The winter of 2009 witnessed the concurrent spread of 2009 pandemic H1N1 with 2009 seasonal H1N1. It is clinically important to develop knowledge of the key features of these two different viruses that make them unique. A robust pattern recognition technique, Random Forests, was employed to uncover essential amino acid markers to differentiate the two viruses. Some of these markers were also part of the previously discovered genomic signature that separate avian or swine from human viruses. Much research to date in search of host markers in 2009 pandemic H1N1 has been primarily limited in the context of traditional markers of avian-human or swine-human host shifts. However, many of the molecular markers for adaptation to human hosts or to the emergence of a pandemic virus do not exist in 2009 pandemic H1N1, implying that other previously unrecognized molecular determinants are accountable for its capability to infect humans. The current study aimed to explore novel host markers in the proteins of 2009 pandemic H1N1 that were not present in those classical markers, thus providing fresh and unique insight into the adaptive genetic modifications that could lead to the generation of this new virus. Random Forests were used to find 18 such markers in HA, 15 in NA, 9 in PB2, 11 in PB1, 13 in PA, 10 in NS1, 1 in NS2, 11 in NP, 3 in M1, and 1 in M2. The amino acids at many of these novel sites in 2009 pandemic H1N1 were distinct from those in avian, human, and swine viruses that were identical at these positions, reflecting the uniqueness of these novel sites.
Samji, T. (2009) Influenza A: Understanding the viral life cycle. Yale Journal of Biology Medicine, 82(4), 153- 159.
Gavin, J.D., Smith, D.V., Bahl, J., Lycett, S.J., et al. (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature, 459, 1122-1125.
Chang, Y.S., van Hal, S.J., Spencer, P.M, Gosbell, I.B. and Collett, P.W. (2010) Comparison of adult patients hospitalised with pandemic (H1N1) 2009 influenza and seasonal influenza during the PROTECT phase of the pandemic response. The Medical Journal of Australia, 192(2), 90-93.
Dhiman, N., Mark, J.E., Irish, C., Wright, P., Smith, T.F. and Pritt, B.S. (2010) Mutability in the matrix gene of novel influenza A H1N1 virus detected using a fret probe-based real-time reverse transcriptase PCR assay. Journal of Clinical Microbiology, 48(2), 677-679.
Zheng, X., Todd, K.M., Yen-Lieberman, B., Kaul, K., Mangold, K. and Shulman, S.T. (2009) Unique finding of a 2009 H1N1 influenza virus－positive clinical sample suggests matrix gene sequence variation. Journal of Clinical Microbiology, 48(2), 665-666.
Shen, J., Ma, J. and Wang, Q. (2009) Evolutionary trends of A (H1N1) influenza virus hemagglutinin since 1918. PLoS One, 4(11), e7789.
Soundararajan, V., Tharakaraman, K., Raman, R., Raguram, S., Shriver, Z., Sasisekharan, V. and Sasisekharan, R. (2009) Extrapolating from sequence—the 2009 H1N1 ‘swine’ influenza virus. Nature Biotechnology, 27, 510- 513.
Childs, R.A., Palma, A.S., Wharton, S., Matrosovich, T., Liu, Y., Chai, W.G., Campanero-Rhodes, M.A., et al. (2009) Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nature Biotechnology, 27, 797-799.
Igarashi, M., Ito, K., Yoshida, R., Tomabechi, D., Kida, H. and Takada, A. (2009) Predicting the antigenic structure of the pandemic (H1N1) 2009 influenza virus hemagglutinin. PLoS One, 5(1), e8553.
Cosic, I. (1997) The resonant recognition model of macromolecular bioreactivity, theory and application. Birkhauser Verlag, Berlin.
Veljkovic, V., Niman, H.L., Glisic, S., Veljkovic, N., Perovic, V. and Muller, C.P. (2009) Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor. BMC Structural Biology, 9, 62.
Hu, W. (2010) Identification of highly conserved domains in hemagglutinin associated with the receptor binding specificity of influenza viruses: 2009 H1N1, avian H5N1 and swine. Journal of Biomedical Science and Engineering, 3, 114-123.
Hu, W. (2010) Quantifying the effects of mutations on receptor binding specificity of influenza viruses. Journal of Biomedical Science and Engineering, 3, 227-240.
Janies, D.A., Voronkin, I.O., Studer, J., Hardman, J., Alexandrov, B.B., Treseder, T.W. and Valson, C. (2010) Selection for resistance to oseltamivir in seasonal and pandemic H1N1 influenza and widespread co-circulation of the lineages. International Journal of Health Geogra- phics, 9(1), 13.
Deyde, V.M., Sheu, T.G., Trujillo, A.A., Okomo- Adhiambo, M., Garten, R., Klimov, A.I. and Gubareva, L.V. (2010) Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing. Antimicrob Agents Chemother, 54(3), 1102-1110.
Hurt, A.C., Holien, J.K., Parker, M., Kelso, A. and Barr, I.G. (2009) Zanamivir-resistant influenza viruses with a novel neuraminidase mutation. The Journal of Virology, 83(20), 10366-10373.
Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., Sessions, W.M., Xu, X., et al. (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science, 325(5937), 197-201.
Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., et al. (2009) In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature, 460, 1021-1025.
Uhlendorff, J., Matrosovich, T., Klenk, H.D. and Matrosovich, M. (2009) Functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses. Archives of Virology, 154(6), 945-957.
Sung, J.C., van Wynsberghe A.W., Amaro, R.E., Li, W.W. and McCammon, J.A. (2010) Role of secondary sialic acid binding sites in influenza N1 neuraminidase. Journal of the American Chemistry Society, 132(9), 2883-2885.
Steel, J., Lowen, A., Mubareka, S., Palese, P. and Baric, R. (2009) Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog, 5, e1000252.
Subbarao, E.K., London, W. and Murphy, B.R. (1993) A single amino-acid in the Pb2-gene of influenza-A virus is a determinant of host range. Journal of Virology, 67, 1761-1764.
Mehle, A. and Doudna, J.A. (2009) Adaptive strategies of the influenza virus polymerase for replication in humans. Proceedings of the National Academy of Sciences of the United States of America, 106(50), 21312-21316.
Rolling, T., Koerner, I., Zimmermann, P., Holz, K., Haller, O., Staeheli, P. and Kochs, G. (2009) Adaptive mutations resulting in enhanced polymerase activity contribute to high virulence of influenza A virus in mice. Journal of Virology, 83 (13), 6673-6680.
Liu, X. and Zhao, Y.P. (2010) Switch region for pathogenic structural change in conformational disease and its prediction. PLoS One, 5(1), e8441.
Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O'Neill, R., Schickli, J., Palese, P., Henklein, P., Bennink, J.R. and Yewdell, J.W. (2001) A novel influenza A virus mitochondrial protein that induces cell death. Nature Medicine, 7, 1306-1312.
Lamb, R.A. and Takeda, M. (2001) Death by influenza virus protein. Nature Medicine, 7, 1286-1288.
Zell, R., Krumbholz, A., Eitner, A., Krieg, R., Halbhuber, K.J. and Wutzler, P. (2007) Prevalence of PB1-F2 of influenza A viruses. Journal of General Virology, 88, 536- 546.
McAuley, J.L., Zhang, K. and McCullers, J.A. (2010) The effects of influenza A virus PB1-F2 protein on polymerase activity are strain specific and do not impact pathogenesis. Journal of Virology, 84(1), 558-564.
Ramakrishnan, M.A., Gramer, M.R., Goyal, S.M. and Sreevatsan, S. (2009) A Serine12Stop mutation in PB1- F2 of the 2009 pandemic (H1N1) influenza A: A possible reason for its enhanced transmission and pathogenicity to humans. Journal of Veterinary Science, 10(4), 349-351.
Trifonov, V. and Rabadan, R. (2009) The contribution of the pb1-f2 protein to the fitness of influenza a viruses and its recent evolution in the 2009 influenza A (H1N1) pandemic virus. PLoS Current: Influenza, 21, RRN1006.
Hale, B.G., Randall, R.E., Ortín, J. and Jackson, D. (2008) The multifunctional NS1 protein of influenza A viruses, Journal of General Virology, 89, 2359-2376.
Zhang, C.F., Yang, Y.T., Zhou, X.W., Liu, X.L., Song, H.B., He, Y.X. and Huang, P.T. (2010). Highly pathogenic avian influenza A virus H5N1 NS1 protein induces caspase-dependent apoptosis in human alveolar basal epithelial cells. Virology Journal, 7, 51.
Jackson, D., Hossain, M.J., Hickman, D., Perez, D.R. and Lamb, R.A. (2008) A new infl uenza virus virulence determinant: The NS1 protein four C-terminal residues modulate pathogenicity. Proceedings of the National Academy of Sciences of the United States of America, 105, 4381-4386.
Seo, S.H., Hoffmann, E. and Webster, R.G. (2002) Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nature Medicine, 8, 950-954.
Salomon, R., Franks, J., Govorkova, E.A., Ilyushina, N.A., Yen, H.L., Hulse-Post, D.J., Humberd, J., Trichet, M., Rehg, J.E., Webby, R.J., Webster, R.G. and Hoffmann, E. (2006) The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04, Journal of Experimental Medicine, 203(3), 689-697.
Furuse, Y., Suzuki, A., Kamigaki, T. and Oshitani, H. (2009) Evolution of the M gene of the influenza A virus in different host species: Large-scale sequence analysis. Virology Journal, 6, 67.
Furuse, Y., Suzuki A. and Oshitani, H. (2009) Large- scale sequence analysis of M gene of influenza A viruses from different species: Mechanisms for emergence and spread of amantadine resistance. Antimicrobial Agents and Chemotherapy, 53(10), 4457-4463.
Chen, G.W., Chang, S.C., Mok, C.K., Lo, Y.L., Kung, Y.N., et al. (2006) Genomic signatures of human versus avian influenza A viruses. Emerging Infectious Diseases, 12, 1353-1360.
Chen, G.W. and Shih, S.R. (2009) Genomic signatures of influenza A pandemic (H1N1) 2009, Virus. Emerging Infectious Diseases, 15, 1897-1903.
Pan, C., Cheung, B., Tan, S., Li, C., Li, L., et al. (2010) Genomic signature and mutation trend analysis of pandemic (H1N1) 2009, Influenza A virus. PLoS One, 5(3), e9549.
Miotto, O., Heiny, A., Tan, T.W., August, J.T., Brusic, V. (2008) Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis. BMC Bioinformatics, 9, S18.
Miotto, O., Heiny, A.T., Albrecht, R., García-Sastre, A., Tan, T.W., August, J.T. and Brusic, V. (2010) Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains. PLoS One, 5(2), e9025.
Finkelstein, D.B., Mukatira, S., Mehta, P.K., Obenauer, J.C., Su, X., Webster, R.G. and Naeve, C.W. (2007) Persistent host markers in pandemic and H5N1 influenza viruses. Journal of Virology, 81(19), 10292-10299.
Katoh, K., Kuma, K., Toh, H. and Miyata, T. (2005) MAFFT version 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Research, 33, 511- 518.
Breiman, L. (2001) Random Forests. Machine Learning, 45(1), 5-32.
Díaz-Uriarte, R. and Alvarez de Andrés, S. (2006) Gene selection and classification of microarray data using random forest. BMC Bioinformatics, 7, 3.
Archer, K.J. and Kimes, R.V. (2008) Empirical characterization of random forest variable importance measures. Computational Statistics and Data Analysis, 52, 2249- 2260.
Reif, D.M. Motsinger, A.A., McKinney, B.A., Crowe, J.E. and Moore, J.H. (2006) Feature selection using a random forests classifier for the integrated analysis of multiple data types. Proceedings of 2006 IEEE Symposium on Computational Intelligence and Bioinformatics and Computational Biology, Toronto.
Granittoa, P.M., Furlanellob, C., Biasiolia, F. and Gasperia, F. (2006) Recursive feature elimination with random forest for PTR-MS analysis of agroindustrial products. Chemometrics and Intelligent Laboratory Systems, 83, 83-90.
Menze1, B.H., Kelm, B.M., Masuch, R., Himmelreich, U., Bachert, P., Petrich, W. and Hamprecht, F.A. (2009) A comparison of random forest and its Gini importance with standard chemometric methods for the feature selection and classification of spectral data. BMC Bioinformatics, 10, 213.
Gao, D., Zhang, Y.X. and Zhao, Y.H. (2009) Random forest algorithm for classification of multi-wavelength data. Research in Astronomy and Astrophysics, 9(2), 220-226.
Hu, W. (2009) Identifying predictive markers of chemosensitivity of breast cancer with random forests. Journal of Biomedical Science and Engineering, 3(1), 59-64.
KováccaronOVá, A., Ruttkay-Nedecký, G., Karol HaverlíK1, I. and Janecccaronek, S. (2002) Sequence similarities and evolutionary relationships of influenza virus A hemagglutinins. Virus Genes, 24, 57-63.
Colman, P.M., Hoyne, P.A. and Lawrence, M.C. (1993) Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase. Journal of Virology, 67, 2972-2980.
Maurer-Stroh, S. Ma, J.M., Lee, R.T.C., Sirota, F.L. and Eisenhaber, F. (2009) Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites. Biology Direct, 4, 18.
Liu, T. and Ye, Z.P. (2005) Attenuating mutations of the matrix gene of influenza A/WSN/33 Virus. Journal of Virology, 79(3), 1918-1923.
Baudin, F., Petit, I., Weissenhorn, W. and Ruigrok, R.W.H. (2001) In vitro dissection of the membrane binding and RNP binding activities of influenza virus M1 protein. Virology, 281, 102-108.
Dua, Q.S., Wang, S.Q., Huang, R.B. and Chou, K.C. (2010) Computational 3D structures of drug-targeting proteins in the 2009-H1N1 influenza A virus. Chemical Physics Letters, 485, 191-195.
Ye Q., Krug R.M. and Tao Y.J. (2006) The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature, 444, 1078-1082.
Biswas, S.K., Boutz, P.L. and Nayak, D.P. (1998) Influenza virus nucleoprotein interacts with influenza virus polymerase proteins. Journal of Virology, 72, 5493-5501.
Lin, D., Lan, J. and Zhang, Z. (2007) Structure and function of the NS1 protein of influenza A virus. Acta Biochim Biophys Sin (Shanghai), 39(3), 155-162.
Robb, N.C., Smith, M., Vreede, F.T. and Fodor, E. (2009) NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. Journal of General Virology, 90, 1398-1407.
Iwatsuki-Horimoto, K., Horimoto, T., Fujii, Y. and Kawa- oka, Y. (2004) Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence. Journal of Virology, 78(18), 10149-10155.
Yuan, P.W., Bartlam, M., Lou, Z.Y., Chen, S.D., Zhou, J., He, X.J., Lv, Z.Y., Ge, R.W., Li, X.M., Deng, T., Fodor, E., Rao, Z.H. and Liu, Y.F. (2009) Crystal structure of an avian influenza polymerase PAN reveals an endonuclease active site. Nature, 458, 909-913.
Biswas, S.K. and Nayak, D.P. (1994) Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. Journal of Virology, 68, 1819-1826.
Ohtsu, Y., Honda, Y., Sakata, Y., Kato, H. and Toyoda, T. (2002) Fine mapping of the subunit binding sites of influenza virus RNA polymerase. Microbiology and Immunology, 46, 167-175.
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.