Influenza Activity And Variation Of Influenza A Viruses In Shanghai

Influenza is a highly contagious acute respiratory disease caused by influenza virus. As the wide host range and high genetic variation, influenza A virus causes global seasonal epidemics with different scales every year and pandemic per 10 to 15 years.250000 to 500000 people were killed worldwide each year, which brought great challenges to the public health. Vaccination on susceptible people is an important measure to prevent influenza, but flu vaccine will soon lose the protection for people because of frequency virus mutation. Therefore, we study the flu virus variation, immune status against influenza virus in the population, seasonal epidemics and subtype distribution of influenza and time and area series analysis on evolutionary characteristics of influenza virus genes via field epidemiology, laboratory virology and molecular biology, as the following four aspects:1. Influenza activity was surveilled during 2009 to 2010 in Shanghai to study seasonal epidemics and subtype distributions. Seasonal H1N1 and A/H3N2 co-circulated in winter and spring of 2009 (from 1st to 25th week) with seasonal H1N1 being dominated, and the peak occurred from January to February, while influenza B viruses were isolated in the end. Swine origin influenza viruses (A-H1N1) began to co-circulated with seasonal A/H3N2 in August, and then became dominating since October (the 40th week) while seasonal influenza B virus and H1N1 were isolated occasionally. the peak of summer and autumn of 2009 (from 26th to 52nd week) was August to September, and the influenza activity was stronger and lasted longer with positive isolates, mostly A-H1N1 isolates, in the 52nd week, This is because influenza virus A-H1N1 was not associated with seasonal H1N1 in gene and antigen and immunity of population early could not effectively protect from it. From the 1st to 10th week in 2010, though A-H1N1 still co-circulated with influenza B virus, A-H1N1 activity was weaker relatively, and the number of influenza B virus gradually increased until the 5th week of 2010 and finally became dominating. It might relate to the epidemic and vaccination of A-H1N1 in population.2. HAI antibody levels against influenza A virus were detected from 2008 to 2009 in Shanghai population to understand the immune level due to past infection and to forecast the flu epidemic trends. The result showed the positive rate of antibodies to influenza virus A/Guangdong Luohu/219/06(H1N1) is 38.8%(134/345),to A/ Jiangxi Donghu/312/06(H3N2) is 42.3%(115/272)in the general population in 2008; the Shanghai general population have a certain immune barrier to A/Brisbane/59/2007 (H1N1) and A/Brisbane/10/2007 (H3N2) with 70.8%(638/901) and 43.5%(392/901) positive rate of HAI antibody respectively during 2009. In 2008, the positive rate of antibodies to influenza A virus subtype H5 is 0.3%(1/345) and 4%(8/198) in the general population and the contacted population respectively, and the positive rate of antibodies to influenza A virus subtype H9 is 2.6%(9/345) and 17.7%(35/198) respectively; during 2009 the positive rate of antibodies to H5 is 0.3%(1/332) and 4.2 %(15/356), to H9 is 2.4%(8/332) and 34.6%(123/356) in these two groups respectively. There is antibody to avian flu H5 and H9 in people, especially in the contacted population, and the increase of H5 and H9 positive rate indicated possible infected from avian, but no sign of human-human transmission. A-H1N1 influenza viruses outbroke and got pandemic in the world, we tested the antibody levels to A-H1N1 flu in serum collected from the Shanghai general population in January 2009. The positive rate of antibody of A-H1N1 among the whole population in Shanghai was 9.2%(37/404), revealed the whole population in Shanghai generally lacked the immunity against A-H1N1 and were highly sensitive to A-H1N1. Among various age groups the positive rate of antibody of the≥60 age group was the highest with 25%(21/84), then the 25-year group with 10%(8/80), the 5 to 25-year group was lower with 2.5%(2/80).With the epidemic of A-H1N1 in Shanghai, the antibody levels increased in population, until mid-October 2009, the positive rate of antibody of 5 to 15-year group was 26.5%(53/200), showed that people have begun to obtained the immunity against A-H1N1 before vaccination was applied widely.3. We sequenced HA, NA, PB2 and M genes of some influenza A isolates and compared with the WHO reference vaccine strains subsequently, to analyze the genetic evolution of influenza A. During 2005 to 2009, HA genes of seasonal H1N1 were rooted in one trunk, and the sequences of the same year were clustered as similar to each other, but the branches between years were longer revealing notable variation. Compared to A/New Caledonia/20/1999 (H1N1) (recommend as vaccine from 2000 to 2007), A/Solomon slands/3/2006(H1N1) (recommend as vaccine from 2007 to 2008) and A/Brisbane/59/2007(H1N1) (recommend as vaccine from 2008 to 2010) had shorter branch, which indicated the two latter would have better protection to the population of Shanghai. However, all strains isolated in summer and autumn of 2008 and some in winter and spring of 2009 formed a separate branch, and were relatively far away from A/Brisbane/59/2007 (H1N1), which revealed that some large variations on HA gene. Amino acid sequence comparison showed that the variable sites involved in three antigenic regions, and the unique variable sites were up to a dozen.The phylogenetic tree of A/H3N2 in 2004～2009 was characterized as one long trunk with very short brunches. The interlaced influenza viruses isolated from different years made the more remarkable successive connection compared to the seasonal H1N1. A/Brisbane/10/2007(H3N2) recommended as vaccine of 2008 to 2010 had shorter branch, which showed similar to most influenza viruses and better protection to the population of Shanghai. But mutations were found in some HA amino acid sites, and mostly involved in three or four antigenic regions.The phylogenetic trees of HA, NA and PB2 gene of the A-H1N1 strains isolated in 2009 had the similar model with the HA phylogenetic trees of seasonal H1N1 and A/H3N2, the interlaced influenza viruses isolated from different regions and months had some successive connection. Several isolates had some variations with the longer lateral branches far from the trunk since December. Mutations were found in some HA amino acid site, mostly in the HA1 segment, but none of them was in the antigenic determinant region and there was no change in potential glycosylation sites. None changes were observed in the 274 NA amino acid residue which was related to the drug resistance to oseltamivir. PB2 protein analysis showed that the 627 and 701 amino acid residues were Glutamic acid (Glu, E) and Aspartic acid (Asp, D) respectively, which were same encoded amino acid with avian flu PB2 protein.4. We also did time series analysis of the antigenic variation of influenza virus. We chose golden hamster as the animal model and selected influenza virus isolated from sentry hospitals and influenza outbreaks of Shanghai during 2005 to 2009 to get strain specific antiserum, and carried out cross HAI test to study the HA antigenic variation. The results showed that the antigenic evolution of A/H3N2 strains slowed down relatively since 2005, and seasonal H1N1 isolated in first half of 2008 had some antigenic variations than the previous isolated strains and the vaccine strain, while the HA antigen of A-H1N1 strains were very different from human seasonal H1N1.