Abstract
A/chicken/FJ/G9/09 (FJ/G9) is an H9N2 subtype avian influenza virus (H9N2 AIV) strain causing high morbidity that was isolated from broilers in Fujian Province of China in 2009. FJ/G9 has been used as the vaccine strain against H9N2 AIV infection in Fujian Province of China. Here, we report the complete genome sequence of FJ/G9 with natural six-way reassortment, which is the most complex genotype strain in China and even in the world so far. The present findings will aid in understanding the complexity and diversity of H9N2 subtype avian influenza virus.
GENOME ANNOUNCEMENT
The influenza virus (an orthomyxovirus) genome is an eight-segment single-stranded RNA with high potential for reassortant in situ. The reassortment can happen within all of the segments of influenza virus A, HA, NA, NP, M, PB1, PB2, PA, and NS (4). Panorama phylogenetic analysis from recent studies revealed that H9N2 viral genomes include at least 74 lineages (2, 3). Except for CA189-like (Ty/CA/189/66) and WI/1/66-like (Ty/WI/1/66) lineages from North America, several major lineages of H9N2 isolates from Eurasia, such as BJ94-like (Ck/BJ/1/94), G1-like (Qa/HK/G1/97), DE113-like (Dk/DE/113/95), KR323-like (Ck/KR/96323/96), G9-like (Ck/HK/G9/97), SH/F/98-like (Ck/SH/F/98), Y439-like (Dk/HK/Y439/97), DK/ST/163/04, and H5N1-like (Ck/YN/1252/03), have become established in domestic birds (3, 5, 6, 7, 10, 11).
From 2002 to 2011, we have conducted large-scale surveys and phylogenetic analyses of H9N2 subtype avian influenza viruss (AIVs) in China (1, 9). The A/chicken/FJ/G9/09 (FJ/G9) virus strain was isolated from broilers with typical respiratory symptoms and 10 to 35% mortality in Fujian Province of China in 2009. The virus was propagated in 10-day-old specific-pathogen-free embryonated chicken eggs. The whole genome of FJ/G9 was amplified by reverse transcription-PCR (RT-PCR) as reported previously (5). The eight segments were cloned into pMD19-T vector (TaKaRa Bio, Inc., Japan), sequenced three times, and assembled using DNAStar version 7.0 to obtain the complete genome sequence. Multiple-sequence alignment was performed using Clustal X (BioEdit version 7.0). Phylogenetic trees were constructed for each genomic segment using the MEGA 5 program as reported previously (3, 8).
The molecular characterization analysis results indicated that the motif 226L in a receptor binding site (RBS) suggested that the FJ/G9 virus can bind to cellular receptors with a sialic acid α-2,6-NeuAcGal linkage and the virus might have the potential to cross species to infect humans, so continuous monitoring is required. Genotype analysis results showed that the FJ/G9 virus belongs to genotype T, which was a novel genotype in chickens (6, 11). The results of gene-sequence analysis revealed that FJ/G9 virus was a six-way reassortant virus with PB2 that probably originated from homologous genes of DK/ST/163/04 (H9N2); the PB1, HA, and NS genes were most closely associated with CK/BJ/1/94 (H9N2); the most likely origin of the PA gene was DK/HK/Y439/97 (H9N2); the most likely origin of the NP gene was CK/SH/F/98 (H9N2); the most likely origin of the NA gene was CK/HK/G9/97 (H9N2), and the most likely origin of the M gene was Qa/HK/G1/97 (H9N2). The six-way reassortant H9N2 viral genome (FJ/G9) is first reported here and reveals a novel natural reassortant event of H9N2 influenza virus.
Nucleotide sequence accession numbers.
The virus genome sequences of the A/chicken/Fujian/G9/09 strain are available from GenBank, under accession numbers JF715008.1 and JN869514.1 to JN869520.1.
ACKNOWLEDGMENTS
This work was supported by the Fund from Guangdong Momentous Scientific and Technological Project (grant no. 2009B020201008) and Strategic Cooperation Project of Guangdong Province & Chinese Academy (grant no. 2010B090301019).
REFERENCES
- 1. Chen F, et al. 2012. Phylogenetic analysis of hemagglutinin genes of 40 H9N2 subtype avian influenza viruses isolated from poultry in China from 2010 to 2011. Virus Genes 45:69–75 [DOI] [PubMed] [Google Scholar]
- 2. Dong G, et al. 2011. Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis. PLoS One 6:e17212 doi:10.1371/journal.pone.0017212 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Dong G, et al. 2011. Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China. PLoS One 6:e25808 doi:10.1371/journal.pone.0025808 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. He CQ, et al. 2009. Homologous recombination as an evolutionary force in the avian influenza A virus. Mol. Biol. Evol. 26:177–187 [DOI] [PubMed] [Google Scholar]
- 5. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146:2275–2289 [DOI] [PubMed] [Google Scholar]
- 6. Huang Y, et al. 2010. Diversified reassortant H9N2 avian influenza viruses in chicken flocks in northern and eastern China. Virus Res. 151:26–32 [DOI] [PubMed] [Google Scholar]
- 7. Li KS, et al. 2003. Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans? J. Virol. 77:6988–6994 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Tamura K, et al. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Mol. Biol. Evol. 28:2731–2739 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Wu ZQ, et al. 2010. Cloning and phylogenetic analysis of hemagglutinin gene of H9N2 subtype avian influenza virus from different isolates in China during 2002 to 2009. Poult. Sci. 89:1136–1143 [DOI] [PubMed] [Google Scholar]
- 10. Xu KM, et al. 2007. Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000 to 2005. J. Virol. 81:2635–2645 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Zhang P, et al. 2009. A novel genotype H9N2 influenza virus possessing human H5N1 internal genomes has been circulating in poultry in eastern China since 1998. J. Virol. 83:8428–8438 [DOI] [PMC free article] [PubMed] [Google Scholar]