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1 7 H9N2 HA NA H9N2 HA NA PCR,P GEM2T Easy Vector 4, DH210,,,, H9N2 HA 226 L,H9N2,, R2L2S2R,R2S2S2R ;NA 6264, AΠShaoguanΠ408Π 98, AΠSwineΠHong KongΠ9Π98 AΠDuckΠHong Kong Πy 280Π97 (H9N2) ;HA NA,H9N2 HA AΠChickenΠHong KongΠG23Π97 AΠChickenΠHong KongΠG9Π97, NA AΠShaoguanΠ408Π98 H9N2 HA NA H9N2 HA, H9N2 NA, A,; ; ; Characterization of HA and NA genes of swine influenza A ( H9N2) viruses GUO Yuan2ji, WEN Le2ying, WANG Min, LI Zi, ZHANG Ye, GUO Jun2feng1 Department of Influenza, National Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing ,China Abstract Objective To understand the origin of HA and NA genes of swine influenza A ( H9N2) viruses isolated from pigs in the mainland of China and on basis of these to reveal the pathogenecity of them in pigs1 Methods The target gene was amplified by PCR,the PCR product was ligated with P GEM2T Easy Vector (Promega company, U S A) at 4, the recombined plasmid was transferred into DH210bacteria ; positive colonies were selected and identified then digested with restriction enzyme1afterwards,the nucleotide seguence was determined1finally,phylogenetic analysis of the seguencing data was performed with MegAlign (Version 1103) and Editseg (Version 3169) softwares1 Results Two strains of swine influenza A ( H9N2) virus isolated in the mainland had an amino acid residue, leucine (L) at position 226 ( H3 numbering) on HA protein molecule found in H9N2 viruses isolated either in pigs or humans previously ; the amino acid sequence at HA connecting peptide of isolates possessed R2L2S2R, whereas the other H9N2 viruses with virulence in poultry had R2S2S2R at HA connecting peptide1 The two pig H9N2 isolates shared the same three2amino2acids deletion in the NA stalk at position foumd in AΠShaoguanΠ408Π98 and AΠSwineΠHong KongΠ9Π98, as well as AΠDuckΠHong Kong ΠY280Π97 (H9N2) viruses1 The analysis of the phylogenetic tree indicated that the HA and NA genes of new isolates were closely related to those of AΠChickenΠHong KongΠG23Π97 and AΠChickonΠHong KongΠG9Π97 and AΠShaoguanΠ408Π 98 viruses, respectively1 Conclusion The HA and NA genes of swine influenza A(H9N2) viruses isolated in the mainland of China probably wese derived from those of avian influenza A ( H9N2) virus1 The occurrence of substitution of amino acid sequence at HA connecting peptide,could result in the H9N2 virus from non pathogenic to pathogenic in pigs1 However, avian influenza A( H9N2) virus had deletion in the stalk of the NA that resulted in host range transmission1therefore they could infect pigs directly1 Key words Influenza A virus,porcine ; Hemagglutinin ; Neuraminidase ; Genes, :100052, [1 ], [2 ], [3 ],1979, H1N1 [4 ],

2 Chinese J Exp Clin Virol,March 2004,Vol 18,No11, [5 ], [6 ],1991 (H1N1) [7 ], [8 ],2000,,2000, 2002 ( H1N1) [9 ], H9N2, H9N2 [10 ], 1998 H9N2 [11 ], H9N2, H9N2? AΠSwineΠShandongΠ5Π2002 ( H9N2) AΠ SwineΠShandongΠ10Π2002 (H9N2),, HA NA, HA : AF156376, AF156374, AF156373, AF222810, AF156379,AF156380,AF156378,AJ ,AJ , AF156381,AF156383,AF186266,AF156377,AF AF ; NA : AF156399, AF156395, AF156397, AF156391, AF156392, AY043024, AF156398,AF222812,AF AJ RNA [12 ] 113 DNA Star,Meg Align (Version 1103) Editseg (Version 3169) H9N2 HA HA HA, 1 1, AΠSwineΠShandongΠ5Π2002 AΠSwineΠShandongΠ10Π2002 HA H9N2, AΠQuailΠHong KongΠG1Π97 AΠHong KongΠ1073Π99 HA ( HA1) (HA2), 2 2, H9N2, 1 H9N2 HA Tab11 The potential glycosylation sites of HA proteins of H9N2 influenza viruses Position AΠDuckΠHong KongΠy280Π97 ( Hky280297) AΠShantouΠ239Π98 (Shantou239298) AΠChickenΠHong KongΠG23Π97 ( HKG23297) AΠChickenΠHong KongΠG9Π97 (hkg 9297) AΠSwineΠShandongΠ5Π2002 (shondong5202) AΠSwineΠShandongΠ10Π2002 (shandong10202) AΠChickenΠHong KongΠ9Π98 ( HK9298) AΠChickonΠHong KongΠ739Π94 ( HK739294) AΠchickenΠBeijingΠ1Π94 (Beijing1294) AΠQuailΠHong KongΠG1Π97 ( HKG1297) AΠHong KongΠ1073Π99 ( HK ) AΠChickenΠPKΠ2Π99 (PK2299) AQuailΠHong KongΠAF157Π92 ( HKAF157292) AΠDuckΠHong KongΠ784Π79 ( HK784279) AΠTurkeyΠPV Π40Π83 (TYPV40283) AΠDuckΠHong KongΠy439Π97 ( HKY439297) AΠChickenΠKorΠ006Π96 ( Kor006296) AΠChickenΠKorΠ323Π96 ( Kor323296) :+ ; (3) Note :plus sign and blank space in table 1 mean the site with and without potential glycosylation, respectively (These meanings will also be used in Table 3)

3 9 HA 228 ( G) ;,226 (L), AΠQuailΠHong KangΠAF157Π92 ( H),2Π3 (Q),1Π3 L, 2,, :RXXR KXXR (R,X,K ) ; XXXR, R2L2S2R, R2S2S2R (S ) 212 H9 HA 1 1,: Shandong P10202 Shandong P5202 HA G9, G1 (HKG1297) HA HKY280297,Shantou HKG23297 hkg297 AΠDuckΠHong KongΠy280Π97, 7, NA NA 2 2, NA AΠShanguanΠ408Π98, AΠDuckΠHong KongΠy280Π97 AΠSwineΠHong KongΠ9Π98 2 H9N2 NA Fig12 Phyhogenetic tree for HA gene of influenza A (H9N2) viruses 3,(2) 1 H9N2 HA Horizontal distance is proportional to the genetic distance, and the vertical lines are only for spacing the branches (these meanings will be also used in figure 2) Fig11 Phylogenetic tree for HA gene of H9N2 influenza A viruses 213 H9N2 NA NA, AΠHong KongΠ1073Π99 NA (38 39),,AΠShanguanΠ408Π98, AΠSwineΠHong KongΠ9Π98 AΠDuckΠHong KongΠy280Π97 3, HA ,NA H9N2,HA NA,,H9N2, H9N2,,, HA NA HA,, [13 ] NA [14 ] H9N2? HA, 2,, H9N2,NA 3 [10 ] 3 H9N2 [15 ], H9N2,HA 226 ( 2) HA 226 L H9N2 :

4 Chinese J Exp Clin Virol,March 2004,Vol 18,No11 2 H9N2 HA Tab12 Comparison of the amino acid sequences of the HA protein of H9N2 influenza viruses (RBS) HA (HA Conneting peptide) AΠDuckΠHong KongΠy280Π97 L G R S S R AΠShantouΠ239Π98 L G R S S R AΠChickenΠHong KongΠG23Π97 L G R S S R AΠChickenΠHong KongΠG9Π97 L G R S S R AΠSwineΠShandongΠ5Π2002 L G R L S R AΠSwineΠShandongΠ10Π2002 L G R L S R AΠChickenΠHong KongΠ9Π98 L G R S S R AΠChickenΠHong KongΠ739Π94 Q G R S S R AΠChickenΠBeijingΠ1Π94 Q G R S S R AΠQuailΠHong KongΠG1Π97 L G R S S R AΠHong KongΠ1073Π99 L G R S S R AΠChiclenΠRKΠ2Π99 Q G R S S R AΠQuailΠHong KongΠAF157Π92 H G K S S R AΠDuckΠHong KongΠ784Π79 Q G A S D R AΠTurkeyΠPVΠ40Π83 Q G T S G R AΠDuckΠHong KongΠy439Π97 Q G A S N R AΠChickenΠKorΠ006Π96 Q G A S V R AΠChickenΠKorΠ323Π96 Q G A S Y R 3 H9N2 NA Tab13 The potential glycosylation sites of NA proteins of H9N2 influenza viruses Position AΠShaoguanΠ408Π AΠChickenΠBeijingΠ1Π AΠGuangzhouΠ333Π AΠSwineΠHong KengΠ9Π AΠHong KongΠ1073Π AΠQuailΠHong KongΠAF157Π AΠChickenΠHong KongΠG9Π AΠChickenΠHong KongΠG23Π AΠDuckΠHong KongΠy280Π AΠDuckΠHong KongΠy439Π AΠSwineΠShandongΠ5Π AΠSwineΠShandongΠ10Π AΠShantouΠ239Π AΠChickenΠHong KongΠ739Π SA 216Gal, SA 213Gal HA 226 L, Q [16 ]? 1 Castrucci MR, Donatelli I, Siaoli L, et al1 Genetic reassortment between avian and human influenza A virues in Italian pigs1 Virology, 1993, Scholtissek C1 Pigs as mixing vesslls for the creation of new pandemic influenza A viruses1 Med Principles Pracy,1990,2 : Olsen CW, Mc Gregor MW, Cooley AT, et al1 Antigenic and genetic analysis of a recently isolated H1N1 swine influenza virus1 Am J Vet Res,1992, 54 : Pensaert M, Ottis K, Vandeputte J, et al1 Evidence of natural transmission of influenza A virus from wild ducks to swine and its potential importance for man1 Bull WHO, 1981,59 : Guan Y, Shortridge KF, Krauss S, et al1 Emergence of avian H1N1 influenza viruses in pigs in China1 J Virol, 1996, 70 : Ludwig S, Stitz L, Planz O, et al1 European swine virus as a possible source for the next influenza pandemic? Virology, 1995, 212 : ,Webster RG,,1 (H1N1)

5 11 1, 1992,6 : ,Webster RG,,1 1,1995,9 : ,,,1 ( H1N1) 1,2000,17 : Peiris JSM, Guan Y, Markwell D, et al1 Cocirculation of avian H9N2 and contemporary human H3N2 influenza A viruses in pigs in Southeastern China : potential for genetic reassortment? J Virol, 2001, 75 : ,,,1 H9N2 1,1999,13 : ,,,1 H9N2 1,2003, 17 : Kawaoka Y, Naeve CW, Webster RG1 Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from hemagglutinin? Virology, 1984, 139 : Sugiura A, Ueda M1 Neurovirulence of influenza virus in mice1 11 Neurovirulence of recombinants between virulence and avirulent virus strains1 Virology, 1980, 101 : Guo YJ, Krauss S, Senne DA, et al1 Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia1 Virology, 2000,267 : Webster RG, Bean WJ, Gorman OT, et al1 Evalution and ecology of influenza A viruses1 Microbiol Rev, 1992,56 : ( : ) B Sererini, ( EV) 1DDM 2DDM B (CoxB), : (1) 2DDM (), DDM 12 d ( 2 ) 70, 33, 37, 62 (2478 ) (2),40, 20, 62 (2576 ) : (1) RT2PCR ( EV2RNA) (2) CoxB IgM (3) CoxB,CoxB 16 : (1) EV2RNA :70 RNA 11, 1517 %,40, 1, 215 %, ( 2 = P < 0105) (2) CoxB IgM : 70 CoxB IgM 17, 2413 %,40,1, 215 %, ( 2 = P < 0101) (3) CoxB : 70 24, 3413 %,B1 3 B3 10 B4 5 B5 4 B6 2 B3 B4 40 3, 715 %,B3 B5 B6 1, :515031,, ( 2 = P < 0101) (4) CoxB IgM CoxB,70 CoxB IgM CoxB, 14 (20 %),43 (6114 %),8114 % ( IgM 10, IgM 3 ) (5) 3,70 RT2PCR ELISA 3, EV 29 (4114 %), % ; 9 ( PCR + 4 IgM + 5 ) 1219 % ; 15 ( PCR 2 IgM 3 10 ) 2114 % :RT2PCR EV EV IgM,,70 EV 29, 27 CoxB 3816 %( 2 PCR RNA EV) Banatvala 1DDM CoxBIgM 29 %33 % Clements PCR 1DDM 64 % (9/ 14) PCR 1DDM 39 % (17/ 45), B3 B4 1DDM CoxB IgM % (13/ 35) PCR,CoxB IgM CoxB B3 B4,,CoxB 2DDM ( : )

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