Genetic characterization of H1N2 influenza a virus isolated from sick pigs in Southern China in 2010

In China H3N2 and H1N1 swine influenza viruses have been circulating for many years. In January 2010, before swine were infected with foot and mouth disease in Guangdong, some pigs have shown flu-like symptoms: cough, sneeze, runny nose and fever. We collected the nasopharyngeal swab of all sick pigs as much as possible. One subtype H1N2 influenza viruses were isolated from the pig population. The complete genome of one isolate, designated A/swine/Guangdong/1/2010(H1N2), was sequenced and compared with sequences available in GenBank. The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. HA, NP, M and NS were shown to be closely to swine origin. PB2 and PA were close to avian origin, but NA and PB1were close to human origin. It is a result of a multiple reassortment event. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially before the emergence of highly pathogenic FMDs in pigs in Guangdong.

Influenza viruses are members of family Orthomyxoviridae and have segmented, Negative-sense RNA genomes. Swine influenza virus (SIV) belongs to Influenza A Viruses. SIV causes respiratory diseases in pigs and has been disseminated all over the world [1]. At present, there were three main influenza viruses circulating in pigs in the world including H1N1, H1N2 and H3N2. In addition some other unusual subtypes of swine influenza were also reported includingH1N7, H3N1, H4N6, H5N1, H5N2, H6N6 and H9N2 [2][3][4][5][6][7].
The first SIV H1N2 was reported in Japan from 1978 to 1992 [8,9]. From then on, H1N2 was show up in different pigs of different countries, including France from 1987 to 1988 [10], and in the United Kingdom in 1994 [11], the United States in 1999 [12], Germany in 2000 [13], Korea in 2003 and thereafter [14]. Recently it first shows up in Swedish herd [15]. During an influenza virus surveillance programme in Guangdong pigs, we isolated an H1N2 virus from clinically ill pigs, which was genetically characterized as a result of reassortment events between a human H3N2 strain, Classical SIV strain and North America avian-like SIV lineage strain.
In January of 2010, some pigs have a severe outbreak of influenza-like disease occurred in an intensive pig farm of Guangdong province. Many pigs have similar clinical symptoms: cough, sneeze, runny nose. These clinical symptoms last for 3-8 days then some pigs have sick of foot and mouth disease (FMD). Because Swine influenza (SI) was immunosuppressive disease which frequently predisposesed to highly fatal secondary infections. Maybe the SI lowers pig's immunity to common illnesses, some pigs will get FMD. From now on, the FMD caused rampant epidemic diseases in pig population of Guangdong [16].
Initial isolations of the viruses were performed in 10day-old specific pathogen free (SPF) embryonated chicken eggs through the allantoic route, incubated at 35°C for 72 h. Embryonic death was monitored every 12 h, and then allantoic fluid were harvested under aseptic conditions and stored at -70°C for reserved. Subtype identification were conducted through RT-PCR and through standard hemagglutination inhibition and neuraminidase inhibition assays. One influenza virus was isolated and named: A/swine/Guangdong/1/2010(H1N2).
The virus RNA was extracted from allantoic fluid by using TRIzol reagents (Invitrogen). RT-PCR was performed as a one-step reaction with the TAKARA OneStep RT-PCR Kit, according to manufacturer's protocol. The primer of reverse transcription used 12 bp (5-AGC AAA AGC AGG-3). cDNAs were synthesized at 37°C for 1 h using M-MLV reverse-transcription system (Promega). Full-length PCR amplification of eight RNA segments was performed with a set of primer. The genome of this H1N2 SIV was sequenced fully as described previously, with the GenBank accession numbers HQ85339 to HQ853346.
Phylogenetic analysis of A/swine/Guangdong/1/2010 (H1N2) was carried out by analyzing the data obtained here with those of other sequences of influenza viruses from GenBank database. A neighbor-joining nucleic acid tree was constructed in MEGA 4.0 using the Kimura 2parameter model with 1, 000 bootstrap replicates. Clustal W of Lasergene was used for multiple alignments. In this study, the nucleotide sequences used for the phylo-  Table 1 lists the reference viruses with the highest level of sequence identity to A/swine/Guangdong/1/2010 for each gene segment.
The HA deduced amino acid sequences of the A/ swine/Guangdong/1/2010 was analyzed in this study. There were eight potential glycosylation sites in HA, including six in HA1 and two in HA2. Its cleavage site is (PSIQSRGLFGAI), which is not same with the HPAI (RXR/KRGLF) of the 1918 influenza virus which have strong pathogenic effect on the human (Figure 1).
Phylogenetic analysis revealed that the HA gene of A/swine/Guangdong/1/2010 is of classical swine lineage resides in the same clade with pandemic (H1N1) 2009A/ California/04/2009, along with A/swine/Beijing/47/1991 (H1N1) and A/Swine/Indiana/9K035/99(H1N2) (Figure 2). Sequence homology with A/swine/Beijing/47/1991(H1N1) and A/swine/Hong Kong/273/1994(H1N1) was respectively found to be 94.6% and 93.9%. It still has a high homology with A/Swine/Indiana/9K035/99(H1N2) with 92.1%, which is the first reported triple reassortment swine influenza virus H1N2 in United States [12]. A/swine/Cloppenburg/IDT4777/2005(H1N2) is a novel SIV H1N2 isolated from Germany, whose HA gene originated from a human epidemic strain, is clearly distinct in genetic origin compared with those classical SIVs. Therefore, it is obviously that the HA gene of A/swine/Guangdong/1/2010 is a descendent of classical SIV and originated from the HA gene of neither avian origin nor human origin.
The phylogeny of the NA genes (Figure 3) also demonstrated that A/swine/Guangdong/1/2010 is genetically related to A/New York/568/1996(H3N2) and that the NA genes of triple reassortment H3N2(      [20]. The NS1 protein of this virus contains 219 amino acid, it loss the PL motifs (PDZ domain ligand, PL), which located in 227-230aa. This feature is the same with recent classical SIV H1N1 [21]. In China, classical swine influenza virus has been existing since 1996. In the previous epidemiological study, H3N2 infected pigs in this area. The H1N2 was reported in 2004, which is a human-swine reassortment virus [22]. In this study we find the multiple reassortment viruses, it confirmed the swine as a mixing vessel in transmission. This deserves to be paid attention. Especially swine population was seized with FMD after the SIV infected pigs in this area. The 2009 H1N1 pandemic virus is a swine-origin reassortant: HA, NP and NS from classical swine (North American) lineage; PB2 and PA from avian (North American) lineage; PB1 from human seasonal H3N2; and NA and M from Eurasian swine lineage [23]. Southern China is designated as a putative influenza epicenter [24]. Both 1957 and 1968 pandemic influenza emerged from this area. The precursors of currently still ongoing H5N1 HPAIVs were identified also in Southern China. Recently H6 AIVs have been identified in this area [25]. The current situation, therefore, presents continued risk for further reassortment of swine influenza virus in pig populations and continuous monitoring of this virus in swine population will be needed.

Nucleotide sequence accession numbers
Nucleotide sequences from the A/swine/Guangdong/1/ 2010 (H1N2) isolate have been submitted to GenBank with accession numbers HQ85339 to HQ853346.