A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature.
References
[1]
Gubler DJ (2007) The continuing spread of West Nile virus in the western hemisphere. Clin Infect Dis 45: 1039–1046. doi: 10.1086/521911
[2]
Murata R, Hashiguchi K, Yoshii K, Kariwa H, Nakajima K, et al. (2011) Seroprevalence of West Nile virus in wild birds in far eastern Russia using a focus reduction neutralization test. Am J Trop Med Hyg 84: 461–465. doi: 10.4269/ajtmh.2011.09-0714
[3]
Rossini G, Carletti F, Bordi L, Cavrini F, Gaibani P, et al. (2011) Phylogenetic analysis of west nile virus isolates, Italy, 2008–2009. Emerg Infect Dis 17: 903–906. doi: 10.3201/eid1705.101569
[4]
Lupulovic D, Martin-Acebes MA, Lazic S, Alonso-Padilla J, Blazquez AB, et al.. (2011) First Serological Evidence of West Nile Virus Activity in Horses in Serbia. Vector Borne Zoonotic Dis.
[5]
Barrera R, MacKay A, Amador M, Vasquez J, Smith J, et al. (2010) Mosquito vectors of West Nile virus during an epizootic outbreak in Puerto Rico. J Med Entomol 47: 1185–1195. doi: 10.1603/me10038
[6]
Venter M, Swanepoel R (2010) West Nile virus lineage 2 as a cause of zoonotic neurological disease in humans and horses in southern Africa. Vector Borne Zoonotic Dis 10: 659–664. doi: 10.1089/vbz.2009.0230
[7]
Van Den Hurk AF, Craig SB, Tulsiani SM, Jansen CC (2010) Emerging tropical diseases in Australia. Part 4. Mosquitoborne diseases. Ann Trop Med Parasitol 104: 623–640. doi: 10.1179/136485910x12851868779984
[8]
Khan SA, Dutta P, Khan AM, Chowdhury P, Borah J, et al. (2011) West nile virus infection, assam, India. Emerg Infect Dis 17: 947–948. doi: 10.3201/eid1705.100479
[9]
McLean RG, Ubico SR, Docherty DE, Hansen WR, Sileo L, et al. (2001) West Nile virus transmission and ecology in birds. Ann N Y Acad Sci 951: 54–57. doi: 10.1111/j.1749-6632.2001.tb02684.x
[10]
Steele KE, Linn MJ, Schoepp RJ, Komar N, Geisbert TW, et al. (2000) Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet Pathol 37: 208–224. doi: 10.1354/vp.37-3-208
[11]
Nemeth NM, Beckett S, Edwards E, Klenk K, Komar N (2007) Avian mortality surveillance for West Nile virus in Colorado. Am J Trop Med Hyg 76: 431–437.
[12]
Eidson M, Kramer L, Stone W, Hagiwara Y, Schmit K (2001) Dead bird surveillance as an early warning system for West Nile virus. Emerg Infect Dis 7: 631–635. doi: 10.3201/eid0704.017405
[13]
Davis CT, Ebel GD, Lanciotti RS, Brault AC, Guzman H, et al. (2005) Phylogenetic analysis of North American West Nile virus isolates, 2001–2004: Evidence for the emergence of a dominant genotype. Virology 342: 252–265. doi: 10.1016/j.virol.2005.07.022
[14]
Foppa IM, Beard RH, Mendenhall IH (2011) The impact of West Nile virus on the abundance of selected North American birds. BMC Vet Res 7: 43. doi: 10.1186/1746-6148-7-43
[15]
Ladeau SL, Kilpatrick AM, Marra PP (2007) West Nile virus emergence and large-scale declines of North American bird populations. Nature.
[16]
Ward MP, Beveroth TA, Lampman R, Raim A, Enstrom D, et al.. (2010) Field-Based Estimates of Avian Mortality from West Nile Virus Infection. Vector Borne Zoonotic Dis.
[17]
Wheeler SS, Barker CM, Fang Y, Armijos MV, Carroll BD, et al. (2009) Differential Impact of West Nile Virus on California Birds. Condor 111: 1–20. doi: 10.1525/cond.2009.080013
[18]
Jerzak GV, Brown I, Shi PY, Kramer LD, Ebel GD (2008) Genetic diversity and purifying selection in West Nile virus populations are maintained during host switching. Virology 374: 256–260. doi: 10.1016/j.virol.2008.02.032
[19]
Brault AC, Huang CY, Langevin SA, Kinney RM, Bowen RA, et al. (2007) A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet 39: 1162–1166. doi: 10.1038/ng2097
[20]
Botha EM, Markotter W, Wolfaardt M, Paweska JT, Swanepoel R, et al. (2008) Genetic determinants of virulence in pathogenic lineage 2 west nile virus strains. Emerg Infect Dis 14: 222–230. doi: 10.3201/eid1402.070457
[21]
Bakonyi T, Hubalek Z, Rudolf I, Nowotny N (2005) Novel flavivirus or new lineage of West Nile virus, central Europe. Emerg Infect Dis 11: 225–231. doi: 10.3201/eid1102.041028
[22]
Charrel RN, Brault AC, Gallian P, Lemasson JJ, Murgue B, et al. (2003) Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe. Virology 315: 381–388. doi: 10.1016/s0042-6822(03)00536-1
[23]
Papa A, Bakonyi T, Xanthopoulou K, Vazquez A, Tenorio A, et al. (2011) Genetic characterization of West Nile virus lineage 2, Greece, 2010. Emerg Infect Dis 17: 920–922. doi: 10.3201/eid1705.101759
[24]
Bakonyi T, Ivanics E, Erdelyi K, Ursu K, Ferenczi E, et al. (2006) Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerg Infect Dis 12: 618–623. doi: 10.3201/eid1204.051379
[25]
Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff BJ, et al. (2004) Differential virulence of West Nile strains for American crows. Emerg Infect Dis 10: 2161–2168. doi: 10.3201/eid1012.040486
[26]
Kinney RM, Huang CY, Whiteman MC, Bowen RA, Langevin SA, et al. (2006) Avian virulence and thermostable replication of the North American strain of West Nile virus. J Gen Virol 87: 3611–3622. doi: 10.1099/vir.0.82299-0
[27]
Luo D, Xu T, Watson RP, Scherer-Becker D, Sampath A, et al. (2008) Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein. Embo J. doi: 10.1038/emboj.2008.232
[28]
Ebel GD, Carricaburu J, Young D, Bernard KA, Kramer LD (2004) Genetic and phenotypic variation of West Nile virus in New York, 2000-2003. Am J Trop Med Hyg 71: 493–500.
[29]
Koo QY, Khan AM, Jung KO, Ramdas S, Miotto O, et al. (2009) Conservation and variability of West Nile virus proteins. PLoS ONE 4: e5352. doi: 10.1371/journal.pone.0005352
[30]
Nemeth NM, Thomsen BV, Spraker TR, Benson JM, Bosco-Lauth AM, et al. (2011) Clinical and Pathologic Responses of American Crows (Corvus Brachyrhynchos) and Fish Crows (C ossifragus) to Experimental West Nile Virus Infection. Vet Pathol. doi: 10.1177/0300985811398249
[31]
Langevin SA, Brault AC, Panella NA, Bowen RA, Komar N (2005) Variation in virulence of West Nile virus strains for house sparrows (Passer domesticus). Am J Trop Med Hyg 72: 99–102.
[32]
Murgue B, Murri S, Zientara S, Durand B, Durand JP, et al. (2001) West Nile outbreak in horses in southern France, 2000: the return after 35 years. Emerg Infect Dis 7: 692–696. doi: 10.3201/eid0704.010417
[33]
Misra M, Schein CH (2007) Flavitrack: an annotated database of flavivirus sequences. Bioinformatics 23: 2645–2647. doi: 10.1093/bioinformatics/btm383
[34]
Zimmerman E (2009) House sparrow history. Sialis.
[35]
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539. doi: 10.1038/msb.2011.75
[36]
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9: 772. doi: 10.1038/nmeth.2109
[37]
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704. doi: 10.1080/10635150390235520
[38]
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755. doi: 10.1093/bioinformatics/17.8.754
[39]
Milne I, Wright F, Rowe G, Marshall DF, Husmeier D, et al. (2004) TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics 20: 1806–1807. doi: 10.1093/bioinformatics/bth155
[40]
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, et al. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59: 307–321. doi: 10.1093/sysbio/syq010
[41]
Kosakovsky Pond SL, Posada D, Gravenor MB, Woelk CH, Frost SD (2006) Automated phylogenetic detection of recombination using a genetic algorithm. Mol Biol Evol 23: 1891–1901. doi: 10.1093/molbev/msl051
[42]
Kinney R, Huang Y-H, Whitman MC, Bowen RA, Langevin SA, et al. (2006) Avian virulence and thermostable replication of the North American strain of West Nile virus. Journal of General Virology in press. doi: 10.1099/vir.0.82299-0
[43]
Andrade CC, Maharaj PD, Reisen WK, Brault AC (2011) North American West Nile virus genotype isolates demonstrate differential replicative capacities in response to temperature. Journal of General Virology 92: 2523–2533. doi: 10.1099/vir.0.032318-0
[44]
Deardorff E, Estrada-Franco J, Brault AC, Navarro-Lopez R, Campomanes-Cortes A, et al. (2006) Introductions of West Nile virus strains to Mexico. Emerg Infect Dis 12: 314–318. doi: 10.3201/eid1202.050871
[45]
Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, et al. (1999) Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286: 2333–2337. doi: 10.1126/science.286.5448.2333
[46]
Huang CY, Butrapet S, Pierro DJ, Chang GJ, Hunt AR, et al. (2000) Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 74: 3020–3028. doi: 10.1128/jvi.74.7.3020-3028.2000
[47]
Beasley DW, Li L, Suderman MT, Barrett AD (2002) Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 296: 17–23. doi: 10.1006/viro.2002.1372
[48]
Bera AK, Kuhn RJ, Smith JL (2007) Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease. J Biol Chem 282: 12883–12892. doi: 10.1074/jbc.m611318200
[49]
Wu J, Bera AK, Kuhn RJ, Smith JL (2005) Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing. J Virol 79: 10268–10277. doi: 10.1128/jvi.79.16.10268-10277.2005
