[Analysis of pathogenic features and infection status of human parainfluenza virus type 3 among children in Hangzhou].

OBJECTIVE: To determine the level of genetic variation of human parainfluenza virus type 3 (HPIV-3), and to describe infection and co-infection characteristics of HPIV-3 in children. METHODS: Single respiratory samples from 856 pediatric patients with acute respiratory tract infection (ARI) in Hangzhou were collected from December 2009 to March 2013. All samples were screened for HPIV-3 by real-time RT-PCR and followed by HN sequencing and phylogenetic analysis. In all RSV positive specimens, we screened for the other pathogens, and co-infection characteristics were evaluated. RESULTS: A total of 9.6% of 856 samples were positive for HPIV-3, the nucleotide among the strains ranged from 96.9% to 100%. All Hangzhou strains were placed in C3 subgroup based on HN gene analysis. 49% (n=41) of all HPIV-3-positive children with ARI were found to be co-infected with at least one of the other pathogen. The highest co-infection rate of HPIV-3 was with HRV (n=17). Children in the younger groups (≤12 months old) were significantly more prone to be co-infected with other pathogen (χ(2)=4.78, P=0.029). Pneumonia infection rate was significantly higher in the mono-infection group than the co-infection group (χ(2)=3.92, P=0.048). CONCLUSION: HPIV-3 was an important pathogen in children with ARI in Hangzhou. HN gene variation rate was low, but showed a more local pattern. The co-infections with other respiratory viruses were popular. Except for pneumonia, no significant differences in other clinical presentation between the HPIV-3 mono-infection and co-infection groups were observed.

Influenza H7N9 and H9N2 viruses: coexistence in poultry linked to human H7N9 infection and genome characteristics.

UNLABELLED: Avian influenza virus A of the novel H7N9 reassortant subtype was recently found to cause severe human respiratory infections in China. Live poultry markets were suspected locations of the human H7N9 infection sources, based on the cases' exposure histories and sequence similarities between viral isolates. To explore the role of live poultry markets in the origin of the novel H7N9 virus, we systematically examined poultry and environmental specimens from local markets and farms in Hangzhou, using real-time reverse transcription-PCR (RT-PCR) as well as high-throughput next-generation sequencing (NGS). RT-PCR identified specimens positive for the H7 and N9 genomic segments in all of the 12 poultry markets epidemiologically linked to 10 human H7N9 cases. Chickens, ducks, and environmental specimens from the markets contained heavily mixed subtypes, including H7, N9, H9, and N2 and sometimes H5 and N1. The idea of the coexistence of H7N9 and H9N2 subtypes in chickens was further supported by metagenomic sequencing. In contrast, human H7N9 infection cases (n = 31) were all negative for H9N2 virus according to real-time RT-PCR. The six internal segments were indistinguishable for the H7N9 and H9N2 viruses. The H9, N2, and internal-segment sequences were very close to the sequence of the H9N2 virus circulating in chickens in China recently. Our results provide direct evidence that H9N2 strains coexisted with the novel human-pathogenic H7N9 influenza virus in epidemiologically linked live poultry markets. Avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus and continues to do so. IMPORTANCE: Our results suggest that avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus, a novel reassortant avian influenza virus A subtype, and continues to do so. The finding helps shed light on how the H7N9 virus emerged, spread, and transmitted to humans. It is of considerable interest for assessing the risk of the possible emergence of novel reassortant viruses with enhanced transmissibility to humans.

[Analysis of infection status and pathogenic features of human metapneumovirus among children in Hangzhou between year 2009 and 2011].

OBJECTIVE: To study the infection status and pathogenic features of human metapneumovirus (hMPV) among children with acute respiratory tract infection in Hangzhou. METHODS: A total of 372 children less than 14 years old with acute respiratory tract infections were recruited as subjects from the pediatric clinic or intensive care unit (ICU) of 3 hospitals in Hangzhou during November 2009 to January 2010, and November 2010 to January 2011. A total of 372 specimens were collected, including 351 respiratory swab, 9 nasopharyngeal aspirate material, 8 endotracheal aspirate material and 4 sputum. The total nucleic acid was then extracted from the specimens, and the nucleoprotein (N) gene of hMPV was amplified by RT-PCR, whose positive products were sequenced and analyzed. Africa green monkey kidney cells (Vero-E6) were applied to culture hMPV among the positive samples; meanwhile fluorescence quantitative RT-PCR was adopted to test other respiratory virus infection. RESULTS: Out of 372 patients, 42 (11.2%) were positive for N gene of hMPV. The positive rate of hMPV among boys was 11.5% (26/226), and correspondingly 10.9% (16/146) among girls. The difference showed no statistical significance (χ(2) = 0.026, P > 0.05). The youngest patient was only 2 month-old and the eldest patient was 14 years old. The median of the patients' age was 24 months. Fifteen positive samples amplified by RT-PCR were sequenced, and all turned out to be subtype B1; whose similarity to GD165 found in Guangdong was 98.1% - 99.5% and similarity to BJ1897 in Beijing was 87.8% - 89.2%. The co-infection rate between hMPV and other respiratory virus was 45.2% (19/42); most of which was between hMPV and respiratory syncytial virus, whose rate at 26.1% (11/42). CONCLUSION: hMPV was the single genotype relevant with the acute respiratory tract infection disease among children in Hangzhou district; however, the co-infection with other respiratory virus did exist.

Human respiratory syncytial virus in children with lower respiratory tract infections or influenza-like illness and its co-infection characteristics with viruses and atypical bacteria in Hangzhou, China.

BACKGROUND: Human respiratory syncytial virus (RSV) is the most important viral pathogen in children. However, its epidemic patterns and co-infection characteristics are not fully understood. OBJECTIVES: We attempted to determine the level of genetic variation of RSV, and describe the prevalence and co-infection characteristics of RSV in Hangzhou during two epidemic seasons. STUDY DESIGN: Single respiratory samples from 1820 pediatric patients were screened for RSV and genotyped by RT-PCR and sequencing. In all RSV positive specimens, we screened for viruses and atypical bacteria. Demographic and clinical information was recorded and analyzed. RESULTS: A total of 34.5% and 3.8% of samples from acute lower respiratory tract infections (ALRI) and influenza-like illness (ILI) were positive for RSV, respectively. Phylogenetic analysis revealed that 61.1% of the selected 167 RSV strains were NA1, 31.1% were BA, 3.6% were ON1, 2.4% were CB1, and 1.8% were NA3. A new genotype, BA11 was identified, which comprised 98.1% of BA strains in this study, while the rest were BA10. A total of 36.4% and 9.1% of RSV-positive children with ALRI and ILI respectively were found to be co-infected. Rhinovirus was the most common additional respiratory virus, followed by human metapneumovirus. Except for fever, no significant differences in other clinical presentation between the RSV mono-infection and co-infection groups were observed. CONCLUSIONS: The circulating RSV strains had high genetic variability with RSV-B showing a more local pattern. In ALRI cases, co-infection of RSV with other viruses or atypical bacteria has no significant effect on the clinical presentation except fever.

[Genetic Diversity and Evolution of the M Gene of Human Influenza A Viruses from 2009 to 2013 in Hangzhou, China].

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.

Interim estimates of divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance (2010-2015) in Hangzhou, southeast of China.

OBJECTIVES: In the post-pandemic period 2010-2015, seasonal influenza A(H3N2) virus predominated in Hangzhou, southeast of China, with an increased activity and semi-annual seasons. This study utilized HA virus gene segment sequences to analyze the divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance in Hangzhou. METHODS: Virological and serological analyses of 124 representative A(H3N2) viruses from prospective studies of systematic surveillance samples were conducted to quantify the genetic and antigenic characteristics and their vaccine strain match. RESULTS: Bayesian phylogenetic inference showed that two separate subgroups 3C.3 and 3C.2 probably diverged from group 3C in early 2012 and then evolved into groups 3C.3a and 3C.2a, respectively, in the 2014/15 influenza season. Furthermore, high amino acid substitution rates of the HA1 subunit were found in A(H3N2) group 3C.2a variants, indicating that increased antigenic drift of A(H3N2) group 3C.2a virus is associated with a vaccine mismatch to the 2015/16 vaccine reference strain Switzerland/9715293/2013 (group 3C.3a). CONCLUSIONS: A portion of the group 3C.2a isolates are not covered by the current A(H3N2) vaccine strain. These findings offer insights into the emergence of group 3C.2a variants with epidemic potential in the imminent influenza seasons.

Progressive antigenic drift and phylogeny of human influenza A(H3N2) virus over five consecutive seasons (2009-2013) in Hangzhou, China.

Vaccine efficacy (VE) can be affected by progressive antigenic drift or any new reassortment of influenza viruses. To effectively track the evolution of human influenza A(H3N2) virus circulating in Hangzhou, China, a total of 65 clinical specimens were selected randomly from outpatients infected by A(H3N2) viruses during the study period from November 2009 to December 2013. The results of reduced VE and antigenic drift of the correspondent epitopes (C-D-E to A-B) suggest that the current vaccine provides suboptimal protection against the A(H3N2) strains circulating recently. Phylogenetic analysis of the entire HA and NA sequences demonstrated that these two genes underwent independent evolutionary pathways during recent seasons. The H3-based phylogenetic tree showed that a special strain A/Hangzhou/A289/2012 fell in a cluster among viruses with reduced VE predominantly circulating in 2013. Our findings underscore a possible early warning for the circulation of A(H3N2) variants with antigenic drift during the previous seasons.

[Molecular epidemiology of human metapneumovirus in children with respiratory tract infection in Hangzhou].

OBJECTIVE: To understand the molecular epidemiologic features of human metapnenmovirus (hMPV) in children with respiratory tract infection in Hangzhou. METHODS: 2 593 throat swabs were collected from patients with respiratory tract infections who visited the hospitals with sentinel surveillance programs from January 2011 to December 2013, including 1 676 outpatients and 917 inpatients. Total nucleic acid was extracted from the specimens and the fusion (F) protein gene of hMPV was amplified by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), with positive samples picked to compare with the sequence of hMPV in GenBank, after the sequence of amplification products were determined. Other two types of common respiratory virus were tested using RT-PCR. RESULTS: The overall positive rate in this study was 6.51% (169/2 593), with 6.62% (111/1 676) in outpatients and 6.32% (58/917) in inpatients, but no statistically significant difference was found (χ(2) = 0.086, P = 0.769). The rates was 7.01% in males and 5.72% in females, with no statistically significant difference in different sex (χ(2) = 1.676, P = 0.195). The positive rate was 14.14% (28/198)in the 2-year-olds, 14.01% (22/158)in 3-year olds. The rate in 2-year olds was higher than in other groups, with statistically significant differences between the groups (χ(2) = 38.654, P = 0.000). Of the 169 positive cases, 153 (90.53%) in the younger than 5 years olds. The rates of infection with hMPV in winter and spring were statistically higher than in summer and autumn (χ(2) = 67.032, P = 0.000). The rate of co-infection was 19.52% (33/169). 88 amplified productions were selected for gene sequence analysis, and the F gene homology were 81.6%-100.0% with reference strains in GenBank. Data showed that all the 4 viral subtypes: A2 (52.27% , 46/88), B1 (37.51%, 33/88), B2 (9.09%, 8/88) and A1 (1.13%, 1/88) co-circulated during the study. However, different subtypes appeared predominant in different years:hMPV subtype B1 was in 2011 and 2012, subtype A2 in the end of 2012 and in 2013. Of the 88 specimens, gene sequences were determinate, with A genotype accounted for 67.56% (25/37), B genotype for 32.43% (12/37)in children younger than 1-year olds, and A genotype accounted for 43.13% (22/51), B genotype for 56.86% (29/51)in children above 1-year olds. Significant differences between the two groups (χ(2) = 5.143, P = 0.023) were noticed. CONCLUSION: It was confirmed that hMPV was one of the substantial pathogens causing the respiratory tract infections. Data from our study suggested that the peak time of hMPV infection predominated during winter and spring in Hangzhou. Both hMPV subtype B1 and subtype A2 were found popular in this study, with hMPV genotype A dominating in children younger than 1-year olds.

Trimeric coiled-coil domain of human pulmonary surfactant protein D enhances zinc-binding ability and biologic activity of soluble TRAIL.

Unlike other TNF ligand family members, the homotrimeric tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contains a buried zinc atom coordinated by three Cys230 residues from each subunit. The bound zinc ion is essential for maintaining the structure stability and bioactivity of TRAIL. To improve characteristics of TRAIL by modification to enhance its zinc-binding ability, we constructed a new variant of TRAIL in which the extracellular region of the ligand was N-terminally fused with a trimeric coiled-coil domain derived from human pulmonary surfactant-associated protein D (ST), and compared its characteristics with that of native TRAIL. Circular dichroism (CD) studies and metal assays showed that two versions of TRAIL folded as predicted into secondary and tertiary structures, and contained stoichiometric Zn(2+) through optimizing bacterial expression and purification. The addition of the human trimeric coiled-coil domain, however, significantly increased the antitumor activity of soluble TRAIL in vitro and in vivo. The accelerated thermal stability studies demonstrated that human serum album (HSA) promoted the aggregation and degradation of native TRAIL, but not ST, and the addition of ZnSO(4) to the solution of native TRAIL with HSA partially inhibited its aggregation, suggesting ST is more difficult to lose its bound zinc ion than native TRAIL. The issue was further confirmed by dialysis assess. This is the first example of modified TRAIL with enhanced zinc-binding ability.