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Genetic characterization of Indian type O FMD virus 3A region in context with host cell preference
Infection, Genetics and Evolution 10 (2010) 703–709
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Short communication
Genetic characterization of Indian type O FMD virus 3A region in context with host cell preference V. Maroudam a, S.B. Nagendrakumar a, P.N. Rangarajan b, D. Thiagarajan a, V.A. Srinivasan a,* a b
Research and Development Centre, Indian Immunologicals Limited, Rakshapuram, Gachibowli Post, Hyderabad 500 032, Andhra Pradesh, India Department of Biochemistry, Indian Institute of Sciences, Bangalore 560 080, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2009 Received in revised form 9 March 2010 Accepted 10 March 2010 Available online 17 March 2010
The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. For example, amino acid deletions in the porcinophilic strain (O/TAW/97) at 93–102 aa of the 153 codons long 3A protein have been recognized as the determinant of species specificity. In the present study, 18 type O FMDV isolates from India were adapted in different cell culture systems and the 3A sequence was analyzed. These isolates had complete 3A coding sequence (153 aa) and did not exhibit growth restriction in cells based on species of origin. The 3A region was found to be highly conserved at Nterminal half (1–75 aa) but exhibited variability or substitutions towards C-terminal region (80–153). Moreover the amino acid substitutions were more frequent in recent Indian buffalo isolates but none of the Indian isolates showed deletion in 3A protein, which may be the reason for the absence of host specificity in vitro. Further inclusive analysis of 3A region will reveal interesting facts about the variability of FMD virus 3A region in an endemic environment. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Foot-and-mouth disease virus Serotype O Cell culture 3A region Genomics
1. Introduction Foot-and-mouth disease (FMD) is a highly infectious viral disease affecting domestic cloven-hoofed cattle, pigs, sheep, goats as well as more than 70 species within 20 families of mammals (Barnett and Cox, 1999; Davies, 2002; Brown, 2003; Grubmann and Baxt, 2004). The disease is a serious threat to livestock industry and creates severe economic consequences for domestic livestock production and international trade. FMD is endemic in India and occurs both in cattle and buffalo (Dutta et al., 1983; Maroudam et al., 2008). The outbreaks in India are ascribed to serotypes A, O and Asia-1 of which serotype O is predominant (Nagendrakumar et al., 2006). The FMD viral genome is a positive-stranded RNA consisting of about 8500 nucleotides. Its single long open reading frame encodes a polypeptide that is cleaved to mature polypeptide products by virally encoded proteinases. In addition to four capsid proteins (VP1–VP4), the RNA-dependent RNA polymerase (3D) and three proteinases (L, 2A and 3C), the FMDV genome encodes several other mature proteins including 3A, a highly conserved protein of 153 amino acids (Mason et al., 2003; Belsham, 1993). Although 3A was found to be associated with intracellular membranes during
* Corresponding author. Tel.: +91 40 23000211/2000894; fax: +91 40 23005958. E-mail address: [email protected] (V.A. Srinivasan). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.03.004
the proliferation of picornavirus, the functions of nonstructural protein 3A in the life cycle of FMDV is less understood (Bienz et al., 1983; Towner et al., 1996). However, alteration in 3A protein, such as point mutations and deletions, was linked to altered host specificity, adaptation, attenuation and virulence in FMDV (Giraudo et al., 1990; Beard and Mason, 2000; Knowles et al., 2001; Nunez et al., 2001, 2007; Pacheco et al., 2003; Nobiron et al., 2005) in hepatoviruses (Graff et al., 1994; Morace et al., 1993), in rhinoviruses (Heinz and Vance, 1996) and in enteroviruses (Lama et al., 1998). Genomic investigation of FMD virus (FMDV) isolated from pigs in Taiwan during the outbreak in 1997 (O/TAW/97) demonstrated that altered nonstructural protein 3A is a primary determinant of restricted growth of the isolate on bovine cells in vitro and significantly contributed to bovine attenuation in vivo (Beard and Mason, 2000). Importance of 3A in host range and virulence have been studied by many investigators for isolates exotic to India (O’Donnell et al., 2001; Nunez et al., 2007), Indian type A and Asia 1 isolates (Mittal et al., 2005; Mohapatra et al., 2009). However the 3A region of Indian FMDV type O isolates has not been studied in detail. Here, we report the results of a study using 18 FMDV Indian type O isolates adapted to different host cells (primary cattle and buffalo cells and Instituto Biologico Renale Swine-2 (IBRS-2)) cell line and comparison of complete sequence of 3A region of these Indian type O isolates with exotic isolates.
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Table 1 List of field viruses used in the study with their place of origin, type of epithelium and vaccination status. Lab ref. no.
Year of isolation
District
State
Species
Epitheilium
Vaccinated status
GenBank accession no.
MPC 29/84 APP 33/88 MASg 9/90 GUSk 26/90 MAB 43/90 MAB 28/01 HAJh 53/00 GuP 06/02 APR 36/02 KAB 11/04 KAK 12/04 APP 17/04 APMb 67/04 APMb22/05 HAS 34/05 APRR 84/05 APNg 86/05 APNg 87/05
1984 1988 1990 1990 1990 2001 2000 2002 2002 2004 2004 2004 2004 2005 2005 2005 2005 2005
Rajpur Prakasam Sangli Sabarkanta Bombay Mumbai Jhajjar Pachanmahal Rangareddy Bangalore Kolar Prakasam Mahboobnagar Mahboobnagar Sirsi Rangareddy Nalgonda Nalgonda
Madhya Pradesh Andhra Pradesh Maharashtra Gujarat Maharashtra Maharashtra Haryana Gujarat Andhra Pradesh Karnataka Karnataka Andhra Pradesh Andhra Pradesh Andhra Pradesh Haryana Andhra Pradesh Andhra Pradesh Andhra Pradesh
Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Pig Cattle Cattle Cattle Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo
Tongue epithelium Tongue epithelium Tongue epithelium Tongue epithelium Tongue epithelium Feet epithelium Feet epithelium Tongue epithelium Snout epithelium Tongue epithelium Tongue epithelium Tongue epithelium Feet epithelium Feet epithelium Feet epithelium Buccal Feet epithelium Feet epithelium
Unvaccinated Unvaccinated Vaccinated Vaccinated Vaccinated Vaccinated Unvaccinated Unvaccinated Unvaccinated Vaccinated Unvaccinated Unvaccinated Unvaccinated Unvaccinated Unvaccinated Vaccinated Unvaccinated Unvaccinated
– GU181359 GU181366 – GU181365 GU181364 GU181361 – GU181360 GU181362 GU181363 GU181358 – – – – – –
Linked Immuno Sorbant Assay (ELISA) (Hamblin et al., 1984) and confirmed by reverse transcription polymerase chain reaction (RTPCR) (Reid et al., 1998).
2. Materials and methods 2.1. FMDV isolates FMDV serotype O virus isolates from buffaloes (n = 14), cattle (n = 3) and swine (n = 1) collected during the period 1984–2005 from different geographical locations of the country were used in this study (Table 1). Indian vaccine strains (OTNN 24/84 and O R2/ 75) were also used in this study. Complete 3A amino acid sequences of type O isolates (n = 12) were obtained from GenBenk for genetic comparisons (Table 2). 2.2. Buffalo and cattle cell cultures Primary cell cultures from cattle and buffalo thyroid and kidney were prepared as described by Freshney (2004). Cells were seeded with a density of 106 cells/ml in growth medium containing 8% fetal bovine serum (BiochromTM) in 25 cm2 flasks and were used for virus isolation and viral bank preparation.
2.4. RNA isolation, RT-PCR amplification and nucleotide sequencing Genomic RNA was extracted from the FMDV infected cell culture supernatants of all passages and clinical epithelial tissue suspension (10%, w/v) using TRIzol reagent (Reid et al., 1998). Ten microliters of total RNA was subjected to one step RT-PCR (Qaigen, USA) using primer pair IIL-3AF (CCC CTC CAG AAT GTG TAC CAG CTTG) and IIL-3AR (GCT TTG TGT TGC CCA TGA CCA TCTT). The amplified RT-PCR products were gel purified with Qiaquick1 Gel Extration Kit (Qiagen, USA) and reconstituted in 30 ml of elution buffer and sequenced in automated DNA sequencer (Applied Biosystems, USA) using Big-Dye Terminator Cycle Sequencing kit v3.0 (Applied Biosystems) in accordance with the manufacturer’s instructions. 2.5. Genetic sequence analysis
2.3. Virus isolation Epithelial tissues (10%, w/v) were inoculated into primary cells buffalo thyroid (BTY), cattle thyroid (CTY), buffalo kidney (BK) and cattle kidney (CK) cells and IBRS-2 cell line. After three consequent passages in these primary cells, the virus was inoculated in BHK-21 cell line. Master seed banks were stored as 50% glycerol stocks at 20 8C until further use. Virus serotyping of epithelial samples and cell culture supernatants was carried out by antigen Enzyme
Overlapping molecular sequences generated by the forward and the reverse primers in this study were manually processed in the GeneDoc Multiple Sequence Alignment version 2.6.002 (Nicholas and Nicholas, 1997). The 3A sequences of exotic isolates were retrieved from GenBank and their accession numbers are given in Table 2. The 3A sequences were aligned with ClustalX algorithm (Thompson et al., 1997) and the aligned amino acid sequences
Table 2 Details of 3A sequences of virus obtained from GenBank. S. no.
Virus ID
Geographic origin
Date of collection date/month/year
Host
Deletion
GenBank accession no.
1 2 3 4 5 6 7 8 9 10 11 12
O/HKN/19/73 O/HKN/6/83 O/TAW/97 O/CAM/1/98 O/CAM/2/98 O/CAM/3/98 O/CAM/12/94 O1 kaufbeuren O1 bfs46 O1 Campos94 O1 Manisa O5 India
Ha Cheung Sha, Lantau Isaland N.T. Kowloon, Hong Kong Pokfulam, Hong Kong Island Yunlin prefecture, Taiwan, POC Borset, Kg. Speu, Cambodia Kg. Speu, Cambodia Kg. Speu, Cambodia Mong Russey District, Battambang Cambodia Germany United Kingdom Argentina Turkey India
19/06/1973 18/12/1982 04/1997 05/01/1998 05/01/1998 05/01/1998 09/1994 1996 Not known Not known 1969 1962
Bovine Bovine Porcine Porcine Bovine Bovine Porcine Bovine Bovine Bovine Bovine Bovine
93–102 93–102 93–102 133–143 133–143 133–143 133–143 None None None None None
AJ294986 AJ294992 AJ308157 AJ294981 AJ294982 AJ294983 AJ294980 X00871 AY593816 AY593819 AY593823 AY593828
V. Maroudam et al. / Infection, Genetics and Evolution 10 (2010) 703–709
were analyzed for the presence of any mutations in clinical samples and during cell culture passages. 2.6. Phylogenetic analysis Deduced amino acid sequences of 3A region from isolates of present study were compared with 3A sequences of FMDV serotype O available in the GenBank. Phylogenetic comparison was done using MEGA version 4 (Tamura et al., 2007). The evolutionary history was inferred using the neighbour-joining tree method (Saitou and Nei, 1987). The robustness of the tree topology was assessed with 1000 bootstrap replicates as implemented within the program. The evolutionary distances used to infer the phylogenetic tree was computed using the Tamura–Nei method (Tamura and Nei, 1993) and are in the units of the number of nucleotide substitutions per site. 3. Results
705
Table 4 Titres of FMDV field isolates in different cell culture. S. no.
Lab ref. no.
Host
CTY
CK
BuTY
BK
IBRS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
MPC 29/84 APP 33/88 MASg 09/90 GUSk 26/90 MAB 43/90 HAJh 53/00 MAB 28/01 GuP 06/02 APR 36/02 KAB 11/04 KAK 12/04 APP 17/04 APMb 67/04 APMb22/05 HAS 34/05 APRR 84/05 APNg 86/05 APNg 87/05
Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Pig Cattle Cattle Cattle Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo
3.95 4.95 4.60 1.74 4.56 2.20 2.1 4.80 1.80 4.60 4.25 3.24 2.98 3.25 4.25 4.30 4.56 4.60
1.95 1.96 1.96 1.82 3.46 2.94 1.56 1.80 2.5 3.76 3.52 2.74 1.85 1.67 4.20 2.4 2.10 1.8
3.1 4.25 4.50 4.20 4.80 3.80 3.75 4.56 4.20 4.16 4.50 3.60 2.35 2.56 4.50 3.56 2.68 3.10
2.9 2.1 1.78 2.1 3.26 1.92 3.26 3.42 3.70 3.3 3.10 3.42 2.10 1.78 4.52 3.68 3.45 1.67
3.1 4.80 3.76 5.10 2.40 3.10 2.84 4.56 3.10 2.96 2.86 3.91 3.90 4.25 1.80 4.95 4.35 4.85
3.1. FMDV adaptability in different cell culture systems Primary cattle and buffalo kidney, primary cattle and buffalo thyroid cells and IBRS-2 cells were used to adapt and propagate the field virus samples. The ability of FMDV field isolates (n = 18) to multiply in different cell culture systems was evaluated and cytopathic effects (CPE) were recorded 48 h post-infection in each of the three consecutive passages (Table 3). All the eighteen isolates (buffaloes = 14; cattle = 3; pig = 1) could replicate in buffalo and cattle primary cells. All the FMDV isolates included in the study could be adapted to IBRS 2 cell line. However, the O HAS 34/05 isolate produced low virus titers when titrated in IBRS 2 cell line (1.8 TCID50/ml) compared to buffalo and cattle primary cells (>4.1 TCID50/ml). The O HAS 34/05 isolate produced comparatively less CPE in IBRS 2 cell line. A marginal increase in virus titers was noticed when the buffalo isolates were grown in Buffalo kidney cells compared to cattle kidney cells. Similar variation was not noticed in buffalo isolates when they were grown in buffalo and cattle thyroid cells (Table 4). After adaptation to primary cells and IBRS-2 cells, the FMDV isolates were propagated in BHK 21 monolayer cultures. All the 18 FMDV isolates grew well in BHK 21 monolayer cells. Thus, none of the Indian type O FMDV buffalo isolates were confined to grow in a particular host in vitro. However, minor differences in the growth
Table 3 Cytopathic effect of field virus recorded after 48 h post-inoculation in cell culture at third passage level. S. no.
Lab ref. no.
CTY3
CK3
BuTY3
BK3
IBRS3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
MPC 29/84 APP 33/88 MASg 09/90 GUSk 26/90 MAB 43/90 HAJh 53/00 MAB 28/01 GuP 06/02 APR 36/02 KAB 11/04 KAK 12/04 APP 17/04 APMb 67/04 APMb22/05 HAS 34/05 APRR 84/05 APNg 86/05 APNg 87/05
3+ 5+ 4+ 1+ 4+ 2+ 1+ 5+ 1+ 4+ 4+ 2+ 2+ 3+ 2+ 4+ 4+ 4+
1+ 1+ 1+ 1+ 3+ 2+ 1+ 1+ 2+ 3+ 3+ 2+ 1+ 1+ 2+ 1+ 1+ 1+
2+ 4+ 4+ 4+ 4+ 3+ 3+ 5+ 4+ 3+ 4+ 2+ 1+ 2+ 4+ 3+ 1+ 1+
2+ 2+ 1+ 1+ 3+ 1+ 2+ 3+ 3+ 3+ 2+ 3+ 2+ 1+ 3+ 3+ 3+ 3+
2+ 5+ 2+ 5+ 3+ 3+ 3+ 5+ 3+ 3+ 3+ 3+ 3+ 5+ 1+ 5+ 4+ 4+
Note: + indicates presence of cytopathic effect with increment of 20% for each +.
characteristics in terms of virus titer and CPE were noticed when grown in different cell cultures. 3.2. Deduced amino acid sequence comparison of 3A region of FMDV isolates Sequences of 3A coding region were obtained directly from the tissue samples of FMDV infected animals and also from the three consequent passages in different cell cultures (CK, CTY, BuTY, BuK and IBRS-2). 3A sequence from original clinical samples had 100% homology when compared with the 3A sequences of three consequent passages in cell culture systems in all field isolates. The deduced amino acid sequences of 3A region from 18 FMDV field isolates were compared with the 3A sequences of vaccine strains and exotic isolates. Deduced amino acid sequences of 3A region from all Indian type O isolates were 153 aa in length and they lacked deletions or insertions (Fig. 1). The N-terminal half (1–75 amino acid residues) of the 3A region of Indian type O isolates was well conserved whereas all the substitutions were noticed in the C-terminal region (75–153 amino acid residues). Within the C-terminal region 14.6% and 2.4% of amino acid positions had single and multiple substitutions, respectively, between residue numbers 80 and 120. Similarly 40.6% and 9.37% of amino acid positions had single and multiple substitutions, respectively, between residue numbers 121 and 153. Thus, the substitutions were moderate in the region surrounding deletions observed in Taiwan isolates (93–102 amino acid positions) and high in the region surrounding deletions observed in Cambodian isolates (133–143 amino acid positions). Out of the three linear B-cell epitopes identified in 3A (amino acid positions 91–104, 101–114 and 126–139; Hohlich et al., 2003), the first two stretches were almost conserved whereas the last one was highly variable. Hence the first two peptide regions are more suitable for designing diagnostics to detect FMDV infection than the highly variable third B-cell epitope. 3.3. Phylogenetic analyses of FMDV 3A The NJ tree analysis of the predicted amino acids sequence of 3A showed that the selected viruses can be subdivided into two major groups. One group contained viruses with the 93–102 codons deletion such as HKN 1973, HKN 683 and TAW 97 and it was placed distantly from the Indian type O isolates (Fig. 2). The other group contained the Cambodian isolates (CAM 198, CAM 298, CAM 398
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Fig. 1. Alignment of predicted amino acid sequences of 3A coding regions from FMDV type O viruses. All Indian viruses are shown in the chronological order with the predicted amino acids shown in one letter codes; 3A sequences of HKN 1973, HKN 683 and TAW 97 had deletion between 93 and 102 residues. Similarly the 3A sequences of CAM 198, CAM 298, CAM 398 and CAM 1294 showed deletion between 133 and 143 amino acids. All the Indian type O isolates are 153 amino acids in length.
and CAM 1294) of bovine origin with 133–143 codon deletions along with the Indian type O isolates which had full-length 3A. Within the group majority of the earlier Indian isolates formed a separate lineage (highlighted blue in Fig. 2) and recent Indian isolates formed two different lineages (highlighted yellow in Fig. 2). (For interpretation of the references to colour in this
sentence, the reader is referred to the web version of the article.) These later isolates had many changes in and around 133–143 amino acid positions (the region of deletion in Cambodian isolates). Especially few of the recent Indian buffalo isolates (bottom highlighted yellow in Fig. 2) grouped nearer the Taiwan isolates compared to other Indian isolates (Fig. 2) and these isolates
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Fig. 2. Dendrogram showing relationship among selected virus based on predicted amino acids sequences of FMDV 3A protein. Phylogenetic tree was constructed using the NJ algorithm of MEGA version 4 (Tamura et al., 2007).
showed additional substitutions before 93–102 amino acid positions (the region of deletion in Taiwanese isolates). But none of the Indian isolates were closely related to Taiwanese isolates. However, the grouping was not strictly based on the year of isolation. O1 Manisa (exotic isolate) and APP 33/88 which showed higher substitution in and around 133–143 amino acid positions grouped along with the recent isolates, though they were older isolates. Similarly, few of the newer isolates like, APR 36/02 (swine isolate), GuP 06/02 and HAJh 69/00 grouped along with the older isolates. Therefore, it is evident that the viruses containing substitutions in and around the regions of deletions were circulating for long time. However, these viruses have become dominant in recent times. A further, in depth analysis is needed to find out the reason. 4. Discussion Conventionally FMDV was grown in bovine primary cells as well as in IBRS 2 and BHK 21 cell lines without any major host cell restriction in vitro (Ferris and Dawson, 1988; Mowat and Chapman, 1962). However, Beard and Mason (2000) reported that the FMDV isolate O/Taiwan/97 from swine did not grow in bovine thyroid
cells in vitro and replicated well in swine cells. In the present study, all the FMDV type O isolates could grow in cattle and buffalo primary cells and IBRS 2 cell line though there were minor differences in the growth characteristics. The viruses could infect both the bovine and swine cells irrespective of the species from which the virus was isolated. Therefore, the Indian type O isolates of present study were not confined to grow in a particular host in vitro. The 3A region of the Indian isolates was sequenced as 3A was indicated to play a role in host adaptation. None of the isolates had deletion in 3A, which may be the possible reason for the absence of host adaptation. As per Pacheco et al. (2003) complete and stable 3A and 3B copies across all seven serotypes of FMDV are essential for efficient virulence and transmission of the disease in bovines. The appearance of porcinophilic virus (O/Taiwan/97) may be a very atypical and rare adaptation of the viruses with 3A deletions to new intensive pig raising region (Pacheco et al., 2003). Deduced amino acid sequences of 3A region from all Indian type O isolates were 153 amino acids in length and none of the Indian isolates had deletions in their 3A coding region unlike the Taiwan isolates and few of the Southeast Asian isolates (Fig. 1). The Nterminal 75 residues were well conserved and all the amino acid substitutions were noticed in the C-terminal half of the 3A. Within
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the C-terminal half the substitutions were high in and around the region where the deletion was observed in Cambodian isolates (133–143 amino acid residues). Thus, the C-terminal end region surrounding 133–143 amino acid residues of 3A may tolerate mutation without affecting the host adaptation. Interestingly, the region from 85 to 103 where deletions and extensive substitutions observed in egg adapted attenuated virus (Giraudo et al., 1990) and O/HKN/21/70 lineages (procinophilic lineage) did not undergo changes in Indian isolates. Q44R mutation is associated with adaptation and a pathogenic phenotype in guinea pigs (Nunez et al., 2001) and Q44 is found to be conserved in the Indian type O isolates of present study. Except for the deletion between 133 and 143 amino acids, the Cambodian isolates were similar to Indian isolates in their 3A amino acid sequence. Majority of the amino acid substitutions in Indian isolates were observed in and around the region where the amino acid deletion noted in Cambodian isolates (133–143). Few more substitutions were present before the region where the amino acid deletion was observed in Taiwan isolate (93–102). Recent buffalo isolates (2000–2005) had many substitutions in these two regions compared to earlier (1984–1989) buffalo isolates (Fig. 1). The reason for this higher rate of substitutions in recent isolates is not clear. All reported 3A sequences (n = 18) of Indian field FMDV isolates irrespective of the mild differences in 3A region replicated well in BHK cells with viral titers ranging between 2.3 and 4.5 TCID50/ml. This is similar to the findings of Pacheco et al. (2003). They showed that FMD viruses irrespective of size and alteration in 3A produced plaques of indistinguishable shape and size in BHK monolayers with virus titers between 1 108 and 5 108 pfu/ml. The NJ tree analysis of the predicted amino acids sequence of 3A showed that the Indian type O isolates are placed distantly from viruses with the 93–102 codons deletion such as HKN 1973, HKN 683 and TAW 97. However Cambodian isolates (CAM 198, CAM 298, CAM 398 and CAM 1294) of bovine origin with 133–143 codons deletions grouped along with the Indian type O isolates. This coincides with Knowles et al. (2001) findings that Cambodian isolates (O/CAM/11/94, O/CAM/12/94, O/CAM/1/98, and O/CAM/3) are closely related to the Burma viruses (BUR/89), which contain full-length 3A coding regions. In summary, the 3A region of Indian type O isolates exhibited variability in their amino acid sequences and the substitutions were concentrated towards C-terminal end. The non-existence of deletion in 3A region of Indian FMDV isolates may be responsible for the absence of host specificity. Our observation also indicates that Indian buffalo isolates showed higher amino acid substitutions in the 3A region when compared with cattle and swine isolates. In the neighbour-joining tree few of the recent buffalo isolates are on the branch that contain exotic isolates with deletion in 3A. Therefore, it will be interesting to study the 3A region continuously to verify whether this is an indication for deletion in 3A in future. However, none of the Indian isolates are closely related to Taiwanese lineage. Further studies may help to elucidate how new viruses with altered pathogenesis and host range emerge. Acknowledgement The authors thank Dr. B. Mohana Subramanian for the technical help and also for critically reviewing the manuscript. References Barnett, P.V., Cox, S.J., 1999. The role of small ruminants in the epidemiology and transmission of foot-and-mouth disease. Br. Vet. J. 158, 6–13. Beard, C.W., Mason, P.W., 2000. Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus. J. Virol. 74, 987–991.
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Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host. J. Virol. 81, 8497–8506. O’Donnell, V.K., Pacheco, J.M., Henry, T.M., Mason, P.M., 2001. Subcellular distribution of the foot-and-mouth disease virus 3A protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3A. Virology 287, 151–162. Pacheco, J.M., Henry, T.M., O’Donnel, V.K., Gregory, J.B., Mason, P.W., 2003. Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-andmouth disease virus. J. Virol. 77, 13017–13027.
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Short communication
Genetic characterization of Indian type O FMD virus 3A region in context with host cell preference V. Maroudam a, S.B. Nagendrakumar a, P.N. Rangarajan b, D. Thiagarajan a, V.A. Srinivasan a,* a b
Research and Development Centre, Indian Immunologicals Limited, Rakshapuram, Gachibowli Post, Hyderabad 500 032, Andhra Pradesh, India Department of Biochemistry, Indian Institute of Sciences, Bangalore 560 080, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2009 Received in revised form 9 March 2010 Accepted 10 March 2010 Available online 17 March 2010
The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. For example, amino acid deletions in the porcinophilic strain (O/TAW/97) at 93–102 aa of the 153 codons long 3A protein have been recognized as the determinant of species specificity. In the present study, 18 type O FMDV isolates from India were adapted in different cell culture systems and the 3A sequence was analyzed. These isolates had complete 3A coding sequence (153 aa) and did not exhibit growth restriction in cells based on species of origin. The 3A region was found to be highly conserved at Nterminal half (1–75 aa) but exhibited variability or substitutions towards C-terminal region (80–153). Moreover the amino acid substitutions were more frequent in recent Indian buffalo isolates but none of the Indian isolates showed deletion in 3A protein, which may be the reason for the absence of host specificity in vitro. Further inclusive analysis of 3A region will reveal interesting facts about the variability of FMD virus 3A region in an endemic environment. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Foot-and-mouth disease virus Serotype O Cell culture 3A region Genomics
1. Introduction Foot-and-mouth disease (FMD) is a highly infectious viral disease affecting domestic cloven-hoofed cattle, pigs, sheep, goats as well as more than 70 species within 20 families of mammals (Barnett and Cox, 1999; Davies, 2002; Brown, 2003; Grubmann and Baxt, 2004). The disease is a serious threat to livestock industry and creates severe economic consequences for domestic livestock production and international trade. FMD is endemic in India and occurs both in cattle and buffalo (Dutta et al., 1983; Maroudam et al., 2008). The outbreaks in India are ascribed to serotypes A, O and Asia-1 of which serotype O is predominant (Nagendrakumar et al., 2006). The FMD viral genome is a positive-stranded RNA consisting of about 8500 nucleotides. Its single long open reading frame encodes a polypeptide that is cleaved to mature polypeptide products by virally encoded proteinases. In addition to four capsid proteins (VP1–VP4), the RNA-dependent RNA polymerase (3D) and three proteinases (L, 2A and 3C), the FMDV genome encodes several other mature proteins including 3A, a highly conserved protein of 153 amino acids (Mason et al., 2003; Belsham, 1993). Although 3A was found to be associated with intracellular membranes during
* Corresponding author. Tel.: +91 40 23000211/2000894; fax: +91 40 23005958. E-mail address: [email protected] (V.A. Srinivasan). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.03.004
the proliferation of picornavirus, the functions of nonstructural protein 3A in the life cycle of FMDV is less understood (Bienz et al., 1983; Towner et al., 1996). However, alteration in 3A protein, such as point mutations and deletions, was linked to altered host specificity, adaptation, attenuation and virulence in FMDV (Giraudo et al., 1990; Beard and Mason, 2000; Knowles et al., 2001; Nunez et al., 2001, 2007; Pacheco et al., 2003; Nobiron et al., 2005) in hepatoviruses (Graff et al., 1994; Morace et al., 1993), in rhinoviruses (Heinz and Vance, 1996) and in enteroviruses (Lama et al., 1998). Genomic investigation of FMD virus (FMDV) isolated from pigs in Taiwan during the outbreak in 1997 (O/TAW/97) demonstrated that altered nonstructural protein 3A is a primary determinant of restricted growth of the isolate on bovine cells in vitro and significantly contributed to bovine attenuation in vivo (Beard and Mason, 2000). Importance of 3A in host range and virulence have been studied by many investigators for isolates exotic to India (O’Donnell et al., 2001; Nunez et al., 2007), Indian type A and Asia 1 isolates (Mittal et al., 2005; Mohapatra et al., 2009). However the 3A region of Indian FMDV type O isolates has not been studied in detail. Here, we report the results of a study using 18 FMDV Indian type O isolates adapted to different host cells (primary cattle and buffalo cells and Instituto Biologico Renale Swine-2 (IBRS-2)) cell line and comparison of complete sequence of 3A region of these Indian type O isolates with exotic isolates.
V. Maroudam et al. / Infection, Genetics and Evolution 10 (2010) 703–709
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Table 1 List of field viruses used in the study with their place of origin, type of epithelium and vaccination status. Lab ref. no.
Year of isolation
District
State
Species
Epitheilium
Vaccinated status
GenBank accession no.
MPC 29/84 APP 33/88 MASg 9/90 GUSk 26/90 MAB 43/90 MAB 28/01 HAJh 53/00 GuP 06/02 APR 36/02 KAB 11/04 KAK 12/04 APP 17/04 APMb 67/04 APMb22/05 HAS 34/05 APRR 84/05 APNg 86/05 APNg 87/05
1984 1988 1990 1990 1990 2001 2000 2002 2002 2004 2004 2004 2004 2005 2005 2005 2005 2005
Rajpur Prakasam Sangli Sabarkanta Bombay Mumbai Jhajjar Pachanmahal Rangareddy Bangalore Kolar Prakasam Mahboobnagar Mahboobnagar Sirsi Rangareddy Nalgonda Nalgonda
Madhya Pradesh Andhra Pradesh Maharashtra Gujarat Maharashtra Maharashtra Haryana Gujarat Andhra Pradesh Karnataka Karnataka Andhra Pradesh Andhra Pradesh Andhra Pradesh Haryana Andhra Pradesh Andhra Pradesh Andhra Pradesh
Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Pig Cattle Cattle Cattle Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo
Tongue epithelium Tongue epithelium Tongue epithelium Tongue epithelium Tongue epithelium Feet epithelium Feet epithelium Tongue epithelium Snout epithelium Tongue epithelium Tongue epithelium Tongue epithelium Feet epithelium Feet epithelium Feet epithelium Buccal Feet epithelium Feet epithelium
Unvaccinated Unvaccinated Vaccinated Vaccinated Vaccinated Vaccinated Unvaccinated Unvaccinated Unvaccinated Vaccinated Unvaccinated Unvaccinated Unvaccinated Unvaccinated Unvaccinated Vaccinated Unvaccinated Unvaccinated
– GU181359 GU181366 – GU181365 GU181364 GU181361 – GU181360 GU181362 GU181363 GU181358 – – – – – –
Linked Immuno Sorbant Assay (ELISA) (Hamblin et al., 1984) and confirmed by reverse transcription polymerase chain reaction (RTPCR) (Reid et al., 1998).
2. Materials and methods 2.1. FMDV isolates FMDV serotype O virus isolates from buffaloes (n = 14), cattle (n = 3) and swine (n = 1) collected during the period 1984–2005 from different geographical locations of the country were used in this study (Table 1). Indian vaccine strains (OTNN 24/84 and O R2/ 75) were also used in this study. Complete 3A amino acid sequences of type O isolates (n = 12) were obtained from GenBenk for genetic comparisons (Table 2). 2.2. Buffalo and cattle cell cultures Primary cell cultures from cattle and buffalo thyroid and kidney were prepared as described by Freshney (2004). Cells were seeded with a density of 106 cells/ml in growth medium containing 8% fetal bovine serum (BiochromTM) in 25 cm2 flasks and were used for virus isolation and viral bank preparation.
2.4. RNA isolation, RT-PCR amplification and nucleotide sequencing Genomic RNA was extracted from the FMDV infected cell culture supernatants of all passages and clinical epithelial tissue suspension (10%, w/v) using TRIzol reagent (Reid et al., 1998). Ten microliters of total RNA was subjected to one step RT-PCR (Qaigen, USA) using primer pair IIL-3AF (CCC CTC CAG AAT GTG TAC CAG CTTG) and IIL-3AR (GCT TTG TGT TGC CCA TGA CCA TCTT). The amplified RT-PCR products were gel purified with Qiaquick1 Gel Extration Kit (Qiagen, USA) and reconstituted in 30 ml of elution buffer and sequenced in automated DNA sequencer (Applied Biosystems, USA) using Big-Dye Terminator Cycle Sequencing kit v3.0 (Applied Biosystems) in accordance with the manufacturer’s instructions. 2.5. Genetic sequence analysis
2.3. Virus isolation Epithelial tissues (10%, w/v) were inoculated into primary cells buffalo thyroid (BTY), cattle thyroid (CTY), buffalo kidney (BK) and cattle kidney (CK) cells and IBRS-2 cell line. After three consequent passages in these primary cells, the virus was inoculated in BHK-21 cell line. Master seed banks were stored as 50% glycerol stocks at 20 8C until further use. Virus serotyping of epithelial samples and cell culture supernatants was carried out by antigen Enzyme
Overlapping molecular sequences generated by the forward and the reverse primers in this study were manually processed in the GeneDoc Multiple Sequence Alignment version 2.6.002 (Nicholas and Nicholas, 1997). The 3A sequences of exotic isolates were retrieved from GenBank and their accession numbers are given in Table 2. The 3A sequences were aligned with ClustalX algorithm (Thompson et al., 1997) and the aligned amino acid sequences
Table 2 Details of 3A sequences of virus obtained from GenBank. S. no.
Virus ID
Geographic origin
Date of collection date/month/year
Host
Deletion
GenBank accession no.
1 2 3 4 5 6 7 8 9 10 11 12
O/HKN/19/73 O/HKN/6/83 O/TAW/97 O/CAM/1/98 O/CAM/2/98 O/CAM/3/98 O/CAM/12/94 O1 kaufbeuren O1 bfs46 O1 Campos94 O1 Manisa O5 India
Ha Cheung Sha, Lantau Isaland N.T. Kowloon, Hong Kong Pokfulam, Hong Kong Island Yunlin prefecture, Taiwan, POC Borset, Kg. Speu, Cambodia Kg. Speu, Cambodia Kg. Speu, Cambodia Mong Russey District, Battambang Cambodia Germany United Kingdom Argentina Turkey India
19/06/1973 18/12/1982 04/1997 05/01/1998 05/01/1998 05/01/1998 09/1994 1996 Not known Not known 1969 1962
Bovine Bovine Porcine Porcine Bovine Bovine Porcine Bovine Bovine Bovine Bovine Bovine
93–102 93–102 93–102 133–143 133–143 133–143 133–143 None None None None None
AJ294986 AJ294992 AJ308157 AJ294981 AJ294982 AJ294983 AJ294980 X00871 AY593816 AY593819 AY593823 AY593828
V. Maroudam et al. / Infection, Genetics and Evolution 10 (2010) 703–709
were analyzed for the presence of any mutations in clinical samples and during cell culture passages. 2.6. Phylogenetic analysis Deduced amino acid sequences of 3A region from isolates of present study were compared with 3A sequences of FMDV serotype O available in the GenBank. Phylogenetic comparison was done using MEGA version 4 (Tamura et al., 2007). The evolutionary history was inferred using the neighbour-joining tree method (Saitou and Nei, 1987). The robustness of the tree topology was assessed with 1000 bootstrap replicates as implemented within the program. The evolutionary distances used to infer the phylogenetic tree was computed using the Tamura–Nei method (Tamura and Nei, 1993) and are in the units of the number of nucleotide substitutions per site. 3. Results
705
Table 4 Titres of FMDV field isolates in different cell culture. S. no.
Lab ref. no.
Host
CTY
CK
BuTY
BK
IBRS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
MPC 29/84 APP 33/88 MASg 09/90 GUSk 26/90 MAB 43/90 HAJh 53/00 MAB 28/01 GuP 06/02 APR 36/02 KAB 11/04 KAK 12/04 APP 17/04 APMb 67/04 APMb22/05 HAS 34/05 APRR 84/05 APNg 86/05 APNg 87/05
Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo Pig Cattle Cattle Cattle Buffalo Buffalo Buffalo Buffalo Buffalo Buffalo
3.95 4.95 4.60 1.74 4.56 2.20 2.1 4.80 1.80 4.60 4.25 3.24 2.98 3.25 4.25 4.30 4.56 4.60
1.95 1.96 1.96 1.82 3.46 2.94 1.56 1.80 2.5 3.76 3.52 2.74 1.85 1.67 4.20 2.4 2.10 1.8
3.1 4.25 4.50 4.20 4.80 3.80 3.75 4.56 4.20 4.16 4.50 3.60 2.35 2.56 4.50 3.56 2.68 3.10
2.9 2.1 1.78 2.1 3.26 1.92 3.26 3.42 3.70 3.3 3.10 3.42 2.10 1.78 4.52 3.68 3.45 1.67
3.1 4.80 3.76 5.10 2.40 3.10 2.84 4.56 3.10 2.96 2.86 3.91 3.90 4.25 1.80 4.95 4.35 4.85
3.1. FMDV adaptability in different cell culture systems Primary cattle and buffalo kidney, primary cattle and buffalo thyroid cells and IBRS-2 cells were used to adapt and propagate the field virus samples. The ability of FMDV field isolates (n = 18) to multiply in different cell culture systems was evaluated and cytopathic effects (CPE) were recorded 48 h post-infection in each of the three consecutive passages (Table 3). All the eighteen isolates (buffaloes = 14; cattle = 3; pig = 1) could replicate in buffalo and cattle primary cells. All the FMDV isolates included in the study could be adapted to IBRS 2 cell line. However, the O HAS 34/05 isolate produced low virus titers when titrated in IBRS 2 cell line (1.8 TCID50/ml) compared to buffalo and cattle primary cells (>4.1 TCID50/ml). The O HAS 34/05 isolate produced comparatively less CPE in IBRS 2 cell line. A marginal increase in virus titers was noticed when the buffalo isolates were grown in Buffalo kidney cells compared to cattle kidney cells. Similar variation was not noticed in buffalo isolates when they were grown in buffalo and cattle thyroid cells (Table 4). After adaptation to primary cells and IBRS-2 cells, the FMDV isolates were propagated in BHK 21 monolayer cultures. All the 18 FMDV isolates grew well in BHK 21 monolayer cells. Thus, none of the Indian type O FMDV buffalo isolates were confined to grow in a particular host in vitro. However, minor differences in the growth
Table 3 Cytopathic effect of field virus recorded after 48 h post-inoculation in cell culture at third passage level. S. no.
Lab ref. no.
CTY3
CK3
BuTY3
BK3
IBRS3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
MPC 29/84 APP 33/88 MASg 09/90 GUSk 26/90 MAB 43/90 HAJh 53/00 MAB 28/01 GuP 06/02 APR 36/02 KAB 11/04 KAK 12/04 APP 17/04 APMb 67/04 APMb22/05 HAS 34/05 APRR 84/05 APNg 86/05 APNg 87/05
3+ 5+ 4+ 1+ 4+ 2+ 1+ 5+ 1+ 4+ 4+ 2+ 2+ 3+ 2+ 4+ 4+ 4+
1+ 1+ 1+ 1+ 3+ 2+ 1+ 1+ 2+ 3+ 3+ 2+ 1+ 1+ 2+ 1+ 1+ 1+
2+ 4+ 4+ 4+ 4+ 3+ 3+ 5+ 4+ 3+ 4+ 2+ 1+ 2+ 4+ 3+ 1+ 1+
2+ 2+ 1+ 1+ 3+ 1+ 2+ 3+ 3+ 3+ 2+ 3+ 2+ 1+ 3+ 3+ 3+ 3+
2+ 5+ 2+ 5+ 3+ 3+ 3+ 5+ 3+ 3+ 3+ 3+ 3+ 5+ 1+ 5+ 4+ 4+
Note: + indicates presence of cytopathic effect with increment of 20% for each +.
characteristics in terms of virus titer and CPE were noticed when grown in different cell cultures. 3.2. Deduced amino acid sequence comparison of 3A region of FMDV isolates Sequences of 3A coding region were obtained directly from the tissue samples of FMDV infected animals and also from the three consequent passages in different cell cultures (CK, CTY, BuTY, BuK and IBRS-2). 3A sequence from original clinical samples had 100% homology when compared with the 3A sequences of three consequent passages in cell culture systems in all field isolates. The deduced amino acid sequences of 3A region from 18 FMDV field isolates were compared with the 3A sequences of vaccine strains and exotic isolates. Deduced amino acid sequences of 3A region from all Indian type O isolates were 153 aa in length and they lacked deletions or insertions (Fig. 1). The N-terminal half (1–75 amino acid residues) of the 3A region of Indian type O isolates was well conserved whereas all the substitutions were noticed in the C-terminal region (75–153 amino acid residues). Within the C-terminal region 14.6% and 2.4% of amino acid positions had single and multiple substitutions, respectively, between residue numbers 80 and 120. Similarly 40.6% and 9.37% of amino acid positions had single and multiple substitutions, respectively, between residue numbers 121 and 153. Thus, the substitutions were moderate in the region surrounding deletions observed in Taiwan isolates (93–102 amino acid positions) and high in the region surrounding deletions observed in Cambodian isolates (133–143 amino acid positions). Out of the three linear B-cell epitopes identified in 3A (amino acid positions 91–104, 101–114 and 126–139; Hohlich et al., 2003), the first two stretches were almost conserved whereas the last one was highly variable. Hence the first two peptide regions are more suitable for designing diagnostics to detect FMDV infection than the highly variable third B-cell epitope. 3.3. Phylogenetic analyses of FMDV 3A The NJ tree analysis of the predicted amino acids sequence of 3A showed that the selected viruses can be subdivided into two major groups. One group contained viruses with the 93–102 codons deletion such as HKN 1973, HKN 683 and TAW 97 and it was placed distantly from the Indian type O isolates (Fig. 2). The other group contained the Cambodian isolates (CAM 198, CAM 298, CAM 398
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Fig. 1. Alignment of predicted amino acid sequences of 3A coding regions from FMDV type O viruses. All Indian viruses are shown in the chronological order with the predicted amino acids shown in one letter codes; 3A sequences of HKN 1973, HKN 683 and TAW 97 had deletion between 93 and 102 residues. Similarly the 3A sequences of CAM 198, CAM 298, CAM 398 and CAM 1294 showed deletion between 133 and 143 amino acids. All the Indian type O isolates are 153 amino acids in length.
and CAM 1294) of bovine origin with 133–143 codon deletions along with the Indian type O isolates which had full-length 3A. Within the group majority of the earlier Indian isolates formed a separate lineage (highlighted blue in Fig. 2) and recent Indian isolates formed two different lineages (highlighted yellow in Fig. 2). (For interpretation of the references to colour in this
sentence, the reader is referred to the web version of the article.) These later isolates had many changes in and around 133–143 amino acid positions (the region of deletion in Cambodian isolates). Especially few of the recent Indian buffalo isolates (bottom highlighted yellow in Fig. 2) grouped nearer the Taiwan isolates compared to other Indian isolates (Fig. 2) and these isolates
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707
Fig. 2. Dendrogram showing relationship among selected virus based on predicted amino acids sequences of FMDV 3A protein. Phylogenetic tree was constructed using the NJ algorithm of MEGA version 4 (Tamura et al., 2007).
showed additional substitutions before 93–102 amino acid positions (the region of deletion in Taiwanese isolates). But none of the Indian isolates were closely related to Taiwanese isolates. However, the grouping was not strictly based on the year of isolation. O1 Manisa (exotic isolate) and APP 33/88 which showed higher substitution in and around 133–143 amino acid positions grouped along with the recent isolates, though they were older isolates. Similarly, few of the newer isolates like, APR 36/02 (swine isolate), GuP 06/02 and HAJh 69/00 grouped along with the older isolates. Therefore, it is evident that the viruses containing substitutions in and around the regions of deletions were circulating for long time. However, these viruses have become dominant in recent times. A further, in depth analysis is needed to find out the reason. 4. Discussion Conventionally FMDV was grown in bovine primary cells as well as in IBRS 2 and BHK 21 cell lines without any major host cell restriction in vitro (Ferris and Dawson, 1988; Mowat and Chapman, 1962). However, Beard and Mason (2000) reported that the FMDV isolate O/Taiwan/97 from swine did not grow in bovine thyroid
cells in vitro and replicated well in swine cells. In the present study, all the FMDV type O isolates could grow in cattle and buffalo primary cells and IBRS 2 cell line though there were minor differences in the growth characteristics. The viruses could infect both the bovine and swine cells irrespective of the species from which the virus was isolated. Therefore, the Indian type O isolates of present study were not confined to grow in a particular host in vitro. The 3A region of the Indian isolates was sequenced as 3A was indicated to play a role in host adaptation. None of the isolates had deletion in 3A, which may be the possible reason for the absence of host adaptation. As per Pacheco et al. (2003) complete and stable 3A and 3B copies across all seven serotypes of FMDV are essential for efficient virulence and transmission of the disease in bovines. The appearance of porcinophilic virus (O/Taiwan/97) may be a very atypical and rare adaptation of the viruses with 3A deletions to new intensive pig raising region (Pacheco et al., 2003). Deduced amino acid sequences of 3A region from all Indian type O isolates were 153 amino acids in length and none of the Indian isolates had deletions in their 3A coding region unlike the Taiwan isolates and few of the Southeast Asian isolates (Fig. 1). The Nterminal 75 residues were well conserved and all the amino acid substitutions were noticed in the C-terminal half of the 3A. Within
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the C-terminal half the substitutions were high in and around the region where the deletion was observed in Cambodian isolates (133–143 amino acid residues). Thus, the C-terminal end region surrounding 133–143 amino acid residues of 3A may tolerate mutation without affecting the host adaptation. Interestingly, the region from 85 to 103 where deletions and extensive substitutions observed in egg adapted attenuated virus (Giraudo et al., 1990) and O/HKN/21/70 lineages (procinophilic lineage) did not undergo changes in Indian isolates. Q44R mutation is associated with adaptation and a pathogenic phenotype in guinea pigs (Nunez et al., 2001) and Q44 is found to be conserved in the Indian type O isolates of present study. Except for the deletion between 133 and 143 amino acids, the Cambodian isolates were similar to Indian isolates in their 3A amino acid sequence. Majority of the amino acid substitutions in Indian isolates were observed in and around the region where the amino acid deletion noted in Cambodian isolates (133–143). Few more substitutions were present before the region where the amino acid deletion was observed in Taiwan isolate (93–102). Recent buffalo isolates (2000–2005) had many substitutions in these two regions compared to earlier (1984–1989) buffalo isolates (Fig. 1). The reason for this higher rate of substitutions in recent isolates is not clear. All reported 3A sequences (n = 18) of Indian field FMDV isolates irrespective of the mild differences in 3A region replicated well in BHK cells with viral titers ranging between 2.3 and 4.5 TCID50/ml. This is similar to the findings of Pacheco et al. (2003). They showed that FMD viruses irrespective of size and alteration in 3A produced plaques of indistinguishable shape and size in BHK monolayers with virus titers between 1 108 and 5 108 pfu/ml. The NJ tree analysis of the predicted amino acids sequence of 3A showed that the Indian type O isolates are placed distantly from viruses with the 93–102 codons deletion such as HKN 1973, HKN 683 and TAW 97. However Cambodian isolates (CAM 198, CAM 298, CAM 398 and CAM 1294) of bovine origin with 133–143 codons deletions grouped along with the Indian type O isolates. This coincides with Knowles et al. (2001) findings that Cambodian isolates (O/CAM/11/94, O/CAM/12/94, O/CAM/1/98, and O/CAM/3) are closely related to the Burma viruses (BUR/89), which contain full-length 3A coding regions. In summary, the 3A region of Indian type O isolates exhibited variability in their amino acid sequences and the substitutions were concentrated towards C-terminal end. The non-existence of deletion in 3A region of Indian FMDV isolates may be responsible for the absence of host specificity. Our observation also indicates that Indian buffalo isolates showed higher amino acid substitutions in the 3A region when compared with cattle and swine isolates. In the neighbour-joining tree few of the recent buffalo isolates are on the branch that contain exotic isolates with deletion in 3A. Therefore, it will be interesting to study the 3A region continuously to verify whether this is an indication for deletion in 3A in future. However, none of the Indian isolates are closely related to Taiwanese lineage. Further studies may help to elucidate how new viruses with altered pathogenesis and host range emerge. Acknowledgement The authors thank Dr. B. Mohana Subramanian for the technical help and also for critically reviewing the manuscript. References Barnett, P.V., Cox, S.J., 1999. The role of small ruminants in the epidemiology and transmission of foot-and-mouth disease. Br. Vet. J. 158, 6–13. Beard, C.W., Mason, P.W., 2000. Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus. J. Virol. 74, 987–991.
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