The Surveillance And Molecular Epidemiology Research Of Influenza Viruses In Qingdao During2005-2011

Influenza viruses belong to the Orthomyxoviridae family. They are the major respiratory pathogens that can cause amount of morbidity and mortality worldwide. Three types of influenza viruses, influenza A, B, and C, could be identified based on the different antigenic characters of matrix protein (M) and nuclear protein (NP). Influenza viruses infect5%～15%of the global population annually, resulting in～500,000deaths.Among these, influenza A virus are characterized by high mutation rates with consequent antigenic changes, thus cause epidemics even pandemics through immune escape. Influenza A virus could be further divided into different subtypes, based on antigenic differences in the two main surface glycoproteins:the hemagglutinin (HA) and neuraminidase (NA). Currently16HA and9NA subtypes of influenza A virus have been identified, while three subtypes, seasonal A/H1N1, seasonal A/H3N2, and2009pandemic influenza A virus establishes transmission on humans at present.Based on the knowledge obtained through studies carried over half a century, two classes of antiviral agents that block the M2ion channel and neuraminidase activity have been developed to treat infection, and a trivalent influenza vaccine is updated twice annually to account for frequent antigenic changes. However, the rapid and unpredictable evolutionary dynamics of the influenza A virus result in (a) sporadic extreme antigenic changes associated with severe disease and vaccine-epidemic mismatches,(b) the proliferation of new variants that are resistant to antiviral agents, and (c) occasional lethal infection of humans with avian influenza A viruses. The evolutionary basis for these events is still not well understood. While all the institutes are focusing on the capacity of avian influenza A viruses to cause pandemics in humans, during April2009a novel A(H1N1) influenza virus emerged in Mexico and since then has spread worldwide.As the influenza A virus poses a continual, but unpredictable threat to human health, the evolution of the influenza A virus is of particular interest. The extensive genetic diversity is largely produced by the genomic reassortment between two genetically distinct viruses that co-infect a host cell. A particularly notable reassortment event was associated with one of the most important influenza epidemics of the twentieth century, the major epidemic of1947. These findings confirm the importance of studying influenza A virus evolution at a whole-genome level, taking into consideration genomic reassortment and the critical evolutionary dynamics of genes other than the main surface antigen HA.In the present study, pharyngeal swab samples were collected from influenza-like illness (ILI) patients treated at three Qingdao sentinel hospitals during2005～2011for routine influenza surveillance, while an emergency surveillance targeting four defined groups were carried out during the2009influenza pandemic. Whole-or part-genome viral sequences were amplified by reverse-transcription polymerase chain reaction and determined. A chemical luminal method was applied to determine the virus sensitivity to oseltamivir, a kind of NAI antiviral drug, and the M2amino acid sequences were analyzed to determine the virus sensitivity to ion channel blockers, adamantanes. MEGA5.0software was explored to perform bioinformatic analysis on the genome sequences, thus constitute a primary study on the evolution disciplines, drug-resistant mechanism, and characters of molecular changes of influenza A virus in Qingdao. Furthermore, to investigate if the Qingdao influenza virus have an international representation, a phylogenetic analysis was performed on the HA sequences of seasonal H3N2virus from Qingdao and those randomly selected from the whole world. Six major conclusions were drawn as following:1. A total of2560pharyngeal swab samples were collected in Qingdao during2005～2011,245of which were tested as influenza virus positive. The general situation of influenza prevalence in Qingdao during the recent6years was thus illuminated:except for the2007-2008epidemic season, which presented an influenza B virus prevalence, influenza A virus was generally predominant in Qingdao over the recent years. Seasonal HlNl and H3N2influenza virus alternated with each other before the2009pandemic, while the co-circulation of seasonal H3N2and the pandemic H1N1virus was established after the pandemic, as seasonal H1N1virus haven’t been isolated since then. Whole-or part-genome sequences of influenza A virus were meanwhile determined in the present study, and the obtained data should be of great significance for the prevention and control of influenza, as well as provide important information for the molecular epidemiology of influenza virus in China.2. An emergency surveillance targeting four defined groups were carried out during the2009influenza pandemic, providing banking for the city to establish its coping strategy and risk evaluation for the pandemic. A mutant with S128P substitution in the viral HA gene was detected in the domestic pandemic virus, as compared with those isolated from the imported cases. Further analysis on the pandemic virus sequences available on the GenBank revealed the abnormal transmission of the mutant in a defined Eurasian area including China, Russia, Mongolia, and South Korea in2009. However, the mutant was subsequently eliminated in the next year together with the weakening of the pandemic virus activity. The transitional transmission advantage of the mutant virus revealed our insufficient knowledge in predicting viral development and evolution. Therefore, intensified influenza surveillance should be applied for mutant searching as to possibly reduce viral damages, and further studies should be taken on the gene mutation, especially the whole-genome mutation of influenza virus. Moreover, co-circulation of pandemic virus and seasonal influenza virus was determined, which could facilitate reassortment between different subtypes. This emergency surveillance targeting defined risk groups should be of significance for the city to establish its early detection system for acute respiratory diseases in the future.3. All the influenza A virus isolated in Qingdao during2005～2011was adamantine-resistant, as they all present the S31N substitution in M2,except for the six NAI-resistant seasonal A/H1N1influenza viruses in2008～2009. NAI sensitivity analysis showed that, seasonal H3N2, pandemic H1N1viruses were all oseltamivir-sensitive. The resistant viruses emerged in seasonal H1N1influenza A virus with a ratio of28.6%in Qingdao during the2008～2009season, and quickly reached50%during the2009～2010season. As the seasonal H1N1influenza viruses haven’t been isolated since2010, NAI antivial agents are still potent to influenza virus prevalent at present, thus should be the preferred drugs for prophylactic or clinical application. Besides, the emerging of double-resistant virus to NAI and adamantine agents should arouse more attention, as the current treatments have no effects on it.4. Sequence analysis revealed a direct relation between the viral antigenic changes and virus prevalence. Compared with those isolated in2005～2006season, multiple mutations in different viral genes was detected in virus of2008～2009season, which facilitate the virus breakout after a two-year interval. Only4seasonal H1N1influenza isolates were obtained during2009-2010season, all collected within late August and the beginning of September in2009. These viruses present no new mutations compared with those isolated in the last season thus didn’t cause a sustaining prevalence, and didn’t exist any longer in2010-2011. Besides the H275Y mutation in NA, the NAI-resistant virus also present multiple "hitch-hiking" substitutions, which could make up the compromised replication and transmission of the H275Y mutant, and facilitate the survival and wide transmission of the drug resistant virus.5. Whole-genome evolution analysis of seasonal H1N1and H3N2influenza A virus revealed the co-circulation of multiple clades of the same subtype and the genome reassoitment events. All the seasonal H1N1influenza virus during2009～2010, and the H3N2virus during2010～2011are evolved with genome reassortment, indicating reassortment as one of the major evolution patterns of influenza virus. The viruses circulating in Qingdao in a given season tend to derive from newly imported genetic material rather than from isolates circulating in Qingdao in the previous season. The NAI-resistant seasonal H1N1virus emerged through a genomic reassortment in addition to the NA H275Y and other additional mutations. Influenza viruses in Qingdao could be served as vaccine candidates, as they evolved earlier than their counterparts from worldwide. On both the HA and NA phylogenies, Qingdao H3N2influenza virus during2006～2008were genetically close to A/Brisbane/10/2007isolate selected for the2008～2009vaccine, other than the2006～2007influenza vaccine strain A/Wisconsin/67/2005, indicating a vaccine mismatch.6. Phylogenetic analysis performed on the HA sequences of Qingdao seasonal H3N2viruses and those randomly selected from the whole world revealed an international representation of the Qingdao isolates, which confirming the possibility of Qingdao influenza viruses as vaccine candidate. Furthermore, it’s reasonable to set Qingdao as an international surveillance sentinel for influenza, which could be of great significance for the national, even worldwide monitoring of the influenza prevalence and changes.Briefly, the influenza surveillance network, especially the emergency surveillance targeting defined groups during pandemic is of great significance for the coping method to acute respiratory diseases. Viral antigenic changes are directly associated to virus prevalence. NAI antiviral agents should be the preferred drugs for prophylactic or clinical application on influenza viruses. Genome reassortment occurs frequently as one of the major evolution patterns of influenza virus. The NAI-resistant influenza virus emerged through a genomic reassortment in addition to the NA H275Y and other additional mutations. Qingdao influenza viruses could be served as vaccine candidate owing to their international representation. Although influenza A virus can probably never be globally eradicated due to the permanent viral reservoir maintained in wild waterfowl, a greater understanding of the evolutionary dynamics of the influenza A virus will improve our capacity to effectively target global surveillance and potentially to predict evolutionary trajectories for appropriate selection of influenza vaccine strains. The present study could be of great scientific significance for further study on the evolution of influenza A virus, as well as provide foundation for the development of more effective influenza vaccine and strategy for prevention and controling of the virus.