Location: Foreign Animal Disease Research2013 Annual Report
1a. Objectives (from AD-416):
Collaborators at the INTA laboratory, Argentina, have previously conducted studies on the nucleotide sequence of the foot-and-mouth disease virus (FMDV) outbreak prototype strain A/Arg/2001 MC267 (VFA A2001). The collaborators have derived a molecular clone of this virus so-called A2001clon. Comparison studies showed 7 amino acid changes in the polyprotein (in viral proteins VP2, VP1, 2C and 3D) and two nucleotide differences in the internal ribosome entry site (IRES) regions between the parental A2001 and A2001clon FMDVs. INTA further demonstrated that VFA A2001 was more pathogenic than VFA A2001clone in mice, however these viruses shared similar growth properties in cell culture. The main objective of this collaborative research project is to describe and characterize new viral factors of pathogenicity and virulence of FMDV. Objectives: 1. Obtain chimeric viruses based on VFA A2001clon containing the different genetic changes identified in FMDV A2001. 2. Evaluate the involvement of various genomic regions on viral virulence and pathogenicity. 3. Determine the genetic diversity of both VFA A2001 and VFA A2001clon. 4. Increase the genetic diversity of FMDV A2001clon quasispecies by performing serial passages in mice. 5. Characterize the genetic diversity and viral pathogenicity of the virus population arisen after serial passages in mice. Amendement I: 6. Characterize the role of FMDV isolates in viral replication and interaction with the host.
1b. Approach (from AD-416):
To identify the differences in pathogenicity and virulence between VFA A2001 and VFA A2001clone, INTA will: 1. Construct four chimeric viruses; one in the internal ribosomal entry site, and the other in the viral proteins VP1, VP2, 2C, and 3D coding regions. 2. Determine virus plaque phenotypes, one-step growth curves in cell culture. Viral inoculation studies in mice will be performed to compare the pathogenicity and virulence among the chimeras and the original viruses. 3. Mutation frequency in both viral populations will be evaluated as a marker of genetic diversity. Nucleotide sequences of each virus will be obtained. 4. If the quasispecies complexity of VFA A2001clon is significantly lower, successive passages of the virus in suckling mice will be conducted. Analysis of the viruses that arise will show the relation between genetic diversity and pathogenicty of the isolates. Collaborators from INTA will perform all bench work and ARS, PIADC will review data and provide counseling/ technical expertise needed for the understanding of the molecular basis for virulence in the clone versus parental FMD viruses.
3. Progress Report:
Researchers at the INTA laboratory, Argentina, have previously conducted studies on the nucleotide sequence of the Foot-and-Mouth Disease Virus (FMDV) outbreak prototype strain A/Arg/2001 MC267 (FMDV A/Arg/01). Two derivatives of this virus were identified: A/Arg/01-L and A/Arg/01-NL. An infectious clone of A/Arg/01-NL was derived, named vA2001c. INTA further demonstrated that FMDV A/Arg/01-L was more pathogenic than A/Arg/01-NL and vA2001c in mice; however, in preliminary studies, viruses shared similar growth properties in cell culture. Previous studies compared the phenotypic and genotypic characteristics of A/Arg/01-NL with A/Arg/00. We observed that the difference in internal ribosome entry site (IRES) function was only observed when the 3’UTR was present. The main objective of this collaborative research project is to describe and characterize new viral factors of pathogenicity and virulence of FMDV. The phenotypic characteristics of the infectious chimeric virus Q-2C-L were assessed and compared with the phenotypic characteristics of A/Arg/01-L, A/Arg/01-NL and vA2001c. None of the viruses evaluated presented a growth curve significantly different from the other viruses. However, Q-2C-L and vA2001c seemed to display higher vRNA values at 2 hours post infection (hpi) when compared with the wild type viruses. In preliminary studies, we determined that Q-2C-L did not present an exacerbation of its pathogenicity in adult C57/Bl6 mice, suggesting that the amino acid changes present in A/Arg/01-L 2C protein would not be responsible for the higher pathogenicity of A/Arg/01-L. However, further experiments are required to determine Q-2C-L pathogenicity in mice. The region including the four amino acid changes present in A/Arg/01-L proteins VP1 and VP2 was cloned and several clones are currently being analyzed. To gain insight into the molecular basis of infectivity differences between FMDVs A/Arg/00 and A/Arg/01, the secondary structure of the IRES elements was analyzed. No technologies were tranferred or publications produced during FY 2013.