Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

Abstract

Background: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV.

Methods: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice.

Results: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain.

Conclusions: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.

Figure 1

Reactivity of Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06 with MAb 1B4 in an indirect immunofluorescence assay. (A) Asia1/1/YZ/CHA/06-infected BHK-21 cells, (B) Asia1/YS/CHA/05-infected BHK-21 cells and (C) normal BHK-21 cells as a negative control.

Figure 2

Comparison of the VP2 amino acid sequence between Asia1 FMDV strains. The amino acids at position 72 are boxed.

Figure 3

Reactivity of the mutant viruses with MAb 4B2 by indirect immunofluorescence. (A) rS154N-infected BHK-21 cells, (B) rD72G-infected BHK-21 cells, (C) rD72N-infected BHK-21 cells, (D) rV107I-infected BHK-21 cells, (E) Asia1/YS/CHA/05-infected BHK-21 cells as a positive control and (F) normal BHK-21 cells as a negative control.

Figure 4

Reactivity of mutant viruses with MAb 1B4 by indirect immunofluorescence. (A) rS154N-infected BHK-21 cells, (B) rD72G-infected BHK-21 cells, (C) rD72N-infected BHK-21 cells, (D) rV107I-infected BHK-21 cells, (E) Asia1/YS/CHA/05-infected BHK-21 cells as a positive control and (F) normal BHK-21 cells as a negative control.

Figure 5

Growth kinetics of rD72G and rD72N mutant viruses. BHK-21 cells were infected and harvested at different times post infection. (A) Single-step growth curves obtained by titration of virus present at each time. Samples were taken in triplicates and standard deviations are shown in the graphic. (B) RNA production curves were obtained by real time RT-PCR assay from Trizol resuspended monolayers. The mean viral load generated by real time RT-PCR is expressed as log₁₀ vRNA copies/ml of virus transport medium. Samples were taken in triplicates and standard deviations are shown in the graphic. (◆) FMDV Asia1/YS/CHA/05,(■) rD72G and (▴) rD72N.