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Indian Immunologicals Limited, Gachibowli, Hyderabad
Department of Biochemistry, Indian Institute of Sciences, Bangalore, India
ABSTRACT
Comparison of nucleotide sequences of the partial 1D region of foot-and-mouth disease type C viruses of Indian origin with those of European, South American, and Southeast Asian viruses revealed that the Indian viruses form a distinct genotype. The vaccine strain C IND/51/79 belongs to this genotype and may be a prototype strain of this genotype.
TEXT
Foot-and-mouth disease (FMD) is an acute contagious viral disease of cloven-footed livestock, and the virus exists as seven serotypes: A, O, C, Asia1, SAT I, SAT II, and SAT III. The type C virus is the least prevalent of all the serotypes, causing about 8% of all FMD outbreaks during 1977 to 1990 and 1.6% during 1991 to 1994 (8). Since 1996, type C outbreaks have not been recorded in India, and the World Reference Laboratory for FMD (Pirbright, United Kingdom) has not received any isolates during the past 8 years (10).
The 1D region in the viral RNA codes for a major antigenic site consisting of the GH loop (140 to 160 amino acids) and a minor antigenic site (205 to 210 amino acids) at the C terminus of VP1 protein. These two sites are considered important for phylogenetic analysis, because genetic and antigenic variations in these sites are ascribed to differences among strains (9). Until recently two vaccine strains, C Bombay/64 and C IND/51/79, have been used in India; either of these strains was included in quadrivalent vaccines by different vaccine manufacturers. Hemadri et al. reported that the vaccine strain C Bombay/64 belonged to the European genotype (2); their study did not include the strain C IND/51/79. In the present study, the partial 1D sequences of various field isolates (1991 to 1994) and both the vaccine strains were studied to compare the relationship of these vaccine strains with the field isolates along with GenBank sequences available for type C viruses of European, South American, and Southeast Asian origin.
FMD viruses from independent outbreaks in different parts of India (Table 1) were obtained as tongue or foot epithelial tissue samples. A total of 11 isolates were included in this study, and all the isolates, except one (C MPH/12/92), were from nonvaccinated crossbred cattle. The virus isolates were passaged once in cells of the baby hamster kidney-21 (BHK-21) cell line, and antigenic comparison with the C IND/51/79 was performed using a Micro Neutralization test (MNT) as per the procedure of Rweyemamu and Hingley (7). The viruses were declared homologous when the r value was >0.23 and heterologous when the r value was <0.23.
Total RNAs from the tongue or foot lesions were extracted using TRIzol reagent (Invitrogen), and a region of the 1D sequence was amplified using a One-Step RT-PCR kit (QIAGEN, Hilden, Germany) with specific oligonucleotide primers (sense,5'-AAAGACTTTGAGCTCCGGCTACC-3'; antisense, 5'-AGCTTGTACCAGGGTTTGGC) (3) following the manufacturers' instructions. The purified PCR products were sequenced using an ABI Prism 310 genetic analyzer with the antisense primer 5'-GCCCAGGGTTGGACTC-3' (3) using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit per the manufacturer's protocol. Table 1 lists the GenBank accession numbers of the partial 1D sequences of the Indian isolates. The sequences were compared with those of type C viruses of European, South American, and Southeast Asian origin (Table 2) by use of the GrowTree program (GCG software; Accelrys, San Diego, Calif.). The phylogenetic tree was drawn using the DRAWTREE program of PHYLIP, version 3.5c (1). The deduced amino acid sequences were aligned and analyzed using GeneDoc software (5).
The one-way antigenic relationship determined using MNT showed that the Indian field isolates are homologous to the C IND/51/79 vaccine strain (data not shown). The phylogenetic tree drawn using the neighbor-joining method on aligned sequences showed that the type C viruses were distributed in seven distinct lineages (Fig. 1). The Indian type C viruses formed a distinct genotype.
The nucleotide sequence analyses of the field isolates indicated that they are genetically similar. Hemadri et al. (2) had reported earlier that all the Indian type C strains showed similarity in nucleotide sequences and that the vaccine strain C Bombay/64 is homologous to the European isolates. The present study confirms their findings and extends those findings to include the C IND/51/79 vaccine strain and field samples from different parts of the country. Most of the Indian field isolates had less than 5% divergence at the nucleotide level from the vaccine strain C IND/51/79 except two isolates (6 to 8% divergence). The phylogenetic analysis of genomic sequences revealed that all the Indian isolates showed >10% divergence from C Bombay/64. The Indian isolates were grouped in a separate lineage or genetic group that is distinct from those of any of the previously described genetic lineages (4).
Amino acid comparisons made with those reported by Hemadri et al. (2) also revealed sequence similarity between the Indian serotype C isolates. However, C Bombay/64 strain has a deletion of one amino acid (R) in front of the RGD motif, in similarity to the European isolates. This may indicate that the C Bombay/64 strain is closer to the European isolates in the genetic makeup of the 1D region studied here than to the Indian isolates.
The GH loop near the C-terminal region and the BC loop near the N-terminal region of the VP1 region have been reported as the antigenic regions for the FMD viruses. Alignment of the deduced amino acid sequence revealed that there were 10 distinct substitutions in the field isolates compared to the results seen with the vaccine strain around the GH loop and in the C-terminal end of VP1. The substitutions were 131 TS/N, 145 ML, 149 AV, 153 KS, 188 VI/H, 189 SL, 190 FP, 191 HF/L/I, 198 YH, 202 IL/S, and 203 IV. Three substitutions were noticed in the GH loop (positions 138 to 156), and two were noticed in the carboxyl end (positions 195 to 200) of the 1D region. The residue H-147 in the RGD loop remained unchanged. Few isolates have shown distinct changes in their amino acid sequences. Changes in residues A-149 (AT/V) and A-151 (AT) have been noticed in few isolates. Residue T-194 of minor antigenic site C also remained unchanged except in one isolate. However, a change at the residue 203 is evident in four isolates. In spite of these changes the field isolates were found by use of the MNT to be homologous to the vaccine strain C IND/51/79.
Another interesting observation is that unlike other serotypes, classification of the C-type lineages is similar to the classification made on the basis of subtypes identified by Pereira (6). The phylogenetic analysis revealed seven distinct genotypes for the type C viruses (Fig. 1). The Southeast Asian and Indian isolates are grouped under two distinct genotypes—genotypes VI and VII, respectively. In the phylogenetic tree the C Bombay/64 strain is grouped with the European strains (genotype III). The C IND/51/79 is grouped with the Indian isolates but as a distinct lineage (genotype VII). The type C viruses in the subcontinent are not clearly descendants of the vaccine strains. The Indian type C viruses have a distinct evolution pattern and require further scrutiny to determine the origin of the type C viruses in India. The vaccine strain C IND/51/79 has a wide antigenic spectrum, as is evident from the MNT results, and is genetically related to the type C Indian isolates. This strain was characterized at the World Reference Laboratory, Pirbright, United Kingdom, after it was isolated from an outbreak in the state of Tamil Nadu, India. This vaccine strain was a part of the quadrivalent vaccine manufactured by Indian Immunologicals Ltd., Hyderabad, India, with technical support from the Wellcome Research Foundation, London, United Kingdom. Because of its homology to the Indian isolates, the C IND/51/79 may be considered a prototype strain of the type C viruses of Indian origin. Although type C outbreaks have been minimal, data presented in this study show that future type C FMD outbreaks in India and South Asia could be controlled by the use of C IND/51/79 vaccine strain.
ACKNOWLEDGMENTS
We thank Syed Hasnain, Director, Centre for DNA Fingerprinting and Diagnostics, and Nagarajan Ram, Director, and S. Swaminathan, Technical Officer, Department of Bio-informatics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India, for help with the GCG software.
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