Title page for ETD etd-02192009-165513


Document Type Master's Dissertation
Author Chitray, Melanie
Email chitraym@arc.agric.za
URN etd-02192009-165513
Document Title Investigating potential factors affecting foot-and-mouth disease virus internalisation
Degree Msc
Department Veterinary Tropical Diseases
Supervisor
Advisor Name Title
Dr F F Maree Co-Supervisor
Dr W Vosloo Supervisor
Keywords
  • FMD virus
  • Picomaviridae family
  • foot-and-mouth disease
Date 2008-11-28
Availability unrestricted
Abstract

Foot-and-mouth disease (FMD) is a highly contagious disease caused by the FMD virus (FMDV) belonging to the Picornaviridae family. The virus affects cloven-hoofed animals and occurs as seven immunologically distinct serotypes where six of the seven serotypes occur in Africa. This fact, as well as the role of wildlife in virus maintenance, makes eradication and control of FMDV in Africa difficult. Thus, it is imperative to attain more information regarding the genetic diversity of FMD viruses prevalent on the African continent to further our knowledge of the virus as well as to enable better control strategies and the development of improved vaccines. Sufficient genetic information regarding the Leader (L) and complete capsid-coding, P1, region of serotype A and O viruses prevalent on the African continent is lacking, although the SAT isolates have been extensively characterised in the past.

In this study the sequence of the L/P1-coding region was successfully determined using a genome-walking approach for a small number of A and O viruses recovered from outbreaks isolated from various species in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions 1A, 1B, 1C and 1D revealed that the African isolates grouped strictly according to serotype and geographic region which indicated the possibility of transboundary spread of the virus within East and West African countries respectively. In contrast, phylogenetic analysis of the non-structural, Lpro-coding region revealed a different tree topology compared to the capsid-coding regions for the A and O isolates with sub-grouping according to serotype and geographic regions was less apparent. The relatedness between the serotype A and O L region might be the result of genetic recombination.

The inter and intratypic nucleotide and amino acid variation of the A and O isolates revealed that the most variable capsid-coding region was the externally located VP1 whilst the internally located VP4 capsid protein was the most conserved. The observed variation is in agreement with other studies and reflects the selective pressures on these proteins which either allow or prevent the occurrence of genetic changes for structural constraints or immune escape. Surprisingly, the L protease-encoding region also displayed a high degree of variation. A detailed analysis of the L/P1 amino acid alignment of the A and O isolates revealed that although the extent of variation is high in these regions, the amino acids identified in previous studies as important for FMDV structure (for the capsid-coding regions) and function were found to be conserved, indicating that the virus has adapted itself to elude the host immune response without affecting its vital functional and structural abilities. Additionally, it was observed that the amino acid residues identified as being important for FMDV attaching to the host cell receptors e.g. the RGD amino acid motif of VP1 was highly conserved for all isolates.

To further investigate the FMDV-receptor interaction, RT-PCRs were developed to examine the mRNA expression level of the known FMDV receptors. The ‚ integrins that facilitate FMDV cell entry i.e. ‚1, ‚3, ‚6, ‚8 and heparan sulphate proteoglycans (HSPG) were investigated in susceptible cell lines used for FMDV vaccine production i.e. IB-RS-2 and BHK-21. The RT-PCRs were successfully developed and optimised. The results showed that the mRNA expression levels were variable for all receptor cDNAs tested across 36 passage levels of IB-RS-2 and BHK-21 cells. No distinct differences in virus susceptibility for three FMDV strains with continuous cell passage of IB-RS-2 and BHK-21 cells at passage levels 5, 21 and 36 could be found.

The information gained from this study regarding the viral L and P1 region genetic diversity, and phylogenetic analysis has indeed impacted on our understanding of FMDV African viruses. Additionally, the investigation of the FMDV receptor mRNA expression levels and virus susceptibility on two cell lines with continuous cell passage has proved a vital starting point to determine the possible receptors expressed on the surface of cells used by the vaccine production division at the ARC-OVI-TADP and forms the basis for further investigations of the FMDV receptors on the protein level and the development of a real-time RT-PCR for FMDV receptor expression.

©University of Pretoria 2008

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