Progress 07/01/04 to 06/30/09
Outputs
OUTPUTS: Identify WN viral genetic determinants responsible for differential crow virulence. Elucidate differences in avian pathogenesis among WN viruses Assess the role of viral adaptation in avians and gauge the potential for the emergence of epizootic strains from alternative WNV genotypes. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Synergistic interaction between the NS1-2B and the NS3-249P of the NY99 genotype results in complete avian virulence in the Kenyan genetic backbone. Positive selection models indicate the single NS3-249 genetic loci to be under strong selective pressure. This same residue (Thr-Pro) has been involved in at least three emergence events of West Nile virus. The single NS3-249P mutation is the dominant genetic substitution associated with increased virulence of the WNV strain introduced into North America. Variation in avian virulence exists depending upon the amino acid residue at NS3-249. (Pro>Asp>His>Ala>Thr). No significant increase in helicase or ATPase activity was identified for the NS3-249Pro over Ala, Thr, His or Asp acid mutants. Transcriptional inhibition of the KEN strain in duck embryonic fibroblast cells (DEF) incubated at 44C limits viral production and cytopathic effect. Genetic residues within the NS1-2B gene region modulate the resistance to transcriptional inhibition of the NY99 virus as well as serve as accessory modulating determinants for avian virulence. Leukocytes appear to be the primary site for viral replication for West Nile viruses in AMCRs. Differential replication of the NY99 and KEN WNV strains are responsible for the large disparity in circulating titers in AMCRs following infection. The NS3-249P substitution is not sufficient to imbibe an avian virulent phenotype in the KUN (MRM-61C) genetic backbone. Rapid selection for virulence of a lineage II West Nile virus strain from South Africa further indicates the importance of corvids for the emergence of epiornitic genotypes and the capacity of these viruses for adaptation to new niches. NS1 and NS3 mutations appear tom modulate increased replication of a lineage II WNV in AMCRs.
Publications
- Brault, Aaron C. 2009. Changing Patterns of West Nile Virus Transmission: altered Vector Competence and Host Susceptibility in Adaptive strategies of vector-borne pathogens to vectorial transmission, Bruno Chomel and Joelle Charley-Poulain ed. Veterinary Research 40:43.
- Brault, Aaron C., Huang, C.Y.-H., Langevin, Stan A., Kinney, Richard M., Bowen, R.A., Ramey, W.N., Panella, Nicholas A., Holmes, Edward C., Powers, Ann M., Miller and Barry R. 2007. A single positively selected helicase mutation confers increased avian virogenesis in American crows. Nature Genetics 39 1162-1166.
- Kinney, Richard M., Huang, Claire Y.H, Whitman, Melissa, Bowen, Richard A., Langevin, Stanley A., Miller, Barry R. and Brault, Aaron C. 2006. Avian virulence and thermostable replication of the North American strain of West Nile virus. Journal of General Virology 87 (12) 3611-3622.
- Langevin, Stanley A., Brault, Aaron C., Panella, Nicholas A., Bowen, Richard A., and Komar, Nicholas 2005. Kunjin and West Nile virus strains vary in virulence for house sparrows (Passer domesticus) The American Journal of Tropical Medicine and Hygiene 72(1) 99-102.
- Brault, Aaron C., Langevin, Stanley A., Bowen, Panella, Nicholas A., Richard A., Biggerstaff, Brad J., Miller, Barry R. and Komar, Nicholas 2004. Differential virulence of West Nile viral strains for American crows (Corvus brachyrhynchos). Emerging Infectious Diseases 10(12) 2161-2168.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Specific aims: We have made extensive progress in year three of the funded project AI061822 entitled "West Nile Viral Determinants of Avian Pathogenesis" in all three of the outlined specific aims: (1) Identify WN viral genetic determinants responsible for differential crow virulence. (2) Elucidate differences in avian pathogenesis among WN viruses (3) Assess the role of viral adaptation in avians and gauge the potential for the emergence of epizootic strains from alternative WNV genotypes. The aims have not been modified. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
AIM 1-Identify WN viral genetic determinants responsible for differential crow virulence. In our previous report, we had generated fourteen chimeric viruses between the avian virulent NY99 West Nile viral (WNV) genome and the avian avirulent Kenya-3829 (KEN) strain. Viruses rescued from all fourteen of these constructs have been sequenced in their entirety to confirm their genetic identity (end of year 1). In addition to these constructs, in year two of this project we completed a full-length infectious cDNA clone of the avian avirulent WNV strain in which we have further identified genetic determinants associated with virulence in American crow (AMCRs). Results of these experiments have demonstrated the role of the nonstructural genetic elements in the pathogenesis phenotype. Recombinant viruses generated with alternate nonstructural gene elements in which the 3'UTR were exchanged between the two viruses failed to demonstrate alterations in the parental virulence phenotype. These data indicated the role of one or a combination of the seven nonstructural non-synonymous substitutions. We were capable of generating an intermediate virulence phenotype when we incorporated three amino acid substitutions from the NS1-2B (one substitution from each gene) of NY99 virus into the Kenyan infectious cDNA. We have further demonstrated that incorporation of a single amino acid substitution (Thr→Pro) within the NS3 gene within the viral helicase domain at amino acid position 249 of the Kenyan WNV strain increased the virulence of this recombinant virus from 30 to 95%. Interestingly, the survival time as well as the viremia magnitude were prolonged and suppressed respectively as compared to the wild type NY99 genotype. When the NS3-Pro substitution was incorporated into the Kenyan infectious cDNA clone in addition to three amino acid substitutions from the NS1-2B, a fully virulent phenotype was achieved. This fact indicates that modulating substitutions within the NS1-2B act synergistically with the NS3-Pro to generate an avian virulent phenotype. In year three of funding, we have focused on assessing the role of the NS1-2B substitutions on AMCR virulence. This has been performed through the insertion of individual point mutations individually as well as in combination in a KEN-IC backbone as well as a KEN-IC backbone in which we have incorporated the NS3-249P substitution. The relative mortality rates of chimeras versus the derived peak viremias induced in AMCRs were positively correlated Inclusion of the Pro NS3-249 substitution into the Kenyan infectious cDNA clone failed to increase the thermostability of the Kenyan construct. As was previously described for the avian virulence studies, the constructs that are currently being generated for identification of the NS3-Pro synergism will be included in the temperature sensitivity studies. We have also generated a corvid cell line from a yellow-billed magpie (kidney fibroblast) that we intend on using for temperature sensitivity studies in addition to the DEF cells.
Publications
- Brault, A. C., S. A. Langevin, R. A. Bowen, N. A. Panella, B. J. Biggerstaff, B. R. Miller, and K. Nicholas. 2004. Differential virulence of West Nile strains for American crows. Emerg Infect Dis 10:2161-8.
- Langevin, S. A., A. C. Brault, N. A. Panella, R. A. Bowen, and N. Komar. 2005. Variation in virulence of West Nile virus strains for house sparrows (Passer domesticus). Am J Trop Med Hyg 72:99-102.
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Progress 05/31/07 to 12/31/07
Outputs
In our previous report, we had generated fourteen chimeric viruses between the avian virulent NY99 West Nile viral (WNV) genome and the avian avirulent Kenya-3829 (KEN) strain. Viruses rescued from all fourteen of these constructs have been sequenced in their entirety to confirm their genetic identity (end of year 1). In addition to these constructs, in year two of this project we completed a full-length infectious cDNA clone of the avian avirulent WNV strain in which we have further identified genetic determinants associated with virulence in American crow (AMCRs). Results of these experiments have demonstrated the role of the nonstructural genetic elements in the pathogenesis phenotype. Recombinant viruses generated with alternate nonstructural gene elements in which the 3'UTR were exchanged between the two viruses failed to demonstrate alterations in the parental virulence phenotype. These data indicated the role of one or a combination of the seven nonstructural
non-synonymous substitutions. We were capable of generating an intermediate virulence phenotype when we incorporated three amino acid substitutions from the NS1-2B (one substitution from each gene) of NY99 virus into the Kenyan infectious cDNA. We have further demonstrated that incorporation of a single amino acid substitution (Thr to Pro) within the NS3 gene within the viral helicase domain at amino acid position 249 of the Kenyan WNV strain increased the virulence of this recombinant virus from 30 to 95%. Interestingly, the survival time as well as the viremia magnitude were prolonged and suppressed respectively as compared to the wild type NY99 genotype. When the NS3-Pro substitution was incorporated into the Kenyan infectious cDNA clone in addition to three amino acid substitutions from the NS1-2B, a fully virulent phenotype was achieved. This fact indicates that modulating substitutions within the NS1-2B act synergistically with the NS3-Pro to generate an avian virulent
phenotype. In year three of funding, we have focused on assessing the role of the NS1-2B substitutions on AMCR virulence. This has been performed through the insertion of individual point mutations individually as well as in combination in a KEN-IC backbone as well as a KEN-IC backbone in which we have incorporated the NS3-249P substitution. The relative mortality rates of chimeras versus the derived peak viremias induced in AMCRs were positively correlated.
Impacts
Since its introduction in 1999, West Nile virus (WNV) has become the leading cause of arboviral encephalitis in the United States. The most notable epidemiological factors associated with the rapid emergence of WNV in North America have been the development of high viral titers within birds and mortality among wild bird populations. Despite the use of crow mortality as a sentinel for WNV activity, the underlying viral genetic basis for WNV pathogenicity in birds and the role of increased avian virulence in the transmission of WNV are poorly understood. Generation of fundamental data on the basis of replication within the WNV avian reservoir will fill a critical gap in our knowledge of WNV transmission and aid in the improvement of existing surveillance strategies and predictive emergence models for the prevention of human and veterinary disease. Our data thus far indicated that monitoring of the genetic polymorphisms found at the NS3-249 position could be used to assess
the risk of the introduction of viruses with alterative avian virulence phenotypes that could be key indicators of rapidly disseminating disease agents. Furthermore, we are investigating the cellular and virological factors associated with early replication in the birds and the factors associated with increased replication that appears to be involved in increased virulence as well as more efficient transmission to humans.
Publications
- Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff BJ, Miller BR, Komar N, 2004. Differential virulence of West Nile strains for American crows. Emerg Infect Dis 10: 2161-8.
- Langevin, S. A., A. C. Brault, N. A. Panella, R. A. Bowen, and N. Komar. 2005. Variation in virulence of West Nile virus strains for house sparrows (Passer domesticus). Am J Trop Med Hyg 72:99-102.
- Kinney, Richard M., Huang, Claire Y.H, Whitman, Melissa, Bowen, Richard A., Langevin, Stanley A., Miller, Barry R. and Brault, Aaron C. 2006. Avian virulence and thermostable replication of the North American strain of West Nile virus. Journal of General Virology 87 (12) 3611-3622.
- Brault AC, Huang CY, Langevin SA, Kinney RM, Bowen RA, Ramey WN, Panella NA, Holmes EC, Powers AM, Miller BR, 2007. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet 39: 1162-1166.
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