Epidemiology And Whole Genome Sequence Analysis Of Human Infected With H7N9 Avian Influenza In Shandong Province

BackgroundAvian influenza was a kind of human disease caused by the cross-species transmission of avian influenza virus (AIV). The first case of human infected with novel H7N9 avian influenza virus was appeared in east China in February 2013 after the H5N1, H9N2, H7N7, H7N2, H7N3 and H10N8 avian influenza virus infecting humans. It has attracted global concern immediately because of the clinical intensive and high mortality. Although the cases showed a sporadic situation, there were still family clusters cases reported from part of several provinces in China. The epidemic potential remains a serious public health problem.More than 80% of the confirmed H7N9 avian influenza cases have the experiences of poultry or poultry related environmental (live poultry markets) exposure. The main mode of transmission is from the poultry to human. In early epidemic stages of human infected with H7N9 avian influenza, the possible reduction of cases occurrence could be up to the most if the H7N9 avian influenza virus contamination places could be found in the external environment. And then the corresponding control measures such as taking off the live poultry markets could be effective.One of the necessary conditions for the spread of AIV is that it should be able to infect human host cells, and in which to effectively reproduce. The receptor binding preference changes must be occurred if the avian influenza viruses break the species barrier to infect human, of which the preference depends on key amino acid of HA receptor binding site. Differences on the critical functional site of the amino acid viral protein NA, polymerase PB1/PB2/PA, etc. is to determine the molecular basis of virulence and interspecies communication. The current anti-influenza virus drugs mainly directed against NA neuraminidase inhibitors and M2 ion channel inhibitors. These two proteins key amino acid sites of resistance mutations can lead to the emergence of resistant strains.In this study, we use epidemiological investigation data of the seven avian influenza cases in Shandong province from 2013 to 2015 and the outside environmental investigation to analyze the incidence characteristics of cases as well as the source of infection. This result can help us to find out the prevention focus. Through the analysis of strain genetic evolution and key amino acid sites, we can grasp the characteristics of strain evolution and its pathogenicity, and then make a good guidance of the early clinical treatment. And also this can help to provide a scientific basis for clarifying the disease pathogenesis of the H7N9 avian influenza.Objectives1、To analysis the epidemiological characteristics through the epidemiological data. To analyze the infection sources, transmission modes, prevention and control measures of the familial aggregation infected cases. This study can provide the scientific basis for the effective prevention and control of human infected H7N9 avian flu.2、To analysis the environment surrounding the live bird markets and poultry farms where the epidemic broke. And then make a preliminary explore of the outer environment of H7N9 avian influenza virus contamination. The found of sensitive indicators to monitor could be used to help the early detection and the epidemic prevention.3、To full understand the evolution directions of virus through the analysis of phylogenetic and key amino acid sites. The information could be used to guide the early clinical medication. And also this can help to provide a scientific basis for clarifying the disease pathogenesis of the H7N9 avian influenza.Methods1、Epidemiological data:Epidemiological data of seven human infected with the H7N9 avian influenza cases in Shandong Province from 2013 to 2015 to analyze the incidence characteristics of cases as well as the source of infection. Investigation and detection data of the environment contamination surrounding the live bird markets and poultry farms where the epidemic broke, and then make a preliminary explore of the outer environment of H7N9 avian influenza virus.2、Avian influenza virus detection:H7N9 avian influenza virus nucleic acid in the specimens is detected through the method of qReal-time PCR. Ct value in the samples which not above 40.0 is defined as the positive.3、Avian influenza virus isolation:The virus in seven patients and fifty-eight environmental samples (Ct≤40.0)are isolated by the SPF chicken embryos.4、Phylogenetic and key amino acid sites analysis:We get the whole genome sequence from the help of Chinese National Influenza Center. Sequence alignment is implemented with the help of the BLAST programs provided by NCBI. The homology of gene of the eight segments is calculated by the MegAlign program in DNAStar software. The phylogenetic trees are made by Mega 6.0 software. HA and NA protein N-glycosylation sites are predicted in the PROSITE database of ExPASy website.Results1、Epidemiological characteristics of human infected with H7N9 avian influenza:(1) Seven cases of human infected with H7N9 avian influenza in Shandong province are all incidence in winter and spring season. Most of the cases are concentrated in the male population over the age of 50.(2) The longest interval from exposure to the onset stage is about nine days while the shortest is one day with five days incubation period. The interval from onset to the diagnosis time is between one to sixteen days of which the median is seven days.(3) The main source of human infected with H7N9 avian influenza cases in Shandong province is poultry exposed and visiting to live poultry markets.(4) There is sufficient evidence of transmission between human and H7N9 avian influenza virus from the one case of family clusters of disease even though the ability to communicate is limited.2、H7N9 environmental pollution situation:(1)All the H7N9 avian influenza virus detected in Shandong province are derived from live poultry markets. The samples of outside environment in poultry farms were not detected.(2) Detection rate H7N9 AIV is 12.8%. The outside environment samples in Zaozhuang city, Yantai city and Tai’an city were all detected out of the H7N9, H5 and H9 subtype of avian influenza virus.3、Analysis of phylogenetic and key amino acid sites:(1) H7N9 AIVs separated from 2013 to 2015 are highly homologous (99% to 100%) with the samples in Jiangxi, Zhejiang, Jiangsu and Hunan province in the same period. The virus separated from the indigenous cases are highly homologous to the virus in the live poultry market environment around the cases. The viral gene of the living poultry market environment or the case has a high similarity with the neighboring provinces(2) The H7 hemagglutinin gene in the first three isolated H7N9 avian influenza virus in Shandong is come from Zhejiang duck’s H7N3 avian influenza virus HA gene. And at the same time, the NA gene is come from wild birds’H7N9 avian influenza virus in South Korea. The other six internal genes are from the domestic H9N2 avian influenza virus.(3) H7N9 avian influenza virus genes evolve constantly and then they differentiate into different branches from 2013 to 2015. Six internal gene fragments from part of the H7N9 avian influenza virus recombine with the H9N2 virus.(4) Seven in fifteen of the virulence-associated site involved in this study are enhanced virulence locus. Five strains of human infected with H7N9 avian influenza have occurred drug-resistant ion channel inhibitors variation, but they all still sensitive to the neuraminidase inhibitors.Conclusion1、Winter and spring is the season with high incidence of human infected with H7N9 avian influenza. Middle-aged male population might be the dangerous crowd of the disease. H7N9 can cause limited human transitions.2、Poultry market is also a major source of the AIV human infected. There are many subtypes of avian influenza virus in outside environment of Shandong province. It is more possible for the population exposed in the outside environment to be infected. The possibility of restructuring cannot be ruled out.3、There are multiple origins of H7N9 bird flu virus in Shandong province. H7N9 avian influenza virus continues to occur in the dissemination of variation and evolution, and then differentiate into different small branches. H7N9 avian influenza virus is input virus in Shandong province. H7N9 avian influenza virus isolated in Shandong province has not yet obtained sufficient conditions to spread by droplets, so there is less likelihood of human transmission. We cannot ignore the fact of the virus’s development direction to infect the mammals at the same time. The early onset of clinical cases should be treated timely with the type of neuraminidase inhibitors (such as Tamiflu).