| Background: Public health emergencies are the sudden events which cause or may cause serious hazards to public health. They are composed of major infectious diseases, mass unexplained diseases, major food and occupational poisoning and other serious events that affect public health. It poses a severe challenge to public health and safety when the existing infectious diseases outbreak occasionally and the diseases which have been eliminated or controlled resurgence and new infectious disease emerges and outbreaks. Hemorrhagic fever with renal syndrome (HFRS) is a natural focal disease which is caused by hantavirus. It has a more than a half century history in mainland China. HFRS is endemic in China except Qinghai and Taiwan provinces, and rodents are the main reservoir and a source of human infection. In China, HFRS is mainly caused by Hantaan virus (HTNV) and Seoul virus(SEOV). The clinical disease caused by HTNV is more severe than that caused by SEOV. One object of our research is Ningbo City which is located in China's southeast coast, with a semi-humid climate siutable to the propagation and growth of rodents. HFRS has been recognized as a notable public health in Ningbo for a long time. With the improvement of disease surveillance system and public health level, the city's outbreak of hemorrhagic fever with renal syndrome showed a declining trend since 1997. But in recent years, HFRS occurrence has rised and in 2009-2010 the disease occered in relatively high status. The novel H1N1/2009, a major emerging infectious disease causing a widespread concern around the world, shows a strong ability to transmit from human to human and has caused influenza A pandemic worldwide since its first emergence in Mexico and the US in March 2009. According to World Health Organization, as of August 1, 2010, 214 countries had reported laboratory confirmed cases of novel H1N1/2009 in the world, killed at least 18,449 people. According to the Ministry of Health, as of January 10, 2010, mainland China had 124,764 cases of confirmed cases of influenza A H1N1 influenza, of which 744 cases died. The reassortment of swine, avian, and human influenza viruses in swine possibly results in interspecies transmission of influenza and generation of novel progeny viruses to which humans are immunologically naive and highly susceptible. But between the 1930s and the 1990s, classic swine H1N1 influenza A underwent little change. However, by the late 1990s, multiple strains and subtypes (H1N1, H3N2, and H1N2) of triple-reassortant swine influenza A (H1) viruses whose genomes included combinations of avian, swine, and human influenza virus gene segments had emerged in pigs. Evolutionary process of swine-origin H1N1 influenza A viruses that infected humans from sporadic to pandemic is of high epidemiological significance but still remains obscure.Objective: This study is to explore the evolutionary process of hantavirus in Ningbo city and the novel H1N1/2009 by applying principles of epidemiology, site monitoring, laboratory processing and bioinformatics. Revealing the epidemic hantavirus strain in Ningbo the evolutionary process of swine-origin H1N1 influenza A viruses that infected humans from sporadic to pandemic. To provide a theoretical basis for public health and epidemic prevention.Methods: Firstly, we collected the data in hemorrhagic fever with renal syndrome in Ningbo by Ningbo Disease Prevention and Control Center to explore the characteristics and disciplines of HFRS occurrence in recent years. We also collected the rodents lung samples and serum samples of HFRS patients. Total RNA was extracted from the lung tissue samples with the TRIzol reagent (Invitrogen, Beijing, China), according to the manufacturer's instructions and from the serum samples with the QiAamp Viral RNA Mini Kit . cDNAs were synthesized with avian myelobalastosis virus reverse transcriptase (Promega Biotech, Beijing, China) in the presence of primer P14. Then we conducted a RT-PCR to amplifed the M segment of hantaviruses. MEGA4 software (Molecular Evolutionary Genetics Analysis VERSION 4) was used to construct phylogenetic trees by using the neighbor-joining and maximum-likelihood methods. Secondly, we searched the PubMed database up to March 2009, the time point before the outbreak of novel influenza A viruses, using the searching terms"swine","influenza", and"human", to identify articles describing sporadic cases of human infection thought to be caused by swine influenza virus strains. Then we performed phylogenetic, bootscan, and adaptive evolution analyses using the sequences of the 8 gene segments from swine-origin H1N1 influenza A viruses that infected humans and the reference viruses.Results: We totally collected 20 serum samples of HFRS patients and 4 hantavirus antigen positive rodent lung tissues. Of the 20 serum samples, 4 were amplified successfully, along with all the 4 rodent lung tissues. In another, A total of 49 sporadic influenza patients caused by swine-origin influenza viruses were reported in North America, of which 14 submitted the gene sequences.1. Evolutionary characteristics of hantaviruses in Ningbo(1) Occurrence of HFRS in Ningbo CityAt least 2 major HFRS epidemics had occurred in Ningbo since 1980. The first peak was observed during 1985–1987, when 600-800 cases were registered. The second epidemic peaked in the early 1990s when 542-567 HFRS cases were reported. After this peak, the number of HFRS cases declined and was relatively stable after 1997. 131 HFRS cases were reported in 2010, the occurrence reported was 1.96/10. Compared with the data in 2009, the occurrence of HFRS rised by 8.89%. HFRS cases occurred throughout the year, forming a bimodal incidence curve. December had the most cases and April for the second. August to October had fewer cases. The characteristic of the occurrence was consistent with the rules in south China. Male cases and female cases were 96 and 35 respectively. Occupational distribution of the top three were farmers, workers, migrant workers and Rattus norvegicus were the dominate reservoirs.(2) Genetic analyses by RT-PCRWe totally collected 20 serum samples of HFRS patients and 63 rodent lung tissues from Ningbo Disease Prevention and Control Center. Of the 20 serum samples, 4 were amplified patial M segment successfully named E1,E9,E19,E20. Among the 63 rodent lung tissues, 4 were tested hantavirus antigen positive named D005, D008, D009, E002. Patial M segment were recovered from all the 4 lung tissue samples successfully. All the 4 positive lung tissues were belong to Rattus norvegicus.(3) Phylogenetic analysesA total of 18 SEOV and 4 HNTV patial M segments from different years and locations were downloaded from GenBank to conduct phylogenetic analyses. All partial M-segment sequences from humans (E1, E9, E19, E20) and wild rats (D005, D008, D009, E002) fell into the SEOV genetic clade, well separated from other hantaviruses, thus indicating that the HFRS outbreak was caused by SEOV. Homologous analysis showed that partial M-segments recovered from Ningbo had a 94.7%-99.6% identities. Compared with L99 which was isolated in Jiangxi province in 1984, it had a 5.3% difference, with HLJ3-23 which was isolated in Heilongjiang province in 2007 the difference was only 0.4%. But with the passage of time, evolution of M-segments did not show significant differences (P>0.1). Compared with M-segments recovered from serum samples and long tissue samples , E20 had a 1.4% difference, the remaining had a 100% identity. This result showed that hantavirus in Ningbo did not have mutant. But continuous observation was needed to determine whether E20 was a mutant strain.2. Evolutionary characteristics of swine-origin H1N1 influenza virus that infected humans from sporadic to pandemic(1) Genetic characteristics of swine-origin influenza viruses that sporadically infected humans before 2009 All of the 8 gene segments of swine H1N1 viruses that sporadically infected humans in 1970s-1990s clustered with those of classic swine influenza viruses of North American lineage, except A/Wisconsin/10/98, an unreported isolate from a human case in Wisconsin in 1998. Partial PB2 (1,573 bp) and PA (1,470 bp) segments and other 6 full-length gene segments of A/Wisconsin/10/98 were retrieved from GenBank. Phylogenetic analyses with the partial sequences indicated that the PB2 and PA segments of A/Wisconsin/10/98 were closely related to that of A/Iowa/CEID23/2005(H1N1). The full-length PB1 gene of A/Wisconsin/10/98 was closely related to that of A/Iowa/CEID23/2005(H1N1). These results indicated that the triple-reassortant swine-origin H1N1 viruses that caused sporadic human infection were initially generated as early as late 1990s.(2) Evolutionary relationship of swine-origin H1N1 influenza viruses that infected humans from sporadic to pandemicthe A/H1N1/2009 pandemic strains clustered in a unique clade for each of the 8 gene segments, respectively. Within the clades of the nearest genetic neighbors, the PB2, PB1, PA, HA, NP, and NS gene segments of the novel H1N1/2009 strains were most closely linked to those of A/Iowa/CEID23/2005 (H1N1) and those of other triple-reassortant swine-origin viruses that sporadically infected humans since 2005. The NA and MP were closely related to those of influenza A H1N1 viruses circulating in swine populations in Eurasia. Bootscan analysis showed that the A/H1N1/2009 pandemic strain shared great homology with A/Iowa/CEID23/2005 (H1N1) in the PB2 (95.3%), PB1 (95.1%), PA (95.2%), HA (90.3%), NP (95.2%), and NS (94.7%) genes. The NA and MP genes of the A/H1N1/2009 pandemic strain were most closely related to those of A/Swine/Spain/50047/2003 (H1N1), a swine virus of Eurasian lineage, sharing 90.2% and 94.4% identities, respectively.(3) Positive selection drives the evolution of swine-origin H1N1 viruses that infected humans from sporadic to pandemic A HI value of 0.817 was observed in the HA segment between the classic swine H1N1 viruses that occasionally infected humans before 1998 and the triple-reassortant H1N1 viruses that sporadically infected humans in or after 2005. However, positive selection was not observed on this gene between the triple-reassortant H1N1 viruses and A/H1N1/2009 pandemic viruses. Moreover, a HI value of 0.773 was observed acting on the NA gene between A/H1N1/2009 viruses and their nearest genetic neighbors (Euroasian swine H1N1 viruses).Conclusions: Hantavirus and A/H1N1/2009 were the two major causes of Public health emergencies we concerned in this study. Epidemic strain of hantavirus in Ningbo was SEOV and the M segment did not have significant mutation. The reassortment of swine-origin H1N1 with avian influenza virus of North America lineage and human seasonal influenza virus before 2005 in North America was found to be an important step during evolutionary process of A/H1N1/2009 pandemic virus. The monitoring of virus host is of high epidemiological significance.
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