Genetic diversity of swine influenza viruses in Thai swine farms, 2011-2014.

Influenza A viruses cause respiratory diseases in human and animals. In pigs, swine influenza virus (SIV) subtype H1N1, H1N2 and H3N2 can infect pigs worldwide. In avian, avian influenza virus (AIV) subtype H1-H16 and N1-N9 can infect several avian species. This dissertation entitled “Genetic diversity and evolution of swine influenza viruses in Thailand during 2012-2015” contains 3 parts. Part 1, a cross-sectional surveillance of swine influenza virus (SIV) was conducted during September 2011 to December 2015 in 46 commercial swine farms in 11 provinces, Thailand. The results showed that the occurrence of SIVs in Thailand was 3.81% (139/3,646). The SIV isolates in this study could be subtyped as H1N1 (n=77), H1N2 (n=13) and H3N2 (n=49). Subsequently, twelve SIVs (H1N1; n=5, H1N2; n=2, H3N2; n=5) were subjected to for whole genome sequencing and genetic characterization. Genetic analysis revealed that three SIV-H1N1 were endemic Thai SIVs circulating in Thai pig population. On the other hand, nine SIVs were reassotant viruses between pandemic H1N1 2009 and endemic Thai SIV. This results suggested that reassortant SIVs were predominant genotypes in Thai pig population. Part 2, mix-genotype of SIVs were investigated in four commercial swine farms. In total, 145 nasal swab samples were collected and 18 samples were successfully performed virus isolation. The samples were subsequently subjected to whole genome sequencing by next-generation sequencing, illumina Miseq. The results showed that mix-genotypes could be observed in this study. For example, swine farms A, B and D infected with mix-genotypes of SIVs (H1, H3, N1 and N2). Moreover, SIV-H3N1, an uncommon subtype in Thailand, was identified in this study. This results indicated that illumina sequencing is able to discriminate mix-genotypes of SIVs. Part 3, highly pathogenic avian influenza (HPAI) H5N2 in USA in 2015 was investigated. Forty-six samples from turkeys and their environment were collected during the outbreak and directly subjected to illumina sequencing. The viruses were then analyzed to identify origins and evolutionary changes. The results showed that the time to most recent common ancestor analysis suggest two likely possibilities of reassortant HPAI-H5N2 origins: either a reassortment in Alaska area or multiple reassortments with North American low pathogenic avian influenza strains. In summary, this dissertation provided useful information that swine influenza and avian influenza are fast evolving of the viruses especially after the introduction of new genetic pool of the viruses into population or interspecies transmission. Thus, influenza A virus surveillance should be continuously conducted to promote awareness and prevention and control of influenza A virus infection in human and animals in the future.

Influenza A virus infections in pigs is a disease of concern to the swine industry and to the ecology and epidemiology of influenza viruses in humans. Pigs have been proposed as a “mixing vessel” for generation of pandemics via reassortment between avian and mammalian viruses. The H1N1pdm 2009 virus probably emerged from swine into humans though reassortment between the recent North American triple reassortant H1N2 swine viruses and Eurasian avian-like swine viruses.
\nSwine influenza viruses of H1N1, H1N2 and H3N2 subtypes have been regularly detected in pigs in most parts of the world. Nevertheless, ecological and virological data on swine influenza is not available in Sri Lanka, and indeed, little documented data is available in the South Asian continent. The swine population in Sri Lanka is about 80,000, and live pigs are not regularly imported to the country. Swine husbandry is largely confined to four neighboring administrative districts in the country. 
\nSystematic virological and serological surveillance carried in swine abattoirs in Sri Lanka during 2009-2013 detected H1N1pdm 2009 like virus in local herds. Infection in pigs followed each of the H1N1pdm 2009 outbreaks in humans; October 2009 – January 2010, October 2010 – February 2011 and November 2012 – March 2013, respectively. Genetic, phylogenetic, and epidemiologic analysis of the human, and swine influenza viruses indicated spillover events of H1N1pdm 2009 from humans into pigs, with self-limited transmission and extinction within pig herds. The data also indicated that although H1N1pdm 2009 was able to spill over from humans to swine, it is not ideally adapted to establish sustained transmission among swine in the absence of further reassortment with other swine influenza virus lineages. 
\nTheses finding might reflect characteristics of swine husbandry in Sri Lanka, which has a low density pig population and remains isolated from global swine influenza viruses because of the absence of regular cross-border and cross-continental movements of swine. In contrast to some other parts of the world, we failed to isolate established lineages of swine influenza viruses, viz. Classical, North American triple reassortant and European Avian lineages. Sero prevalence to these endemic swine viruses was largely absent in local swine herds. 
\nIn vitro replicative kinetic study indicated that H1N1pdm 2009 viruses isolated from swine have undergone some adaptation to swine led to decreased fitness for replication in human cells.

BackgroundHuman-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.MethodsNasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.ResultsInfluenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.ConclusionsThe present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.

Background Recent studies have revealed the existence of genetic diversity in swine influenza viruses (SIVs) in the world. In Thailand, there has been a little information on the molecular characteristics of the SIVs since the first isolation of viruses of H1N1 and H3N2 subtypes in the late 1970s. Our previous study demonstrated that Thai H1N1 SIVs possessed the classical swine H1 and avian‐like swine N1 genes (Takemae et al., Proceedings of the Options for the Control of Influenza VI.2007;350–353).Objectives In the present study, we genetically characterized 12 SIVs including those of H1N1, H1N2 and H3N2 subtypes isolated between 2000 and 2005.Methods We determined the entire nucleotide sequences of the eight gene segments of those isolates.Results Phylogenetic analysis revealed the existence of nine distinct genotypes amongst the Thai SIVs. These genotypes arose from multiple introductions of classical swine, avian‐like swine and human viruses. The existence of two distinct sublineages within classical swine H1 and NS, avian‐like swine PA and M and human H3 and N2 genes of the Thai SIVs suggested that introduction of viruses of classical swine, avian‐like swine and human origins occurred twice respectively into the Thai pig population. The predominance of avian‐like swine genes amongst the Thai SIVs was evident. In particular, three polymerase (PB1, PB2 and PA) and matrix genes of avian‐like swine origin were retained in all the Thai SIVs examined.Conclusions These observations may suggest that genes of avian‐like swine lineages have some advantages to be maintained in pigs as seen in the SIVs established through multiple introductions in other regions.

Swine are a mixing vessel for the emergence of novel reassortant influenza A viruses (IAV). Interspecies transmission of swine-origin IAV poses a public health and pandemic risk. In the United States, the majority of zoonotic IAV transmission events have occurred in association with swine exposure at agricultural fairs. Accordingly, this human-animal interface necessitates mitigation strategies informed by understanding of interspecies transmission mechanisms in exhibition swine. Likewise, the diversity of IAV in swine can be a source for novel reassortant or mutated viruses that pose a risk to both swine and human health. In an effort to better understand those risks, here we investigated the epidemiology of IAV in exhibition swine and subsequent transmission to humans by performing phylogenetic analyses using full genome sequences from 272 IAV isolates collected from exhibition swine and 23 A(H3N2)v viruses from human hosts during 2013-2015. Sixty-seven fairs (24.2%) had at least one pig test positive for IAV with an overall estimated prevalence of 8.9% (95% CI: 8.3-9.6, Clopper-Pearson). Of the 19 genotypes found in swine, 5 were also identified in humans. There was a positive correlation between the number of human cases of a genotype and its prevalence in exhibition swine. Additionally, we demonstrated that A(H3N2)v viruses clustered tightly with exhibition swine viruses that were prevalent in the same year. These data indicate that multiple genotypes of swine-lineage IAV have infected humans, and highly prevalent IAV genotypes in exhibition swine during a given year are also the strains detected most frequently in human cases of variant IAV. Continued surveillance and rapid characterization of IAVs in exhibition swine can facilitate timely phenotypic evaluation and matching of candidate vaccine strains to those viruses present at the human-animal interface which are most likely to spillover into humans.

The risk for HEV infection through transfusions of donated blood emerged in West Africa in a similar way as described in European countries.Further assessment of the transfusion risk associated with HEV-positive donors will require an evaluation of HEV RNA in prospective donors and posttransfusion surveillance of occurrence of hepatitis.

Porcine circovirus type 2 (PCV2) is the major swine pathogen associated with Porcine circovirus associated disease (PCVAD) including post-weaning multisystemic wasting syndrome (PMWS). Currently, there are 4 subtypes of PCV2 (PCV2a, b, c and d) and some epidemiological evidences demonstrated that virulence of PCV2 may relate to its subtypes. Recently, PMWS was observed more frequently in swine farms in Thailand; however, the information regarding to PCV2 subtype involved was limited. Therefore, this study was aimed to determine the association between occurrence of PMWS and PCV2 subtypes as well as genetically characterize PCV2 in Thailand. PCV2 DNA was isolated from faecal swabs and whole blood of piglets from PMWS-affected and -negative farms. The full length ORF2 sequences were compared using multiple alignment. The results showed that PCV2 DNA was detected more frequently in PMWS-affected farms. The nucleotide identities of the ORF2 from 9 PCV2 isolates representing each PMWS-affected farm and one from the negative farm ranged from 92.4 to 99.5% suggesting that there is some genetic variation of PCV2 in Thai swine. The 10 PCV2 isolates were classified into 2 clusters, in which the 7 isolates from PMWS-positive farms were in PCV2b cluster 1 A/B. The remaining isolates were separated in the new subtype called PCV2e. The results suggest the presence of new PCV2 subtypes in addition to PCV2a and PCV2b in Asian swine population. However, correlation between subtypes and virulence of PCV2 infection is not conclusive due to limited number of the PCV2 sequences from PMWS negative farms.

Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

Pandemic outbreaks of H1N1 swine influenza virus have been reported since 2009. Reassortant H1N2 viruses that contain genes from the pandemic H1N1 virus have been isolated in Italy and the United States. However, there is limited information regarding the molecular characteristics of reassortant H1N2 swine influenza viruses in eastern China. Active influenza surveillance programs in Zhejiang Province identified a novel H1N2 influenza virus isolated from pigs displaying clinical signs of influenza virus infection. Whole-genome sequencing was performed and this strain was compared with other influenza viruses available in GenBank. Phylogenetic analysis suggested that the novel strain contained genes from the 2009 pandemic human H1N1 and swine H3N2 viruses. BALB/c mice were infected with the isolated virus to assess its virulence in mice. While the novel H1N2 isolate replicated well in mice, it was found to be less virulent. These results provide additional evidence that swine serve as intermediate hosts or 'mixing vessels' for novel influenza viruses. They also emphasize the importance of surveillance in the swine population for use as an early warning system for influenza outbreaks in swine and human populations.

Influenza A viruses (IAVs) pose a major public health threat due to their wide host range and pandemic potential. Pigs have been proposed as “mixing vessels” for avian, swine, and human IAVs, significantly contributing to influenza ecology. In the United States, IAVs are enzootic in commercial swine farming operations, with numerous genetic and antigenic IAV variants having emerged in the past two decades. However, the dynamics of intensive swine farming systems and their interactions with ecological factors influencing IAV evolution have not been systematically analysed. This review examines the evolution of swine IAVs in commercial farms, highlighting the role of multilevel ecological factors. A total of 61 articles published after 2000 were reviewed, with most studies conducted after 2009 in Midwestern US, followed by Southeast and South-central US. The findings reveal that ecological factors at multiple spatial scales, such as regional transportation networks, interconnectedness of swine operations, farm environments, and presence of high-density, low-genetic diversity herds, can facilitate virus transmission and enhance virus evolution. Additionally, interactions at various interfaces, such as between commercial swine and feral swine, humans, or wild birds contribute to the increase in genetic diversity of swine IAVs. The review underscores the need for comprehensive studies and improved data collection to better understand the ecological dynamics influencing swine IAV evolution. This understanding is crucial for mitigating disease burden in swine production and reducing the risk of zoonotic influenza outbreaks.

Influenza A virus (IAV) can infect avian and mammalian species, including humans. The genome nature of IAVs may contribute to viral adaptation in different animal hosts, resulting in gene reassortment and the reproduction of variants with optimal fitness. As seen again in the 2009 swine-origin influenza A H1N1 pandemic, pigs are known to be susceptible to swine, avian, and human IAVs and can serve as a ‘mixing vessel’ for the generation of novel IAV variants. To this end, the emergence of swine influenza viruses must be kept under close surveillance. Herein, we report the isolation and phylogenetic study of a swine IAV, A/swine/Korea/PL01/2012 (swPL01, H3N2 subtype). After screening nasopharyngeal samples from pigs in the Gyeongsangnam-do region of Korea from December 2011 to May 2012, we isolated the swPL01 virus and sequenced its all of 8 genome segments (polymerase basic 2, PB2; polymerase basic 1, PB1; polymerase acidic, PA; hemagglutinin, HA; nucleocapsid protein, NP; neuraminidase, NA; matrix protein, M; and nonstructural protein, NS). The phylogenetic study, analyzed with reference strains registered in the National Center for Biotechnology Information (NCBI) database, indicated that the swPL01 virus was similar to the North American triple-reassortant swine strains and that the HA gene of the swPL01 virus was categorized into swine H3 cluster IV. The swPL01 virus had the M gene of the triple-reassortant swine H3N2 viruses, whereas that of other contemporary strains in Korea was transferred from the 2009 pandemic H1N1 virus. These data suggest the possibility that various swine H3N2 viruses may co-circulate in Korea, which underlines the importance of a sustained surveillance system against swine IAVs.

The global anxiety and a significant threat to public health due to the current COVID-19 pandemic reiterate the need for active surveillance for the zoonotic virus diseases of pandemic potential. Influenza virus due to its wide host range and zoonotic potential poses such a significant threat to public health. Swine serve as a “mixing vessel” for influenza virus reassortment and evolution which as a result may facilitate the emergence of new strains or subtypes of zoonotic potential. In this context, the currently available scientific data hold a high significance to unravel influenza virus epidemiology and evolution. With this objective, the current systematic review summarizes the original research articles and case reports of all the four types of influenza viruses reported in swine populations worldwide. A total of 281 articles were found eligible through screening of PubMed and Google Scholar databases and hence were included in this systematic review. The highest number of research articles (n = 107) were reported from Asia, followed by Americas (n = 97), Europe (n = 55), Africa (n = 18), and Australia (n = 4). The H1N1, H1N2, H3N2, and A(H1N1)pdm09 viruses were the most common influenza A virus subtypes reported in swine in most countries across the globe, however, few strains of influenza B, C, and D viruses were also reported in certain countries. Multiple reports of the avian influenza virus strains documented in the last two decades in swine in China, the United States, Canada, South Korea, Nigeria, and Egypt provided the evidence of interspecies transmission of influenza viruses from birds to swine. Inter-species transmission of equine influenza virus H3N8 from horse to swine in China expanded the genetic diversity of swine influenza viruses. Additionally, numerous reports of the double and triple-reassortant strains which emerged due to reassortments among avian, human, and swine strains within swine further increased the genetic diversity of swine influenza viruses. These findings are alarming hence active surveillance should be in place to prevent future influenza pandemics.

Real-time reverse-transcription PCR (rRT-PCR) assays are currently the method of choice in many laboratories for the detection and subtyping of influenza A virus (IAV) in swine. Traditionally, nasal swabs and lung tissues (sometimes bronchoalveolar lavage and tracheal tissues) are the primary specimens for IAV testing. However, oral fluids are becoming more common for IAV prognostic profiling. In this chapter, we describe (1) procedures of RNA extraction from the common clinical specimens, (2) two rRT-PCR assays for detection of IAV in swine, and (3) an rRT-PCR assay for subtyping swine IAV. RNA extraction procedures include a magnetic bead method optimized for extraction from nasal swabs and tissue homogenates and a magnetic bead method optimized for extraction from oral fluids. Two rRT-PCR assays for detection of swine IAV include a USDA-validated IAV rRT-PCR targeting the matrix gene and the USDA-licensed VetMAX™-Gold Swine Influenza Virus Detection rRT-PCR kit (Thermo Fisher Scientific) targeting the nucleoprotein and matrix genes. The swine IAV subtyping assay described here is VetMAX™-Gold Swine Influenza Virus Subtyping rRT-PCR kit (Thermo Fisher Scientific) which distinguishes swine IAV H1 from H3 and N1 from N2.

Serological evidence of pig-to-human influenza virus transmission on Thai swine farms

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