (RT-PCR). Adenovirus (ADV) and human bocavirus (HBoV) were detected by standard polymerase chain reaction (PCR). WU polyomaviruses (WUPyV) and KI polyomaviruses(KIPyV) were detected by nested PCR. The positive samples further underwent genetic sequencing. RESULTS: Among the 1165 nasopharyngeal aspirates, viruses were detected in 871 samples (74.76%), among which RSV (27.03%) was the most common virus, followed by HRV (17.33%), PIV3 (13.73%), HBoV (8.67%) and hMPV (6.52%). The overall positive rate of viral detection showed no significant differences between males and females (X2=2.241, P=0.134), whereas the positive rates of PIV3, hMPV, and HBoV in males were higher than in females. The positive rate of viral detection showed significant differences among different age groups (X2=10.934, P=0.027), and the highest positive rate was noted in the age group of 6 months to 1 year. Furthermore, the overall positive rate of viral detection showed a significant difference in term of seasonal distribution, with a peak prevalence in winter. CONCLUSIONS: Virues predominate in the etiology of pediatric ALRTI in Changsha, and RSV, HRV and PIV3 are the main viruses for ALRTI. HBoV and hMPV have become increasingly important. Viral infection-associated ALRTI shows a prevail in the age group of 6 months to 1 year as well as in winter.

Novel human bocavirus in children with acute respiratory tract infection.

Human bocavirus (HBoV) and HBoV2, two human bocavirus species, were found in 18 and 10 of 235 nasopharyngeal aspirates, respectively, from children hospitalized with acute respiratory tract infection. Our results suggest that, like HBoV, HBoV2 is distributed worldwide and may be associated with respiratory and enteric diseases.

Human respiratory syncytial virus in children with acute respiratory tract infections in China.

There are limited data on the prevalence and clinical and molecular characterization of human respiratory syncytial virus (HRSV) in children with acute respiratory tract infections (ARTIs) in China. From December 2006 to March 2009, 894 nasopharyngeal aspirates (NPAs) were collected from children under 14 years of age with ARTIs. Samples were screened for HRSV and genotyped by reverse transcription-PCR (RT-PCR) and sequencing. Demographic and clinical information was recorded. A total of 38.14% (341/894) of samples were positive for HRSV. Phylogenetic analysis revealed that 60.4% of the selected 227 RSV strains were GA2, 34.4% were BA, 4.8% were GB2, and 0.4% were GB3. A total of 40.47% of all of the RSV-positive samples were coinfected with other respiratory viruses, and adenovirus was the most common additional respiratory virus. No statistical differences were found in the frequency of diagnosis and symptoms between the coinfection group and monoinfection group. Additionally, no statistical differences were found in epidemiological characterizations or disease severity between genotype BA- and GA2-positive patients, except for a greater frequency of lower respiratory tract infections (LRTIs) (mostly bronchitis)with BA. HRSV is the most important viral pathogen in Chinese children with ARTIs. Four genotypes (i.e., GA2, BA, GB2, and GB3) circulate locally, and the predominant genotype may shift between seasons. Coinfection with other viruses does not affect disease severity. HRSV genotypes were not associated with different epidemiological characterizations or disease severity.

Human bocavirus infection in young children with acute respiratory tract infection in Lanzhou, China.

Human bocavirus (HBoV) is a recognized human parvovirus associated with acute respiratory tract infection. However, HBoV has yet to be established as a causative agent of respiratory disease. In this study, the epidemiological and virological characteristics of HBoV infection were studied in children with acute respiratory tract infection in China. In total, 406 children younger than 14 years of age with acute respiratory tract infection were included in this prospective 1-year study. HBoV was detected in 29 (7.1%) of the 406 children. No clear seasonal fluctuation was observed in infection rates of HBoV. Of the 29 children infected with HBoV, 16 (55.2%) were coinfected with other respiratory viruses, most commonly respiratory syncytial virus (RSV). Viral coinfection with HBoV did not affect the severity of the respiratory disease (P = 0.291). The number of HBoV genome copies ranged from 5.80 x 10(2) to 9.72 x 10(8) copies/ml in nasopharyngeal aspirates among HBoV-positive specimens by real-time PCR, and neither coinfection nor the severity of disease correlated with the viral load (P = 0.148, P = 0.354, respectively). The most common clinical features were cough and acute upper respiratory infection, and acute bronchopneumonia. Additionally, the NP-1 gene of HBoV showed minimal sequence variation. These data suggest that HBoV is frequent in young children with acute respiratory tract infection in Lanzhou, China, and RSV is the most common coinfecting virus. There was no apparent association between the viral load of HBoV and coinfection or disease severity. The NP-1 gene was highly conserved in HBoV.

Prevalence and clinical characterization of a newly identified human rhinovirus C species in children with acute respiratory tract infections.

Human rhinovirus C (HRV-C) is a newly identified genotype of HRV found in patients with respiratory tract infections (RTIs); however, its epidemiological profile and clinical characteristics are not well understood. In this study, Chinese children with RTIs were screened for HRV-C and their epidemiological and clinical characteristics were analyzed. From December 2006 to November 2007, 406 nasopharyngeal aspirates from children younger than 14 years of age with RTIs were screened for HRV and other common respiratory viruses by PCR or reverse transcription-PCR. Two-hundred twenty-four (55.2%) of the specimens were infected with at least one virus, including 53 patients with HRV (13%). HRV-A, HRV-B, and HRV-C were detected in 22, 12, and 19 specimens, respectively. HRV-C was detected mainly from December 2006 to April 2007 and from October to November 2007, with peaks in December and April (10/19). Acute upper respiratory infection and bronchopneumonia were observed in 53 and 37% of the cases, respectively. The most common symptoms were cough (82%), runny nose (53%), and fever (37%). Wheezing and bronchiolitis were less common in patients infected with HRV-C than in those infected with respiratory syncytial virus (RSV). Partial sequencing of the genes coding for VP4 and VP2 revealed that the HRV-C strains were 56 to 62% identical at the amino acid level to HRV-B and HRV-A reference strains and 80 to 99% identical to HRV-C reference strains. In conclusion, HRV-C is an important cause of RTIs in children, and highly diversified strains of HRV-C are prevalent in China. HRV-C may produce different epidemiological features, and patients infected with HRV-C may exhibit different clinical features from patients infected with RSV or HRV-A/B.

Phospholipase A2-like activity of human bocavirus VP1 unique region.

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A2 (PLA2) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA2 enzymatic activity and how these critical residues contribute to its PLA2 activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca2+-dependent secreted PLA2-like (sPLA2) activity, which was inhibited by sPLA2-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA2 activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA2-like enzymatic activity, and these residues are crucial for its sPLA2-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.

Prevalence of human KI and WU polyomaviruses in children with acute respiratory tract infection in China.

The KI and WU polyomaviruses were found in 11 (2.7%) and 17 (4.2%) of 406 nasopharyngeal aspirates, respectively, from children with acute respiratory tract infection (ARTI). The phylogenetic analysis indicates that they are all in the same cluster as the prototype strains. Our findings suggest that they are common in children with ARTI in China.

[hEPO transfer and expression by a recombinant adeno-associated virus vector].

In order to achieve sustained human erythropoietin (hEPO) expression in vivo for the treatment of anemias, recombinant adeno-associated virus (rAAV)-mediated hEPO transfer was studied in this report. rAAV vector plasmid carrying hEPO was constructed, and rAAV vector cell line for production of rAAV was also established. By using the one helper virus-one vector cell line strategy, which we reported previously, rAAV containing the hEPO expression cassette was produced in large-scale. The results showed that the hEPO expressed effectively by rAAV-mediated hEPO transfer into cultured BHK-21 cells. Intramuscular injection of rAAV-hEPO to Balb/c mice resulted in the in vivo expression of hEPO for at least ten weeks, along with the significant elevation of the hematocrits. This report indicates the potential use of rAAV-mediated gene transfer for the treatment of various anemias.

Functional change of human peripheral blood monocyte-derived dendritic cells after recombinant adeno-associated virus type 2-mediated HBsAg gene infection.

OBJECTIVE: To observe the changes in the functions of human peripheral blood monocyte-derived dendritic cells (DCs) after hepatitis B virus surface antigen (HBsAg) gene infection mediated by recombinant adeno-associated virus type 2 (rAAV). METHODS: The levels of both interleukin (IL)-12 in the supernatant of in vitro cultured DCs infected with rAAV-HbsAg and interferon gamma (IFN-gamma) in the supernatant of the lymphocyte populations co-cultured with DCs were determined by ELISA. The functions of the rAAV-HbsAg-infected DCs were assessed by mixed leukocyte reaction (MLR), and the changes of the surface markers (including CD80, CD83, CD86 and HLA-DR) in response to the infection were detected by flow cytometry. RESULTS: After rAAV-HBsAg infection, the IL-12 secretion of DCs was significantly enhanced (P <0.05), while IFN-gamma production by the lymphocyte populations co-cultured with rAAV-HbsAg-infected DCs was reduced (P <0.01). rAAV-HBsAg infection of DCs did not affect the surface marker expressions and stimulation ability of the DCs in allogeneic lymphocytes reaction. CONCLUSION: DCs infected by rAAV-HBsAg are more efficient than naive DCs in eliciting the differentiation of the lymphocytes toward T helper type I cells, but the functions and the surface markers of DCs remain unaffected after the infection, suggesting the applicability of rAAV as a potentially useful vector for HBsAg gene transfer into the DCs for HBV immunotherapy.

[Construction of recombinant adeno-associated virus carrying hepatitis B surface antigen gene and preliminary study of the gene expression and function].

OBJECTIVE: To construct recombinant adeno-associated virus (rAAV) carrying hepatitis B surface antigen (HBsAg) gene and study the function of the expressed HBsAg. METHODS: HBsAg gene (subtype ayw) was amplified from PTHBV-1 by PCR and cloned into the adeno-associated virus vector pSNAV to form the recombinant pSNAV-HBsAg, which was transfected into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell line, BHK-HBsAg, was isolated, which was capable of HBsAg expression and was subsequently infected with HSV-1-HSV1-rc/Delta UL2 that was able to package the rAAV. After purification, rAAV-HBsAg was obtained. The expression of HBsAg in BHK-21 cells and 293 cells infected with rAAV-HBsAg were detected by enzyme-linked immunosorbent assay (ELISA), and the HBsAg antibodies in the sera of rAAV-HBsAg-immunized BalB/C mice were assayed by radio immunoassay. RESULTS: As detected by ELISA, the expressed HBsAg in mixed cell line mounted to 28.6+/-6.72 ng per 5 x 10(6) cells. The BHK-21 cells and 293 cells infected with rAAV-HBsAg were both capable of HBsAg expression, the amount of which augmented with the increase of multiplicity of infection (MOI). BalB/C mice immunized with rAAV-HBsAg produced anti-HBsAg antibodies. CONCLUSIONS: rAAV-HBsAg can induce humoral immune response against HBsAg and therefore can be a promising candidate hepatitis B vaccine, and in addition, it may serve the purpose of exploring possible immunotherapy for chronic HBV infection.

[The detection and clinical feature of HcoV-nL63 in children with acute respiratory tract infection in Lanzhou city].

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.

[Prevalence and clinical characteristics of coronavirus NL63 infection in children hospitalized for acute lower respiratory tract infections in Changsha].

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.

[Detection and clinical study on coronavirus HKU1 with acute lower respiratory tract infections of hospitalized children in Changsha].

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.

Prevalence and clinical and molecular characterization of human metapneumovirus in children with acute respiratory infection in China.

BACKGROUND: Human metapneumovirus (HMPV), a newly discovered paramyxovirus, has been associated with acute respiratory tract infections (ARTIs). However, the prevalence and molecular characteristics of HMPV in China are still unclear. METHODS: A total of 661 nasopharyngeal aspirates (NPA) specimens were collected from 661 children with ARTIs between December 2006 and November 2008. Specimens were screened for HMPV by reverse transcription-polymerase reaction. All positive amplification products were confirmed by sequencing. RESULTS: HMPV was detected in 45 patients (6.80%) of the 661 children. The HMPV-infected patients were from 29 days to 9 years of age. A high incidence of HMPV infection (84.4%) was observed during the winter-spring season. Of the 45 HMPV-positive patients, 25 (55.6%) were co-infected with other respiratory viruses, and respiratory syncytial virus (RSV) was the most common additional respiratory virus. The most common clinical diagnosis was bronchopneumonia (57.8%) and cough (88.9%) was the most common clinical symptom. Phylogenetic analysis of the F gene revealed that 80% of the HMPV detected were A2, 2.2% were A1, and 17.8% were B1. Statistical analyses showed that sex, ages, seasons, and severity of the disease did not correlate with HMPV genotype (P = 0.986, 0.347, 0.660, 0.252), but viral coinfection with HMPV increased hospitalization rates (P = 0.005). CONCLUSIONS: HMPV was frequently detected in the pediatric patients with ARTI in China. RSV was the most common coinfection virus and coinfection increased hospitalization rates. All HMPV subgroups except B2 cocirculated and there was no association found between HMPV genotypes and severity of disease.

Prevalence and clinical characteristics of human CoV-HKU1 in children with acute respiratory tract infections in China.

BACKGROUND: Human CoV-HKU1 (HCoV-HKU1) has been isolated from a 71-year-old man with pneumonia; however, the impact and role of emerging HCoV-HKU1 have not been defined in children with acute respiratory tract infection (ARTI). OBJECTIVE: To investigate the Prevalence and clinical characteristics of HCoV-HKU1 in children with ARTI in Lanzhou, China. STUDY DESIGN: The reverse transcription polymerase chain reaction (RT-PCR) or PCR was employed to screen HCoV-HKU1 and other common respiratory viruses in 645 nasopharyngeal aspirate (NPA) specimens collected from children with ARTI from November 2006 to October 2008. All PCR positive products were sequenced. And the demographic and clinical data were collected for all patients. RESULTS: Nineteen of 645 (2.95%) specimens tested positive for HCoV-HKU1, and all HCoV-HKU1 positive specimens were distributed in the winter and spring season. The HCoV-HKU1 co-infection rate with other respiratory viruses was 47.37% (9/19). There was no statistically significant difference in the detection rate between groups by age or gender, except between patients with and without underlying diseases. The phylogenetic analysis indicated that HCoV-HKU1 genotype B was circulating in the years 2007 and 2008 in children with ARTI in Lanzhou, China. CONCLUSIONS: HCoV-HKU1 is an uncommon virus existing among Chinese children with ARTI. Children with underlying diseases are more vulnerable to viral infection. Only HCoV-HKU1 genotype B circulated locally.

[Construction of human metapneumovirus DNA vaccine and study on its immune response in mice].

OBJECTIVE: To construct human metapneumovirus (hMPV) DNA vaccines and evaluate the cellular and humoral immune response in mice. METHODS: Fusion protein FdeltaTM (without transmembrane domain) gene and M gene of hMPV were amplified from cDNA by PCR, then DNA vaccines pcDNA3.1His-FdeltaTM and pcDNA3.1His-M were constructed to verify the expression of F and M protein by Western blotting and indirect immunofluorescent assay (IFA) respectively. Serum IgG and spleen cell CTL were detected with ELISA and ELISPOT assay after the BALB/c mice were immunized intramuscularly with the vaccines. RESULTS: The candidate DNA vaccines could express FdeltaTM and M protein as detected with Western blotting and IFA. The IgG antibody titers of mice was 1:44 when immunized with pcDNA3.1His-FdeltaTM, but could increase to 1:64 when co-immunized with pcDNA3.1His-M. ELISPOT assay demonstrated that IFN-gamma-secreting effector T cells reached 42 +/- 8.9 in co-immunization group, higher than single vaccine pcDNA3.1His-FdeltaTM group (32 +/- 7.4). CONCLUSION: DNA vaccine pcDNA3.1His-FdeltaTM could induce specific cellular and humoral immune responses, and the immune response could increase when co-immunization with pcDNA3.1His-M was carried out.

[Evaluation of reverse transcription loop-mediated isothermal amplification for detection of avian influenza A H5N1 virus].

A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was established to provide a new alternative for clinical diagnosis of Avian influenza A H5N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty-one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT-LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A H5N1 virus.

[WU polyomavirus and KI polyomavirus detected in specimens from children with acute respiratory tract infection in China].

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.