Location: Foreign Animal Disease Research2011 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.
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
1. The construction of a chimeric virus based on FMDV A2001 clone containing 2C region of FMDV A2001. Ten clones were obtained and one completely sequenced. RNA was prepared and in vivo mice studies were conducted. FMDV A2001 infected mice perished at 24 hours, however FMDV A2001 clone infected animals survived. Both however, showed similar growth curves in cell culture, 2. Studies were done on the involvement of internal ribosome entry site (IRES) region to determine the virulence in a viral model in which a chimeric FMDV A2001 was obtained by replacement of its counterpart in FMDV A2000. The infectious virus production, viral RNA and viral protein synthesis were evaluated. Results showed that IRES play an important role in determining the differential virulence between both isolates; however the differences are observed in the viral context of the full-length RNA and do not depend only on the IRES sequence. This project was monitored through email and telephone exchange.