Journal of Clinical Virology

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1 Journal of Clinical Virology 49 (2010) Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: Genetic correlation between current circulating H1N1 swine and human influenza viruses Lu Lu a,b, Yanbo Yin c, Zhongsheng Sun d, Lei Gao e, George F. Gao a,f, Sidang Liu e, Lei Sun a,, Wenjun Liu a,f, a Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China b Graduate University of Chinese Academy of Sciences, Beijing , China c College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao, Shandong , China d Qingdao Oland-better Bioengineering Ltd., Co., Qingdao, Shandong , China e College of Animal Science, Shandong Agricultural University, Taian, Shandong , China f China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China article info abstract Article history: Received 8 February 2010 Received in revised form 16 July 2010 Accepted 27 July 2010 Keywords: Influenza A virus H1N1 subtype Swine Human Genetic correlation Background: H1N1 is the main subtype influenza A virus circulating in human and swine population, and has long been a threat to economy and public health. Objective: To explore the genetic correlation between current circulating H1N1 swine and human influenza viruses. Study design: Three new H1N1 swine influenza viruses (SIVs) were isolated and genomes sequencing were conducted followed by phylogenetic and molecular analysis of all swine and human H1N1 influenza viruses isolated in China in the past five years. Results: Homology and phylogenetic analysis revealed that the three isolates possessed different characteristics: the genome of A/Swine/Shandong/1112/2008 was closely related to that of classical H1N1 SIV, while A/Swine/Shandong/1123/2008 was a reassortant with NS gene from the human-like H3N2 influenza virus and other genes from the classical H1N1 SIV, and A/Swine/Fujian/0325/2008 fell into a lineage of seasonal human H1N1 influenza viruses. Genetically, 2009 H1N1 influenza A viruses (2009 H1N1) in China were contiguous to the SIV lineages rather than the seasonal H1N1 human influenza virus s lineage. Furthermore, molecular analysis among human and swine influenza viruses provided more detail information for understanding their genetic correlation. Conclusions: These results suggested that in China in the past five years, the classical, avian-like and human-like H1N1 SIV existed in swine herds and the reassortment between H1N1 swine and H3N2 human influenza viruses was identified. In addition, the present data showed no evidence to support a strong correlation between the 2009 H1N1 and the swine influenza virus circulating in China Elsevier B.V. All rights reserved. 1. Background Three predominant subtypes of swine influenza viruses (SIVs) circulating in pigs globally are H1N1, H3N2 and H1N2. The worldwide H1N1 pandemic in 1918 also affected swine, then the classical H1N1 SIV remains endemic in swine globally. In 1970s, avian-like H1N1 SIV lineages became established in Europe and Asia. In North America, a triple reassortant H1N1 influenza virus which emerged in 1998 co-circulated with the classical SIV. 1,2 In Southern China, avian-like H1N1 viruses were detected co- Corresponding authors at: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beichenxi Road, Chaoyang District, Beijing , China. Tel.: ; fax: addresses: sunlei362@im.ac.cn (L. Sun), liuwj@im.ac.cn (W. Liu). circulating with classical H1N1 viruses in pigs in early 1990s and the reassortant H1N2 viruses were isolated later in 2004, 3 but cases of human-like H1N1 SIV were reported infrequently. 4 Early studies on SIV in China also revealed the co-circulation of H1N1 and human-like H3N2 SIV, and the reassortments between them were characterized. 5 Swine influenza virus has been proved as a zoonotic agent in humans. 6 A typical example was the outbreak of the swine-origin reassortant H1N1 influenza virus in It is also indicated that lack of surveillance for SIV led to the nearest common ancestor of 2009 H1N1 circulating among pig populations somewhere in the world for a period of time. 7,8 2. Objectives In the present study, surveillance in swine herds was carried out to understand the epidemiology and evolution of H1N1 SIV, /$ see front matter 2010 Elsevier B.V. All rights reserved. doi: /j.jcv

2 L. Lu et al. / Journal of Clinical Virology 49 (2010) and phylogenetic and molecular analysis were made to explore the genetic correlation between current circulating H1N1 swine and human influenza viruses in China with the aim of further prediction and preparation for the potential human-infected influenza A virus. 3. Study design 3.1. Virus isolation and identification In 2008, nasal swabs and lung tissue samples were collected from pigs with acute respiratory symptoms in several pig farms of Shandong and Fujian provinces of China. The clinical specimens were incubated into both specific pathogen free (SPF) eggs (Beijing Merial Vital Laboratory Animal Technology Company) and MDCK cells after tissue homogenization. Virus isolation and subtyping were performed in previous description. 9 Virus was plaque-purified three times on primary MDCK cells and then grown in 10-day-old SPF chicken embryonated eggs. The allantoic fluids were harvested and stored at 70 C Viral gene sequencing Viral RNA was extracted from infected MDCK cells using Trizol reagents (GIBCO-BRL). Reverse transcription (RT) were carried out using Uni12 (AGCAAAAGCAGG) primers and specific primers were designed for subtyping and get full length of eight genes before performing PCR. The PCR products were purified and cloned to pmd19-t (Promega), then sequenced using synthetic oligonucleotides by Bio-med Company. Sequencing of three independent clones of each PCR product was performed in order to eliminate errors resulting from the RT-PCR or cloning steps Sequence analysis All sequences of H1N1 human influenza virus, including all available 2009 H1N1 and seasonal H1N1 strains circulating in China in the past 5 years ( ) as well as SIV were obtained from NCBI and GISAID databases. BLASTn analysis was carried out on NCBI. Nucleotide phylogenetic trees were generated with MEGA program (version 4) using neighbor-joining analysis. The consensus sequences of each lineage were obtained using MegAlign and then compared with MEGA (version 4) using Clustal W Method. Bootstrap value was calculated on 1000 replicates of the alignment. 4. Results 4.1. Genetic origin of new isolated SIV strains Three SIV strains were identified as H1N1 subtype and were designated as A/Swine/Shandong/1123/2008, A/Swine/Shandong/ 1112/2008, and A/Swine/Fujian/0325/2008 (GenBank accession no. GU GU646035, HM and HM176665). Genetic homology of the three strains with related gene sequences was analyzed by BLASTn and, viruses with the highest identity in each segment were listed (Table 1). Phylogenetic trees for HA, NA, M, NS, PB2, PB1, PA and NP genes of H1N1 swine and human influenza viruses were showed in Fig. 1. It was indicated that all eight genes of A/Swine/Shandong/ 1112/2008 were originated from the circulating classical H1N1 SIV in China. Seven gene segments of A/Swine/Shandong/1123/2008 also have the highest identity to those of the classical H1N1 SIV, however its NS gene exhibited higher similarity to that of A/swine/Guangdong/164/06 (H3N2), which was described as the descendant of a human H3N2 virus, A/Moscow/10/ On the other hand, each gene segment of A/Swine/Fujian/0325/2008 fell into the cluster of seasonal H1N1 human influenza virus Phylogenetic analysis of circulating H1N1 swine and human influenza viruses in China Phylogenetic analysis revealed that the H1N1 viruses circulating in swine and human could be clearly separated into four lineages, including the classical SIV, avian-like H1N1 SIV, 2009 H1N1 and seasonal H1N1 human influenza viruses. It was indicated that classical SIV and avian-like SIV were two predominant lineages of H1N1 SIV circulating in swine herds recently in China. In our studies, a human-like SIV A/Swine/Fujian/0325/2008 was isolated, which did not belong to the subcluster of the seasonal human H1N1 influenza viruses circulating in recent years ( ) but fell into the subcluster in relatively early time (around early 2000s). Another human-like H1N1 SIV shown in the phylogenetic tree is A/swine/Henan/01/06, which had lower similarity to A/Swine/Fujian/0325/2008. Gene segments of 2009 H1N1 in China were genetically close to the SIV, the HA, NS and NP segments were in the sister lineages of classical SIV, while the NA, M, PB2, PB1and PA genes were close to the avian-like H1N1 SIV. Phylogenetic analysis also suggested the main lineage of the 2009 H1N1 formed two sublineages according to HA or NA (Fig. 2). The nucleotide and amino acid mapping of all outbreak influenza virus strains indicated that some residues were specific for the two clusters for nucleotide replacements at HA T658A and C1408T, NA G316A and A742G Molecular analysis of HA and NA protein Amino acid sequence mapping of HA (Fig. 3a) and NA (Fig. 3b) gene was conducted to further understand the molecular correlation among the different lineages of swine and human influenza viruses. In HA1 domain, the receptor-binding site (RBS) of H1N1 influenza virus consisted of two loops and one helix. 11 The loop (aa ) and helix (aa ) in HA1 of 2009 H1N1 lineage were identical with classical swine lineage while the loop (aa ) was identical with the seasonal H1N1 human lineage. Moreover, the cleavage site (aa ) in each lineage was of the typical H1 characteristic which may indicate a relatively low pathogenicity. Five epitopes in HA1 (Cb, Sa, Sb, Ca1 and Ca2) were highly variable. The 2009 H1N1 lineage shared the same Sa with avian-like swine lineage and the same Sb with the classical swine lineage. The epitopes in HA2 were correspondingly conservative in all five lineages. Additionally, the glycosylation sites have a significant effect on the antigenic and receptor-binding properties of the influenza virus. Molecular analysis showed that the avian-like swine lineage had seven glycosylation sites while the other four lineages had eight. Four potential glycosylation sites (27NNST, 28NSTD, 498NGT and 557NGDL) were conservative in all five lineage, and 103NGT could be found in four lineages expect avian-like SIV lineage. In addition, two sites at position 304NTSL and 293NTT were unique to 2009 H1N1 and classical SIV lineage and the seasonal H1N1 human lineage lacked one aa at residue 147. The increase of glycosylation sites in NA protein may reduce the ability of affinity between influenza virus and the host receptor as well as the release of the virus particles. 12 In our study, seven glycosylation sites in NA protein were found in all SIV lineages. Four were in linker region (N44, N58, N63 and N68) and the other three were in the NA domain (N88, N146 and N235). However, the outbreak human 2009 lineage contained a new glycosylation site N386 compared to the other lineages, and this glycosylation site was also in the antigenic region which exposed on the surface, indicated a probable impact to the genetic and biological characteristics of the newly virus. Compared to other lineages, the NA protein of 2009

3 188 L. Lu et al. / Journal of Clinical Virology 49 (2010) Fig. 1. Phylogenetic trees for all eight gene segments of selected H1N1 influenza virus. The unrooted nucleotide phylogenetic trees were generated with MEGA program (version 4) using neighbor-joining analysis. Bootstrap values were calculated on 1000 replicates of the alignment and only those above 80% were shown. Three new isolated strains were marked with circles. The human H1N1 influenza vaccine virus strain was marked with square.

4 L. Lu et al. / Journal of Clinical Virology 49 (2010) Fig. 1. (Continued ). H1N1 shared most of the same antigen regions with the avianlike SIV lineage. 13 In addition, the seasonal H1N1 human influenza lineage had one more Thr at residue Discussion A/Swine/shandong/1123/2008 was found as a probable swinehuman influenza virus reassortant between two different subtypes. Pigs contain both sialic acid 2,6 and 2,3 receptors and therefore have been hypothesized to serve as intermediate hosts for the adaptation of avian influenza viruses to humans or as mixing vessels for the generation of genetically reassortant viruses. 14 The reassortant influenza virus strains have the epidemic potential. The human influenza pandemics of 1957 and 1968 were caused by reassortant viruses that possessed internal gene segments from avian and human strains. 14 In 1998, a strain of avian and human influenza virus recombination caused influenza outbreak in swine population in US, and also the origin of the 2009 global outbreak was reassortant swine-origin H1N1 influenza virus. In China, reassortants between human and swine influenza viruses

5 190 L. Lu et al. / Journal of Clinical Virology 49 (2010) Fig. 2. Phylogenetic trees for HA, NA of 2009 H1N1 strains. Bootstrap values were calculated on 1000 replicates of the alignment and only those above 50% were shown. Fig. 3. Molecular analysis of HA (a) and NA (b) amino acid sequences of four lineages. Potential glycosylation sites were marked with dotted line. Previously defined antigenic sites were indicated by shades with different colors. The solid boxes marked receptor-binding sites and the cleavage sites were in the dotted box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

6 L. Lu et al. / Journal of Clinical Virology 49 (2010) Table 1 Genetic homology of the three strains with related sequences available in GenBank. Virus Gene Virus with the highest identity Identity GenBank accession no. A/swine/Shandong/1123/2008 PB2 A/Swine/Shanghai/2/2005(H1N1) 99.6% FJ PB1 A/swine/Guangdong/103/2002(H1N1) 99.7% GQ PA A/swine/Guangdong/103/2002(H1N1) 99.8% GQ HA A/Swine/Shanghai/2/2005(H1N1) 99.9% EU NA A/Swine/Shanghai/2/2005(H1N1) 99.5% EU NP A/swine/Guangdong/103/2002(H1N1) 99.4% GQ M A/swine/Guangdong/103/2002(H1N1) 99.9% GQ NS A/Swine/Guangdong/1/2003(H3N2) 99.7% EU A/swine/Shandong/1112/2008 PB2 A/swine/Guangdong/103/2002 (H1N1) 98.7% GQ PB1 A/swine/Guangdong/103/2002 (H1N1) 99.9% GQ PA A/swine/Guangdong/103/2002(H1N1) 99.7% GQ HA A/swine/Shanghai/2/2005(H1N1) 99.8% EU NA A/Swine/Shanghai/2/2005(H1N1) 99.3% EU NP A/Swine/Shanghai/2/2005(H1N1) 98% FJ M A/swine/Guangdong/103/2002(H1N1) 99.5% GQ NS A/Swine/Shanghai/2/2005(H1N1) 99.9% FJ A/Swine/Fujian/0325/2008 PB2 A/Dunedin/2/2000 (H1N1) 99.6% CY PB1 A/Dunedin/2/2000 (H1N1) 99.6% CY PA A/Dunedin/2/2000 (H1N1) 99.9% CY HA A/Wellington/18/2000 (H1N1) 99.5% CY NA A/Canterbury/43/2000 (H1N1) 99.2% CY NP A/Dunedin/2/2000 (H1N1) 98.4% CY M A/NewCaledonia/20/1999 (H1N1) 99.5% CY NS A/NewYork/443/2001 (H1N1) 99.3% CY were successively reported in recent years, probably due to their co-existence in swine herds, which provided more reassortment opportunities. 5,10 Pigs can be infected with human influenza A virus under natural conditions. 15,16 Though it was proved that the humanlike H3N2 viruses were isolated frequently in recent years, the H1N1 human-like SIV isolates were few. In the present study, A/Swine/Fujian/0325/2008 had the highest identity with H1N1 seasonal human strains mainly circulating in Northern Hemisphere around the year Besides, human H1N1 influenza vaccine virus strains that were used in the Northern Hemisphere in the last five years were A/New Caledonia/20/99 ( ), A/Solomon Islands/3/2006 ( ), and A/Brisbane/59/2007 ( ), as recommended by WHO. In the phylogenetic analysis, A/swine/Fujian/0325/2008 had a highly similarity to A/New Caledonia/20/1999, which was representative of the predominant human H1N1 viruses during 2000 and It suggested that this human-like H1N1 SIV might drive from human influenza vaccine virus through human-to-swine transmission. In summary, the current analysis contributes interesting data to understand the swine influenza viruses circulating in China, and also correlation between H1N1 human and swine influenza virus, which might encourage further research on this topic. Though our current data showed no evidence to support a strong correlation between 2009 H1N1 and SIV circulating in China, the future surveillance work of influenza virus should be addressed far more importance. Conflict of interest The group declares no financial or personal relationship with other people or organizations that could inappropriately influence their work. Acknowledgements This study was funded by the Ministry of Science and Technology of China Program (2006BAD06A04/3, and 2010BAD04B01),Chinese Academy of Sciences Innovation Projects (KSCX2-YW-R-158 and KSCX2-YW-N-054), 948 Program of Ministry of Agricultry (2004- Z40), and the Major Special Program of the Ministry of Science and Technology of China (2009ZX ). References 1. Subbarao K, Swayne DE, Olsen CW. Epidemiology and control of human and animal influenza. In: Kawaoka Y, editor. Influenza virology: current topics. Caister Academic Press; p Van Reeth K. 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