Human Infection with a Triple- Reassortant Swine Influenza A(H1N1) Virus Containing the Hemagglutinin and Neuraminidase Genes of Seasonal Influenza Virus

Influenza A viruses infect a broad range of species, with avian and swine strains presenting the most potential for interspecies transmission. Since the 1990s, H3N2 triple-reassortant viruses with genes derived from human (hemagglutinin [HA], neuraminidase [NA], and polymerase basic protein 1 [PB1]), swine (nucleoprotein [NP], matrix [M], and nonstructural [NS]), and avian (polymerase basic protein 2 [PB2] and polymerase acidic [PA]) influenza viruses have caused outbreaks of respiratory disease in pigs throughout North America [1, 2]. These H3N2 viruses, through additional reassortment events with classical H1N1 swine influenza virus, generated further swine reassortant virus strains—namely, H1N2 and H1N1 viruses containing genes from the human, classical swine, and avian lineages [3]. There have been reports of symptomatic human infection caused by swine influenza virus (SIV) after exposure to pigs, and seroepidemiological studies have also demonstrated elevated titers of antibody to SIV in people with occupational exposure [4]. Triple-reassortant SIVs (H1N1 and H1N2) in humans have also been reported in the United States [5]. Until recently, all reported cases of SIV infection in humans presented limited human-to-human transmission. These recurrent events highlighted the need for increased surveillance of influenza in swine and among swine workers to allow for early detection of strains with the potential for human-to-human transmission and the establishment of proper infection control measures and vaccine development.

In April 2009, a novel swine-origin influenza A(H1N1) virus was identified as the cause of outbreaks of human respiratory infection [6]. This novel virus was contagious and spread easily from one person to another; in June 2009, the World Health Organization declared an influenza pandemic. The PB2, PB1, PA, HA, NP, and NS genes of the pandemic H1N1 2009 strain were similar to those previously found in triple-reassortant SIVs, whereas the NA and M genes were more closely related to those of the Eurasian swine lineage [6]. In previously reported SIV H1N1 triple reassortants, the NA and M genes belonged to the classic North American lineage. Here, we describe the genetic and antigenic characterization of yet another novel influenza A(H1N1) triple-reassortant virus, which was isolated from humans in June 2009 in Saskatchewan, Canada.

Methods. Molecular diagnostic testing was performed using primer sets developed by the Centers for Disease Control and Prevention for a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay to detect seasonal influenza A, B, H1, and H3 as well as swine influenza A and H1 [7]. A set of primers based on the published sequences of the H1 gene (FJ966082) of A/California/04/2009 (AH1-Swine-F, 5'-89-CAGACACTGTAGACACAGTAC- 109-3'; AH1-Swine-R, 5'-605- CTAGTAGATGGATGGTGAATGC-584-3') was also used in a conventional RT-PCR assay.

Antigenic characterization was done by hemagglutinationinhibition (HI) assay, using 4 HA units of virus, 0.5% (vol/vol) turkey red blood cells, and postinfection ferret antisera against specific strains of influenza A(H1N1) virus. HI titers were defined as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination.

All 8 RNA segments of A/Saskatchewan/5350/2009, A/Saskatchewan/ 5351/2009, and A/Saskatchewan/5131/2009 were amplified by RT-PCR and sequenced. A combination of inhouse primers and a universal primer set for the full-length amplification of all influenza A viruses was used for RT-PCR [8]. The DNA sequences were assembled and analyzed with the SeqMan, EditSeq, and MegAlign programs in the Lasergene software package (version 8; DNAStar). Phylogenetic trees were generated by the neighbor-joining method, using the MEGA program (version 4) [9].

Drug susceptibility was determined by analyzing the M and NA genes of the influenza virus isolates for point mutations that have been associated with amantadine or oseltamivir and zanamivir resistance. Susceptibility to oseltamivir and zanamivir was also determined by a chemiluminescent enzymatic activity inhibition assay, using the NA-Star Influenza Neuraminidase Inhibitor Detection kit (Applied Biosystems).

Results. Three patients presented in June 2009 with influenza- like illnesses. Symptoms included cough, fever, runny nose, nasal congestion, rhinorrhea, sneezing, malaise, and dizziness (Table 1). No household members were ill at the time. All patients worked at the same hog operation and were at work during the week before the onset of symptoms. Patient 1 received the influenza seasonal vaccine in the fall of 2008. Patients 2 and 3 were not immunized. Mild respiratory illness was detected in <1% of the affected swine herd, but there was no laboratory confirmation that the novel influenza A(H1N1) triple-reassortant virus was present in the herd. Nasopharyngeal swab samples obtained from the 3 patients were tested at the Saskatchewan Disease Control Laboratory for influenza viruses by RT-PCR, and atypical results were obtained. Specimens tested positive for influenza A, swine influenza A, and seasonal H1 but were negative for pandemic swine 2009 H1. The results suggested the occurrence of a novel reassortant virus composed of seasonal H1N1 and SIV. Viral isolates obtained from the inoculation of the swabs on monkey kidney cell culture were sent to the National Microbiology Laboratory for influenza subtyping and complete genome sequencing. The isolates were designated A/Saskatchewan/5350/2009, A/Saskatchewan/5351/ 2009, and A/Saskatchewan/5131/2009. Patients made uneventful recoveries at home and returned to work after 7 days.

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Figure 1.

Phylogenetic analysis of the hemagglutinin (HA), nucleoprotein (NP), and matrix (M) genes of A/Saskatchewan/5350/2009, A/Saskatchewan/5351/2009, and A/Saskatchewan/5131/2009. Phylogenetic analysis was performed using the neighbor-joining method of the MEGA program [9]. Numbers at the nodes of the phylograms denote the bootstrap confidence values. The isolates presented in this study are shown in blue. Previously reported human and swine influenza virus isolates with an H1 gene of human lineage are shown in green, and the reference sequence for the triple-reassortant virus (A/Swine/Ontario/333853/2005[H3N2]) is shown in red. GenBank accession numbers for the sequences of all reference viruses are provided after the virus names. Bars indicate the number of nucleotide substitutions per site.

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Figure 2.

Phylogenetic analysis of the neuraminidase (NA), nonstructural (NS), polymerase acidic (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) genes of A/Saskatchewan/5350/2009, A/Saskatchewan/5351/2009, and A/Saskatchewan/5131/2009.

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Table 1.

Clinical Characteristics of Patients Infected with A/Saskatchewan/5350/2009, A/Saskatchewan/5351/2009, or A/Saskatchewan/ 5131/2009

Nucleotide sequences of the full-length coding region of all 8 segments were deposited in GenBank (accession nos.GQ457544- GQ457567). The sequences revealed that the 3 isolates were similar, with nucleotide and amino acid identities ranging from 97.3% to 100%. Phylogenetic analysis determined that the M, NP, NS, PB1, PB2, and PA genes of the isolates were most closely related to those of triple-reassortant viruses that emerged in North American swine during the 1990s (Figures 1 and 2). The HA and NA genes of A/Saskatchewan/5350/2009, A/Saskatchewan/ 5351/2009, and A/Saskatchewan/5350/2009 clustered with seasonal human H1N1 influenza isolates and were more closely related to those of A/Brisbane/59/2007 (Figures 1 and 2). Because all 3 isolates were so similar, A/Saskatchewan/5350/2009 was selected as the representative isolate for detailed comparative analysis. The M, NP, NS, PB1, PB2, and PA genes were aligned to the reference sequences for triple-reassortant virus (A/Swine/Ontario/ 333853/2005[H3N2]) and revealed 98.1% (PA) to 99.4% (M) nucleic acid homologies and amino acid identities ranging from 99.2% (PB2) to 100% (M). In contrast, the HA and NA genes of A/Saskatchewan/5350/2009 revealed nucleotide identities of 99.0% and 98.9%, respectively, with the seasonal H1N1 genotype represented by A/Brisbane/59/2007 (Figure 1). Amino acid identities were 98.9% (HA) and 99.4% (NA) for the 3 Saskatchewan strains relative to A/Brisbane/59/2007.

A/Saskatchewan/5350/2009 was significantly different from the pandemic H1N1 2009 strain, with nucleic acid and amino acid identities ranging from 72.1% (HA) to 94.9% (NS) and from 79.1% (HA) to 98.6% (NP), respectively.

HI testing of the isolates further determined that the HA proteins were antigenically related to those of seasonal H1N1 influenza A isolates, with HI titers of 160 against A/Brisbane/ 59/2007. None of the Saskatchewan isolates were inhibited by antisera raised against the pandemic H1N1 2009 strain (A/ California/07/2009).

Genetic and phenotypic drug-susceptibility analysis of the isolates demonstrated that they were sensitive to zanamivir and resistant to oseltamivir, with median inhibitory concentrations (mean ± standard deviation) of 144.9±25.6, 182.0±28.3, and 116.9±27.3 nmol/L, respectively. Congruently, the NA sequences of A/Saskatchewan/5350/2009, A/Saskatchewan /5351/ 2009, and A/Saskatchewan/5131/2009 encoded the H274Y amino acid substitution known to confer resistance to oseltamivir [10]. Genetic analysis only was performed to determine whether the isolates were susceptible to amantadine. Unlike the pandemic H1N1 2009 strain, all the Saskatchewan isolates were sensitive to amantadine by sequence analysis of the M gene.

Discussion. In this study, we report 3 cases of recent H1N1 SIV infection in humans. Genomic characterization of the isolates revealed that the internal genes (NS, NP, M, PA, PB1, and PB2) were similar to those of a contemporary North American SIV triple reassortant, whereas the HA and NA genes were most similar to those of A/Brisbane/59/2007(H1N1)-like viruses. Antigenic characterization of the Saskatchewan isolates has demonstrated that they react with reference antisera against the current seasonal vaccine strain and that they are closely related antigenically to A/Brisbane/59/2007(H1N1)-like viruses. The isolates also encoded the H274Y amino acid substitution in NA, which is known to confer resistance to oseltamivir and has been found in seasonal H1N1 influenza viruses since the fall of 2007 [10].

These findings indicate that this recent H1N1 SIV is detectable by means of molecular diagnostic assays designed for seasonal influenza detection. Moreover, isolates could be subtyped by routine antigenic characterization and, thus, may not be correctly identified as novel reassortants by public health laboratories' standard approaches for seasonal influenza surveillance, including viral culture and characterization. In fact, these recent Saskatchewan reassortants were originally detected using a combination of molecular diagnostic assays for the current seasonal H1N1 and pandemic swine-origin H1N1 2009 strain. Specimens tested positive for influenza A (M gene), swine influenza A (NP gene), and seasonal H1 but were negative for pandemic swine 2009 H1. Continued surveillance using molecular assays directed at multiple targets is required to minimize the likelihood of missing additional variant influenza viruses. The need for systematic surveillance of influenza in swine because of the frequency with which reassortment may occur has been emphasized recently [11].

The close antigenic relationship between the recent H1N1 SIV and A/Brisbane/59/2007 suggests that a portion of the human population would likely have preexisting immunity via previous infection with seasonal influenza H1N1 and/or vaccination.

Several cases of SIV infection in humans have been reported [4]. Most recently, Shinde et al [5] reported 11 cases of infections in humans with triple-reassortant SIV (H1N1 [10 cases] and H1N2 [1 case]) in the United States from 2005 to 2009. These isolates also shared internal genes of the North American SIV triple reassortant, with HA and NA genes from the classical swine North American lineage (the H1N1 cases) or the human lineage (the H1N2 case). The H1 gene from the human lineage that Shinde et al reported differed considerably from the those of the isolates reported here and was more closely related to those of A/New Caledonia/20/1999(H1N1)-like viruses. Influenza H1 SIVs with HA genes of human lineage have also been reported among pigs in Canada [12] and the United States [13]. The internal genes of the US isolates were similar to those of North American triple-reassortant SIV [11, 13], whereas the Canadian isolates were wholly human or human-swine doublereassortant viruses [11, 12]. The HA gene of these previous Canadian isolates were also more closely related to those of A/ New Caledonia/20/1999(H1N1)-like viruses, indicating that the emergent H1N1 described here resulted from a more recent novel genetic reassortment between seasonal A/Brisbane/ 59/2007(H1N1)-like virus and triple-reassortant virus that emerged in North American swine during the late 1990s. Similar to all characterized SIV triple reassortants that have become endemic in North American swine, this recent H1N1 reassortant contains the triple-reassortant internal gene (TRIG) cassette. It has been suggested that the TRIG cassette may accept multiple HA and NA types, thereby providing selective advantage to swine viruses containing this gene constellation [3]. An increase in genetic and antigenic diversity appeared coincident with the emergence of the triple-reassortant H3N2 in 1998 and acquisition of the TRIG cassette. The combination of avian PA and PB2 genes and the human PB1 gene seems to have increased the rate of antigenic drift and reassortment, thereby increasing viral capacity for evading established herd immunity [3]. The resultant increased evolutionary rate may in turn facilitate the emergence of viruses that are capable of cross-species transmission and causing widespread disease in humans. In congruence with other reported cases of swine influenza virus infection in humans, the recent H1N1 presented limited human- to-human transmission [4, 5]. The cases were limited to workers of the same large hog operation in Saskatchewan, and no spread in the community has been observed to date. One of the 3 patients received the influenza vaccine before infection with the novel H1N1. Sequence analysis revealed 4 amino acid changes between the H1N1 vaccine strain (A/Brisbane/59/2007) and the recent H1N1. Two of these amino acid changes were situated in a known antigenic site, which may have had an effect on the efficacy of the vaccine [14]. Several studies have shown that persons who work with swine are at increased risk of zoonotic influenza virus infection [4, 15]. The 2009 H1N1 pandemic provides evidence that influenza virus of swine origin has the potential to cause epidemics in humans. The findings of this study highlight once again the urgent need for increased surveillance of influenza in swine and among swine workers to enable early detection of reassortant viruses with the potential for human-to-human transmission, the establishment of proper infection control measures, and vaccine development for SIV.

• Received September 17, 2009.
• Accepted November 24, 2009.

• © 2010 by the Infectious Diseases Society of America