Susceptibility to foot and mouth disease virus infection in vaccinated cattle, and host BoLA A and BoLA DRB3 genes polymorphism

Abstract

The vaccination of the susceptible animal population against FMDV remains the most important measure to control the virus and prevent economic loss. Occurrence of infection in vaccinated animals is well-known in some diseases and is termed as breakthrough infection. The reasons include host genetic factors which can play an important role resulting in differences in susceptibility of animals to virus infection even with vaccine induced protective immune response. The Major Histocompatibility Complex (MHC) of bovines i.e. Bovine Leukocyte Antigen (BoLA) is important for antigen presentation. The BoLA DRB3 allele, which codes for the beta chain in Class II antigen, has been extensively studied and numerous reports have previously shown association of polymorphism in the gene with resistance/ susceptibility to several bacterial and viral diseases. In addition, previous studies have shown relationship between BoLA Class I and resistance or susceptibility to different diseases in cattle. The present study investigated the polymorphism in BoLA DRB3 and BoLA gene sequences of host and their relation with breakthrough FMDV infection in vaccinated animals. The study has identified three polymorphic sites each in both the genes which correlate with evidence of recent infection indicating their role in determining susceptibility of vaccinated animals to FMDV infection. Our limited study was performed on a relatively small samples size collected from one region of country. Further validation would require more detailed investigations on larger sample size.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13337-021-00754-8.

Introduction

Foot and Mouth Disease (FMD) is a highly contagious disease of cloven-hoofed animals [7]. The causative virus, Foot and Mouth Disease Virus (FMDV), belongs to the family Picornaviridae, genus Apthovirus. The positive-sense ssRNA genome of the virus codes for four structural (VP4, VP2, VP3, and VP1) and 10 nonstructural (L pro, 2A pro, 2B, 2C, 3A, 3B3, 3C pro, and 3D pol) proteins. Two non-translated regions (NTRs), 5′- and 3′- NTRs flank the coding portion of the genome [13]. The symptoms of the disease appear as lesions on foot and mouth region of the infected animal along with profuse salivation and sometimes lameness. India, endemic to FMDV, is home to three different FDMV serotypes, Serotype O, Asia 1 and A, in the decreasing order of frequency of outbreaks [15]. FMDV outbreaks cause huge livestock losses affecting the national economy [13]. The economic loss during the FMD outbreak can be up to 80% related to reduced milk production. Therefore, vaccination of the susceptible animal population remains the most important measure to control the virus and prevent economic loss. Currently in India, a trivalent binary ethylenimine (BEI) inactivated vaccine containing all the three serotypes are used for vaccination [6, 21]. ELISA-based techniques are commonly used for studying the prevalence of this disease, which either detects the presence of viral antigen or host immune response as antibodies produced against the virus. The presence of antibodies in vaccinated animals can result in false positivity in sero-surveillance studies. Therefore, the ELISA tests which can differentiate infected from vaccinated animals (DIVA) are frequently used. The DIVA test detects the presence of antibodies against non-structural proteins (NSPs) of FMDV in serum of animals, which indicates a recent infection. The DIVA test exploits the fact that antibodies against the NSPs appear in host system after 8–10 days post infection [3]. The antibody which is detected in this test is against 3AB protein, which is an intermediary protein synthesized only during successful infection of the host. The inactivated viral particles used in vaccine contain only one NSP, VPg protein. Hence, presence of anti-3AB antibodies in serum of animal is an indicator of recent infection. The annual report from ICAR-Directorate of Foot and Mouth Disease (DFMD) for year 2019 shows the overall seropositivity (DIVA positive) rate is ~ 20% regardless of the vaccination status [15].

The Major Histocompatibility Complex (MHC) of bovines i.e. Bovine Leukocyte Antigen (BoLA) is important for antigen presentation and plays critical role in immune response against FMDV [2, 19]. The BoLA DRB3 allele, which codes for the beta chain in Class II antigen, has been extensively studied and numerous reports have previously shown association of polymorphism in the gene with resistance/ susceptibility to several bacterial and viral diseases [8, 17, 26]. Different studies have reported several of DRB3 alleles linkage to FMD occurrence. One study performed using PCR–RFLP analysis reported 16 different alleles of DRB3 from 46 different cattle [8]. This study also suggested that T cell responsiveness to FMDV is dependent on the BoLA class II haplotype and thereafter the B cell response and immunity. Upon exposure to FMDV derived peptide A and ACT, alleles DRB3.2*1, 3, and 7 were found to be associated with protection while DRB3.2* 12 and 18 exhibited non-protective properties. Another study analyzed DRB3 gene of hundred FMDV challenged cattle and showed allele HaeIII A and HaeIII C were associated with susceptibility and resistance to FMD, respectively [17]. Another study investigated the genetic polymorphism of BoLA-DRB3 gene in Egyptian buffalo and identified 5 genotypes of BoLA-DRB3 exon 2 as a candidate genetic marker of FMD resistance/susceptibility [20].

BoLA Class I gene is a heterodimer which contains a α-chain and β-microglobulin chain which is polymorphic. The arrangement of Class I MHC molecule is more complex than the Class II MHC molecule. BoLA A gene is the majorly expressed gene in Class I MHC genes. The level of polymorphism reported is very high in certain regions of BoLA class I molecule, primarily within alpha 1 and alpha 2 regions. These regions contain certain specific amino acids that play an important role in peptide binding and T-cell receptor recognition. BoLA Class I region has been described as complex due to the interlocus recombination which had led to the increased diversity amongst alleles [14]. The number of alleles are also high in BoLA Class I region but due to the complexity not many studies have been done to determine all the alleles. However, some reports have mentioned alleles responsible for providing protection against infection. A study has reported that resistance against tick infection is due to BoLA Class I alleles w6.1 and w7 [22]. Several studies have shown a relationship between BoLA Class I and resistance or susceptibility to mastitis and Mycobacterium bovis [18]. The present study was performed to analyze the role of polymorphism in BoLA DRB3 and BoLA A gene in FMDV infection in vaccinated cattle. Our study has identified three polymorphic sites each in both the genes that show relation with recent infection in vaccinated animals.

Materials and methods

Sample collection

Forty-one cattle (Bos indicus) with owner provided history of FMDV vaccination (Raksha Triovac™, Indian Immunologicals) in villages near Leh town in Ladhak valley were investigated in this study (Table 1). The animals had received vaccinations at-least more than one month before samples were collected. The complete record of prior vaccination history was not available. Whole blood (5 ml) was collected aseptically from all the animals in Lithium Heparin (Heparin/LH) tubes (Peerless Biotech, India), as well as in clot activator tubes for serum separation (Peerless Biotech, India). After collection, the samples were transported on ice to the lab where they were further processed. The serum samples as well as whole blood samples were stored at -80 °C in deep freezer till further use. For subsequent experiments, frozen whole blood samples were thawed on ice for genomic DNA isolation, and serum samples were used for Sd-LPBE and 3AB₃ DIVA ELISA.

Table 1.

Summary of results of serum samples tested by DIVA assay and Sd-LPB ELISA for detection of antibodies against FMDV NSP 3AB₃, and structural proteins specific for serotype O, A, and Asia 1, respectively

Sample ID	Species	Sex	Location	FMDV 3AB3 DIVA*	Sd−LPBE			AGE (years)	Sequenced BoLA DRB3 and BoLA A
					Serotype O#	Serotype A#	Serotype Asia 1#
01	Bos indicus	Female	Thiskey	−	+	+	+	7	-
02	Bos indicus	Female	Thiskey	E	+	+	+	3	-
03	Bos indicus	Female	Thiskey	−	+	+	+	1	-
04	Bos indicus	Female	Thiskey	−	+	+	+	5	-
05	Bos indicus	Female	Thiskey	−	+	+	+	7	-
06	Bos indicus	Female	Thiskey	−	+	+	+	11	YES
07	Bos indicus	Female	Thiskey	−	+	+	+	1	YES
08	Bos indicus	Female	Thiskey	−	−	−	−	1	YES
09	Bos indicus	Female	Thiskey	−	+	+	+	6	-
10	Bos indicus	Female	Thiskey	−	+	+	+	6	-
11	Bos indicus	Female	Thiskey	−	+	+	+	10	-
12	Bos indicus	Female	Thiskey	−	+	+	+	3	-
13	Bos indicus	Female	Thiskey	−	+	+	+	7	YES
14	Bos indicus	Female	Thiskey	−	+	+	+	5	YES
15	Bos indicus	Female	Thiskey	−	−	−	−	7	YES
16	Bos indicus	Female	Tangyar	−	−	+	+	NA	-
17	Bos indicus	Female	Tangyar	−	+	+	+	NA	-
18	Bos indicus	Female	Tangyar	−	+	+	+	NA	-
19	Bos indicus	Female	Turtuk	−	+	+	+	NA	-
20	Bos indicus	Female	Turtuk	+	+	+	+	NA	-
21	Bos indicus	Female	Turtuk	+	+	+	+	NA	YES
22	Bos indicus	Female	Turtuk	−	+	+	+	NA	-
23	Bos indicus	Female	Turtuk	+	+	+	+	NA	YES
24	Bos indicus	Female	Turtuk	E	+	+	+	NA	-
25	Bos indicus	Female	Turtuk	−	+	+	+	NA	-
26	Bos indicus	Female	Turtuk	+	+	+	+	NA	YES
27	Bos indicus	Female	Turtuk	+	+	+	+	NA	YES
28	Bos indicus	Female	Diskit	+	+	+	+	NA	YES
29	Bos indicus	Female	Diskit	−	+	+	+	NA	YES
30	Bos indicus	Female	Diskit	−	+	+	+	NA	YES
31	Bos indicus	Female	Diskit	−	+	+	+	NA	YES
32	Bos indicus	Female	Diskit	+	+	+	+	NA	YES
33	Bos indicus	Female	Diskit	−	+	+	+	NA	YES
34	Bos indicus	Female	Diskit	−	+	+	+	NA	-
35	Bos indicus	Female	Diskit	−	+	+	+	NA	-
36	Bos indicus	Female	Diskit	−	+	+	+	NA	YES
37	Bos indicus	Female	Diskit	+	+	+	+	NA	YES
38	Bos indicus	Female	Diskit	+	+	+	+	NA	YES
39	Bos indicus	Female	Diskit	+	+	+	+	NA	YES
40	Bos indicus	Female	Diskit	−	−	+	+	NA	YES
41	Bos indicus	Female	Diskit	+	+	+	+	NA	YES

^* The PP values of 0–20, 20–40 and > 40 were designated as negative, elevated, and positive, respectively for DIVA test

^# For sd-LPBE, the antibody titers were designated as positive or negative as per manufacturer recommendation using Excel utility provided by manufacturer

Single dilution-Liquid Phase Blocking ELISA (Sd-LPBE) and 3AB₃ DIVA ELISA

The serum samples were tested for presence of antibodies against structural proteins specific for FMDV serotypes O, A and Asia1, using single dilution-Liquid Phase Blocking ELISA (Sd-LPBE) (Arsh Biotech, Cat. No. AB-SdLPBE-045). The antibody titers were calculated using Excel utility downloaded from manufacturer website as per manufacturer recommendation. The antibody titer were designated as positive or negative for sd-LPBE results as per manufacturer recommendation using Excel utility provided by manufacturer. The antibodies against viral NSP 3AB₃ were assayed using 3AB₃ DIVA ELISA (Arsh Biotech, Cat. No. AB-3AB3-002). Optical density (OD) was measured using ELISA reader at a wavelength of 492 nm (Reference 620 nm). The percent positivity was calculated using the formula as per manufacturer recommendation {PP value = (Test sample mean OD / Positive control mean OD) * 100}. The PP values of 0–20, 20–40 and > 40 were designated as negative, elevated, and positive, respectively for antibodies against AB3.

PCR amplification and sequencing of host BoLA DRB3 and BoLA A genes

Genomic DNA was extracted from the frozen blood samples using Purelink™ Genomic DNA kit (Invitrogen, cat no. K182000). Using the gDNA as template, PCR amplifications were carried out for BoLA DRB3 and BoLA A genes using nested PCR and conventional PCR strategies, respectively. The amplification was carried out in 25 µl reaction mixture which consisted of 2.5 µl of 1X buffer, 250 µM of each dNTPs (Thermo Fischer Scientific, cat no. R0186), 20 pmol of each forward and reverse primer, 1 µl of DNA template, and 1 IU of Taq polymerase (NEB, cat no. M0273). The details of the primers and the PCR conditions have been listed in the Table 2. The PCR products were purified using QIAquick PCR purification kit (Qiagen, cat no. 28104). The PCR products were then got sequenced at the Central Instrumentation Facility, University of Delhi South Campus, using forward primers of respective genes.

Table 2.

Details of target genes, primers, annealing temperature, and their amplicon size

Primers	Nucleotide sequence	Target gene	Amplicon size (bp)	Annealing temperature (oC)	Reference
BoLA DRB3-F′	5′-CTCTCACTCTCTGCACTTCAAT- 3′	BoLA DRB3	1698	60.1	This study
BoLA DRB3-R′	5′-AAATGGACAACCTGGAAGAAATG- 3′	BoLA DRB3	1698	59.2	This study
BoLA DRB3-F	5′ -ATCCTCTCTCTGCAGCACATTTCC- 3′	BoLA DRB3	287	65.2	Lei et al., 2012
BoLA DRB3-R	5′ -TCGCCGCTGCACAGTGAAACTCTC-3′	BoLA DRB3	287	68.5	Lei et al., 2012
BoLA A-F	5′-GGTCCCACTCCCTGAGGTATTTC- 3′	BoLA A	709	66.6	This study
BoLA A-R	5′-CACCAGGTATCTGCGGAGC- 3′	BoLA A	709	61.6	This study

DNA and Protein sequence alignment, and structural analysis of proteins

The obtained gene sequences and predicted protein sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) verison.6 tool [25]. For protein alignment, the DNA sequences were first translated into protein sequences using Expasy translate tool (https://web.expasy.org/translate/) [9]. The sequence logo was created for protein alignment using the online available tool (https://weblogo.berkeley.edu/logo.cgi) [4]. Protein structure models were predicted from existing model of MHC Class I and Class II molecules using Swiss model online prediction tool (https://swissmodel.expasy.org/) [27] which were used for further analysis.

Result

Majority of the vaccinated animals were positive for protective antibodies levels against all the three major serotypes of FMDV

The results of Sd-LPB ELISA for quantization of protective antibody levels against FMD serotypes have been shown in Fig. 1a. Out of 41 samples which were tested, 36 samples (88%) showed presence of antibodies against all three serotypes above protection levels, whereas 3 (7%) had protective antibodies against at least two serotypes above protection levels (Fig. 1a). Only 2 serum samples (5%) were found to be negative for antibodies against all 3 serotypes. Our data indicate that vaccination of animals resulted in generation of protective immune response in majority (95%) of the animals which were tested. The protective immune response confers protection to animals from developing clinical signs of the disease if they get infected with a closely related vaccine strain. However, it may not prevent sub-clinical infection or virus persistence even leading to a carrier state [23, 24]. We then wanted to test if any of these vaccinated and protected animals did get infected recently. The results of DIVA test show that 11 out of 41 samples tested positive and 3 showed elevated levels of antibodies against 3AB₃ NSP indicating recent sub-clinical infection in 27% of vaccinated animals (Fig. 1b). To further investigate the role of host genetic factors in modulating the susceptibility of vaccinated animals for virus infection, we sequenced specific host genes of the all of the 11 DIVA positive animals and randomly selected 12 DIVA negative animals. However due to deterioration in quality of blood sample collected from one of the DIVA positive animal, that sample could not be processed further. Therefore, a total of 22 samples were analyzed, which included 10 DIVA positive and 12 DIVA negative animals.

Fig. 1.

A fraction of vaccinated cattle serum samples which had protective antibody titers against FMDV Serotype O, A and Asia 1 above protection levels, also tested positive for antibodies against 3AB₃ non-structural proteins of FMD virus. Antibody titres (log₁₀) of protective antibody levels against FMD Serotype O, A and Asia 1 in animal serum samples were investigated using sd-LPBE test. Most of the samples tested positive for antibodies against all 3 serotypes with titres above protection level of 1.8 (Fig. 1a). The serum samples were then tested for presence of antibodies against 3AB₃ non-structural proteins of FMD virus using 3AB₃ DIVA ELISA. Eleven samples tested positive with titres above threshold (PP > 40) indicating evidence of recent infection (Fig. 1b)

BoLA DRB3 Exon 2 and BoLA A genes sequences showed nucleotide polymorphism which showed correlation with their DIVA status

The outcome of host–pathogen interaction can be dependent on the genetic polymorphisms in genes which are important for modulation of cellular pathways critical for disease pathogenesis and progression. The major histocompatibility complex (MHC) is one of the most important genetic systems for infectious disease resistance in vertebrates. In cattle, the MHC genes are referred to as Bovine lymphocyte antigen (BoLA). Previous studies have found linkages between BoLA-DRB3 gene polymorphism and resistance or susceptibility to FMD in cattle and buffaloes [17, 20]. In present study, we investigated the link between polymorphism in BoLA DRB3 and BoLA A genes and susceptibility of vaccinated animals to sub-clinical FMDV infection. The exon 2 of the BoLA DRB3 gene which code for peptide binding cleft of the BoLA Class II molecule was analyzed for polymorphism. This region was amplified using a nested PCR method to improve the specificity of the PCR product [17]. A 1698 bp region containing the exon 2 was amplified using two outer primers BoLA DRB3 F’ and BoLA DRB3 R’ (Table 2). The product of this PCR was used as template to amplify exon 2 using primers BoLA DRB3 F and BoLA DRB3 R (Table 2). The 287 bp product obtained was got sequenced using BoLA DRB3 F primer. The 709 bp region of BoLA A gene was amplified using primers and conditions listed in Table 2.

The analysis of BoLA DRB3 gene sequences revealed three different sites of polymorphism between DIVA positive and DIVA negative animals (Fig. 2a). The first site of polymorphism was at nucleotide position 79–81, where 80% of the DIVA positive animals had TAC whereas 67% of the DIVA negative animals had TTC (Fig. 2a). The second site of polymorphism was at nucleotide position 213–215 where 50% of the DIVA positive animals had AAG and rest either had AGG or GAG. On the other hand, 50% of the DIVA negative animals had GCG, approximately 33% had GAG and the rest either had AGG or GGG. The third site of polymorphism was observed at nucleotide position 223–225. Among DIVA positive animals, 80% of them had TCG or AAT and rest had GCT. In contrast, approximately 58% of DIVA negative animals had GCG and rest either had GAG or TCG or GCT.

Fig. 2.

The BoLA DRB3 Exon 2 and BoLA A genes sequences of vaccinated cattle showed nucleotide polymorphism which showed correlation with their DIVA status. The nucleotide sequences of BoLA DRB3 Exon 2 gene of vaccinated and DIVA positive animals were compared with sequences in vaccinated and DIVA negative animals. The analysis showed nucleotide polymorphism at three sites which strongly correlated with DIVA status of animal (Fig. 2a). The nucleotide sequences of BoLA A gene also showed nucleotide polymorphism at three sites which also strongly correlated with DIVA status of animal (Fig. 2b)

The 709 bp gene product of BoLA A gene was successfully PCR amplified in all the samples and sequenced. The analysis of sequences from DIVA positive and DIVA negative animals also showed polymorphism in 3 distinct locations that were linked with their DIVA status (Fig. 2b). The first site of polymorphism was observed at nucleotide position 128–130 where almost 90% of DIVA positive animals had ATG and remaining 10% had GTA. On the other hand, 66% of DIVA negative animals had ATA whereas 33% had ATG. The second site of polymorphism was observed at nucleotide position 194–196 wherein among the DIVA positive animals, 50% had AGA, 30% had ATC and 20% had AAG and ATA. However, 75% of the DIVA negative animals had ATC, and 25% had AAA, ATA, or AAC. The third site of polymorphism was identified at nucleotide position 397–399 where 70% of the DIVA positive animals had either GAG or GAA, 20% had AAG and 10% had AAC. Among the DIVA negative animals approximately 37% had AGC, 27% GAG, 18% AAG, 9% GAA, 9% AAC.

The predicted amino acid sequences of BoLA DRB3 Exon 2 and BoLA A proteins in vaccinated cattle showed amino acid polymorphism which correlated with their DIVA status

The translated protein sequences of BoLA DRB3 Exon 2 were aligned and analyzed. The three sites of nucleotide polymorphism corresponded to amino acid changes at position 27, 72 and 75 (Fig. 3a). At amino acid position 27, more than 70% of the DIVA positive animals had tyrosine and rest either had leucine or phenylalanine. However, approximately 41% of the DIVA negative animals had phenylalanine and rest had tryptophan or leucine or isoleucine. At amino acid position 72, more than 50% of the DIVA positive animals had lysine, barring some which had arginine and glutamic acid. In case of DIVA negative animals, 41% of them had alanine, 33% had glycine and rest either had arginine or leucine. At amino acid position 75, about 90% of the DIVA positive animals had either serine or asparagine and rest had alanine. However, DIVA negative animals had either alanine or glutamic acid. Amino acid could not be translated for sample 44 & 48 at 72^nd position and sample 77 at 72^nd and 75^th could not be deciphered due to lack of accurate nucleotide sequence.

Fig. 3.

The predicted amino acid sequences of BoLA DRB3 Exon 2 and BoLA A proteins in vaccinated cattle showed amino acid polymorphism which correlated with their DIVA status. The sequence logo representation of the predicted amino acids sequence of BoLA DRB3 protein show polymorphism at position 27, 72 and 75 (Fig. 3a), and of predicted BoLA A amino acid sequence show polymorphism at position 44, 65 and 112 of BoLA A protein (Fig. 3b)

For BoLA A gene, the polymorphism at the three nucleotide polymorphisms sites resulted in polymorphism in predicted amino acid sequence at 3 sites (Fig. 3b). At amino Acid Position 44, approximately 90% of the DIVA positive animals had methionine and 10% had isoleucine. On the other hand, approximately 66% of the DIVA negative animals had isoleucine except for 33% which had methionine. At amino Acid Position 65, among all the DIVA positive animals 50% had arginine, 40% had isoleucine and 10% had lysine. However, 75% of the DIVA negative animals had isoleucine and 30% had either lysine or asparagine. At amino Acid Position 112, 90% of the DIVA positive animals had aspartic acid and 10% had tyrosine. In contrast, 75% of the DIVA negative animals either had phenylalanine or tyrosine and remaining 25% had either isoleucine or glycine or leucine.

Predicted protein structure models of BoLA DRB3 protein show that amino acids polymorphism sites associated with DIVA status of host are spatially proximal

The predicted amino acid sequence of partial BoLA DRB3 protein of DIVA positive animals and DIVA negative animals were used for structural modeling as described in material and methods sections. The three sites of polymorphism were observed to be present in different structural motiffs in the model (Fig. 4a). One site (aa27) was located in the β-strands and two sites (aa72, aa75) were located in the α-helix region. Analysis of surface properties of the protein in the predicted model presented two interesting facts. First, these three amino acids were predicted to be part of a narrow pocket on the surface of the protein, indicating a distinct possibility of these polymorphisms acting in sync to result in modifications of structural properties of this protein. Second, in case of DIVA negative samples, the protein surface was predicted to form a deep groove lined by these 3 sites of amino acid polymorphism, which was not the case of DIVA positive samples.

Fig. 4.

The predicted protein structure models of BoLA DRB3 protein and BoLA A protein show location and structural relation between amino acids at sites of DIVA status associated polymorphism. The predicted structure of BoLA DRB3 protein showed that the amino acids involved in DIVA status associated polymorphism were structurally closely located and formed part of a close pocket on the protein surface (Fig. 4a). The predicted structure of BoLA A protein showed that the amino acids involved in DIVA associated polymorphism were distributed at different positions with no structural proximity (Fig. 4b)

Similar structural modeling analysis was done for BoLA A protein. A 3-D structure of the protein was built using the reference sequence from Bos indicus Nellore species. The model was built using Swiss model online tool and the sites of polymorphism were analyzed (Fig. 4b). The three sites of polymorphism in BoLA A protein were predicted to be present in different structural motifs. Two of the sites (aa44, aa112) were present in the β-strands and one site was present in the α-helix region (aa65). The template used for this gene to build a model using Swiss model showed the presence of these sites in the inner surface of the antigen binding groove.

Discussion

Vaccination against FMDV is one the important strategy to manage the disease. Detection of FMDV is commonly done by ELISA based tests detecting antibodies generated against the virus. To avoid detecting false positive results due to vaccine induced antibodies, ELISA tests have been developed which can differentiate vaccinated and infected animals (DIVA). The data published by Project Directorate on FMD (ICAR, India) has reported an average DIVA positivity of 20–25% over previous 10 years indicating FMD virus exposure regardless of vaccination status of the animal (http://www.pdfmd.ernet.in/). The present study was performed on vaccinated animals, out of which 95% had antibodies against all 3 major FMDV serotypes above protection levels. Only 27% of the animals under study were DIVA positive which is very close to national average over last 10 years.

As FMDV is a highly contagious virus, infection in even one animal in a herd may results in quick spread of infection to other animals present in close contact. In present study, it was observed that even though the animals belonging to same owner or farm were being reared in very close quarters, only a proportion of those tested DIVA positive. Occurrence of infection in vaccinated animals is well-known in some diseases and is termed as breakthrough infection. Most common reasons for these are mutation in virus thereby rendering the vaccine induced antibodies ineffective. Other reasons include host genetic factors which can also play an important role resulting in differences in susceptibility of animals to virus infection even with vaccine induced protective immune response. Several genetic factors have been previously studied for their role in immune response, which include polymorphism in immune response related genes of host [10, 11]. The role of BoLA DRB3 alleles in immune response in cattle vaccinated for FMD has previously shown to suggest that amino acid substitutions could lead to unique conformational changes in protein structure associated with immune response to FMDV vaccine [12].

Our study identified 3 sites of nucleotide polymorphism in BoLA DRB3 Exon2 which codes for protein cleft binding region. The presence of specific nucleotides correlated with DIVA status of vaccinated animals, and all these three sites showed synonymous mutations at corresponding amino acid positions. The DIVA positive animals had tyrosine (polar aa) instead of phenylalanine (non-polar aa) at 27 position. Although both amino acids have bulky side chains, however the presence of -OH group in tyrosine can result in modifications in protein functions [1]. At amino acid position 72 in BoLA DRB3, the DIVA positive animals had lysine or arginine (polar amino acids), whereas the DIVA negative animals had alanine (less polar amino acid). Similarly at position 75, DIVA positive animals had either serine or aspargine, whereas DIVA negative had alanine. The mutations at these 3 sites in BoLA DRB3 which correlate with the DIVA status of animal indicate the possible role of these motifs in structural changes resulting in modulation of protein functions. BoLA DRB3 is involved in antigen presentation which requires extensive protein–protein interactions with antigenic peptide, therefore such changes in amino acid at critical positions in protein could affect these interactions. The fact that these 3 amino acids form part of a small pocket provides a plausible explanation for their role in determining DIVA status of vaccinated animals. The presence of a deep groove in the peptide binding cleft may influence the peptide binding affinity of the molecule. It has been previously reported that the binding affinities of peptide decide the T_H cell type selected and affects the type of cytokine produced. Peptide binding with stronger affinity induces interferon-γ production while peptides which exhibit weaker binding induce IL-4 production [16]. The amino acid at β86 position select for peptides of different nature with respect to amino acid present in the antigenic peptide [5]. Studies have shown that the stimulation of T cell with peptide binding with high affinity DRB3 molecule showed greater IFN gamma production, but T cell stimulated with peptide binding with low affinity did not show any significant change on IFN gamma production [16]. Therefore, such amino acid changes as identified in present study in BoLA DRB3 could be a possible reason for FMDV susceptibility in a vaccinated group of cattle. However, further studies will be required to confirm the role of these amino acids in influencing the affinity of peptide binding to the BoLA Class II molecule.

Our studies on BoLA A gene sequence also identified three distinct sites of nucleotide polymorphism which correlated with DIVA status of the animals. All these three sites showed synonymous mutations at corresponding amino acid positions. The predicted structural model analysis showed that two of the sites were present in the β-sheets and one site in the α-helix region. The variations in Class I MHC molecules are concentrated in the three or four discrete hypervariable regions within α1 and α2 domains, while the rest of the molecule is highly conserved and shows little variation. In BolA A protein, the antigen binding groove is formed by α1 and α2 in Class I MHC molecules. The superdomain is comprised of two α-helices and eight β-pleated sheets. The polymorphism in BoLA A identified in present study is predicted to be in the amino acids present in the peptide binding groove. The peptide binding affinity is affected by both the residues in the peptide binding cleft and those in the antigenic peptide. This affinity influences the immune response and eventually the progression of infection. Our study suggests a possibility that these changes in the amino acid residues of BoLA A gene may also have some role in determining susceptibility of vaccinated animals to FMDV infection. Our limited study was performed on a relatively small samples size collected from one region of country. Further validation would require more detailed investigations on larger sample size.

Supplementary Information

Below is the link to the electronic supplementary material.