臺灣博碩士論文加值系統

肥胖已經成為全世界重要的課題，世界衛生組織預測到了2015年全球會有23億人過重，其中更有7億人達到肥胖的標準。有許多研究指出肥胖的人在感染2009新型流感病毒pandemic H1N1(pH1N1)的預後是比較差的，甚至有較高的死亡率。除此之外，也有研究發現季節流感甚至是其他感染症像是肺炎，也和肥胖有密切關係。目前有少數研究提出可能影響的原因，但是大部分都是探討肥胖對於免疫系統的影響，關於病毒學的研究卻是鮮少提及。高脂血症在肥胖是非常常見的疾病，但是高膽固醇環境對於流感病毒的影響卻依然未知。因此，本篇研究想要建立一個高膽固醇細胞培養的模式，並進而了解pH1N1感染細胞之後，病毒性質及宿主免疫反應的變化。
首先測試膽固醇對於MDCK細胞的毒性，來決定添加於培養液中的劑量。使用MTT測試細胞活性之後，在4個不同濃度膽固醇存在之下，細胞都還能維持在70%以上的存活率，最後選定0.04 mM以及0.08 mM來進行後續的實驗。當細胞在高膽固醇環境下培養24小時之後感染pH1N1病毒，觀察病毒吸附及進入細胞的能力。結果發現以膽固醇前處理培養24小時之後的細胞會使pH1N1病毒吸附能力增強，不過病毒進入細胞的效率卻變差。接著觀察病毒在高膽固醇處理的細胞對於生長的影響，發現在感染後12、24以及30小時的病毒量受到膽固醇的影響很小。但是其所產生的病毒卻有著較高的細胞結合力。除了對病毒的影響之外，也觀察在高膽固醇培養下對細胞造成的變化，以及此變化對於病毒感染後細胞激素的影響，可以發現膽固醇對於細胞的影響比病毒更為顯著。在高膽固醇培養之下，細胞表面的唾液酸明顯增加，細胞sphingosine kinase的mRNA表現量也提高。在被病毒感染之後，不論是感染後12小時或是24小時，細胞激素IFN-β、TNF-α以及IL-6的mRNA表現量都顯著提高。最後為了確定這些變化是膽固醇所造成，我們選擇了一種常見的降膽固醇藥物Lovastatin抑制膽固醇的作用以觀察是否可以反轉前述的現象，結果顯示以上所觀察到的現象都可以藉由添加Lovastatin來達到抑制的效果。
總結而言，膽固醇藉由改變細胞的組成而影響了病毒對於MDCK細胞的吸附及進入，病毒的生長雖不受膽固醇的影響，但子代病毒的細胞結合力會增加。而細胞組成的改變，可能使sphingosine kinase mRNA表現量提高，進而造成細胞被感染之後，細胞激素的mRNA表現量顯著提高。膽固醇在動物或人體内對流感病毒真正之影響，仍需更進一步之研究。

Obesity has become an important topic in the world, the World Health Organization predicted that there will be 2.3 billion people overweight on 2015, and 700 million people will surpass the obesity standard. Many studies have pointed out that obese people infected with 2009 pandemic influenza virus H1N1 (pH1N1) had poor prognosis and increased mortality. In addition, studies have found that seasonal flu or other infectious diseases like pneumonia are closely related with obesity. Only a few studies point out the condition of obesity to the outcome of infectious diseases and most of the studies were related to the impact of obesity on immune system. Scared research is focus on the virology entity. Hyperlipidemia in obesity is a very common disease, yet the impact of high cholesterol environment to the growth of influenza viruses is still unclear. Therefore, a high-cholesterol cell cultured model was established to analyze the effects on virus nature and cellular immune responses upon pandemic H1N1 infection.
After enriched with high cholesterol for 24 hours, the enriched cells were infected with pH1N1, and the virus adsorption and viral entry were analyzed. First, pH1N1 virus had higher adsorption ability in cholesterol enriched cells than the virus in un-enriched cells. Nevertheless, the virus entry was decreased in cholesterol enriched cells than the virus in un-enriched cells. Similar findings were also observed in viruses generated in the cholesterol enriched cells. The effect of cholesterol on the sialic acid expression and cellular cytokines production in viruses infected cells were also investigated. The cell surface sialic acid and cellular sphingosine kinase mRNA expression were significantly increased in cells cultured under high cholesterol environment. When the cholesterol enriched cells were infected with pH1N1 viruses, the cytokine mRNA expression of IFN-β、TNF-α and IL-6 mRNA expression was significantly increased than those from virus infected , un-enriched cells. In order to confirm the above findings, a cholesterol-lowering drug Lovastatin was used to inhibit cholesterol synthesis and the effects of cholesterol on virus adsorption、entry and cytokine expression could be reversed by Lovastatin.
In conclusion, cholesterol affected the virus adsorption and entry in MDCK cells by changing the composition of cells. Cholesterol did not affect the growth of the virus, but the cell binding ability in cholesterol enriched virus was increase. These results suggested that sphingosine kinase mRNA expression increased, thereby causing the cytokine mRNA expression significantly increased after infection. The association of cholesterol and the influenza virus pathogenesis in vivo needs further clarification.

第一章 緒論.....................................................................................................................1
第一節 流行性感冒病毒的分類.............................................................................1
第二節 A型流行性感冒病毒歷史..........................................................................1
第三節 A型流行性感冒病毒介紹..........................................................................2
第四節 A型流行性感冒病毒流行概況..................................................................3
第五節 A型流行性感冒病毒之生長......................................................................4
第六節 病毒感染之危險因子.................................................................................5
第七節 流行性感冒的症狀及宿主免疫反應.........................................................6
第八節 病毒及脂筏(lipid raft).................................................................................7
第九節 鞘脂(Sphingolipid)......................................................................................8
第十節 實驗目的.....................................................................................................8
第二章 實驗材料與方法.................................................................................................9
第一節 實驗材料.....................................................................................................9
第二節 實驗方法....................................................................... ...........................12
1. 試劑之製備........................................................................................................12
2. 細胞之繼代培養................................................................................................14
3. 臨床A型流感病毒H1N1分離株之繼代培養................................................14
4. 病毒RNA萃取..................................................................................................14
5. 反轉錄即時定量聚合酶連鎖反應（qRT-PCR）.............................................15
6. qRT-PCR標準品製備.........................................................................................16
7. 病毒溶斑試驗....................................................................................................18
8. Sialic Acid Quantitation.......................................................................................18
9. 病毒結合細胞試驗............................................................................................19
10. 病毒進入細胞試驗..........................................................................................19
11. 細胞RNA萃取................................................................................................20
12. 細胞免疫螢光染色..........................................................................................20
13. 血球凝集試驗..................................................................................................21
14. 細胞毒性試驗..................................................................................................21
15. 西方墨點法......................................................................................................21
16. 統計方法與分析軟體......................................................................................22
第三章 結果...................................................................................................................23
第一節 膽固醇對病毒之影響...............................................................................23
1. 細胞存活率試驗................................................................................................23
2. 病毒結合與進入細胞能力分析........................................................................23
3. 病毒的生長曲線................................................................................................24
4. 膽固醇存在下培養之病毒細胞吸附力試驗....................................................24
5. 病毒HA蛋白質表現量.....................................................................................24
第二節 膽固醇對宿主細胞影響研究....................................... ...........................25
1. 細胞表面唾液酸的變化....................................................................................25
2. 細胞激素比較....................................................................................................25
第三節 使用Lovastatin抑制膽固醇合成..............................................................26
1. 細胞存活率試驗................................................................................................26
2. 病毒結合與進入細胞能力分析........................................................................26
3. 病毒的生長曲線................................................................................................26
4. 病毒HA蛋白質表現量.....................................................................................27
5. 細胞表面唾液酸的變化....................................................................................27
6. 細胞激素比較....................................................................................................27
第四章 討論...................................................................................................................28
第五章 參考文獻...........................................................................................................32

1. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka. 1992. Evolution and ecology of influenza A viruses. Microbiol Rev. 56: 152-79.
2. Tong, S., Li, Y., Rivailler ,P., Conrardy, C., et al. 1992. A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci U S A. 109(11): 4269-74.
3. Johnson, N.P., and Mueller, J. 2002. Updating the accounts: global mortality of the 1918-1920 ‘‘Spanish’’ influenza pandemic. Bull. Hist. Med. 76(1): 105–115.
4. WHO. 2009a. World Health Organization. Pandemic (H1N1) 2009- update 64. Accessed 5 September 2009. Available from www. who. int/csr/don/2009_09_04/ en/index.html.
5. Horimoto, T., and Kawaoka, Y. 2001. Pandemic threat posed by avian influenza A viruses. Clin. Microbiol. Rev. 14(1): 129–149.
6. Steinhauer, D.A., and Skehel, J.J. 2002. Genetics of influenza viruses. Annu. Rev. Genet. 36(1): 305–332.
7. Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., et al. 2001. A novel influenza A virus mitochondrial protein that induces cell death. Nat. Med. 7(12): 1306–1312.
8. Garcı’a-Sastre, A. 2006. Antiviral response in pandemic influenza viruses. Emerg. Infect. Dis. 12(1): 44–47.
9. Kawaoka, Y., Krauss, S., and Webster, R.G. 1989. Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J. Virol. 63(11): 4603–4608.
10. Scholtissek, C. 1985. Stability of infectious influenza A viruses to treatment at low pH and heating. Arch. Virol. 85(1-2): 1–11.
11. Ma, W., Lager, K.M., Vincent, A.L., Janke, B.H., Gramer, M.R., et al. 2009. The role of swine in the generation of novel influenza viruses. Zoonoses Public Health. 56(6-7): 326–337.
12.Ito, T., Couceiro, J.N., Kelm, S., Baum, L.G., Krauss, S., Castrucci, M.R., et al. 1998. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J. Virol. 72(9): 7367–7373.
13. Xu, X., Subbarao, Cox, N.J., and Guo, Y. 1999. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology. 261(1): 15–19.
14. Vijaykrishna, D., Bahl, J., Riley, S., Duan, L., Zhang, J.X., Chen, H., et al. 2008. Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses. PLoS Pathog. 4(9): e1000161.
15. Taubenberger, J.K., Reid, A.H., Lourens, R.M., Wang, R., Jin, G., and Fanning, T.G. 2005. Characterization of the 1918 influenza virus polymerase genes. Nature. 437(7060): 889–893.
16. Zimmer, S.M., and Burke, D.S. 2009. Historical perspective –Emergence of influenza A (H1N1) viruses. N. Engl. J. Med. 361(3): 279–285.
17. Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., et al. 2009. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science. 325(5937): 197–201.
18. Smith, G.J., Vijaykrishna, D., Bahl, J., Lycett, S.J., Worobey, M., Pybus, O.G., et al. 2009. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. 459(7250): 1122–1125.
19. Strauss J, Strauss E. 2008. Minus-strand RNA viruses. In: Viruses and Human Disease. 2nd ed. Oxford, UK: Elsevier; pp.137e175.
20. Palese P, Shaw M. 2006. Orthomyxoviridae : the viruses and their replication. In: Knipe D, Howley P, eds. Fields Virology. 5th ed. Philadelphia: Lippincott Williams &Wilkins; pp. 1647e1689.
21. Ma W, Lager KM, Vincent AL, et al. 2009. The Role of Swine in the Generation of Novel Influenza Viruses. Zoonoses Public Healt. 56(6-7):326-37.
22. Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., Lindstrom, S., et al. 2009. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 360(25): 2605–2615.
23. Centers for Disease Control and Prevention (CDC). 2009a. Neurologic complications associated with novel influenza A (H1N1) virus infection in children - Dallas, Texas, May 2009. Morb. Mortal. Wkly. Rep. 58(28): 773–778.
24. Centers for Disease Control and Prevention (CDC). 2009c. Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1) - United States, May-August 2009. Morb. Mortal. Wkly. Rep. 58(38): 1071–1074.
25. Jamieson, D.J., Honein, M.A., Rasmussen, S.A., Williams, J.L., Swerdlow, D.L., Biggerstaff, M.S., et al.; Novel Influenza A (H1N1) Pregnancy Working Group. 2009. H1N1 2009 influenza virus infection during pregnancy in the USA. Lancet. 374(9688): 451–458.
26. Centers for Disease Control and Prevention (CDC). 2009b. Intensive-care patients with severe novel influenza A (H1N1) virus infection - Michigan, June. Morb. Mortal. Wkly. Rep. 58(27): 749–752.
27. Rello, J., Rodrı’guez, A., Iban˜ez, P., Socias, L., Cebrian, J., et al. 2009. Intensive care adult patients with severe respiratory failure caused by Influenza A (H1N1)v in Spain. Crit. Care. 13(5): R148.
28. Alexia G. Smith, Patricia A. Sheridan, Joyce B. Harp, and Melinda A. Beck., et al. 2010. Diet-Induced Obese Mice Have Increased Mortality and Altered Immune Responses When Infected with Influenza Virus. J. Nutr. 137: 1236–1243.
29. Erik A. Karlsson, Patricia A. Sheridan, and Melinda A. Beck., et al. 2010. Diet-Induced Obesity in Mice Reduces the Maintenance of Influenza-Specific CD8+ Memory T Cells. J. Nutr. 140: 1691–1697.
30. Mauad, T., Hajjar, L.A., Callegari, G.D., da Silva, L.F., Schout, D.,
Galas, F.R., et al. 2010. Lung pathology in fatal novel human influenza A (H1N1) infection. Am. J. Respir. Crit. Care Med. 181(1): 72–79.
31. Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., et al. 2009. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature. 460(7258): 1021–1025.
32. Julkunen I, Melen K, Nyqvist M, et al. 2000. Inflammatory responses in influenza A virus infection. Vaccine. 19: S32–37.
33. Ronni T, Sareneva T, Pirhonen J, et al. 1995. Activation of IFN-alpha, IFN-gamma, MxA, and IFN regulatory factor 1 genes in influenza A virus-infected human peripheral blood mononuclear cells. J Immunol. 154:2764–2774.
34. Ronni T, Matikainen S, Sareneva T, et al. 1997. Regulation of IFN-alpha/beta, MxA, 20,50-oligoadenylate synthetase, and HLA gene expression in influenza A-infected human lung epithelial cells. J Immunol. 158:2363–2374.
35. Stetson DB, Medzhitov R. 2006. Type I interferons in host defense. Immunity.
25:373–381.
36. Falagas ME, Kompoti M. 2006. Obesity and infection. Lancet Infect Dis. 6:438–446.
37. Tsatsanis C , Margioris A. , Kontoyiannis D. 2010. Association between H1N1 Infection Severity and Obesity—Adiponectin as a Potential Etiologic Factor. J. Infect Dis. 202(3):459–460.
38. Nayak, D. P., and S. Barman. 2002. Role of lipid rafts in virus assembly and
budding. Adv. Virus Res. 77:1977–1983.
39. Nayak, D. P., E.-K. W. Hui, and S. Barman. 2004. Assembly and budding of
influenza virus. Virus Res. 106:147–165.
40. Ono, A., and E. O. Freed. 2005. Role of lipid rafts in virus replication. Adv.
Virus Res. 64:311–358.
41. Schmitt, A. P., and R. A. Lamb. 2004. Escaping from the cell: assembly and
budding of negative-strand RNA viruses. Curr. Top. Microbiol. Immunol.
283:145–196.
42. Zhang, J., A. Pekosz, and R. A. Lamb. 2000. Influenza virus assembly and
lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins.
J. Virol. 74:4634–4644.
43. Schroeder, C., H. Heider, E. Moncke-Buchner, and T. I. Lin. 2005. The
influenza virus ion channel and maturation cofactor M2 is a cholesterol binding
protein. Eur. Biophys. J. 34:52–66.
44. Schmitt, A. P., and R. A. Lamb. 2005. Influenza virus assembly and budding
at the viral budozone. Adv. Virus Res. 64:383–416.
45. Colman, P. M., and M. C. Lawrence. 2003. The structural biology of type I
viral membrane fusion. Nat. Rev. Mol. Cell Biol. 4:309–319.
46. Scheiffele, P., A. Rietveld, T. Wilk., and K. Simons. 1999. Influenza viruses
select ordered lipid domains during budding from the plasma membrane. J. Biol. Chem. 274:2038–2044.
47. Choppin, P. W., and R. W. Compans. 1975. The structure of influenza virus,
p. 15–47. In E. D. Kilbourne (ed.), Influenza and influenza viruses. Academic
Press, New York, N.Y.
48. Lenard, J., and R. W. Compans. 1974. The membrane structure of lipid containing viruses. Biochim. Biophys. Acta. 344:51–94.
49. Brown, E. L., and D. S. Lyles. 2003. Organization of the vesicular stomatitis
virus glycoprotein into membrane microdomains occurs independently of
intracellular viral components. J. Virol. 77:3985–3992.
50. Lu, Y. E., and M. Kielian. 2000. Semliki Forest virus budding: assay, mechanisms, and cholesterol requirement. J. Virol. 74:7708–7719.
51. Luan, P., L. Yang, and M. Glaser. 1995. Formation of membrane domains
created during the budding of vesicular stomatitis virus. A model for selective
lipid and protein sorting in biological membranes. Biochemistry. 34:9874–9883.
52. Scheiffele, P., M. G. Roth, and K. Simons. 1997. Interaction of influenza
virus haemagglutinin with sphingolipid-cholesterol membrane domains via
its transmembrane domain. EMBO J. 16:5501–5508.
53. Nguyen, D. H., and J. E. Hildreth. 2000. Evidence for budding of human
immunodeficiency virus type 1 selectively from glycolipid-enriched membrane
lipid rafts. J. Virol. 74:3264–3272.
54. Ono, A., and E. O. Freed. 2001. Plasma membrane rafts play a critical role in
HIV-1 assembly and release. Proc. Natl. Acad. Sci. USA 98:13925–13930.
55. Phalen, T., and M. Kielian. 1991. Cholesterol is required for infection by
Semliki Forest virus. J. Cell Biol. 112:615–623.
56. Waarts, B. L., R. Bittman, and J. Wilschut. 2002. Sphingolipid and cholesterol
dependence of alphavirus membrane fusion. Lack of correlation with
lipid raft formation in target liposomes. J. Biol. Chem. 277:38141–38147.
57. Viard, M., I. Parolini, M. Sargiacomo, K. Fecchi, C. Ramoni, S. Ablan, F. W.
Ruscetti, J. M. Wang, and R. Blumenthal. 2002. Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells. J. Virol. 76:11584–11595.
58. Bender, F. C., J. C. Whitbeck, M. Ponce de Leon, H. Lou, R. J. Eisenberg,
and G. H. Cohen. 2003. Specific association of glycoprotein B with lipid rafts
during herpes simplex virus entry. J. Virol. 77:9452–9552.
59. A.H. Merrill Jr., E.M. Schmelz, D.L. Dillehay, S. Spiegel, J.A. Shayman,
J.J. Schroeder, R.T. Riley, K.A. Voss, E. Wang. 1997. Sphingolipids — the
enigmatic lipid class: biochemistry, physiology, and pathophysiology,
Toxicol. Appl. Pharmacol. 142:208–225.
60. Y.A. Hannun. 1994. The sphingomyelin cycle and the second messenger
functions of Ceramide, J. Biol. Chem. 269:3125–3128.
61. T.H. Kee, P. Vit, A.J. Melendez. 2005. Sphingosine kinase signalling in immune
cells, Clin. Exp. Pharmcol. Physiol. 32:153–161.
62. T. Baumruker, E.E. Prieschl. 2002. Sphingolipids and the regulation of the
immune response, Semin. Immunol. 14:57–63.
63. S. Spiegel. 1999. Sphingosine 1-phospate: a prototype of a new class of
second messengers, J. Leuoc. Biol. 65:341–344.
64. S. Spiegel, S. Milstien. 2002. Sphingosine 1-phosphate, a key cell signaling
molecule, J. Biol. Chem. 277:25851–25854.
65. J.R. Van Brocklyn, M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O.
Cuvillier, D.M. Thomas, P.J. Coopman, S. Thangada, C.H. Liu, T. Hla, S.
Spiegel. 1998. Dual actions of sphingosine-1-phosphate: extracellular through
the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation
and survival, J. Cell Biol. 142:229–240.
66. Seo YJ, Blake C, Alexander S, Hahm B. 2010. Sphingosine 1-phosphate-metabolizing enzymes control influenza virus propagation and viral cytopathogenicity, J Virol. 84:8124–31.
67. Marsolais D, Hahm B, Edelmann KH, Walsh KB, Guerrero M, Hatta Y, et al. 2008. Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus specific immune responses to influenza. Mol Pharmacol. 74:896–903.
68. Marsolais D, Hahm B, Walsh KB, Edelmann KH, McGavern D, Hatta Y, et al. 2009. A critical role for the sphingosine analog AAL-R in dampening the cytokine
response during influenza virus infection. Proc Natl Acad Sci USA. 106:1560–5.
69. Ahmad S, Ahmad A, Schneider KB, White CW. 2006. Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells. Int J Toxicol. 25(1):17-23.
70. O''Brien KB, Vogel P, Duan S, Govorkova EA, Webby RJ, McCullers JA, Schultz-Cherry S. 2012. Impaired wound healing predisposes obese mice to severe influenza virus infection. J Infect Dis. 205(2):252-61.
71. de Vries E, Tscherne DM, Wienholts MJ, Cobos-Jimenez V, Scholte F, Garcia-Sastre A, Rottier PJ, de Haan CA. 2011. Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway. PLoS Pathog. 7(3):e1001329.
72. Keller P, Simons K. 1998. Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol. 140(6):1357-67.
73. Barman S, Nayak DP. 2007. Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells. J Virol. 81(22):12169-78.
74. Takahashi M, Kobayashi T. 2009. Cholesterol regulation of rab-mediated sphingolipid endocytosis. Glycoconj J. 26(6):705-10.
75. Shin HW, Kim D, Lee Y, Yoo HS, Lee BJ, Kim JS, Jang S, Lim H, Lee Y, Oh S. 2009. Alteration of sphingolipid metabolism and pSTAT3 expression by dietary cholesterol in the gallbladder of hamsters. Glycoconj J. 26(6):705-10.
76. Frost FJ, Petersen H, Tollestrup K, Skipper B. 2007. Influenza and COPD mortality protection as pleiotropic, dose-dependent effects of statins. Chest. 131:1006–12.
77. Kwong JC, Li P, Redelmeier DA. 2009. Influenza morbidity and mortality in elderly patients receiving statins: a cohort study. PLoS One. 4:e8087.
78. Vandermeer ML, Thomas AR, Kamimoto L, Reingold A, Gershman K, Meek J, Farley MM, Ryan P, Lynfield R, Baumbach J, Schaffner W, Bennett N, Zansky S. 2012. Association between use of statins and mortality among patients hospitalized with laboratory confirmed influenza virus infections: a multistate study. J Infect Dis. 205(1):13-9.