Open Access

Isolation and complete genomic characterization of H1N1 subtype swine influenza viruses in southern China through the 2009 pandemic

  • Yizhi Liu1,
  • Jun Ji1,
  • Qingmei Xie1Email author,
  • Jing Wang1,
  • Huiqin Shang1,
  • Cuiying Chen1,
  • Feng Chen1, 2,
  • Chunyi Xue3,
  • Yongchang Cao3,
  • Jingyun Ma1 and
  • Yingzuo Bi1
Contributed equally
Virology Journal20118:129

https://doi.org/10.1186/1743-422X-8-129

Received: 31 December 2010

Accepted: 20 March 2011

Published: 20 March 2011

Abstract

Background

The swine influenza (SI) is an infectious disease of swine and human. The novel swine-origin influenza A (H1N1) that emerged from April 2009 in Mexico spread rapidly and caused a human pandemic globally. To determine whether the tremendous virus had existed in or transmitted to pigs in southern China, eight H1N1 influenza strains were identified from pigs of Guangdong province during 2008-2009.

Results

Based on the homology and phylogenetic analyses of the nucleotide sequences of each gene segments, the isolates were confirmed to belong to the classical SI group, with HA, NP and NS most similar to 2009 human-like H1N1 influenza virus lineages. All of the eight strains were low pathogenic influenza viruses, had the same host range, and not sensitive to class of antiviral drugs.

Conclusions

This study provides the evidence that there is no 2009 H1N1-like virus emerged in southern China, but the importance of swine influenza virus surveillance in China should be given a high priority.

Background

Swine influenza (SI) is an acute respiratory disease caused by influenza A virus, a member of the Orthomyxoviridae family. Influenza A viruses have been isolated from a number of animals including humans, birds, dogs, seals, horses and swine [1]. The causative agent is RNA virus with a segmented genome comprised of eight negative-sense, single-stranded RNA segments encoding eleven proteins [2].

Hemagglutinin (HA) and neuraminidase (NA) are two major surface glycoproteins of influenza viruses and important for host range, antigenicity, pathogenesis and diagnostic detection. Influenza A viruses are classified into a number of subtypes based on antigenic differences in haemagglutinin (HA; 16 subtypes) and neuraminidase (NA; 9 subtypes) [3, 1]. Although other subtypes have been sporadically identified, there are three major subtypes of SIV (H1N1, H1N2, and H3N2) circulating in the swine population throughout the world currently [4, 5].

Pigs have the susceptibility of infecting avian and human influenza, for the cells respiratory tract express both sialic acid-a2,3-galactose (SAa2,3Gal) receptors and sialic acid-a2,6-galactose (SAa2,6Gal) receptors, preferred by avian influenza viruses and human influenza viruses, respectively [6, 7]. Therefore, pigs play an important role in supporting co-infection, replication, and reassortment among human, avian, and swine influenza viruses, acting as an intermediate host or mixing vessels thereby creating novel reassortant viruses with pandemic potential [4, 6, 8].

Since Influenza was first described as a disease of swine in 1918, the viruses had continued to cause diseases in populations of pigs worldwide [9]. Much like human influenza [10], SIVs have been shown to be evolving by both antigenic drift and frequent reassortment [11, 12]. Although infection of humans with swine influenza is a relatively uncommon occurrence, H1N1 and H3N2 of swine origin have been reported previously in North America, Europe and Asia, and have caused sporadic human infections over the past decades [1318].

As one of the mainly representative subtypes, swine H1N1 influenza viruses have been reported widely throughout the world. The world-wide H1N1 pandemic in1918 also affected swine, and another classical swine H1N1 virus strain caused the well-known outbreak at Ft. Dix in New Jersey in 1976 [19]. In March and early April 2009, a new swine-origin influenza A (H1N1) Virus (S-OIV) emerged in Mexico and the United States, had developed into the first pandemic of the 21st century [20, 21]. China is generally regarded as an epicenter of pandemic influenza viruses throughout history. In 2009 pandemic, 71 of the first 426 imported cases with confirmed cases of infection were from Guangdong province which was one of the most severely disaster area in China. It is documented that SIVs have become more human-like as well as more avian-like, the probability of generating human-adaptive viruses increase. Co-infections have allowed intermixing of these genomes to produce triple reassortants with genes derived from human, swine and avian influenza strains [22].

For a better understanding the influenza A virus lineages and evolution influenced by the 2009 H1N1, the study were focused on H1N1 SIVs in Guangdong province from 2008 to 2009. The whole genome sequence of each strain were analyzed, intending to learn more epidemiology information about the prevalence of SIVs, the possible correlations with the 2009 H1N1 strains in southern China, and provide useful guides for SI control.

Results

Virus isolation and identification

According to the detection results, eight isolates were positive for HA tests, subtyped to be H1N1 by RT-PCR assays. The eight H1N1 isolates obtained in this study were listed in Table 1.
Table 1

SIV strains isolated from Guangdong province of southern China.

Strain Name

PB1

PB2

PA

NA

HA

NP

NS

M

A/swine/Guangdong/11/2009/H1N1

HM145744 a

HM145745

HM145743

HM145741

HM135403

HM145742

HM145747

HM145746

A/swine/Guangdong/09/2009/H1N1

HM210858

HM210859

HM210857

HM210854

HM210852

HM210855

HM210856

HM210853

A/swine/Guangdong/07/2009/H1N1

HM210866

HM210867

HM210865

HM210862

HM210860

HM210863

HM210864

HM210861

A/swine/Guangdong/06/2009/H1N1

HM215157

HM215158

HM215156

HM215153

HM215151

HM215154

HM215155

HM215152

A/swine/Guangdong/05/2009/H1N1

HM215165

HM215166

HM215164

HM215161

HM215159

HM215162

HM215163

HM215160

A/swine/Guangdong/09/2008/H1N1

HM215173

HM215174

HM215172

HM215169

HM215167

HM215170

HM215171

HM215168

A/swine/Guangdong/07/2008/H1N1

HM223592

HM223593

HM223591

HM223588

HM223586

HM223589

HM223590

HM223587

A/swine/Guangdong/02/2008/H1N1

HM223600

HM223601

HM223599

HM223596

HM223594

HM223597

HM223598

HM223595

a accession number of each segmented gene

Sequence identity analysis

The nucleotide sequence identities among the eight genes of each isolate were 99-100%, suggesting that the eight isolates might evolve from the same progenitor viruses. The genotypes and genetic origins of isolates were initially inferred from BLAST analysis and pairwise comparisons of each gene segment to the corresponding sequences of reference viruses. Taking into account the high levels of sequence identity between the eight isolates, isolate A/swine/Guangdong/11/2009 was used as the representative strain for blast search in the Genbank. Each of the eight genes of the eight isolates showed the highest nucleotide sequence identities to those of classical swine H1N1 virus, with homologies ranging from 98.8 to 99.6%.

Phylogenetic relationship and distribution

To characterize the gene segments of the isolated viruses more precisely, the phylogenetic trees using the nucleotide sequences of eight genes were constructed with genes available in GenBank respectively. Three lineages correlative with 2009 human strains, classical swine strains and European swine strains were observed in the phylogenetic trees (Figures 1, 2, 3, 4, 5, 6, 7 & 8). All the eight genes of the eight isolates, belonged to the same sublineage closely related to the classical swine H1N1 viruses, especially those isolated in China. The phylogenetic trees revealed that the HA, NP and NS segments of the isolated strains with the 2009 H1N1 strains were listed in the sister lineages of classical SIV (Figures 1, 3, 4), while the PB1, PB2, PA, NA and M genes of the eight genes were closed to the European H1N1 SIV (Figures 2, 5, 6, 7, 8). Meanwhile, two H3N2 viruses were also closely relative to the classical swine lineage of PA, M and NS, suggested the reassortment with the classical swine H1N1 viruses (Figures 4, 7, 8).
Figure 1

Phylogenetic trees of the HA genes of influenza A viruses. Analysis was based on the nucleotide sequences in open reading frames of the HA genes. The subtypes of the viruses were marked in the brackets. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 2

Phylogenetic trees of the NA genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 3

Phylogenetic trees of the NP genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 4

Phylogenetic trees of the NS genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 5

Phylogenetic trees of the PB1 genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 6

Phylogenetic trees of the PB2 genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 7

Phylogenetic trees of the PA genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Figure 8

Phylogenetic trees M genes of influenza A viruses. The virus marked with black triangle is the swine H1N1 virus isolated and sequenced in this study.

Key site analyses

The deduced amino acid sequences of the segemented genes were aligned and compared with the reference H1N1 strains. These isolates contained an amino acid motif PSIQSR↓G at the HA cleavage sites, which met the characteristic of low pathogenic influenza viruses. The predicted antigenic epitopes of the HA protein of the isolates were similar to the classical H1N1 SIVs, revealed five major changes located at the position 36, 120, 138, 170 and 278 between different lineages (Figure 9). The specific mutation was located at the position 36 between the isolated strains and the 2009 H1N1 strains, and the mutation located 138 compared to the avian like strains. In the study, all of the isolates displayed the mutations located at 120 and 170 between all of the avian like, 2009 H1N1-like and the classical H1N1 strains, but shared the same antigenic epitope at 278 with 2009 H1N1-like strains only.
Figure 9

Antigenic epitopes prediction (as showed by the upper peaks) of the HA1 of A/swine/Guangdong/11/2009 (represented the isolated strains). The mutated sites were pointed with the arrow.

The eight isolates maintained amino acids typical of swine viruses at residues 68E, 134A, 183P, 187D, 191L and 222G (according to H1 number) of HA genes, which were previously defined receptor-binding sites [23, 24]. Only the European swine strains possessed D at position 68, A/swine/Zhejiang/1/2007/H1N1 possessed V at position 134, all the strains in this study possessed P at position 183, the 2009 H1N1 strains possessed S at position 183. The eight strains shared the same amino acid at the receptor-binding sites, which suggests that these isolates might have the same host range. As amino acid position 222 is in the receptor binding cavity, changes can potentially influence receptor binding of the influenza virus as well. In this study, the eight isolated strains, classical strains and a European strain possessed G at the position 222. The 2009 H1N1 strains possessed D, while the other European swine strains possessed E at the position 222.

The N-linked glycosylation sites are relatively conserved, and the number of potential glycosylation site (PGS) changes may influence the protein folding, oligomerization, quality control, sorting and transport [25]. Six potential glycosylation sites (PGSs) with an N-X-T/S motif (where X can be any amino acid except proline) were conserved at positions 10, 11, 23, 87, 276, and 287 in the HA1 and two were conserved at positions 154, 213 in the HA2 protein of A/Swine/Guangdong/11/2009. The isolated strains, 2009 H1N1 strains and the strains from Shanghai, Maryland and Kentucky of Classical swine had the same PGSs. The Wisconsin and Tennessee of Classical swine strains as the avian-like strains did not have the potential glycosylation sites at the 276 position.

A balance between the activities of HA in virus attachment and NA in virus release needs to be maintained for optimal viral replication [1]. No amino acid substitutions (H274Y and N294S) conferring resistance to oseltamivir were observed at the conserved residues in the NA proteins of the eight isolates, which suggest that they are sensitive to NA inhibitors. Eight PGSs in NA protein were found in the isolated strains and 2009 H1N1 strains, located at positions 44,58,63,68, 88,146,235 and 386 in linker region and the NA domain. The eight isolates shared the same PGSs distributions with the 2009 H1N1 strains, had two extra PGSs at the position 386 compared to the Wisconsin and Tennessee of Classical swine strains. The avian like H1N1 (A/swine/England/WVL7/1992/H1N1) carried different eight PGSs located amino acid positions 36, 44, 49, 54, 74, 132, 221 and 372 in the NA molecule.

Pathogenicity of influenza virus was polygenic. Internal proteins (M, PB1, PB2 and NS) also harbor determinants for host range and virulence, as demonstrated by genetic studies on avian-human reassortant viruses [26]. Anti-influenza drugs amantadine and rimantadine target the M2 protein, and the substitution S31N have been reported to confer resistance to these drugs in recent European swine viruses, including H1N1, H1N2, and H3N2 viruses [27]. Interestingly, the strains of same lineage had difference at the position of 31. The substitution S31N observed in the M2 protein genes of our isolates suggested not sensitive to the primary class of antiviral drugs.

PB1-F2 has been shown to localize to the mitochondria and induce apoptosis, enhance viral virulence in a mouse model [28, 29]. Similar to most classical swine H1N1 viruses, the eight isolates contained truncated PB1-F2 proteins because of in-frame stop codon after residue 11. The highly conserved regions in the PB1 protein that may be functionally important and the amino acid residues at 375 might be critical for host specificity of the protein. Some difference appeared at the position of 375 between different strains. All of the eight isolates, 2009 H1N1 strains and Shanghai, Maryland strains possessed S at position 375, European swine strains were D or N, but other classical strains were G.

Previous study showed that residue 627 of the polymerase basic protein 2 (PB2) was associated with host range adaptation and recognized as one of the most important determinants [3033]. In this study, each of the eight isolates possessed a K at position 627 of PB2 which was characteristic of mammalian influenza viruses, the classical swine strains possessed a K at position 627 of PB2, and the 2009 Human strains and avian-like strains were E.

The NS1 protein is also important in determining the pathogenicity of influenza A virus in different hosts. No deletions were observed at positions 80-84 of NS1 proteins in the isolates. All eight isolates possessed D rather than E at position 92 of NS1, which possesed a mutation might increase the virulence of H5N1 viruses in pigs. The exceptional A/swine/England/WVL7/1992/H1N1 (avian like H1N1) possessed T at position 92 of NS1.

Discussion

Influenza virus infection is an important cause of respiratory disease among pigs throughout the swine producing regions of the world [34]. Pigs have been considered as mixing vessel for human and avian influenza viruses, allowing swine viruses to acquire avian and human virus gene segments to generate novel SIVs. In China, various subtypes of SIVs were present in pig populations. Four subtypes (H1N1, H1N2, H3N1, and H3N2) were circulating in pig populations, and the classical swine H1N1 viruses were the predominant influenza virus infecting and circulating in swine population [35]. In southern China, reassortment of classical swine H1N1 and human-like H3N2 viruses had been reported from 1976 to 1982 [3638] and avian-like swine H1N1 viruses co-circulated with classical swine H1N1 viruses were also detected in pigs [39]. In last several years, many studies reported the co-circulation and reassortment of classical swine H1N1, avian-like H1N1 viruses and human-like H3N2 viruses in most area of China [37, 38]. Some serological surveillance had also indicated that avian H4, H5 and H9 influenza viruses had been transmitted to pig populations in southeastern China [40, 41].

Influenza virus genomes are well known to undergo antigenic drift or antigenic shift that enable escape from preexisting immunity and cause new outbreaks of influenza in animals and even humans [4244]. In China, pigs have a short lifespan (approximately 6 months) and are not vaccinated with any type of swine influenza vaccine. Therefore, the viruses circulating in swine population might appear to have been under less immune selection pressure and all genes evolved more slowly than in humans and poultry [45]. The classical H1N1 remained highly conserved genetically until the 1990s, with only a limited number of nucleotide changes [46, 47]. However, since the introduction of H3N2 viruses with genes of human and avian origin into pig populations and the resultant reassortment with classical H1N1 viruses, the dynamics of clinical disease and prevention of outbreaks has changed dramatically only recently.

In the present study, the phylogenetic and molecular property of the H1N1 SIV strains isolated at Guangdong province in southern China through the 2009 pandemic were described in detail. The whole genome sequences were analyzed, and the genetic relatedness of these isolates with 2009 H1N1 viruses, classical SIV and European SIV were studied. Based on the phylogenetic trees and homology of the nucleotide sequence of gene segments, the isolates were confirmed to be the classical swine virus. The nucleotide sequence identities among the eight genes were 99%-100%, indicated that the classical SIV H1N1 circulating in Guangdong of Southern China were resistant and might derive from the same ancestor. Interestingly, PA, M, and NS genes of two H3N2 strains from Guangdong province were located in similar lineages of the strains in the study, indicated the reassortment had occurred in classical H1N1 viruses. The isolated strains contained genes from the neighboring lineages with 2009 H1N1 which genetically distant from the avian-like clusters, provided further evidences that isolates in the study and the 2009 H1N1 viruses might be related with a finite extent.

Some key amino acids of the segmented gene have been implicated to play the vital role in host range adaptation, pathogenicity of influenza A virus in different hosts, receptor binding and antiviral drug resistance, and these traits are polygenic. Based on whole genome sequence analysis, the characteristics of each isolated strain were deeply and profoundly studied, revealed some resemblance and difference between different lineages. Generally, low pathogenic influenza viruses raise lowly concerning made these strains might have a greater opportunity to become widespread. The isolated strains had the same S at position 375 of PB1, as 2009 human strains, suggested that these strains should be raised more concern for the potentiality to infect human. It should be further studied to determine whether the PGSs changed in glucoprotein of the virion surface could alter the pathogenicity. Compared to the avian-like strains, the extra PGSs in HA protein at the 276 position supposed to enhance the pathogenicity for wide prevalence, and the additional PGSs in NA protein might reduce the ability of affinity between influenza virus and the host receptor as well as the release of the virus particles.

The special environment and lifestyle in southern China provide more chances for wild aquatic birds, domestic poultry, pigs and humans to contact closely, and create the opportunity for interspecies transmission and generation of new reassortment influenza viruses. Although the 2009 H1N1 has not been identified circulating endemically in swine, it is virtually impossible to prevent new outbreaks of influenza in human and animals. It had been recognized that influenza viruses in pigs may form the basis for newly emerging influenza pandemic after further adaptation to the human host or reassortment with other influenza A viruses. Therefore, carrying out swine influenza virus surveillance would be of great significance.

Conclusions

In conclusion, the present study has demonstrated that the circulating SIV strains in commercial farms in southern China are mainly classical swine H1N1. In spite of the low pathogenicity, the strains in the study might have limit evolutionary correlations with the 2009 H1N1 viruses, but also provides the evidence that pigs served as reservoirs of ancestor influenza viruses for emergence of pandemic strains. We hope this study could provide some useful information for the prediction and preparedness of possibly future influenza pandemics.

Materials and methods

Viruses

Nasal swabs were collected from diseased swine in different pig farms from Guangdong province of southern China. Initial isolation was performed in10-day-old specific-pathogen-free embryonated chicken eggs. The Allantoic fluids were harvested after 3-4 days of incubation, and primarily diagnosed by HA test. The viruses were further confirmed and subtyped by 2 different multiplex RT-PCR as previously described. The remains were preserved at -70°C for the subsequent trials.

RNA extraction and gene sequence

RNA was extracted from allantoic fluids using the QIAamp viral RNA Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The full length of all eight genes (HA, NA, PA, PB1, PB2, NP, NS, M) of each isolates were amplified by RT-PCR using universal primer sets [48, 49]. Reverse transcription polymerase chain reaction (RT-PCR) was carried out by PrimeScriptTM One-Step RT-PCR Kit (TaKaRa Biotechnology, Dalian, China) in 25 μl reaction volume containing 20 μl of RT-PCR PreMix (reaction buffer, dNTPs, 2 μl of enzyme mix), 2 μl of extracted viral RNA and the specific primer pair. The PCR products were cloned into pMD19-T vector (TaKaRa Biotechnology, Dalian, China) for later sequencing (AuGCT Biotechnology, Beijing, China).

Sequence analysis and phylogenetic analysis

Sequences were assembled using the Lasergene Seqman package IV (DNAStar 6.0) and aligned using multiple sequence alignment tool CLUSTAL W 1.83. The phylogenetic trees were constructed using the MEGA 4.1 software with neighbor-joining method [50] and each tree was produced using a consensus of 1000 bootstrap replicates. All the other H1N1 viral sequences of eight genes for reference were from the GenBank database.

Nucleotide sequence accession numbers

The segmented sequences determined in this study were available from GenBank database and the corresponding accession numbers were listed in Table 1.

Antigenicity and surface probability analysis

DNA Star software was used to analyze the antigenicity epitopes of the HA proteins of each isolates, using Jameson-Wolf methods (DNAStar Inc., Madison, WI, USA). Mature peptide sequences of HA and NA genes of the H1N1 SIV strains were submitted for prediction of potential N-glycosylation sites using the CBS on-line server 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/).

Notes

Declarations

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant no. 31072152).

Authors’ Affiliations

(1)
College of Animal Science, South China Agricultural University
(2)
Guangdong Wen's Foodstuffs Group Co. Ltd.
(3)
State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University

References

  1. Webster RG, Bean WJ, Gorman OT, Chamber TM, Kawaoka Y: Evolution and ecology of influenza A viruses. Microbiol Rev. 1992, 56: 152-179.PubMed CentralPubMedGoogle Scholar
  2. Brockwell-Staats C, Webster RG, Webby RJ: Diversity of Influenza Viruses in Swine and the Emergence of a Novel Human Pandemic Influenza A (H1N1). Influenza Other Respi Viruses. 2009, 3: 207-213. 10.1111/j.1750-2659.2009.00096.x.PubMed CentralView ArticleGoogle Scholar
  3. Fouchier RAV, Munster A, Wallensten TM, Bestebroer S, Herfst D, Smith GF, Rimmelzwaan B, Olsen CW, Osterhaus AD: Characterization of a novel influenza virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol. 2005, 79: 2814-2822. 10.1128/JVI.79.5.2814-2822.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Brown IH: The epidemiology and evolution of influenza viruses in pigs. Vet Microbiol. 2000, 74: 29-46. 10.1016/S0378-1135(00)00164-4.View ArticlePubMedGoogle Scholar
  5. Jung K, Chae C: Phylogenetic analysis of an H1N2 influenza A virus isolated from a pig in Korea. Arch Virol. 2004, 149: 1415-1422. 10.1007/s00705-004-0324-9.View ArticlePubMedGoogle Scholar
  6. Ito T, Couceiro JN, Kelm S, Baum LG, Krauss S, Castrucci MR, Donatelli I, Kida H, Paulson JC, Webster RG, Kawaoka Y: Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol. 1998, 72: 7367-7373.PubMed CentralPubMedGoogle Scholar
  7. Peiris JSM, Guan Y, Markwell D, Ghose P, Webster RG, Shortridge KF: Cocirculation of avian H9N2 and contemporary ''human'' H3N2 influenza A viruses in pigs in southeastern China: potential for genetic reassortment?. J Virol. 2001, 75: 9679-9686. 10.1128/JVI.75.20.9679-9686.2001.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Landolt GA, Karasin AI, Phillips L, Olsen CW: Comparison of the pathogenesis of two genetically different H3N2 influenza A viruses in pigs. J Clin Microbiol. 2003, 141: 1936-1941. 10.1128/JCM.41.5.1936-1941.2003.View ArticleGoogle Scholar
  9. Vincent AL, Ma W, Lager KM, Janke BH, Richt JA: Swine influenza viruses a North American perspective. Adv Virus Res. 2008, 72: 127-154. 10.1016/S0065-3527(08)00403-X.View ArticlePubMedGoogle Scholar
  10. Rambaut A, Pybus OG, Nelson MI, Viboud C, Taubenberger JK, Holmes EC: The genomic and epidemiological dynamics of human influenza A virus. Nature. 2008, 453: 615-619. 10.1038/nature06945.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Brown IH, Ludwig S, Olsen CW, Hannoun C, Scholtissek C, Hinshaw VS, Harris PA, McCauley JW, Strong I, Alexander DJ: Antigenic and genetic analyses of H1N1 influenza A viruses from European pigs. J Gen Virol. 1997, 78: 553-562.View ArticlePubMedGoogle Scholar
  12. Dunham EJ, Dugan VG, Kaser EK, Perkins SE, Brown IH, Holmes EC, Taubenberger JK: Different evolutionary trajectories of European avian-like and classical swine H1N1 influenza A viruses. J Virol. 2009, 83: 5485-5494. 10.1128/JVI.02565-08.PubMed CentralView ArticlePubMedGoogle Scholar
  13. Class EC, Kawaoka Y, de Jong JC, Masurel N, Webster RG: Infection of children with avian-human reassortant influenza virus from pigs in Europe. Virology. 1994, 204: 453-457. 10.1006/viro.1994.1553.View ArticleGoogle Scholar
  14. Gregory V, Lim W, Cameron K, Bennett M, Marozin S, Klimov A, Hall H, Cox N, Hay A, Lin YP: Infection of a child in Hong-Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs. J Gen Virol. 2001, 82: 1397-1406.View ArticlePubMedGoogle Scholar
  15. Gray GC, McCarthy T, Capuano AW, Setterquist SF, Olsen CW, Alavanja MC: Swine workers and swine influenza virus infections. Emerg Infect Dis. 2007, 13: 1871-1878. 10.3201/eid1302.060680.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Myers KP, Olsen CW, Gray GC: Cases of swine influenza in humans: a review of the literature. Clin Infect Dis. 2007, 44: 1084-1088. 10.1086/512813.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Vincent AL, Swenson SL, Lager KM, Gauger PC, Loiacono C, Zhang Y: Characterization of an influenza A virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the United States. Vet Microbiol. 2009, 137: 51-59. 10.1016/j.vetmic.2009.01.003.View ArticlePubMedGoogle Scholar
  18. Wells DL, Hopfensperger DJ, Arden NH, Harmon MW, Davis JP, Tipple MA, Schonberger LB: Swine influenza virus infections. Transmission from ill pigs to humans at a Wisconsin agricultural fair and subsequent probable person-to-person transmission. J Am Med Assoc. 1991, 265: 478-481. 10.1001/jama.265.4.478.View ArticleGoogle Scholar
  19. Gaydos JC, Top FH, Hodder RA, Russell PK: Swine influenza a outbreak, Fort Dix, New Jersey, 1976. Emerg Infect Dis. 2006, 12: 23-28.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, Sessions WM, Xu X, Skepner E, Deyde V, Okomo-Adhiambo M, Gubareva L, Barnes J, Smith CB, Emery SL, Hillman MJ, Rivailler P, Smagala J, de Graaf M, Burke DF, Fouchier RA, Pappas C, Alpuche-Aranda CM, Lopez-Gatell H, Olivera H, Lopez I, Myers CA, Faix D, Blai PJ, Yu C, Keene KM, Dotson PD, Boxrud D, Sambol AR, Abid SH, St George K, Bannerman T, Moore AL, Stringer DJ, Blevins P, Demmler-Harrison GJ, Ginsberg M, Kriner P, Waterman S, Smole S, Guevara HF, Belongia EA, Clark PA, Beatrice ST, Donis R, Katz J, Finelli L, Bridges CB, Shaw M, Jernigan DB, Uyeki TM, Smith DJ, Klimov AI, Cox NJ: Antigenic and genetic characteristics of swine-origin 2009 A (H1N1) influenza viruses circulating in humans. Science. 2009, 325: 197-201. 10.1126/science.1176225.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JS, Guan Y, Rambaut A: Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. 2009, 459: 1122-1125. 10.1038/nature08182.View ArticlePubMedGoogle Scholar
  22. Olsen CW, Karasin AI, Carman S, Li Y, Bastien N, Ojkic D, Alves D, Charbonneau G, Henning BM, Burton DE: Triple reassortant H3N2 influenza A viruses, Canada, 2005. Emerg Infect Dis. 2006, 12: 1132-1135.PubMed CentralView ArticlePubMedGoogle Scholar
  23. Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR, Donatelli I, Kawaoka Y: Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol. 2000, 74: 8502-8512. 10.1128/JVI.74.18.8502-8512.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  24. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC: Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988, 333: 426-431. 10.1038/333426a0.View ArticlePubMedGoogle Scholar
  25. Igarashi M, Ito K, Kida H, Takada A: Genetically destined potentials for N-linked glycosylation of influenza virus hemagglutinin. Virology. 2008, 376: 323-329. 10.1016/j.virol.2008.03.036.View ArticlePubMedGoogle Scholar
  26. Peiris JSM, de Jong MD, Guan Y: Avian Influenza Virus (H5N1): a Threat to Human Health. Cli Microbiol Rev. 2007, 20: 243-267. 10.1128/CMR.00037-06.View ArticleGoogle Scholar
  27. Marozin S, Gregory V, Cameron K, Bennett M, Valette M, Aymard M, Foni E, Barigazzi G, Lin Y, Hay A: Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe. J Gen Virol. 2002, 83: 735-745.View ArticlePubMedGoogle Scholar
  28. Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, Basta S, O'Neill R, Schickli J, Palese P, Henklein P, Bennink JR, Yewdell JW: A novel influenza A virus mitochondrial protein that induces cell death. Nat Med. 2001, 7: 1306-1312. 10.1038/nm1201-1306.View ArticlePubMedGoogle Scholar
  29. Zamarin D, Ortigoza MB, Palese P: Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. J Virol. 2006, 80: 7976-7983. 10.1128/JVI.00415-06.PubMed CentralView ArticlePubMedGoogle Scholar
  30. Gabriel G, Abram M, Keiner B, Wagner R, Klenk HD, Stech J: Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J Virol. 2007, 81: 9601-9604. 10.1128/JVI.00666-07.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Hatta M, Gao P, Halfmann P, Kawaoka Y: Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science. 2001, 293: 1840-1842. 10.1126/science.1062882.View ArticlePubMedGoogle Scholar
  32. Labadie K, Dos Santos Afonso E, Rameix-Welti MA, van der Werf S, Naffakh N: Host-range determinants on the PB2 protein of influenza A viruses controlthe interaction between the viral polymerase and nucleoprotein in human cells. Virology. 2007, 362: 271-282. 10.1016/j.virol.2006.12.027.View ArticlePubMedGoogle Scholar
  33. Subbarao EK, London W, Murphy BR: A single amino acid in the PB2 gene ofinfluenza A virus is a determinant of host range. J Virol. 1993, 67: 1761-1764.PubMed CentralPubMedGoogle Scholar
  34. Karasin AI, Schutten MM, Cooper LA, Smith CB, Subbarao K, Anderson GA, Carman S, Olsen CW: Genetic characterization of H3N2 influenza viruses isolated from pigs in North America 1977-1999: evidence for wholly human and reassortant virus genotypes. Virus Res. 2000, 68: 71-85. 10.1016/S0168-1702(00)00154-4.View ArticlePubMedGoogle Scholar
  35. Guo Y, Webster RG, Zhuge YH: Swine (H1N1) viruses isolated from pigs in China and studies on the origin of isolates. Chin J Clin Exp Virol. 1992, 6: 347-353.Google Scholar
  36. Shortridge KF, Webster RG: Geographical distribution of swine (Hsw1N1) and Hong Kong(H3N2)influenza virus variants in pigs in southeast Asia. Intervirology. 1979, 11: 9-15. 10.1159/000149006.View ArticlePubMedGoogle Scholar
  37. Sun L, Zhang GH, Shu YL, Chen XM, Zhu YP, Yang LM, Ma GP, Kitamura Y, Liu WJ: Genetic correlation between H3N2 human and swine influenza viruses. J Clin Virol. 2009, 44: 141-144. 10.1016/j.jcv.2008.10.011.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Xu C, Fan W, Wei R, Zhao H: Isolation and identification of swine influenza recombinant A/Swine/Shandong/1/2003(H9N2) virus. Microbes Infect. 2004, 6: 919-925. 10.1016/j.micinf.2004.04.015.View ArticlePubMedGoogle Scholar
  39. Guan Y, Shortridge KF, Krauss S, Webster RG: Emergence of avian H1N1 viruses in pigs in China. J Virol. 1996, 70: 8041-8046.PubMed CentralPubMedGoogle Scholar
  40. Li HY, Yu KZ, Xin XG, Yang HL, Li YB, Qin YN, Bi YZ, Tong GZ, Chen HL: Serological and virologic surveillance of swine influenza in China from 2000 to 2003. Int Congress Series. 2004, 1263: 754-757. 10.1016/j.ics.2004.04.002.View ArticleGoogle Scholar
  41. Ninomiya A, Takada A, Okazaki K, Shortridge KF, Kida H: Seroepidemio-logical evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China. Vet Microbiol. 2002, 88: 107-114. 10.1016/S0378-1135(02)00105-0.View ArticlePubMedGoogle Scholar
  42. Chi XS, Bolar TV, Zhao P, Tam JS, Rappaport R, Cheng SM: Molecular evolution of human influenza A/H3N2 virus in Asia and Europe from 2001 to 2003. J Clin Microbiol. 2005, 43: 6130-6132. 10.1128/JCM.43.12.6130-6132.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  43. Potter CW: A history of influenza. J Appl Microbiol. 2001, 91: 572-579. 10.1046/j.1365-2672.2001.01492.x.View ArticlePubMedGoogle Scholar
  44. Subbaral K, Joseph T: Scientific barriers to developing vaccines against avian influenza viruses. Nature. 2007, 7: 267-278.Google Scholar
  45. Yu H, Zhou YJ, Li GX, Zhang GH, Liu HL, Yan LP, Liao M, Tong GZ: Further evidence for infection of pigs with human-like H1N1 influenza viruses in China. Virus Res. 2009, 140: 85-90. 10.1016/j.virusres.2008.11.008.View ArticlePubMedGoogle Scholar
  46. Luoh SM, McGregor MW, Hinshaw VS: Hemagglutinin mutations related to antigenic variation in H1 swine influenza viruses. J Virol. 1992, 66: 1066-1073.PubMed CentralPubMedGoogle Scholar
  47. Noble S, McGregor MS, Wentworth DE, Hinshaw VS: Antigenic and genetic conservation of the haemagglutinin in H1N1 swine influenza viruses. J GenVirol. 1993, 74: 1197-1200.Google Scholar
  48. Chan CH, Lin KL, Chan Y, Wang YL, Chi YT, Tu HL, Shieh HK, Liu WT: Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction. J Virol Method. 2006, 136: 38-43. 10.1016/j.jviromet.2006.03.027.View ArticleGoogle Scholar
  49. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR: Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol. 2001, 146: 2275-2289. 10.1007/s007050170002.View ArticlePubMedGoogle Scholar
  50. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007, 24: 1596-1599. 10.1093/molbev/msm092.View ArticlePubMedGoogle Scholar

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