Surveillance for seasonal influenza virus prevalence in hospitalized children with lower respiratory tract infection in Guangzhou, China during the post-pandemic era.

BACKGROUND: Influenza A(H1N1)pdm09, A(H3N2) and B viruses have co-circulated in the human population since the swine-origin human H1N1 pandemic in 2009. While infections of these subtypes generally cause mild illnesses, lower respiratory tract infection (LRTI) occurs in a portion of children and required hospitalization. The aim of our study was to estimate the prevalence of these three subtypes and compare the clinical manifestations in hospitalized children with LRTI in Guangzhou, China during the post-pandemic period. METHODS: Children hospitalized with LRTI from January 2010 to December 2012 were tested for influenza A/B virus infection from their throat swab specimens using real-time PCR and the clinical features of the positive cases were analyzed. RESULTS: Of 3637 hospitalized children, 216 (5.9%) were identified as influenza A or B positive. Infection of influenza virus peaked around March in Guangzhou each year from 2010 to 2012, and there were distinct epidemics of each subtype. Influenza A(H3N2) infection was more frequently detected than A(H1N1)pdm09 and B, overall. The mean age of children with influenza A virus (H1N1/H3N2) infection was younger than those with influenza B (34.4 months/32.5 months versus 45 months old; p<0.005). Co-infections of influenza A/ B with mycoplasma pneumoniae were found in 44/216 (20.3%) children. CONCLUSIONS: This study contributes the understanding to the prevalence of seasonal influenza viruses in hospitalized children with LRTI in Guangzhou, China during the post pandemic period. High rate of mycoplasma pneumoniae co-infection with influenza viruses might contribute to severe disease in the hospitalized children.

Clinical, virological and immunological features from patients infected with re-emergent avian-origin human H7N9 influenza disease of varying severity in Guangdong province.

BACKGROUND: The second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region. METHODS: Five patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery. RESULTS: Four patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/ß, IL-1ß and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%). CONCLUSION: Expectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.

Epidemiological and viral genome characteristics of the first human H7N9 influenza infection in Guangdong Province, China.

BACKGROUND: The first H7N9 human case in south of China was confirmed in Guangdong Province on August 2013, outside of the typical influenza season. For investigating the H7N9 virus source and transmission in the local community, we analyze the epidemiology and genome features of the virus isolated from the first human infection detected in Guangdong Province. METHODS: The data including medical records, exposure history and time line of events for the H7N9 patient and close contacts was collected. Variation and genetic signatures of H7N9 virus in Guangdong was analyzed using ClustalW algorithm and comparison with mutations associated with changes in biological characteristics of the virus. RESULTS: The female patient had a history of poultry exposure, and she was transferred from a local primary hospital to an intensive care unit (ICU) upon deterioration. No additional cases were reported. Similar to previous infections with avian influenza A (H7N9) virus, the patient presented with both upper and lower respiratory tract symptoms. Respiratory failure progressed quickly, and the patient recovered 4 weeks after the onset of symptoms. Genome analysis of the virus indicated that the predicted antigen city and internal genes of the virus are similar to previously reported H7N9 viruses. The isolated virus is susceptible to neuraminidase (NA) inhibitors but resistant to adamantine. Although this virus contains some unique mutations that were only detected in avian or environment-origin avian influenza A (H7N9) viruses, it is still quite similar to other human H7N9 isolates. CONCLUSIONS: The epidemiological features and genome of the first H7N9 virus in Guangdong Province are similar to other human H7N9 infections. This virus may have existed in the environment and live poultry locally; therefore, it is important to be alert of the risk of H7N9 re-emergence in China, including emergence outside the typical influenza season.

[Development and characterization of a stable cell line expressing respiratory syncytial virus non-structural protein NS1].

To develop a stable cell line that could express the RSV NS1, the full-length RSV NS1 gene was generated by RT-PCR amplification from respiratory syncytial virus. NS1 gene was ligated with pBABE-puro to construct the recombinant retroviral expression plasmid pBABE-NS1, which was cotransfected into 293FT packaging cells with PIK packaging plasmid by calcium phosphate co-precipitation. The supernatant of 293FT was collected to infect HEp-2 cells, the resulting cell clones stably expressing NS1 were screened by puromycin. Using QPCR, CPE staining method and indirect immunofluorescence assay, the expression of NS1 at both gene and protein levels was identified. The recombinant plasmid pBABE-NS1 was identified by EcoRI and BamHI endonuclease digestion and the sequence analysis. QPCR results showed that the NS1 gene amplification in HEp-2-NS1 cells was 8483 fold higher than that in HEp-2 cells. Although the exogenous interferon was added, all cells were destroyed after 48 hours post infection using CPE staining method, showing that HEp-2-NS1 cells remained sensitive to the VSV virus. The results of RT-PCR and indirect immunofluorescence assay showed that the NS1 gene in HEp-2 cells could not only transcribe mRNA, but also express NS1 protein steadily. We had successfully established HEp-2-NS1 cell lines with stable expression of respiratory syncytial virus non-structural protein NS1.