Location: Foreign Animal Disease Research2012 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:
Previously studies at INTA provided the nucleotide sequence of the foot-and-mouth disease virus (FMDV) outbreak prototype strain A/Arg/2001. An infectious clone of this virus was derived; the so-called A2001 clone. Comparison studies showed seven amino acid changes in the between the parental A2001 and A2001 clone FMDVs. INTA further demonstrated that FMDV A/Arg/2001 was more pathogenic than A2001 clone in mice; however, 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. During FY 2012, studies continued on the role of the internal ribosome entry site (IRES) region of FMDV A/Arg/2001 and FMDV A/Arg/2000. Using the chimeric ARN genome obtained during the past period, we performed in vitro experiments assessing the viral translation efficiency. The results obtained allowed us to compare and study the role of the 3’ untranslated region (UTR) in IRES function. Thus, viral genomes lacking the 3’UTR from both parental and clone sequence were prepared. We observed that the difference in IRES function was only displayed when the whole genome was available. Then, bicistronic plasmids were constructed containing both IRES and both 3’UTR, getting all possible combinations and IRES activity was studied in baby hamster kidney (BHK)-21 cells through the reporter genes CAT and luciferase expression analysis. Similar results were obtained. In addition, we demonstrated that FMDV IRES enhancing is independent of 3’UTR identity. Results obtained by in silico analysis predicted different conformational structures between the apical region of the 3rd domain of the FMDV IRES A2000 and A2001. Chimeric virus FMDVA2001/FMDV 2C was obtained by transfection of BHK-21 with in vitro transcribed RNA. Virus identity was corroborated by nucleotide sequencing. Animal experiments are being conducted in order to determine if the aminoacid changes in 2C coding region of A2001clone are responsible for the differences in mice lethality. We have also performed the cloning of the different regions in intermediate vectors. No technologies have been transferred do date. Publications for this reporting period include; Meeting abstract: “IRES element as a virulence determinant of two serotype A FMDV field isolates X Argentinean Congress of Virology. September 26-29, 2011. Argentinean Society of Virology. PhD thesis: “Bases moleculares de la patogenia del Virus de la Fiebre Aftosa (FMDV molecular basis of pathogenesis)”. 2011. Soledad García Núñez. University of Buenos Aires.