Journal of Microbes and Infection, June 2009, Vol. 4, No. 2

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1 76 H3N2,,, : H3N2 ( SIV) SIV ( PAM), SIV PAM 96 h, 64, DNA ;, 24 h DNA, 72 h 25%,, SIV PAM, PAM SIV : ; H3N2 ; ; ; Swine influenza type A H3N2 virus-induced apoptosis in porcine pulmonary alveolar macrophages in vitro QI Xian, YAO Huo-Chun, LU Cheng-Ping College of Veterinary Medicine, Nanjing Agricultural University, Nanjing , China Abstract: Porcine pulmonary alveolar macrophages ( PAMs) were infected with A/Swine/ Guangdong/1/2004, a swine influenza virus ( SIV) isolate. Results showed that A/Swine/ Guangdong/1/2004 was able to carry out productive replication in PAMs. DNA laddering in SIV-infected cells showed subdiploid DNA peaks at 24, 48, and 72 h post-infection by flow cytometry. Under light and electron microscopes, morphological changes in SIV-infected cells were observed, including cell shrinkage, plasma membrane blebbing, nuclear condensation, and cytoplasm vacuolization. These results indicate that SIV may induce apoptosis in PAMs in vitro, and apoptosis may play an important role in death of PAMs infected with SIV. Key words: Swine influenza virus; H3N2 subtype; Porcine pulmonary alveolar macrophage; Apoptosis; In vitro ( ),, 16 ( hemagglutinin, HA) [ 1, 2 ], H1N1 H3N2 H1N2 [ 2, 3 ] ( swine influenza virus, SIV),, :, lucp@ njau. edu. cn Corresponding author: LU Cheng-Ping, lucp@ njau. edu. cn [ 1-3 ] SIV, [ 1, 3] SIV, SIV [ 2, 3 ],,, SIV SIV,,,,,,, SIV

2 Journal of Microbes and Infection, June 2009, Vol. 4, No ( pulmonary alveolar macrophage, PAM),, ;,, 1( interleukin 1, IL-1) IL-6 IL IL-18 ( tumor necrosis factor-, TNF- ), T ( helper T lymphocyte, Th) SIV PAM, PAM SIV A/Swine/Guangdong/1/2004 ( H3N2) ; ( MDCK) ( ) ; 2 7, ( porcine reproductive and respiratory syndrome virus, PRRSV) SIV 1. 2 ( propidium iodide, PI) TPCK- Sigma, RPMI 1640 DNA DNzol Promiga 1. 3 PAM PBS, PBS ml, 2 min, 2 3, 239 g 10 min, PBS, 239 g 10 min, ; 1 RPMI 1640 ( 10% ), 100 ml, 37 5% CO2 5 h, D-Hank s 2,,, 0. 02% EDTA, min RPMI 1640, RPMI 1640 ( 10% ) /ml, 1. 4 SIV PAM PAM 24, /, 12 h RPMI 1640 ( 0. 1 g/ ml TPCK- ) 2 3, 100 TCID50 SIV,37 1 h; h, , 425 g 5 min DNA ml DNzol, 106 g 10 min, 0. 5 ml 100%, 1 3 min g 5 min, 0. 8 ml 70% 2, 8 mmol/l NaOH DNA, 1. 5% 1. 6 PI ( /L), g 5 min, PBS 1, 106 g 5 min, 70% 1 24 h,, PBS 2 3, 1 ml PI ( 100 g/ml, 100 g/ml RNase), min, ( BD FACSCalibur Cell Sorting System) 1. 7 Giemsa, 5 min, Giemsa, 5 10 min, 95%, 5 min 1. 8, PBS 2 3, 425 g 10 min, 2. 5% 2 4 h 1% 2 h,, SIV PAM h, 2 3, 1% 96 V, 50 l PBS, 50 l PAM2, 50 l,, 30 min,, SIV PAM, 12 h, 96 h ( 2 6 ) ( 1)

Hemagglutination activity of normal PAMs : negative. 2. 2 DNA SIV 96 h PAM DNA 1. 5%, ( 1) A: PAMs infected for 24 h. B: PAMs infected for 48 h. C: PAMs infected for 72 h.

3 78 1 SIV Tab 1. Hemagglutination activity of pulmonary alveolar macrophages infected with SIV, Post-infection time ( h) Hemagglutination activity of infected PAMs Hemagglutination activity of normal PAMs : negative DNA SIV 96 h PAM DNA 1. 5%, ( 1) A: PAMs infected for 24 h. B: PAMs infected for 48 h. C: PAMs infected for 72 h. D: Normal PAMs at 72 h post-infection. 2 SIV Fig 2. Flow cytometric DNA fluorescence profiles of SIV-infected pulmonary alveolar macrophages at different post-infection time 1: normal PAMs; 2: infected PAMs at 96 h post-infection 1 SIV DNA Fig 1. Analysis of DNA fragmentation in pulmonary alveolar macrophages infected with SIV by agarose gel electrophoresis 2. 3 SIV h, PI, DNA ( ), 2, G 0 / G1 1 DNA ( subdiploid DNA peak), 24 h 5. 6%, h15% 25% ; 72 h DNA,, PAM SIV, 2. 4 Giemsa 48 h, Giemsa, ( ) 3,, ; SIV A: Mock-infected PAMs ( 60). B: SIV-infected PAMs at 48 h postinfection ( 100). 3 GiemsaSIV Fig 3. Morphological changes in SIV-infected PAMs under light microscope after Giemsa staining h, ( 4),,,,

2009 6 4 2 Journal of Microbes and Infection, June 2009, Vol. 4, No. 2 79, ;, A: Mock-infected PAMs ( 5 000 ). B: SIV-infected PAMs at 48 h post-infection ( 6 000 ).

4 Journal of Microbes and Infection, June 2009, Vol. 4, No. 2 79, ;, A: Mock-infected PAMs ( ). B: SIV-infected PAMs at 48 h post-infection ( ). C, D: SIV-infected PAMs at 48 h postinfection ( ). 4 Fig 4. Morphological changes in SIV-infected PAMs under electron microscope 3 SIV PAM, SIV PAM, PAM, SIV, [ 4],, [ 5 ],,,, [ 5, 6 ] IL-12 ( ), Noone, HA IL-12 P70 [ 7 ] Kodihalli, 10 d, PAM [ 8] Jung SIV, PAM SIV, SIV PAM [ 9],,,,, PAM SIV, Seo 3 ; A/ Sydney/5/97 ( H3N2 ) A/New Caledonia/1/99 ( H1N1) B/Sichuan/99 < PAM, [ 1 0],, SIV PAM, SIV PAM?? SIV PAM? [ 11, 12 ] [ ], Morri,,, [ 1 1] Ito, ( H5N1 H7N7 H5N3 ), [ 12] NA NS1 PB1-F2 NA ( transforming growth factor, TGF- ) TGF-, [ 19 ] NS1 RNA [ 20], NS1 [ 21] RNA 2 PB1-F2,, [ 22],

5 80,, Fas RNA Bcl-2 [ 12, ] PAM : ; PAM IL-1 IL-10 TNF- ; PAM NO O - 2 PAM, [ 1] Webster RG, Bean WJ, Gorman OT, et al. Evolution and ecology of influenza a viruses [ J]. Microbiol Rev, 1992, 56( 1) : [ 2] Qi X, Lu CP. Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China [ J]. Arch Virol, 2006, 151 ( 11) : [ 3] Brown IH. The epidemiology and evolution of influenza viruses in pigs [ J]. Vet Microbiol, 2000, 74( 1-2) :29-46 [ 4] Hament JM, Kimpen JLL, Fleer A, et al. Respiratory viral infection predisposing for bacterial disease: a concise review [ J]. FEMS Immunol Med Microbiol, 1999, 26( 3-4) : [ 5] Hartshorn KL, Liou LS, White MR, et al. Neutrophil deactivation by influenza A virus. Role of hemagglutinin binding to specific sialic acid-bearing cellular proteins [ J]. J Immunol, 1995, 154( 8) : [ 6] Colamussi ML, White MR, Crouch E, et al. Influenza A virus accelerates neutrophil apoptosis and markedly potentiates apoptotic effects of bacteria [ J]. 1999, 93( 7) : Blood, [ 7] Noone CM, Lewis EA, Frawely AB, et al. Novel mechanism of immunosuppression by influenza virus haemagglutinin: selective suppression of interleukin 12 p35 transcription in murine bone marrow-derived dendritic cells [ J]. J Gen Virol, 2005, 86: [ 8] Kodihalli S, Sivanandan V, Nagaraja KV, et al. Effect of avian influenza virus infection on the phagocytic function of systemic phagocytes and pulmonary macrophages of turkeys [ J]. Avian Dis, 1994, 38( 1) : [ 9] Jung T, Choi C, Chae C. Localization of swine influenza virus in naturally infected pigs [ J]. Vet Pathol, 2002, 39: [ 10] Seo SH, Webby R, Webster RG. No apoptotic deaths and different levels of inductions of inflammatory cytokines in alveolar macrophages infected with influenza viruses. Virology, 329( 2) : [ 11] Mori I, Komatsu T, Takeuchi K, et al. In vivo induction of apoptosis by influenza virus [ J]. J Gen Virol, 1995, 76( Pt 11) : [ 12] Ito T, Kobayashi Y, Morita T, et al. Virulent influenza A viruses induce apoptosis in chickens [ J]. Virus Res, 2002, 84( 1-2) : [ 13] Hinshaw VS, Olsen CW, Dybdahl-Sissoko N, et al. Apoptosis: a mechanism of cell killing by influenza A and B viruses [ J]. J Virol, 1994, 68( 6) : [ 14] Lowy RJ, Dimitrov DS. Characterization of influenza virus-induced death of J macrophages [ J]. Exp Cell Res, 1997, 234( 2) : [ 15] Morris SJ, Price GE, Barnett JM, et al. Role of neuraminidase in influenza virus-induced apoptosis [ J]. J Gen Virol, 1999, 80( Pt 1) : [ 16] Olsen CW, Kehren JC, Dybdahl-Sissoko NR, et al. bcl- 2 alters influenza virus yield, spread, and hemagglutinin glycosylation [ J]. J Virol, 1996, 70( 1) : [ 17] Takizawa T, Matsukawa S, Higuchi Y, et al. Induction of programmed cell death ( apoptosis) by influenza virus infection in tissue culture cells [ J]. J Gen Virol, 1993, 74( Pt 11) : [ 18] Schultz-Cherry S, Krug RM, Hinshaw VS. Induction of apoptosis by influenza virus [ J]. Semin Virol, 1998, 8 ( 6) : [ 19] Schultz-Cherry S, Hinshaw VS. Influenza virus neuraminidase activates latent transforming growth factor beta [ J]. J Virol, 1996, 70( 12) : [ 20] Schultz-Cherry S, Dybdahl-sissoko N, Neumann G, et al. Influenza virus NS1 protein induces apoptosis in cultured cells [ J]. J Virol, 2001,75( 17) : [ 21] Zhirnov OP, Konakova TE, Wolff T, et al. NS1 protein of influenza a virus down-regulates apoptosis [ J]. J Virol, 2002, 76( 7) : [ 22] Chen W, Calvol PA, Malide D, et al. A novel influenza A virus mitochondrial protein that induces cell death[ J]. Nat Med, 2001, 7: ( : )

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