Prevalence And Molecular Epidemiology Of Influenza Viruses Circulating In Shanghai In Children During2009-2012

Influenza viruses are the major cause of acute respiratory disease in human. Humans are generally susceptible to influenza viruses. The antigenic properties of influenza viruses change substantially over time, which causes the annual epidemics of influenza. Influenza spreads around the world in seasonal epidemics, resulting in5to20%of the world’s population infected, about three to five million yearly cases of severe illness and about250,000to500,000yearly deaths during each seasonal epidemic. There have been about3influenza pandemics in the20th century with the1918Spanish flu the most serious pandemic in recent history. The1918Spanish influenza pandemic is estimated to infect one third of the world’s population and the kill approximately50million people or more. The World Health Organization (WHO) established global influenza surveillance and response system in1957, to recognize and manage an influenza pandemic.The influenza viruses are classified into type A, B and C on the basis of their core proteins. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. Influenza B virus is almost exclusively a human pathogen, and is less common than influenza A. influenza C is less common than the other types and usually seems to cause mild disease in children.The accumulating studies have suggested that children are the susceptible to influenza, and also play an important role in the transmission of influenza in the community. Moreover, children are at higher risk of developing influenza-associated complications, resulting in higher rate of hospitalization and higher disease burden. Nevertheless, the epidemiological information about childhood influenza was of scarce in Shanghai and other domestic regions. The main contents of the influenza monitoring include epidemiological characteristics, antigenic evolution and variation and drug resistance. Based on the importance and the gap of knowledge of childhood influenza, the situation of the2009pandemic of novel influenza A (H1N1) influenza in June2009, we carried out a surveillance study of the molecular epidemiology of influenza viruses in Shanghainese children between June2009and May2012, aiming to understand the epidemiological patterns and the genetic evolution of HA epitopes of the circulating influenza viruses, and monitor the antiviral resistance in circulating influenza viruses. This study will provide the important information for prevention and management of childhood influenza in Shanghai.Part OnePrevalence of influenza in Children in Shanghai from June2009to May2012Objective:To understand the epidemiological feature of influenza viruses among children in Shanghai between June2009and May2012and provide the rationale for the preventative strategies of influenza in Shanghai.Method:Nasopharyngeal swab or throat swab samples were collected from each enrolled children who had outpatient visits for influenza-like illness (ILI). One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A/H1N1and A/H3N2viruses, influenza B viruses and2009pandemic A/H1N1influenza viruses.Results:Out of3475enrolled cases, influenza was virologically confirmed in978(28.1%) otherwise healthy children. Five hundred and seventy-seven (59.0%) were influenza A positive (A/H3N2:344, pandemic A/H1N1:222, seasonal A/H1N1:4, A/H3N2co-infected with pandemic A/H1N1:3, and untyped:7), and421(43.0%) were influenza B positive (20co-infected with influenza A). The outbreaks of A/H3N2, pandemic A/H1N1and influenza B took place in fall, winter and spring, alternatively.Conclusion:The annual outbreak of influenza led to a high rate of outpatient visits among children in Shanghai between June2009and May2012. The epidemics of influenza in children usually appeared in autumn, winter and spring with seasonal A/H3N2and influenza B,2009pandemic A/H1N1viruses circulating alternatively in Shanghai. Influenza vaccination should initiate prior to the beginning of autumn semester to prevent influenza outbreaks. Part TwoGenetic analysis of haemagglutinin and antiviral-resistance of influenza A/H3N2viruses circulating in Shanghai in children during the2009-2012seasonsObjective:to understand the genetic evolution of haemagglutinin (HA) antigenic sites and investigate the frequency of antiviral drug resistance of Influenza A/H3N2viruses.Methods:Seventy-second samples A/H3N2viruses were randomly selected for the amplification of partial M, HA1and NA genes. The genetic evolution of antigenic sites in HA1genes was analyzed in comparision with the vaccine strains, and the molecular markers in M2and NA genes conferring resistance to the antiviral drugs were detected.Results:The HA1sequence analysis revealed that A/H3N2viruses underwent constant mutations in HA antigenic sites over3seasons compared to the seasonal vaccine strains, and the amino acid changes involved in at least3epitopes in each season. Phylogenic analyses showed that circulating A/H3N2strains fell into the different clades from the seasonal vaccine strains and the new lineage usually appeared near the end of the seasonal outbreaks. All72A/H3N2viruses obtained from swab samples were genotypically resistant to amantadine but sensitive to oseltamivir.Conclusion:Continued genetic drift occuring in the epitopes of HA1of A/H3N2could lead to the antigen drift of circulating A/H3N2viruses, resulting in the seasonal outbreaks of A/H3N2viruses in Shanghainese children. The circulating A/H3N2viruses were universally resistant to amantadine but sensitive to oseltamivir. Amantadine should be no longer recommended for prophylaxis and treatment of influenza in Shanghai. Part ThreeGenetic analysis of haemagglutinin and antiviral-resistance of influenza A/H1N1viruses circulating in Shanghai in children during the2009-2012seasonsObjective:To investigate the genetic drift in the epitopes of hemagglutinin (HA) of novel influenza A (H1N1) viruses and oseltamivir-resistant variants characterized by H275Y mutation in children in Shanghai.Methods:One-step RT-PCR was performed to amplify the partial M, HA1and NA genes of37novel influenza A (H1N1) viruses. Genetic drift from the vaccine strain in HA epitopes of novel influenza H1N1viruses and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed.Results:The nucleotide sequences of HA1were highly homologous between the vaccine strain A/California/07/2009and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected in the circulating strains. The H275Y amino acid mutation associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains.Conclusion:Two major waves of novel influenza A (H1N1) outbreaks occurred in Shanghainese children during2009-2011. No activity of novel influenza A (H1N1) viruses was observed in2011-2012season. The novel A/H1N1viruses circulating in Shanghai displayed the highly genetic similarity with the vaccine strain in HA1antigenic epitopes. The circulating viruses were not found to contain the oseltamivir-resistant molecular markers. Part FourGenetic analysis of haemagglutinin and antiviral-resistance of influenza Bviruses circulating in Shanghai in children during the2009-2012seasonsObjective:To understand the lineages of influenza B viruses circulating in Shanghainese children, and monitor the genetic drift of the hemagglutinin(HA) epitopes and the variants resistant to the antiviral drugs.Methods:A total of55specimens tested positive for influenza B viruses were randomly selected for sequencing. The partial HA1and NA genes were amplified with the segment-specific primers. The nucleotide sequences of the HA1gene were aligned using MEGA4.1software. The phylogenetic tree was constructed using the neighbor-joining method to determine the best-fitting tree for each gene.Result:Of the55influenza B strains,36were belonged to Victoria lineage strains and19belong to Yamagata lineage. Victoria lineage fell into2clades, including B/Malaysia/2506/04like virus and B/Brisbane60/08like virus. Yamagata lineage belonged to B/Florida/4/06like virus. No molecular marker conferring resistance to osletamivir was detected in the circulating influenza B viruses.Conclusion:Influenza B viruses of Victoria lineage and Yamagata lineage co-circulated in Shanghainese children during the2009-2012seasons. The circulating strains remained sensitive to oseltamivir.