Analysis of influenza B virus lineages and the HA1 domain of its hemagglutinin gene in Guangzhou, southern China, during 2016.

BACKGROUND: Few studies have analyzed influenza B virus lineages based on hemagglutinin A (HA) gene sequences in southern China. The present study analyzed the HA gene and the lineages of influenza B virus isolates from Guangzhou during 2016, and compared our results with the WHO-recommended vaccine strain. METHODS: Ninety patients with influenza B were recruited from the First Hospital of Guangzhou Medical University. Throat swab specimens of 72 patients had high viral loads. Among these 72 isolates, the HA1 domain of the HA gene in 43 randomly selected isolates was sequenced using reverse transcription-polymerase chain reaction (RT-PCR), and analyzed using MEGA 5.05. RESULTS: Eight of the 90 patients (8.9%) also had influenza A virus infections. Analysis of the 43 influenza B virus isolates indicated that 34 (79.1%) were from the Victoria lineage and 9 (20.9%) were from the Yamagata lineage. A comparison isolates in our Victoria lineage with the B/Brisbane/60/2008 strain indicated 12 mutation sites in the HA1 domain, 4 of which (I132V, N144D, C196S, and E198D) were in antigenic epitopes. A comparison of isolates in our Yamagata lineage with the B/Phuket/3073/2013 stain indicated 5 mutation sites in the HA1 domain, none of which was in an antigenic epitope. None of the isolates had a mutation in regions of the neuraminidase gene (NA) that are known to confer resistance to NA inhibitors. CONCLUSION: In Guangzhou during 2016, most influenza B virus isolates were from the Victoria lineage, in contrast to the vaccine strain recommended by the WHO for this period.

Population seroprevalence of antibody to influenza A(H7N9) virus, Guangzhou, China.

BACKGROUND: Since the identification in early 2013 of severe disease caused by influenza A(H7N9) virus infection, there have been few attempts to characterize the full severity profile of human infections. Our objective was to estimate the number and severity of H7N9 infections in Guangzhou, using a serological study. METHODS: We collected residual sera from patients of all ages admitted to a hospital in the city of Guangzhou in southern China in 2013 and 2014. We screened the sera using a haemagglutination inhibition assay against a pseudovirus containing the H7 and N9 of A/Anhui/1/2013(H7N9), and samples with a screening titer ≥10 were further tested by standard hemagglutination-inhibition and virus neutralization assays for influenza A(H7N9). We used a statistical model to interpret the information on antibody titers in the residual sera, assuming that the residual sera provided a representative picture of A(H7N9) infections in the general population, accounting for potential cross-reactions. RESULTS: We collected a total of 5360 residual sera from December 2013 to April 2014 and from October 2014 to December 2014, and found two specimens that tested positive for H7N9 antibody at haemagglutination inhibition titer ≥40 and a neutralization titer ≥40. Based on this, we estimated that 64,000 (95 % credibility interval: 7300, 190,000) human infections with influenza A(H7N9) virus occurred in Guangzhou in early 2014, with an infection-fatality risk of 3.6 deaths (95 % credibility interval: 0.47, 15) per 10,000 infections. CONCLUSIONS: Our study suggested that the number of influenza A(H7N9) virus infections in Guangzhou substantially exceeded the number of laboratory-confirmed cases there, albeit with considerable imprecision. Our study was limited by the small number of positive specimens identified, and larger serologic studies would be valuable. Our analytic framework would be useful if larger serologic studies are done.

Respiratory syncytial virus induces leukotriene C4 synthase expression in bronchial epithelial cells.

BACKGROUND AND OBJECTIVE: Respiratory syncytial virus (RSV) results in acute wheezing in infants and is frequently associated with recurrent wheezing. Although RSV-induced wheezing clinically resembles that of asthma, corticosteroids are not equivalently effective in RSV-associated wheezing. The study sought to determine the mechanisms of RSV-induced wheezing by establishing an in vitro model of RSV-infected human bronchial epithelial cells (16-HBEC). METHODS: Leukotriene C4 synthase (LTC4 S) messenger RNA (mRNA) expression in 16-HBEC was detected using fluorescence quantitative polymerase chain reaction, and the relative level of LTC4 S mRNA was expressed as quotient cycle threshold (qCt) based on the threshold cycle number value compared with that of ß-actin. Cysteinyl leukotrienes (CysLT) in culture supernatant were measured by enzyme-linked immunosorbent assay. RSV-infected 16-HBEC was incubated with gradient concentration of budesonide (BUD) to assess its effects on LTC4 S expression and CysLT secretion. RESULTS: RSV infection resulted in increased LTC4 S mRNA expression between 48 and 96 h post-infection. High level of CysLT was detected in the supernatant of RSV-infected 16-HBEC. BUD at concentrations of 10(-10) to 10(-5) mol/L did not significantly alter LTC4 S mRNA expression. CONCLUSIONS: RSV infection upregulated LTC4 S expression in HBEC leading to increased CysLT secretion. Such induction was not attenuated by BUD, suggesting that CysLT might contribute to the pathogenesis of RSV-induced wheezing.

Detection of CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood of patients with chronic autoimmune urticaria.

BACKGROUND/OBJECTIVES: Compelling evidence indicates a significant role for a population of CD4(+) T regulatory cells in suppressing immune responses and in maintaining immunological homeostasis. This study aims to investigate the potential role of CD4(+) CD25(HIGH) FOXP3(+) T regulatory cells in patients with chronic autoimmune urticaria and to define the characteristics of CD4(+) CD25(HIGH) FOXP3(+) cells in chronic urticaria. METHODS: We used flow cytometry to assess the expression of CD4(+) CD25(HIGH) FOXP3(+) cells in the peripheral blood mononuclear cells of patients with chronic autoimmune urticaria. RESULTS: In this study, we found that patients with chronic autoimmune urticaria have a significantly reduced frequency of CD4(+) CD25(HIGH) FOXP3(+) cells (1.39 ± 0.27% vs 2.09 ± 0.34%; P = 0.001) in their peripheral blood, accompanied by a decreased intensity of FOXP3 expression (50.13 ± 9.79 vs 68.19 ± 6.40; P < 0.001). Notably, although patients with chronic idiopathic urticaria had a reduced frequency of CD4(+) CD25(HIGH) FOXP3(+) cells (1.85 ± 0.46% vs 3.64 ± 0.48%; P < 0.001), their FOXP3 expression levels did not differ from those in healthy controls. CONCLUSIONS: Patients with chronic autoimmune urticaria displayed a reduced percentage of CD4(+) CD25(+) FOXP3(+) regulatory T cells. The results imply CD4(+) CD25(+) FOXP3(+) regulatory T cells may contribute to the autoimmune pathological process of chronic autoimmune urticaria.

[Adamantane resistance among seasonal influenza A viruses between January to October in Guangzhou, 2009].

OBJECTIVE: To study the prevalence of adamantane-resistance among influenza A viruses isolated from Guangzhou between January and October in 2009, and to provide more information for clinical usage of adamantane drugs. METHODS: Totally 311 influenza A strains isolated from 6 hospitals in Guangzhou between January and October in 2009 were selected, and the MP gene of all 311 strains (159 strains of H1 subtype, 152 strains of H3 subtype) was sequenced. The susceptibility of viruses to rimantadine was assayed by biological methods in cells. RESULT: A hundred and forty-eight strains of influenza A (H1) viruses (93.1%, 148/159) were resistant to the adamantanes, and all the 152 influenza A (H3) viruses were resistant to the adamantanes. An amino acid substitution S31N was found in most of the strains except 1 strain with double mutation V27A/S31N. Furthermore, the M gene of influenza A (H1) viruses was divided into genotype B (human) (97/159) and genotype F (European and Australian birds, 62/159), while the M gene of influenza A (H3) viruses was genotype B (human) (152/152). CONCLUSION: Resistance rate of seasonal influenza A viruses isolated from Guangzhou was high. The MP gene of influenza A (H1) may be replaced by a gene from European and Australian birds through a reassortment event.

[A pathogenic and clinical study of 882 cases of adult influenza-like illness in Guangzhou].

OBJECTIVE: This study was undertaken to describe the viral etiology and clinical features in patients with influenza-like illness (ILI) in Guangzhou. METHODS: The nasopharyngeal and throat swabs were collected from 882 patients presenting with ILI between January and September, 2009. Viral pathogens were cultured and identified by immunofluorescence technique using the Shell-Vial method. The clinical data were statistically analyzed. RESULTS: (1) Viral etiology. Of the 882 samples, 385 (43.7%) were confirmed to have at least one of the 9 different respiratory viruses detected. Among these viral isolates, 67.3% (259/385) were seasonal influenza A virus, 27.8% (107/385) were influenza B virus, and 1.3% (5/385) were human parainfluenza virus (PHIV) 1, 2, or 3. In addition, 2 cases (0.5%) of each adenovirus, HSV-1, enterovirus and respiratory syncytial virus (RSV) were also found in the samples. Co-infections with more than one virus were revealed in 8 (2.1%) of 385 samples tested, among them 6 samples were mixture of influenza A and influenza B, 1 sample was positive for both influenza B virus and HPIV-3, and 1 was for both adenovirus and RSV. Seasonal influenza B virus appeared endemic between March and May, and seasonal influenza A virus became dominant between June and August. (2) Clinical features. The percentage of patients aged from 18-30 years was much higher than that of other age groups. The most common symptoms were moderate fever and sore throat, followed by cough. The percentage of upper respiratory infection and pneumonia was 88.4% (727/882) and 10.7% (95/882) respectively. Clinical features did not discriminate between patients with seasonal influenza A and those with influenza B virus infection. The average numbers of leukocytes and lymphocytes were lower in the group positive for influenza viruses than in virus negative group. The patients with adenovirus, HPIV and RSV infection were significantly younger. No rash was observed in patients with enterovirus or HSV infection. CONCLUSIONS: (1) Seasonal influenza virus was the major viral etiologic agent of ILI in Guangzhou during the first 9 months in 2009. Influenza B and A viruses seasonally prevailed in spring and summer, respectively, while other viral etiologic agents appeared to be sporadic. (2) The analysis of clinical features in patients with ILI indicated that fever was the most common symptom, with body temperature varying greatly, and may be associated with evident respiratory and occasionally systemic symptoms. Among the cases with viral infection, the upper respiratory presentation was universal, and pneumonia was frequently noticed.

Inhibitory effect of emodin on bleomycin-induced pulmonary fibrosis in mice.

1. Currently, there is no satisfactory treatment for pulmonary fibrosis. Emodin, a component in Chinese herbs, has been shown to have an antifibrotic effect on pancreatic fibrosis and liver fibrosis. In the present study, we tested the hypothesis that emodin may attenuate the development of pulmonary fibrosis. 2. Mice were randomly divided into five groups (n = 16 in each). One group was a control group; the remaining four groups were treated with intratracheal instillation of 3 mg/kg bleomycin (BLM). The following day, emodin (5, 10 or 20 mg/kg per day, p.o.) treatment was started for three of the BLM-treated groups and was continued for 21 days. The fourth BLM-treated group (and the control group) received daily 0.5% sodium carboxymethyl cellulose (placebo) by gavage over the same period. 3. Bleomycin challenge provoked severe pulmonary fibrosis, with marked increases in fibrosis fraction, hydroxyproline content and myeloperoxidase activity in lung tissue. Emodin treatment (10 and 20 mg/kg per day, p.o.) attenuated all these biochemical indices, as well as histopathological alterations induced by BLM. Furthermore, in mice injected with BLM, elevated levels of transforming growth factor-beta1, interleukin (IL)-4 and IL-13 were found in bronchoalveolar lavage fluid. These increases were significantly inhibited by 10 and 20 mg/kg per day emodin. 4. In cell culture, exposure of cells to 6.25, 12.5, 25 or 50 micromol/L emodin for 24 h decreased fibroblast proliferation. Treatment of cells with the same concentrations of emodin for 72 h decreased collagen production by fibroblasts. In addition, emodin (6.25, 12.5, 25 or 50 micromol/L) inhibited the steady state expression of alpha1 (I) procollagen and alpha2 (I) procollagen mRNA in a dose-dependent manner. 5. The results of the present study suggest that emodin may be effective in the treatment of pulmonary fibrosis.

[In vitro experimental study on the effect of resveratrol against several kinds of respiroviruses].

OBJECTIVE: To observe the cytopathogenic inhibitory effect of resveratrol on vary respiroviruses and explore the mechanism of resveratrol against viruses. METHODS: MDCK, A549, HEp-2 cell and MRC-5 were infected with Influenza virus type A FM1 strain, rhinovirus type R14, RS virus, AD virus type 7 separately, and the antiviral activity of resveratrol were observed. RESULTS: Resveratrol significantly inhibited cytopathogenic effect of AD virus type 7 at the concentration 120 microg/ml. No significant cytopathogenic effect of Resveratrol inhibiting Influenza virus type A FM1 strain, Rhinovirus type R14, RS virus on separate cells was observed. CONCLUSION: It is concluded that resveratrol is effective on inhibiting AD virus type 7 in vitro, however, its mechanism is needed for further study.

Glycomic signatures on serum IgGs for prediction of postvaccination response.

Millions of individuals are vaccinated worldwide each year to stimulate their adaptive immune systems to produce protective antibodies and T-cell response against pathogens. Since glycosylation of the Fc region of immunoglobulin G (IgG) can be influenced by the host's immune status, it was inferred that glycosylation profile of IgG might be altered as a result of the immune response. Therefore, subclass-specific glycosylation profiles of serum IgGs from 26 healthy adults before and after vaccination with a trivalent subunit influenza virus vaccine were comprehensively analyzed to explore glycomic signatures for vaccination. The results showed that no significant changes in the glycosylation of total IgGs took place before and after vaccination, but distinct glycosylation profiles in responders (fourfold or more increase of HI titer after vaccination) and nonresponders (less than fourfold increase of HI titer) were observed. This difference between the responders and nonresponders occurred even in the resting state. On the basis of variable importance parameters, glycosylation markers that distinguish responders from nonresponders were identified. These markers can be used as molecular signatures to predict antibody titers after vaccination. This is the first study of serum IgG glycosylation profiles in healthy adults receiving a trivalent inactivated influenza vaccine.

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.

A prospective comparison of the epidemiological and clinical characteristics of pandemic (H1N1) 2009 influenza A virus and seasonal influenza A viruses in Guangzhou, South China in 2009.

Comparisons of the clinical characteristics of contemporaneous pandemic (H1N1) 2009 influenza A virus (A(H1N1)pdm09)- and seasonal influenza viruses-infected patients are important for both clinical management and epidemiological studies. A prospective multicenter observational study was conducted using a preestablished sentinel surveillance system in Guangzhou, China during 2009. In this study, the clinical presentations of patients with either acute respiratory infection or community-acquired pneumonia were recorded, and nasopharyngeal swab samples were collected for detection of respiratory virus strains using cell cultures or real-time reverse transcription/real-time polymerase chain reaction. Comparisons of the clinical features between A(H1N1)pdm09- and seasonal influenza viruses-infected patients were conducted accordingly. Of the 1,498 patients examined, 265 tested positive for A(H1N1)pdm09, 286 were positive for seasonal influenza A viruses, and 137 for influenza B viruses. The predominant virus was influenza B before the emergence of A(H1N1)pdm09 (epidemiological week [EW] 1-EW 21); then, predominantly non-A(H1N1)pdm09 influenza A and, later, A(H1N1)pdm09, which peaked in EW 46. Compared with the common seasonal influenza-infected patients, A(H1N1)pdm09-infected patients were younger, and had a higher proportion of these patients reported prior contact with infected individuals (P < 0.001, by χ(2) test). However, few significant differences were observed in clinical symptoms and severity among any of the infections caused by the different influenza A strains. Our hospital-based network served as a useful source of information during A(H1N1)pdm09 monitoring. Viral distribution in Guangzhou was characterized by a sharp rise in A(H1N1)pdm09-infected patients in September 2009. Similar to seasonal influenza A-infected cases, A(H1N1)pdm09 cases had a very small proportion of severe cases.

[The effects of a hot water soluble extract (S-03) isolated from Isatis indigotica root on influenza A and B viruses in vitro].

This study was to investigate the antiviral effects of a hot water soluble extract S-03 isolated from Isatis indigotica root on different subtypes of influenza A and B viruses in MDCK cell cultures, using plaque reduction, immunofluorescence and hemo-agglutination inhibition (HAD) assays. Chemical analysis of the extract S-03 showed that it contained high proportion of polysaccharides. The antiviral effects in vitro showed that the S-03 had no effect on different influenza viruses if the drug was used before virus adsorption, but S-03 showed obvious activities against influenza viruses if treatment after virus adsorption or direct reaction of drug and virus before virus adsorption. Hemagglutination inhibition assay showed that S-03 inhibited HA activities of different human influenza viruses (inhibition concentration ranged from 3.12 to 25 mg/mL), avain influenza viruses (inhibition concentration ranged from 25 to 50 mg/mL). The antiviral effects of S-03 on different influenza A and B viruses in vitro might be through the inhibition of the HA to prevent infection.

[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.