Influenza A viruses can infect a variety of animals, including poultry, human, pigs, horses, marine mam-mals, and carnivore animals [i.e. dog and cat], and they are classified into different subtypes on the basis of antigenic properties in the two surface glycop-roteins, haemagglutinin (HA) and neuraminidase (NA) (14, 21). Wild aquatic birds are the primary natural reservoir of influenza A viruses, which harbor all currently known 16 HA and 9 NA subtypes (3, 21). Pigs may also play an important role in the evolution and ecology of the influenza A virus (21). The tracheal epithelium of pigs contain both sialic acid linked to galactose by an α-2, 6 linkage (SA α 2, 6 Gal) and sialic acid linked to galactose by an α-2, 3 linkage (SA α 2, 3 Gal) receptors and can be infected with swine, human and avian viruses (9), therefore, pigs have been hypothesized to serve as an intermediate host for the adaptation of avian influenza viruses to humans or as mixing vessels for the generation of genetically reassortant viruses (21).
Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2, and these include classical swine H1N1, avian-like H1N1, human-like H3N2, reassortant H3N2 and various genotype H1N2 viruses (1, 20). Following the 1968 Hong Kong influenza pandemic (H3N2), H3N2 virus was first isolated from swine in 1970 in Asia. Since that time, human-like H3N2 viruses have been detected frequently throug-hout Europe, Asia and North America, and these viruses continue to co-circulate with H1N1 viruses (1, 2, 20). Unlike human viruses, H3N2 swine viruses have different epizootiological patterns in different areas of world (1, 20). Since 1984, reassortant H3N2 viruses have circulated in pig population in Europe, which contain genes of human (HA and NA) and avian (PB2, PB1, PA, NP, M and NS) (1). Before 1998, classical H1N1 viruses were the exclusive cause of swine in-fluenza in North America. However, around 1997-1998, three different genotypes of H3N2 viruses began to emerge in pigs: wholly human-like, double-reassortant viruses containing genes of human and swine viruses and triple-reassortant viruses containing genes of human, swine and avian viruses, and only the triple-reassortant viruses became established in pigs (20, 25).
Reverse genetics, a term used in molecular virology, describes the generation of viruses possessing a genome derived from cloned cDNAs. Reverse genetics for influenza virus has dramatically changed our under-standing of the replication cycles of these viruses (12). In addition, this methodology has allowed genetic manipulation of viral genomes to generate new viruses, which can be used as live, attenuated vaccines or vectors to express heterologous proteins.
In 2005, we isolated one influenza H3N2 virus from pigs with respiratory disease on a farm in eastern China. Genetic analysis showed that the isolate was a triple-reassortant virus among human-like H3N2, human-like H1N1 and classical swine H1N1 viruses. To manipulate the virus genomes on the level of DNA, we established a reverse genetic system for this H3N2 isolate.
One virus isolate with hemagglutination activity was isolated. The isolate was determined as H3N2 subtype by HI and NI assays and designated as A/-swine/Shandongi3/2005 (Sw/SD/3/2005). RT-PCR was performed with segment-specific primers to amply 8 gene segments of the isolate (Fig 1).The nucleotide sequences of the isolate have been deposited in the GenBank database under accession numbers EU-116038-EU116046. The BLAST and phylogenetic analysis of Sw/SD/3/2005 genomic sequence demon-strated that the HA, NA, NP, M, PB1 and PA genes shared the highest sequence similarity with those of A/Moscow/10/99 or A/Hong Kong/1144/99 (99%), and the PB2 gene shared the highest homology with that of A/Puert Rico/8/34 (99%), while the NS gene share the highest homology with that of A/Swine/-Shanghai/1/2005 (Table 1). These results showed that A/swine/Shandong/3/2005 was a reassortant virus from human-like H3N2, human-like H1N1 and classical swine H1N1 viruses.
Figure 1. Full length amplification of all eight segments of Sw/SD/3/2005 by RT-PCR. M, DNA ladder; 1, PB2; 2, PB1; 3, PA; 4, HA; 5, NP; 6, NA; 7, M; 8, NS.
Table 1. Genetic similarity between Sw/ZJ/1/2004 and reference strains available in GenBank
HA plays an important role in determining tissue tropism, systemic spread, and pathogenicity of in-fluenza A viruses (14, 21). The full-length sequence of HA gene contain 1 778 nucleotides, and have a coding sequence with 1 707 nt. The deduced amino acid sequences from HA gene have 567 amino acid residues, among which there are a 16 aa signal peptide sequence, a 328 aa HA1 sequence, and a 321 aa HA2 sequence. There is an amino acid motif PEKKQTR↓G at their HA cleavage sites. Glycosylation of viral antigens masks and unmasks antigenic sites and there-fore is an important process in the generation of new viruses (11, 17). Ten potential N-linked glycosylation sites were found in the HA protein, nine of which are located in HA1. The amino acid sequence of Sw/SH/ 3/2005 at residues 226 to 228 was Ile-Ser-Ser, a typical motif of human influenza viruses and a site known to affect receptor-binding properties, which implied that the isolate preferentially binds to SA α 2, 6 Gal of human cell receptors rather than SA α 2, 3 Gal of those of avian (11, 22).
A balance between the activity of HA in virus attachment and NA in virus release is crucial for optimal viral replication (8, 21). The entire sequence of HA gene contain 1 467 nucleotides, and has a coding sequence of 1 410 nt. The deduced amino acid sequences from the HA gene have 469 amino acid residues. There are no amino acid substitutions observed at positions 274 [H→Y) or 294 [N→S) which were reported to confer resistance to oseltamivir in clinical influenza isolates (5) in the NA gene of the Sw/SD/3/ 2005 isolate, suggesting that it is sensitive to NA inhibitors. Neither substitution is observed at position 31 [H→Y) of the M2 protein which were reported to confer resistance to anti-influenza drugs Amantadine and rimantadine in the recent Europe swine viruses, including H1N1, H1N2 and H3N2 viruses (10), in isolate Sw/SD/3/2005, suggesting that it is sensitive to this class of antiviral drugs.
The PCR products from the pMD18-T vector con-taining cDNA of Sw/SD/3/2005 were digested with Bsm B Ⅰ or Bsa Ⅰ, and cloned into the vector pHW2000. Eight expression plasmids were constructed containing the eight genomic fragments of Sw/SD/3/2005 (H3N2), designated pHWsd-PB2, pHWsd-PB1, pHWsd-PA, pHWsd-HA, pHWsd-NP, pHWsd-NA, pHWsd-M, and pHWsd-NS, respectively.
The rescued viruses were inoculated into 10-day-old embryonated chicken eggs or MDCK cells. The virus-induced cytopathic effect was observed in MDCK cells (Fig. 2). The virus titer was 1:8 by HA assay in the allantoic fluid. The sequences of HA and NA genes of the rescued virus showed the HA and NA genes were identical to those of Sw/SD/3/2005, demonstrating that the rescues by our constructed eight-plasmid transfection system was successful.
Figure 2. virus-induced cytopathic effect by rescued Sw/SD/3/2005 at post-incubation 48h. A: Mock-infected MDCK cell. B: Post-incubation 48h with rescued Sw/SD/3/2005.
The reverse genetics derived R-swsd3/05 was characterized by its growth potential in chicken embryos and in tissue culture (Table 2). The results showed that R-swsd3/05 was stable after passage 3.
Table 2. Comparison of the hemagglutiation unit of the R-swsd3/05 in chicken embryo and MDCK cell