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Influenza B virus (IBV) is more likely to cause complications than influenza A virus (IAV) and even causes higher disease burden than IAV in a certain season, but IBV has received less attention. In order to analyze the genetic evolution characteristics of the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018), we constructed genetic evolution trees and analyzed the homology and different amino acids of hemagglutinin and neuraminidase referring to the vaccine strains recommended by World Health Organization (WHO). We found that strain B/Guangxi-Jiangzhou/1352/2018 was free of interlineage reassortment and poorly matched with the vaccine strain B/Colorado/06/2017 of the same year. We also determined the median lethal dose ( LD 50 ) and the pathogenicity of strain B/Guangxi-Jiangzhou/1352/2018 in mice. The results showed that the LD 50 was 10 5.9 TCID 50 (median tissue culture infective dose), the IBV titer in the lungs reached peak 1 d post infection and the mRNA level of the most of inflammatory cytokines in the lungs reached peak 12 h post infection. The alveoli in the lungs were severely damaged and a large number of inflammatory cells were infiltrated post infection. The study demonstrated that the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018) could infect mice and induce typical lung inflammation. This will facilitate the research on the pathogenesis and transmission mechanism of IBV, and provide an ideal animal model for evaluation of new vaccines, antiviral and anti-inflammatory drug.

Objective: To analyze the genomic characteristics of human infection with H9N2 avian influenza virus in Gansu province. Methods: The etiological analysis was conducted for human infection with H9N2 avian influenza virus detected in influenza like illness cases in northwestern China in 2016. Molecular bioinformatics Mega 7.0 software was used to analyze the full genomic sequences of the viral isolate. Results: The gene fragments of HA, NA, MP, NP, NS, PA, PB1 and PB2 of the isolate were highly similar (>90%) to those of H9N2 avian influenza virus strain isolated in external environment in Gansu from 2014 to 2019. The HA gene belonged to BJ/94-like branch, PB2 and MP belonged to G1/97-like branch, and the PB1, PA, NS, and NP genes belonged to F/98-like branch. MP and PB2 were closely related to H7N9, H10N8 and H5N6 viruses. Amino acid sequence alignment showed that the HA cleavage site was arranged in PSRSSR ↓ GLF, H183N and Q226L mutated which included 7 HA glycosylated sites; 62-64 sites of NA absented 3 amino acids (ITE); and M2-31N, NS1-42S, PA-356R, and PA-409N mutated. Conclusions: Apparently, this case of human infection with human infection with H9N2 avian influenza virus was an incidental. However, the isolates of H9N2 influenza virus in external environment of Gansu had a series of mammalian adaptive molecular markers, suggesting that the risk of human infection is higher. It is necessary to strengthen the surveillance by multi departments to deal with influenza pandemic.

Objective: To analyze the etiologic and epidemiological characteristics of adult acute respiratory infections in Shanghai during 2015-2017. Methods: Data was collected from outpatients with acute respiratory infections who visited the Fever Clinics in three hospitals of different levels in three administrative regions of Shanghai, from 2015 to 2017. Basic information and nasopharyngeal swabs were collected from cases in line with the inclusion criteria. Multiplex RT-PCR and bacterial cultures were performed to detect the respiratory pathogens. Results: A total of 806 individuals were enrolled from 2015 to 2017. Respiratory pathogens were identified in 73.45% (592/806) of the cases, with the virus detection rate as 66.75% (538/806). It was found that the major respiratory pathogens for virus detection were influenza A in 326 (40.45%), influenza B in 116 (14.39%), rhinovirus/enterovirus in 39 (4.84%) of the cases. The overall detection rate of bacteria was 16.13% (130/806), including Klebsiella pneumoniae in 90 (11.17%) cases, Staphylococcus Aureus in 46 (5.71%) cases. Other kind of bacteria were not detected in our study. The detection rates on Mycoplasma pneumoniae was 5.33% (43/806) and on Chlamydia pneumonia was 0.37% (3/806). Co-infection with multiple pathogens was detected in 18.61% (150/806) of the cases, including 135 with double infection (accounting for 90.00%), 14 with triple infection and 1 with quadruple infection (accounted for 9.33% and 0.67%, respectively). Among the 150 cases with co-infections, the main identified pathogens were influenza A, Klebsiella pneumoniae, Staphylococcus aureus, and Mycoplasma pneumoniae. Pathogens of acute respiratory infections that identified among the outpatients from the Fever Clinics at different time, region or population, the characteristics were different ( P <0.001). Conclusions: In 2015-2017, outpatients with acute respiratory infections in Shanghai were mainly caused by influenza virus or other viruses, however dynamically with its composition, time, region and characteristics of the population. It is necessary to strengthen and combine related medical and preventive services and to develop the appropriate strategies regarding clinical diagnosis and treatment.

Objective: To understand the epidemiological and pathogenic characteristics of hospitalized severe acute respiratory infections (SARI) in Shanghai, China. Methods: From 2015 to 2017, one Tertiary hospital and one Secondary hospital were chosen as the surveillance sites. Two respiratory tract specimens per case were collected from SARI cases aged 15 years and older. One specimen was tested for 22 respiratory pathogens by RT-PCR, and the other specimen was cultured for 6 respiratory bacteria. Results: A total of 287 SARI cases were enrolled for sampling and lab testing. 70.73% of the cases were aged 60 years and older, with 41.46% (119/287) were positive for at least one pathogen. Influenza virus was the predominant pathogen, accounting for 17.77% (51/287) of all SARI cases. Human rhinovirus/Enterovirus and Coronavirus were both accounting for 7.32% (21/287), followed by Mycoplasma pneumoniae (5.57%, 16/287). The positive rates of parainfluenza virus, bocavirus, adenovirus, respiratory syncytial virus and human metapneumo virus were all less than 5%. Bacterial strains were identified in seven SARI cases, including Klebsiella pneumoniae (3 strains), Staphylococcus aureus (2 strains), Streptococcus pneumoniae (1 strain) and Pseudomonas aeruginosa (1 strain). Two or Three pathogens were co-detected from 40 cases, accounting for 33.61% of 119 positive cases. The most common co-detected pathogens were influenza virus and Mycoplasma pneumoniae (10 cases). Influenza cases peaked in winter-spring and summer. Mycoplasma pneumoniae peaked in winter-spring season and overlapped with influenza. The positive rates of pathogens were not significantly different between different age groups. Conclusions: Various respiratory pathogens can be detected from SARI cases aged 15 years and older. Influenza virus was the predominant pathogen and the co-detection of influenza virus with Mycoplasma pneumoniae the most common one.

Objective: To investigate the clinical manifestations, diagnosis, and treatment of H1N1 influenza A-associated encephalopathy (IAE) in children. Methods: The clinical manifestations, laboratory tests, cranial magnetic resonance imaging (MRI), electroencephalography (EEG) examinations and treatments of seven children with H1N1 IAE hospitalized in Guangzhou Women and Children's Medical Center from December 2018 to January 2019 were retrospectively analyzed. Results: Five of the seven children with H1N1 IAE were female. The age at admission was 4 years and 5 months (range 7 months-9 years). Neurological symptoms occurred simultaneously or early (0-3 days) after the flu-like symptom appeared. The main clinical manifestations of neurological symptoms were seizures (repeated seizures in five cases and status convulsion in two cases, including one case of unexpected fever and repeated seizures in a nine-year old girl) accompanied with altered consciousness (drowsiness in five cases and coma in two cases). Cranial MRI in three cases displayed multifocal lesions, mainly in the bilateral thalamus, brainstem and cerebellar hemisphere. MRI also showed reversible splenial lesion in the corpus callusumin in three cases. EEG tracings were characterized by diffuse slow wave activity in four cases, and status epilepticus was monitored in one case. All the 7 cases were treated with oral oseltamivir. Three cases were treated with pulsed methylprednisolone and intravenous immunoglobulin. One case was treated with intravenous immunoglobulin alone and all the patients received oral oseltamivir. All the patients survived, with three patients had minor neurological sequelae at discharge. Conclusions: The main clinical manifestations of H1N1 IAE are seizures and altered consciousness. Cranial MRI combined with EEG is helpful for early diagnosis. Intravenous immunoglobulin and (or) methylprednisolone should be considered for severe cases.

The number of H7N9 bird flu cases was high and the situation was grim in guizhou province in 2017. To understand the molecular characteristics of the hemagglutinin gene (HA) and the risk of human infection with avian influenza virus A(H7N9) in Guizhou Province, 2017. Homology, genetic evolution and pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of 14 avian influenza viruses A(H7N9) were analyzed by a series of bioinformation softwares. It was cleared that there was 95.9%-100% similarity among 14 strains in nucleotide of the HA gene, and there were 96.8%-97.8% and 96.8%-97.9% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Phylogenetic analysis showed that 14 HA genes were directly evolved in the Yangtze River Delta evolution branch, but they could be derived from five diffenrent strains. Then 13 of 14 strains cleavage site sequences of HA protein revealed they were low pathogenic avian influenza viruses, while A/Guizhou-Weining/CSY01/2017 was high pathogenic avian influenza virus. Mutation G186V at the receptor binding sites in the HA was found in all 14 strains, and mutation Q226L in 13 strains besides A/Guizhou-Weining/CSY01/2017. All five potential glycosylation motifs in the HA were conservative.

Objective: To describe the clinical characteristics of acute respiratory distress syndrome (ARDS) caused by severe influenza virus in our hospital. Method: From November 2017 to February 2018, 48 patients were diagnosed with ARDS caused by influenza virus pneumonia. There characteristics, treatment, and outcomes were summarized and analyzed. According to the prognosis, the patients were divided into a survival group and a death group for comparative analysis. The multivariable logistic regression analysis was used for risk factors of hospital mortality. Result: Thirty-one patients were confirmed to have influenza A virus infection, 3 influenza B virus, 2 mixed infection, while 12 patients were diagnosed by clinical criteria. Eighteen patients died, with a mortality of 38%. Twenty-six patients (54%) were co-infected by other pathogenic microorganisms, including 17 cases of invasive aspergillus infection. Sixteen patients (33%) acquired nosocomial infection that significantly correlated with the hospital mortality, P= 0.003. Multivariable logistic regression analysis showed that higher age ( OR= 1.086, 95% CI: 1.016-1.016), P= 0.015), longer duration of flu-like symptoms to RICU time ( OR= 1.196, 95% CI: 1.031-1.031, P= 0.018) and lower PaO(2)/FiO(2) ( OR= 0.973, 95% CI: 0.951-0.951, P= 0.017) were independent risk factors for hospital mortality. Conclusion: In the flu season of 2017-2018, the patients of ARDS caused by severe influenza pneumonia were mainly influenza A virus, with a mortality of 38%. Co-infection was very common, especially aspergillus infection. Nosocomial infection was common and significantly correlated with high mortality. Lower PaO(2)/FiO(2), higher age and the longer duration from influenza-like symptoms to RICU was independent risk factors for high hospital mortality.

Objective: Human infection with avian influenza A (H7N9) is easy to induce severe acute respiratory distress syndrome (ARDS), and traditional mechanical ventilation cannot correct hypoxemia, so patients may die from multiple organ failure (MOF) caused by persistent hypoxia. Extracorporeal membrane oxygenation (ECMO) can provide effective respiratory support and win time for the treatment of severe H7N9. The first case of severe H7N9 in Guangdong Province in 2018 was admitted to Zhongshan Hospital Affiliated to Sun Yat-sen University. The case was insult with severe ARDS caused by H7N9, the traditional mechanical ventilation could not correct hypoxemia, and the lung condition gradually improved with ECMO assistance. After 13 days of ECMO support, the patient was successfully weaned from ECMO and was transferred to a general ward after 55 days. After 102 days of rehabilitation, the patient was discharged from hospital and followed up for 2 months, who was in good health and had a good quality of life. This article states the diagnosis and treatment of severe H7N9 in details, providing experience for the treatment of severe H7N9 in the future.

We report and analyze the clinical data of the first case of severe pneumonia caused by influenza B virus from swine. The patient, a 62 year-old male domestic pig breeder, was admitted to hospital because of fever and muscle pain for 5 days, and anhelation for 3 days. One week before the onset of disease, the patient kept close contact with pigs. CT scan of the chest showed diffuse infiltration in both lungs. Influenza B virus antigen detection (colloidal gold method) was repeatedly positive. These all confirmed influenza B virus infection. Poor appetite, weight loss, cough, poor spirit of pigs, positive influenza B virus antigen test occurred in the pig, while the patient had no history of exposure to influenza B-infected patients. It was likely that influenza B virus was transmitted from domestic pigs to the patient by droplets or close contact. Influenza B virus epidemics always occur every five or six years a time, and patients and carriers are the main source of infection. After searching the Pubmed, Web of science, Elsevier, Wanfang, and CNKI databases, it was found that although there were many studies on influenza B virus infecting seals, ferret, domestic pigs, guinea pigs, and other animals, there was no case report for animal-to-human infection. It is the first case report of type B influenza virus transmission from domestic pigs to people in the world, which provides a new direction for the research and prevention of influenza B virus.

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.

Objective: To understand the epidemiological characteristics of influenza in Guangdong province, during the winter of 2017-2018, to provide evidence for response to the diversity of influenza, in different seasonal patterns. Methods: Data on weekly influenza surveillance from January 2016 to April 2018, were collected in Guangdong. Information on patients with Influenza-like illness (ILI), on influenza virus positive rates and on outbreaks during the winter of 2017 to 2018, was analyzed and compared with those in spring of 2016 and summer of 2017. χ (2) test and Fisher exact test were used. Results: In the above said winter, the average percentage of visits for ILI in 28 hospitals where sentinel surveillance program had been set, was 4.99% (157 235/3 149 656), which was above the level of the same period in the previous five years. The positive rates of influenza virus among samples collected from ILI outpatients and hospitalized cases under severe acute respiratory infection (SARI) were 28.33% (2 137/7 543) and 14.93% (256/1 715), with the proportions of B (Yamagata) as 70.43% (1 505/2 137) and 73.05% (187/256) respectively. A total of 257 influenza outbreaks were reported in the winter period, with 82.49% (212/257) occurred in elementary schools. Cases aged 6-14 years occurred in winter and spring appeared of having higher positive rate than those seen in summer ( P <0.05) whereas elderly cases aged 60 and above showed higher positive rate in summer than those in winter and spring two seasons ( P <0.05). Conclusions: Epidemiological characteristics of influenza appeared in Guangdong province, during the winter from 2017 to 2018, were correlated to Influenza B (Yamagata). Capacity on the implementation of surveillance programs and on the coverage of vaccination should be improved and increased in order to control influenza in different epidemic seasons, in Guangzhou.

Objective: To analyze the reasons for the fluctuations in the percentage of outpatient or emergency visits for influenza-like illness (ILI) during the Spring Festival and National Day in 2014-2018 surveillance season. Methods: ILI surveillance data was collected during the period of Spring Festival and National Day in mainland China, and downloaded from Chinese Influenza Surveillance Information System, during the 2014-2018 surveillance season. Results: There was no significant difference noticed in the number of ILI reports in the festival week with weeks before or after in both the southern and northern provinces. The number of outpatient visits was much less than that of the week before and after, but the number of emergency visits was statistically significantly increased. Conclusion: In the holiday peak of ILI%, the major causes was the impact of holiday-off at sentinel hospitals, resulting in a large reduction in the number of outpatient visits in the consulting room during the festivals.

Every influenza pandemic in history would end up with disastrous outcomes on mankind, of which the most notorious one was the "Spanish flu" pandemic in 1918. In the past century, with advanced knowledge on influenza viruses, laboratory technologies and surveillance methods, human beings were not as helpless when facing the influenza pandemic. In order to control the outbreaks and reducing the negative impacts, programs as: setting up and improving the influenza pandemic preparedness and response plan were recognized as important issues on early detection or prompt warning of any influenza virus strain that might lead to potential pandemics. The scheduled and planned control measures towards the pandemic preparedness and response plan had been considered of key importance in mitigating the peak of pandemic or controlling the transmission of virus. Since the "1918 influenza pandemic" , we had reviewed the evolution and development of plans regarding the preparedness and response on influenza pandemic issued by both WHO and China. We also emphasized on the variety of strategies which were linked to the preparedness and response at different historical stages, to provide reference for the pandemic preparedness of the disease, in the future.

The annual seasonal epidemic of influenza caused serious disease burden around the world, and serious social panic and economic losses. Due to the high variability and uncertainty of influenza virus, prevention and control of influenza faces many challenges. Surveillance is a key strategy to prevent and control influenza, and influenza is the first infectious disease to be monitored globally. More than 60 years, influenza surveillance programs in China has made great contributions to the prevention and control of influenza in China and the world. Especially in the past 10 years, the influenza surveillance network has developed rapidly, the scale has been expanded significantly, the monitoring content and scope have been continuously improved, and the monitoring quality has been rapidly improved. The China Influenza Surveillance Network is one of the early detection systems for emerging infectious diseases in China and the world. It helps to improve the capacity of public health system in prevention and control and early warning of emerging infectious diseases.

Objective: To describe the clinical, chest imaging, pathological manifestations and therapeutic experience of human infection with A/H7N9 virus. Methods: The features of 15 laboratory-confirmed cases of human infection with A/H7N9 virus in Taizhou, Jiangsu Province were retrospectively analyzed. Results: The 15 patients with confirmed viral pneumonia included 12 males and 3 females, with a median age of 61 years(ranging from 33 to 81 years). Twelve patients had a history of exposure to the poultry trading places, or direct contact with ill/dead avian, while 3 patients denied exposure or contact. The most common initial symptoms were fever, coughing, and respiratory distress. The illness progressed rapidly to acute respiratory distress syndrome (ARDS). Lab tests showed normal (8 cases) or decreased (7 cases)white blood cell count , decreased (13 cases) lymphocyte count and proportion , increased creatine kinase (CK, 12 cases) and lactate dehydrogenase (LDH, 15 cases), and respiratory failure (13 cases). Chest radiographic examination showed that the most common features were inflammatory infiltration in the lung, with partial consolidation. The average time of the diagnosis with influenza viral nucleic acid and onset of an oral anti-influenza drug were 7.1 days and 6.5 days. All patients were treated by antiviral drugs (oral oseltamivir 150 mg q12 h and/or intravenous paramivir 600 mg qd), with mechanical ventilation in 9 cases, glucocorticoid therapy in 5 cases (intravenous methylprednisolone in 3 and dexamethasone in 2 patients), extracorporeal membrane oxygenation (ECMO) therapy in 2 cases, continuous renal replacement therapy (CRRT) in 6 cases, and artificial liver therapy in 1 case. The pulmonary pathology was observed from post-mortem biopsy for 2 fatal cases. Patient 1 had diffuse alveolar damage with inflammatory exudation, hyaline membrane formation, and cellular infiltration. Patient 2 had widened alveolar septum, lymphocyte and monocyte cell infiltration in the alveolar septa, and interstitial fibrous proliferation. Nine patients were discharged, and 6 died. Conclusions: Patients with influenza A/H7N9 virus mostly presented with fever, cough, and were prone to progression to viral pneumonia. Once acute respiratory distress and important organ dysfunction occurred, the fatality rate was higher. Early diagnosis and rational treatment were critical for better outcomes.

Objective: To investigate the infection pattern and etiological characteristics of a case of human infection with highly pathogenic avian influenza A (H7N9) virus and provide evidence for the prevention and control of human infection with highly pathogenic avian influenza virus. Methods: Epidemiological investigation was conducted to explore the case's exposure history, infection route and disease progression. Samples collected from the patient, environments and poultry were tested by using real time reverse transcriptase-polymerase chain reaction (RT-PCR). Virus isolation, genome sequencing and phylogenetic analysis were conducted for positive samples. Results: The case had no live poultry contact history, but had a history of pulled chicken processing without taking protection measure in an unventilated kitchen before the onset. Samples collected from the patient's lower respiratory tract, the remaining frozen chicken meat and the live poultry market were all influenza A (H7N9) virus positive. The isolated viruses from these positive samples were highly homogenous. An insertion which lead to the addition of multiple basic amino acid residues (PEVPKRKRTAR/GL) was found at the HA cleavage site, suggesting that this virus might be highly pathogenic. Conclusions: Live poultry processing without protection measure is an important infection mode of "poultry to human" transmission of avian influenza viruses. Due to the limitation of protection measures in live poultry markets in Guangzhou, it is necessary to promote the standardized large scale poultry farming, the complete restriction of live poultry sales and centralized poultry slaughtering as well as ice fresh sale.

Objective: To understand the epidemiological characteristics of human infection with avian influenza A (H7N9) virus in China, and provide evidence for the prevention and control of human infection with H7N9 virus. Methods: The published incidence data of human infection with H7N9 virus in China from March 2013 to April 2017 were collected. Excel 2007 software was used to perform the analysis. The characteristics of distribution of the disease, exposure history, cluster of the disease were described. Results: By the end of April 2017, a total of 1 416 cases of human infection with H7N9 virus were confirmed in China, including 559 deaths, the case fatality rate was 39.5%. In 2016, the case number was lowest (127 cases), with the highest fatality rate (57.5%). The first three provinces with high case numbers were Zhejiang, Guangdong and Jiangsu. The median age of the cases was 55 years and the male to female ratio was 2.3∶1. Up to 66% of cases had clear live poultry exposure history before illness onset, 31% of cases had unknown exposure history and only 3% of the cases had no live poultry exposure history. There were 35 household clusters (5 in 2013, 9 in 2014, 6 in 2015, 5 in 2016, 10 in 2017), which involved 72 cases, accounting for 5% of the total cases. Conclusions: The epidemic of human infection with H7N9 virus in China during 2013-2017 had obvious seasonality and spatial distribution. There was limited family clustering. Infection cases were mostly related to poultry contact.

Objective: To study the epidemiologic characteristics of influenza in Gansu province of China and to optimize the related vaccination program. Methods: Weekly influenza surveillance data from the first week of 2010 to the fortieth week of 2016 were collected, in Gansu province. χ (2) test was used to compare the differences of nucleic acid positive rate and the virus types in the four seasons. Time series seasonal decomposition (TSSD) was used to explore seasonal patterns and characteristics of influenza epidemics in Gansu. Results: 59 791 specimens were tested, with 8 501 positive for influenza virus and positive rates as 14.22 % . Types A(H1N1)pdm09, A(H3N2) and type B were accounted for 98.76 % of all the positive specimens. Proportions of the positive rate of influenza virus appeared in spring, summer, autumn and winter were 15.12 % , 0.98 % , 4.02 % and 24.26 % respectively. The predominant type of virus in autumn and winter was A(H3N2), with B mainly in spring. Influenza in Gansu province showed typical single-peak type distribution, with epidemic peak appeared from December to next January. The type A(H3N2) related peak appeared the earliest, followed by A(H1N1) pdm09, with type B the latest. Conclusions: Peaks and the duration of influenza seasonal epidemics were related to the types of dominant strains. Annual influenza vaccination campaigns should start in October, to provide effective protection during the epidemic period.

We compared the effect of oseltamivir on the hemagglutination (HA) test and hemagglutinin inhibition (HI) test of the influenza A(H3N2) virus in China to obtain the "true" HA titer and antigenic variation. A total of 395 influenza H3N2 strains isolated in mainland China from October 2014 to May 2015 were analyzed with HA and HI tests, with or without oseltamivir.. Gene sequencing was undertaken for selected viruses, and the amino-acid sequence of neuraminidase (NA) protein was compared with the vaccine strain. In the HA test in the presence of oseltamivir, the HA titer was unchanged in 44. 8%, decreased in 43. 8%, and increased in 11. 4% of tested strains. In the presence of oseltamivir, the proportion of viruses similar to A/TX/50/2012 egg isolates was significantly higher, and the proportion of viruses similar to A/SZ/9715293/2013 cell isolates significantly lower, than the proportion obtained from the test without the presende of oseltamivir. A significant difference was detected between the tests with or without oseltamivir. In A/TX/50/2012 cell isolates and A/SZ/9715293/2013 egg isolates, no significant difference was detected between the tests with or without oseltamivir. Nineteen selected strains' of influenza A ( H3N2) were sequenced, and the amino-acid sites were compared with A/TX/50/2012 egg isolates. Five strains had a more-than-fourfold decrease in HA titer when addition of oseliamivir showed no common mutation in amino acids, whereas the A/Shandong Laicheng/119/2015 strain had a D151G mutation and the A/Jilin Tiexi/1194/2015 strain had a V4121 and T434A mutation in the NA protein. The strain had a two-to-fourfold decrease in HA titer when addition of oseltamivir showed 126T, G93S, V1491, N234D, T267K and S416G mutations in NA protein.. These data show that, for recent circulating influenza H3N2 viruses, the presence of oseltamivir can be used to obtain more accurate HA and HI titers for antigenic analysis and vaccine evaluations.

Digital polymerase chain reaction (dPCR) is a new method for absolute quantification. However, an absolute quantification method for influenza A viruses based on droplet digital polymerase chain reaction (dPCR) has not been established. In this study, we found that: (i) the annealing temperature of dPCR was optimized at 64, 4°C; (ii) the detection range of dPCR was 37.7 approximately 8.22 X 10(4) copies/μl for influenza A viruses; (iii) the limit of detection of dPCR was 3. 77 copies/reaction. The liner correlation coefficient (R(2)) was found to be 0. 9988, suggesting the high reliability of our dPCR method. The dPCR method could be used to detect influenza A viruses clinically. In summary, we developed a dPCR method for absolute quantification of influenza A viruses. This method could be used effectively to quantify influenza A viruses in clinical samples. Therefore, our method could be a new tool for the absolute quantification of viral load.

Non-coding RNAs (ncRNAs) are a class of RNAs that have no potential for protein coding. Increasing numbers of studies have provided strong evidence that ncRNAs play important roles in regulation of various biologic processes, including interactions between viruses and the host. Influenza viruses remain a major, threat to human health and animal livestock. Interactions between the host and mutations of influenza viruses are very complicated. Recent data have shown that many ncRNAs play important roles in the interactions between influenza viruses and the host. Understanding the fuiction of these ncRNAs in the infection and replication of influenza viruses is very important to elucidate the pathogenesis of these viruses, and to provide strategies for the prevention and control of influenza. This review summarizes the ncRNAs that act as key regulators of interactions between the host and influenza viruses.

The aim of this study was to explore the influence of mutation of different non-structural (NS) 1 amino-acid residues on the pathogenicity of influenza viruses and the function of NS1 virulence-related sites on the pathogenesis of influenza viruses. We analyzed segments of the NS1 protein gene and key sites related to virulence of influenza viruses based on a literature review. Fragments of the NS1 gene were cloned from the HIN1 subtype PR8F (non-mutated) and preserved by our research team with encoding sequence site-specific mutagenesis at aa42, aa8l, and aa149. Via a reverse genetics system, we rescued the mutant strains PR8F-42, PR8F-81, and PR8F-149, which were inoculated into chick embryos and could replicate stably after five passages. Efficiency of viral replication was measured by testing hemagglutination titers. BALB/c mice were inoculated With mutated or non-mutated PR8F (10(6) TCIDO(50)/100 μl for each mouse), respectively. The typical clinical manifestations (weight change and survival) were recorded. Autopsies, as well as observations of the pathologic features and pulmonary-tissue slices of mice that died after inoculation, were done. RNA of mouse lungs was extracted, and the residual quantity of virus in lungs was detected by quantitative polymerase chain reaction (qPCR). Results showed that mutation of NS1 at aa42 from Ser to Pro did not change the pathogenicity of PR8F in mice, but a low pathogenicity of PR8F occurred after mutation of NS1 at aa8l and aa149. The present study lays the foundation for further investigations of the function of NS1 pathogenicity-related sites in the pathogenesis of influenza viruses.

Objective: To investigate the features and duration of viral nucleic acid shedding in children with influenza A., Methods: The clinical data of 90 children with influenza A with positive influenza A virus nucleic acid in nasopharyngeal swab detected by PCR were collected, and these children were divided into simple influenza A group (n=10), influenza A-pneumonia group (n=61), influenza A-nervous system damage group (n=10), and influenza A-underlying disease group (n=9). A retrospective analysis was performed for clinical features, treatment process, duration of viral nucleic acid shedding, and prognosis., Results: The most common symptoms in these children were fever (89/90, 99%), cough (89/90, 99%), running nose (69/90, 77%), shortness of breath (26/90, 29%), and myalgia (23/90, 26%). The mean duration of viral nucleic acid shedding in 90 children was 9.4±2.9 days. The simple influenza A group had a significantly shorter duration of viral nucleic acid shedding than the influenza A-pneumonia, influenza A-nervous system damage, and influenza A-underlying disease groups (p<0.05), while there were no significant differences between the influenza A-pneumonia, influenza A-nervous system damage, and influenza A-underlying disease groups (p>0.05). The children who received antiviral therapy within 48 hours after disease onset had significantly shorter duration of viral nucleic acid shedding and time to body temperature recovery than those who received antiviral therapy more than 48 hours after disease onset (p<0.05). Of all the children with body temperature recovery, 83% still tested positive for viral nucleic acid., Conclusions: Complications, underlying diseases, and timing of antiviral therapy are influencing factors for the duration of influenza A virus nucleic acid shedding, and whether body temperature returns to normal cannot be used to decide whether to continue antiviral therapy.

Influenza-A viruses were selected as samples. Then, the relationship between synonymous codon bias and mRNA secondary structures was analyzed. Our data will provide a theoretical basis for further research on the synonymous codon and RNA properties of the influenza-A virus. Information on the nucleic-acid sequence of all influenza-A viruses in the National Center for Biotechnology Information was collected. The secondary structure of each nucleic-acid sequence was predicted. Then, the loop structure, stem structure, and free replicating energy of RNA were calculated. Based on this information, the specific flexibility of RNA was worked out. Simultaneously, the synonymous codon bias in each nucleic-acid sequence was counted. A database on the RNA secondary structure of the influenza-A virus was set up. Then, the relationship between the synonymous codon bias and the content of loop structure, stem structure, and flexibility were analyzed.Codon usage of 50% of amino acids was correlated significantly with the content of stem structure or the content of loop structure.Also,60% of amino acids were correlated significantly with average unit folding free energy. In addition, codon usage of 50% of amino acids was correlated significantly with the specific flexibility of RNA. For codons that were correlated significantly with stem structure and loop structure, the correlation between their usage with two types of structural content was completely contrary .Also, the correlation between specific flexibility with synonymous codon bias was better than that observed for other parameters. These results suggest that synonymous codon usage bias has important influences on RNA secondary structure.

This study aims to research the effect of inter-lineage reassortment of type B influenza virus on its biological characteristics. The representative strains isolated in 2013~2015were selected, which included wild type viruses and inter-lineage reassortment viruses. We tested the growth curve of each virus based on the value of TCID50 at different time point. And further detected the Km value of virus to analysis the activity of neuraminidase of each virus. The growth curve of viruses in 2013 and 2014reached peak 48 hours after infection, maintained at high level until 72 hours, then the virus titer declined gradually, however the virus isolated in 2015 reached peak 24 hours after infection. The reassortment strain B/Fujian Tongan/1565/2013 has the similar growth characteristics with the wild type virus on the same year. Meanwhile the growth curve of other inter-lineage viruses in 2014 and 2015is lower than that of wild type viruses. We determined neuraminidase kinetic constants of all viruses. The reassortment strain B/Fujian Tongan/1565/2013 has the strongest affinity of neuraminidase. The results of 2014~2015virus suggest that the stronger affinity of neuraminidase of virus the better growth characteristics of virus. But the virus in 2013 is lack of such relationship between the growth characteristics and activity of neuraminidase. The neuraminidase activity and growth characteristics of inter-lineage reassortments due to surface protein gene exchange is not consistent, suggesting internal virus protein might have affected the growth characteristics of the viruses2013-2015,and pending further study.

To explore the mechanisms of influenza H7N9 virus pathogenesis, influenza H7N9 virus and H1N1 influenza A virus(H1N1pdm09)-infected A549 cellular models were established, and differential protein expression in A549 cells infected with the two strains were investigated.A549 cells were infected with H7N9 and H1N1pdm09influenza virus at a multiplicity of infection(MOI)of 0.001.The temporal response of A549 cells infected with the two strains was evaluated using the proteomics approaches(2DDIGE combined with MALDI-TOF-MS/MS)at 24,48 and 72hours post infection(hpi).There were 11,12 and 33proteins with significantly different expression at 24,48 and 72hpi,respectively.Compared with H1N1pdm09 infection, functional analysis revealed that the down-regulation of proteins in H7N9 infection including F-actin-capping protein subunit alpha-1(CapZ-α1), ornithine aminotransferase(OAT),poly(rC)-binding protein 1(PCBP1)and eukaryotic translation initiation factor 5A-1(eIF5A)produced cytopathic effects. The down-regulation of platelet-activating factor acetylhydrolaseIb subunit beta(PAFAH1B2)in H7N9-infection may be related to the clinical symptoms of patients infected by the influenza H7N9 virus.

Influenza A H1N1pdm09 Virus; Chicken embryo; Phylogenetic tree; Egg-adaptation; Antigenic site; Drug-resistance site; Abstract: To investigate egg-adapted mutation in influenza A H1N1pdm09 viruses isolated from the Hubei influenza surveillance network, a comparative analysis was performed of three influenza A H1N1pdm09 viruses isolated in chicken embryo and the corresponding MDCK cell-derived viruses. Analyses included examination of the phylogenetic tree, evolutionary rates, amino acid substitutions, egg-adapted mutation and homology modeling. We found differences between the egg-adapted viruses and MDCK cell-derived viruses based on phylogenetic trees and evolutionary rates; the viruses showed a trend of " NA>HA>MP". Four amino acid substitutions(Q223R,V527 I,M19Iand H275Y)were found in three egg-adapted viruses.Q223 Rand V527Iwere present in the haemagglutinin protein, while M19I and H275Y were detected in neuraminidase.The Q223R mutation changed the structure of antigenic sites between Sb and Ca2. H275Y is a classic neuraminidase resistance mutation. The results suggest that the egg-adapted mutations were introduced when influenza viruses were isolated in chicken embryos from the Hubei influenza surveillance network. These mutations may affect the selection of vaccine candidates and vaccine efficacy; therefore, monitoring of egg-adapted mutations should be strengthened in the influenza surveillance network.

Objective: To study the antiviral activities of clemastanin B (CB), epigoitrin, phenylpropanoids portion (PEP) and the mixture of phenylpropanoids, alkaloids and organic acid fractions (PEP+ALK+OA) from Banlangen (Radix Isatidis)., Methods: The experiment consisted of four parts: therapeutic action, prophylaxsis action, inhibition of virus attachment, and direct virucidal action. Cytopathic effect (CPE) and 3-(4,5-Dimethylthiazol-2-yI)-2,5-diphenyltetrazolium (MTT) were used to assess antiviral activity., Results: CB, epigoitrin, PEP and PEP + ALK +OA fractions from Banlangen (Radix Isatidis) extract significantly increased the viability of MDCK cells pre-infected with the virus compared with the virus control group in all the dilutions (P < 0.01). Pretreated with either pure compounds or chemical fractions of Banlangen (Radix Isatidis) extract in all the dilutions significantly improved the viability of MDCK cells (P < 0.01). The inhibition of virus absorption to the host cells by CB, epigoitrin and PEP was in a dose dependent manner., Conclusion: CB, epigoitrin, PEP and PEP+ ALK+ OA exert their anti-influenza activity by inhibiting the virus multiplication, prophylaxsis and blocking the virus attachment. The primary mode of action of PEP and PEP + ALK + OA is the inhibition of virus replication. The inhibitory effects on virus attachment and multiplication are the main modes for epigoitrin.

Objective: To investigate the relationship between the intestinal disorder of anaerobic bacteria and influenza virus infection, and the effect on pulmonary inflammatory cytokines in mice., Methods: Totally 36 mice were randomly divided into normal control group, virus-infected group and metronidazole treatment group (12 mice in each group). Mice in the metronidazole group were administrated orally with metronidazole sulfate for 8 days causing anaerobic bacteria flora imbalance; then all groups except the normal control group were treated transnasally with influenza virus (50 μL/d FM1) for 4 days to establish the influenza virus-infected models. Their mental state and lung index were observed, and the pathological morphological changes of lung tissues, caecum and intestinal mucosa were examined by HE staining. The levels of interleukin 4 (IL-4), interferon γ (IFN-γ), IL-10 and IL-17 in the lung homogenates were determined by ELISA., Results: Compared with the virus control group, the metronidazole group showed obviously increased lung index and more serious pathological changes of the lung tissue and appendix inflammation performance. After infected by the FM1 influenza virus, IFN-γ and IL-17 of the metronidazole group decreased significantly and IL-4 and IL-10 levels were raised, but there was no statistically difference between the metronidazole and virus control groups., Conclusion: Intestinal anaerobic bacteria may inhibit the adaptive immune response in the lungs of mice infected with FM1 influenza virus through adjusting the lung inflammatory factors, affect the replication and clean-up time of the FM1 influenza virus, thus further aggravating pulmonary immune pathological injury caused by the influenza virus infection.

Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China.

Objective: To compare the epidemiological and clinical features of lower respiratory tract infection (LRTI) caused by influenza virus A (IVA) and influenza virus B (IVB) in children., Methods: The clinical data of 366 children with LRTI caused by influenza virus (IV), who were hospitalized in Yuying Children&prime;s Hospital of Wenzhou Medical University between 2010 and 2014, were analyzed retrospectively, and there were 272 cases caused by IVA and 94 cases caused by IVB., Results: IV was mainly prevalent from December to March of the next year, with the predominance of IVA. There were small peaks of IVA prevalence in July or September every other year, and IVB was prevalent from December to March of the next year every other year. The children with LRTI caused by IVA alone had a significantly higher white blood cell (WBC) count and significantly higher percentages of children with increased WBC, abnormal serum sodium, and abnormal serum potassium than those caused by IVB alone (P<0.05). However, there were no significant differences in age, sex, underlying diseases, clinical manifestations, and co-infection rate with bacteria or atypical pathogens between the two groups (P>0.05). The rate of co-infection with respiratory syncytial virus (RSV) was significantly higher in the IVB group than in the IVA group (P<0.01)., Conclusions: IVA is prevalent in winter and spring every year and has small peaks in summer every other year, while IVB is prevalent in winter and spring every other year. Compared with IVB, IVA causes more cases of increased WBC and electrolyte disturbance. The children infected with IVB are more likely to be co-infected with RSV. The children with LRTI caused by IVA and IVB have similar clinical manifestations.

Objective: We explored which internal genes of influenza virus that affect the titer of recombinant viruses and contribute to the high yield of Influenza A seed virus in ovo., Methods: Six internal genes or mutant or polymerase complex of A/Puerto Rico/8/1934 (H1N1) (PR8) virus genes were replaced individually by corresponding gene of A/ chicken/ZJ/China/2013 ( H5N1) virus, and the hemagglutination titers of recombinant viruses were compared by HA assay., Results: PB2 gene had the greatest influence, its replace failed to generate recombinant virus. When PB1, PA, or M gene was replaced, the titers of recombinant viruses dropped by 3.7, 3.4, 3.0 (log2), respectively. NS gene had little influence upon HA titer. When polymerase complex genes were replaced, virus titer dropped slightly to 7.6 log2, and it did not confer the same growth characteristics (8.4 log2) found when a complete polymerase complex was of PR8 origin. When amino acids of position 627 of PR8 PB2 gene were mutated to glutamic acid, virus titer rose from 8.4 log2 to 8.7 log2., Conclusion: The optimal gene combinations may facilitate replication through viral RNA and protein interaction with cellular components as well as interaction of viral RNA and protein or protein-protein interactions within the virus. These multi-factorial contributions resulted in selection of a high replication competent reassortant in embryonated chicken eggs in comparison to the respective low yield wild type viruses, and laid the foundation for high

Objective: To obtain non-structural protein 2 (NS2) of influenza A virus (IAV) via the expression system of Escherichia coli, and observe the effect of NS2 on the production of interferon γ (IFN-γ) in mouse lung tissues., Methods: A prokaryotic expression plasmid pET22b(+)/NS2 was constructed and transformed into Rosetta-gami(2) of Escherichia coli. Through the inducible expression and purification, NS2 was prepared. Forty BALB/c mice were divided randomly into 8 groups, normal control group, 200 μg/kg NS2 joint virus group, 200 μg/kg NS2 joint RNA group, 100 μg/kg inactivated virus group, 0.2×lethal dose 50% (LD50) virus group, viral RNA group of 50 μg/kg, 200 μg/kg NS2 group, and 100 μg/kg NS2 group. All mice were subjected to nasal inhalation. Reverse transcription-PCR and Western blotting were respectively performed to detect the mRNA and protein expressions of IFN-γ in lung tissues., Results: Recombinant NS2 was obtained by the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) and purification. After the nasal inhalation of NS2 or/and IAV to mice, reverse transcription-PCR and Western blotting showed that the levels of IFN-γ mRNA and protein in the lung tissues of NS2 joint virus group were lower than those of the virus group., Conclusion: NS2 could inhibit the expressions of IFN-γ mRNA and protein in lung tissues of mice infected by IAV.

To set up a new rapid method for the rapid determination of influenza virus H5N1 and H7N9 basing on the Multi-Analyte Suspension Array (MASA) technology. Sequence analysis and design of degenerate primers and specific probes were set in the comparison and analysis of H5, N1, H7 and N9 genes. In combination with MASA technology, these primers and probes were used for the determination of samples of H5N1 and H7N9 and other subtypes ( H1N1, PH1N1, H5N2, H3N2 and H9N2). We developed a rapid determination method. This method had high specificity and sensitivity that could detect H5N1 and H7N9 at one time, and could detect samples that containing 10 copies of H5N1 and H7N9. This determination method could be used for rapid determination of influenza virus H5N1 and H7N9 at one time.

To analyze influenza pathogen spectrum in Yunnan province during 2009-2014 years, and analyze HA and NA genes of influenza A H1N1. Analysis was made on the monitoring date of influenza cases in Yunnan province in recent 6 years, 23 strains of influenza virus of HA and NA gene was sequenced and analyzed by MEGA 5 software to construct phylogenetic tree. 4 times of influenza AH1N1 epidemic peak were monitored from 2009-2014 years in Yunnan Province, as the nucleic acid detection results of influenza A H1N1 accounted for 28.8% of the total. The sequencing result showed that HA and NA gene were divided into 3 groups, one was detected with H275Y mutation strains. Influenza A H1N1 is one of the important subtypes in Yunnan province and their genes have divided into three branches during the period of 2009-2014 years, the vast majority of influenza a H1N1 are still sensitive to neuraminidase inhibitors.

To study the effect of Yinghua Pinggan granule (YHPG) against influenza A/H1N1 virus in vivo and on the immunologic function of infected mice. The intranasal influenza virus infection was adopted in ICR mouse to establish the influenza virus pneumonia model. At the 3rd and 7th day after the infection, the lung index and pathologic changes in lung tissues of mice were detected. Realtime PCR and flow cytometry were employed to observe the virus load in lung tissues and the levels of CD4+, CD8+, and CD4+/CD8+ in peripheral blood. The result showed that at the 3rd and 7th day after the infection, YHPG (15, 30 g x kg(-1)) can significant decrease in the lung index and virus load in lung tissues of mice infected with influenza virus, alleviate the pathologic changes in lung tissues, significantly increase the levels of CD4+ and CD4+/CD8+ ratio and reduce the levels of CD8+ in whole blood. This indicated that YHPG can inhibit the influenza virus replication, alleviate pulmonary damage and adjust the weak immunologic function of infected mice, with a certain therapeutic effect on mice infected by H1N1 virus in vivo.

Hemagglutinin (HA) contains a head domain with a high degree of variability and a relatively conserved stem region. HA is the major viral antigen on the surface of the influenza virus. To define the biologic activities of chimeric HA bearing different head domains and stem regions or their potential use, a HA chimeric gene containing the head domain of the H7 subtype virus and stem region of the H3 subtype virus was modified and expressed using a baculovirus expression vector. Then, the secreted protein was purified and its biologic activities characterized. Approximately 1.4 mg/mL cH7/3 HA could be obtained, and its molecular weight was ≈ 70 kD. The trimer form of cH7/3 protein had hemagglutination activity and could be recognized by specific antibodies. The method described here can be used for further studies on the screening of HA stem-reactive antibodies or the development of vaccines with conserved epitopes.

Laggera pterodonta is commonly used for treating influenza in Southwest China, especially in Yunnnan province. The main clinical effects of L. pterodonta include anti-influenza, anti-microbial, anti-inflammatory. To investigate the anti-influenza A (H1N1) virus effect of L. pterodonta, neutralization inhibition and proliferation inhibition tests were performed. MDCK culture method was used to observe the cytopathic effect (CPE) of extracts from L. pterodonta in inhibiting influenza A (H1N1) virus and haemagglutination titre of H1N1 virus in vitro. The culture medium were collected at 24 h, 48 h, 72 h, 96 h, and detected by Real time RT-PCR, in order to compare the effect of different extracts from L. pterodonta on in vitro proliferation of H1N1, virus. The result of neutralization inhibition test showed that hemagglutination titer of ethyl acetate extract were 8 times lower at 72 h; in proliferation inhibition test, hemagglutination titer of ethyl acetate extracts reduced by 2 and 4 times. According to the results of Real time RT-PCR test, the H1N1 inhibition ratio of ethyl acetate extract was 72.5%, while the proliferation inhibition ratio of ethyl acetate extract was 25.3%; as for petroleum ether extracts, the H1N1 inhibition ratio was 60.2%, while the proliferation inhibition ratio was 81.4%. In conclusion, both ethyl acetate extract and petroleum ether extract of L. pterodonta have significant neutralization and direct proliferation inhibition effects on influenza A virus.

The influenza virus has evolved numerous mechanisms to overcome host defenses for its benefit. It can also manipulate the immune system to stop it monitoring and clearing the virus. Small ubiquitin-like modifier (SUMO)ylation is emerging as a key post-translational modification that plays an important part in virus replication. This brief review focuses on recent findings on the roles of SUMOylation during infection by the influenza virus. As such, it will aid understanding of the mechanism of action of infection by the influenza virus, and help to provide new strategies for anti-viral treatment.

Objective: To understand the epidemiological characteristics of influenza outbreaks in China from 2005 to 2013., Methods: The data of influenza-like illness outbreaks involving 10 or more cases were collected through Public Health Emergency Management Information System and National Influenza Surveillance Information System in China, and the influenza outbreaks were identified according to the laboratory detection results. Descriptive epidemiological analysis was conducted to understand the type/subtype of influenza virus and outbreak time, area, place and extent., Results: From 2005 to 2013, a total of 3 252 influenza-like illness outbreaks were reported in the mainland of China, in which 2 915 influenza outbreaks were laboratory confirmed, and influenza A (H1N1) pdm09 virus and influenza B virus were predominant. More influenza outbreaks were reported in the influenza A (H1N1) pandemic during 2009-2010. Influenza outbreaks mainly occurred during winter-spring, and less influenza outbreaks occurred in winter and summer vacations of schools. More influenza outbreaks were reported in southern provinces, accounting for 79% of the total. Influenza outbreaks mainly occurred in primary and middle schools, where 2 763 outbreaks were reported, accounting for 85% of the total. Average 30-99 people were involved in an outbreak., Conclusion: A large number of influenza outbreaks occur during influenza season every year in China, the predominant virus type or subtype varies with season. Primary and middle schools are mainly affected by influenza outbreaks.

Objective: To understand the transmission mode of human infection with avian influenza A (H7N9) virus., Methods: Field epidemiological investigation was conducted for a family clustering of human infection with H7N9 virus in Hengxian county, Guangxi Zhuang Autonomous Region in February 2014. Two patients and their 82 close contacts were surveyed. The samples collected from the patients, environments and poultry were tested by using real time reverse transcriptase-polymerase chain reaction (rRT-PCR), and the samples from patients were used for virus isolation. The samples from 5 close contacts were tested with RT-PCR. The clinical data, exposure histories of the patients and the detection results of the isolates and their homology were analyzed., Results: Patient A became ill 4 days after her last exposure to poultry in Zhongshan, Guangdong province, and returned to her hometown in Hengxian 2 days after onset. Patient B was patient A's 5 years old son, who had no known exposure to poultry but slept with patient A for 4 days. He developed symptoms 4 days after last contact with his mother. Two strains of H7N9 virus were isolated from the two patients. The 2 isolates were highly homogenous (almost 100%) indicated by gene sequencing and phylogenetic tree. None of the other 81 close contacts developed symptoms of H7N9 virus infection., Conclusion: Patients B was infected through close contact with patient A, indicating that avian H7N9 virus can spread from person to person, but the transmissibility is limited and non-sustainable.

Objective: To establish a risk early warning model of human infection with avian influenza A (H7N9) virus and predict the area with high risk of the outbreak of H7N9 virus infection., Methods: The incidence data of human infection with H7N9 virus at prefecture level in China from February 2013 to June 2014 were collected, and the geographic and meteorological data during the same period in these areas were collected too. Spatial auto regression (SAR) model and generalized additive model (GAM) were used to estimate different risk factors. Afterwards, the risk area map was created based on the predicted value of both models., Results: All the human infections with H7N9 virus occurred in the predicted areas by the early warning model in February 2014. The early warning model successfully predicted the spatial moving trend of the disease, and this trend was verified by two outbreaks in northern China in April and May 2014., Conclusion: The established early warning model showed accuracy and precision in short-term prediction, which might be applied in the active surveillance, early warning and prevention/control of the outbreak of human infection with H7N9 virus.

Objective: To construct lentivirus siRNA vectors targeting ezrin (E), radixin (R) and moesin (M), transfect rat primary pulmonary microvascular endothelial cells (PMVECs) with the vectors and observe the effects of the transfection on E, R, M gene expressions and influenza virus replication., Methods: According to the principles of siRNA synthesis, siRNA sequences targeting E, R and M genes were synthesized and cloned into lentiviral plasmid GV248 to construct lentiviral siRNA vectors Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. Random Lenti-control-siRNA was also constructed targeting nonsense sequences. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Rat PMVECs were transfected with Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. The levels of ezrin, radixin and moesin mRNA and proteins were detected by real-time quantitative PCR and Western blotting, the distributions of ERM proteins were detected using confocal laser scanning microscope; After lentivirus-transfected PMVECs were infected with influenza virus, the effects of lentiviral vectors on influenza virus replication were measured by real-time quantitative PCR., Results: Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA and Lenti-control-siRNA were successfully constructed according to sequencing identification of recombinant plasmids. The infection efficacy of lentiviral siRNA vectors in rat PMVECs was above 80% at a multiplicity of infection (MOI) of 10. The transcription and expressions of ezrin, radixin and moesin in rat PMVECs were significantly inhibited by Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA respectively. Lentivirus control (containing no target genes) and Lenti-control-siRNA inhibited influenza virus replication in influenza virus-infected PMVECs, while Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA increased the levels of influenza virus mRNA in the infected PMVECs., Conclusion: Lentiviral siRNA vectors targeting E, R and M genes were constructed successfully and could inhibit E, R and M gene expressions specifically and effectively. Lentiviral vectors could inhibit influenza virus replication whereas lentiviral siRNA vectors targeting E, R and M facilitated influenza virus replication.

This study analyzed the genetic evolution of neuraminidases (NAs) of influenza A subtype N9 viruses with the aim of determining the genetic origin of the novel avian A/H7N9 influenza virus. The NA sequences of influenza A subtype N9 viruses available from NCBI were used to construct a phylogenetic tree using the programs ClustalX 2.0 and MEGA 6.0. This analysis indicated that the novel avian A/H7N9 influenza virus is located in the modern Eurasian phylogenetic cluster. This cluster was then further analyzed by estimating the overall rate of evolutionary change and the selective pressure at the nucleotide level using the program BEAST 2.1.2 and the web interface Datamonkey, and by generating an amino acid sequence entropy plot using Bioedit software. In this cluster, the mean rate of nucleotide substitutions in NA was found to be 3.8354 x 10(-3) and the mean ratio of non-synonymous (dN) to synonymous (dS) substitutions per site (dN/dS) was 0.140413. A particularly high level of amino acid mutation entropy was identified at nucleotides 16, 19, 40, 53, 81, 84, 112, 256, 335, 359, and 401. This genetic evolution analysis suggests that the nucleotide substitutions that characterize the novel avian A/H7N9 influenza virus neuraminidase are likely to result from the overall genetic evolution of influenza A subtype N9 virus NAs, and not from selective stress. Phylogenetic analysis suggests that the influenza A virus (A/duck/Siberia/700/1996(H11N9)) isolated in 1996 appears to be the common ancestor of the more recent influenza A subtype N9 viruses NAs.

Data based on the antiviral-resistant phenotyping characteristics of 884 influenza B viruses circulating in mainland China from October 2013 to March 2014 were analyzed to assess the susceptibility of influenza B viruses to neuraminidase inhibitors. All 884 viruses were sensitive to oseltamivir; two viruses (0.23%) had reduced sensitivity to zanamivir and all other viruses were sensitive to zanamivir. Among the 38 viruses with a B/Victoria lineage, B/Shandong-Kuiwen/1195/2014 exhibited a half-maximal inhibitory concentration (IC50) for zanamivir that was elevated by 5. 12-fold (1.78 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D35G, N59D and S402T (39, 64 and 399 with N2 number) amino-acid substitutions in the NA gene were detected with no previously reported antiviral-resistant substitutions. Among viruses with the 846 B/Yamagata lineage, B/Hunan-Lingling/350/2013 exhibited a 7.99-fold elevated IC50 for zanamivir (2.72 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D197N (N2 number), a previously reported antiviral resistant-related amino-acid substitution in the NA gene, was detected in B/Hunan-Lingling/350/2013. These data suggest that recently circulating influenza B viruses in mainland China have retained susceptibility to neuraminidase inhibitors.

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.

To analyze the antigenic and genetic characteristics of the influenza A (H3N2) virus in mainland China during the surveillance year of 2013-2014, the antigenic characteristics of H3N2 virus were analyzed using reference ferret anti-sera. The nucleotide sequences of the viruses were determined by Sanger dideoxy sequencing, phylogenetic trees were constructed with the neighbor-joining method, and the genetic characteristics of the viruses were determined in comparison to current vaccine strains. The results showed that most of the H3N2 viruses were antigenically closely related to the A/Victoria/361/2011 vaccine strain cell-propagated prototype virus (99.6%). Using the A/Texas/50/2012 egg isolate as the reference antigen, 15.1% of the viruses were found to be closely antigenically related to it, while 11.9% of strains were closely antigenically related to the egg-propagated epidemic strain, A/Shanghai-Changning/1507/2012. Phylogenetic analysis of HA genes indicated that the A(H3N2) viruses in this surveillance year were in the same clade, but no drug resistant mutation was identified in the NA genes. During the 2013-2014 influenza surveillance year, no significant genetic change was detected in either the HA or NA genes of the A(H3N2) viruses, while significant mutations were found in egg isolates resulting from their adaptation during propagation in eggs. The antigenic and genetic changes should be investigated in a timely manner to enable the selection of an appropriate vaccine strain in China.

Objective: To predict B cell epitopes of hemagglutinin (HA) of human-infecting H6N1 avian influenza virus and analyze their evolutionary characteristics., Methods: The dataset was downloaded from GISAID and GenBank databases. And the linear and conformational B cell epitopes of HA were predicted separately by various bioinformatic software. Furthermore, the conservation, adaptation and other evolutionary characteristics were also analyzed by some bioinformatic means., Results: Four linear epitopes (A, B, C and D) and two conformational epitopes (E and F) were obtained after consideration of multiple factors. And the C epitope and sites ( 41, 157, 186, 187) mutated easily, but the other epitopes were very conservative and the D epitope was the most conservative. Interestingly, the site 157 was identified under positive selection, suggesting that it may be a particularly important site to make the virus evade the attack from the host immune system., Conclusion: The HA of human-infecting H6N1 avian influenza virus has five conservative B cell epitopes (three linear and two conformational) and one site under positive selection. The findings would facilitate the vaccine development, virus control and pathogenesis understanding.

Influenza poses a great threat to life and health in populations worldwide. Studies regarding the protein components of influenza viruses will facilitate the research and development of vaccines and diag nostic reagents. The influenza virus contains both structural and non-structural proteins. From the outset, it has been accepted that an influenza A virus possesses eight gene segments that encode eight corresponding viral proteins, respectively. Research has demonstrated that the M gene encodes the M2 ion channe! protein and the NS gene encodes the non-structural protein, NS2. In recent years, several novel viral proteins have been identified from influenza A viruses. This article will briefly describe the state of current research into PA-related proteins of influenza A viruses.