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- Materials and methods
Influenza A virus (IAV) infection in swine poses a threat to pig production and has important implications for the epidemiology and control of influenza infection in other species. IAVs have an antisense RNA genome that is divided into eight segments, which allows genetic reassortment between different viruses. The viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are the main targets of the host immune response, and they are important for virulence and host specificity. Three subtypes of swine influenza virus (SIV) are currently circulating in the swine population globally: H1N1, H3N2, and H1N2.
Influenza A infection in pigs has been well characterized both in the United States and in Europe.[4, 5] The classical SIV (cH1N1), which is similar to the human 1918 pandemic virus, remained antigenic and genetically conserved in the United States until the introduction of an H3N2 virus in 1998. The cocirculation of cH1N1 and the H3N2 virus led to reassortment and resulted in the introduction of the reassorted H3N2, H1N1, and H1N2 subtypes. Reassortant viruses currently endemic in North American swine have combinations of different HA and NA genes with a triple-reassortant internal gene (TRIG) cassette formed by the human lineage PB1 gene, the avian lineage PA and PB2 genes, and the classical swine lineage M, NP, and NS genes. A cluster classification best depicts circulating H1 viruses; the α-, β-, and γ-clusters are represented by cH1N1-derived subtypes, and the δ-cluster is represented by H1 strains carrying an HA gene that is most closely related to human seasonal viruses. Both N1 and N2 subtypes can be found in all four clusters. In Europe, an avian-derived virus was the predominant H1N1 strain in swine until the introduction of a human H3N2 virus that led to a genetic reassortment in the mid-1980s. The H1N2 subtype, with human lineage HA and NA genes and other genes from avian-like H1N1 viruses, is also endemic in European pigs. Influenza A virus circulation in Brazilian pigs has been demonstrated by serological studies[10, 11] and some viruses were isolated,[12, 13] but the epidemiology of the infection is still unknown in the country.
After the introduction and spread of the swine-origin pandemic H1N1 virus (pH1N1) in humans in 2009, a number of countries, including Brazil, reported infection in pigs.[12, 14] The pandemic H1N1 has the M and NA genes from avian-like H1N1 Eurasian SIV and the other six genes from the North American triple-reassortant swine lineage.
Although influenza A virus infection has already been detected in swine in Brazil, these circulating viruses have not been genetically characterized and subtyped in most cases.[12, 13] Furthermore, the evolutionary and epidemiologic features of IAV strains circulating in pigs in Brazil are still unknown. Therefore, in this study, we genetically characterized the HA and NA genes of 20 influenza viruses isolated from pigs during 2009 and 2010 in five Brazilian states, which together account for more than 50% of the country's pig population.
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- Materials and methods
Pigs have receptors for both human and avian influenza viruses in their respiratory tracts and thus play an important role for interspecies transmission. They are considered a “mixing vessel” in which reassortment can occur, producing novel viruses to which the human population is naive. In Brazil, aside from a case report of pandemic H1N1 2009 virus infecting pigs in the south region, no detailed genetic or epidemiologic characterization has been reported. Therefore, this is the most comprehensive study on the molecular characterization of influenza viruses infecting pigs in different states in Brazil.
Our results indicate that influenza virus was widely spread in swine in Brazil during 2009–2010 and is associated with clinical illness in pigs of various ages. Isolates were sampled from pigs showing influenza-like signs similar to those observed during seasonal outbreaks and in previous reports of pandemic infection. All of the Brazilian swine influenza isolates described here clustered with the pandemic H1N1 influenza viruses. Thus, pandemic H1N1 virus circulated in pigs at least during 2009 and 2010, and it continued to circulate in the Brazilian swine population even when viral activity in humans was low.
Host immune pressure is thought to be the main selective force driving amino acid substitutions, which may lead to antigenic drift, and HA is the main target of neutralizing antibodies. As expected, genetic diversity in the HA gene of swine isolates was higher than in the NA gene, although all isolates clustered within the H1N1 pandemic clade. Several samples that came from the same herd were identical, indicating within-herd viral dissemination. These findings also support the hypothesis that immune pressure for viral mutation in pigs might be lower due to the constant introduction of naive animals into a herd. However, some amino acid substitutions were found at antigenic sites in Brazilian swine isolates, mainly at Ca and Cb antigenic sites, but the effect on the antigenicity of these viruses is unknown.
The constructed networks were used to provide insights upon the several possible ancestral relationships among haplotypes. Both the HA and NA dendrograms show that the pH1N1 isolates from distinct parts of the world are related to each other with a relatively small distance and share a single common origin, as expected for a pandemic outbreak. The observation that most of the human- and swine-derived isolates appear to have originated from human-derived isolates is also consistent with a higher frequency of human-to-human and human-to-swine transmission compared with zoonotic swine-to-human transmission. Indeed, reports on the 2009 pandemic outbreak suggest that the virus evolved silently in the swine host until its introduction to the human population, after which it disseminated rapidly among humans and frequently spilled over from humans to pigs. Although in line with other reports, the relationships displayed in the dendrograms should be interpreted carefully. The analysis displayed here does not account for the likely possibility that an heterogeneous mixture of viruses at the nucleotide level is produced within an infected host, as only the majority or consensus sequences are generated based on Sanger sequencing methods. Therefore, more detailed studies are needed to confirm the relationships described here.
Nonetheless, the data presented here also provide evidence of a possible swine-to-human influenza virus transmission. A technician became ill after entering a herd during a confirmed influenza outbreak. The onset of the illness was consistent with the influenza incubation period, suggesting that the visited swineherd was the most probable source of infection. Furthermore, both HA and NA genes from the human (21/2009) case show the same origin and have higher genetic relatedness to the animal samples from the same herd (16/2009 and 17/2009) than to any other human- or pig-derived isolate.
Seven distinct clades of pandemic H1N1 viruses were identified globally circulating in the beginning of the pandemic. The HA substitution S203T combined with NA V106I and N248D found in 18 of the 20 swine isolates and in the human isolate described here suggests that these Brazilian isolates might be members of clade 7 of pH1N1. Isolate 7/2009 could be clustered with clade 6 because it did not show the HA substitution. Interestingly, isolate 14/2009, which did not have the N248D NA mutation, cannot be clustered within any of the seven clades. However, a detailed amino acid analysis of all eight viral genes is necessary to confirm these assumptions. It has been demonstrated that pandemic H1N1 2009 evolved and shifted from an initial mixed clade pattern to a clade 7–predominant pattern. The subsequent selection and evolution of clade 7 resulted in the circulation of variants D222G/N or E. Mutation D222G in the hemagglutinin protein has been correlated with the clinical onset of disease, and it was frequently found in severe/fatal cases of the pandemic influenza in humans. Likewise, the substitution D222N, which was observed in isolates 19/2010 and 20/2010, is more frequent among fatal cases of the disease in humans. However, the animals from which those viruses were isolated did not show signs of severe disease.
The genetic and antigenic characterization of influenza viruses in swine populations worldwide is critical for understanding the epidemiology of the infection in that species and allowing the selection of ideal vaccine strains. Monitoring of Brazilian swineherds is necessary for establishing better control measures for swine influenza and reducing the risk of the introduction of novel strains to the susceptible human population that can lead to a pandemic. Furthermore, vaccination against swine influenza is not practiced in Brazilian herds; hence, pigs in Brazil may not have protective immunity. Together, our results suggest that the pandemic H1N1 subtype has become established in Brazilian swine populations and may become endemic in the country.