Virus Research 112 (2005) 5259

Antigenic and genetic analyses of foot-and-mouth disease virus type A isolates for selection of candidate vaccine strain reveals emergence of a variant virus that is responsible for most recent outbreaks in India
Rohit Kumar Jangra, Chakradhar Tosh , Aniket Sanyal, Divakar Hemadri, Santanu Kumar Bandyopadhyay
Project Directorate on Foot-and-Mouth Disease, IVRI Campus, Mukteswar, Nainital-263138, Uttaranchal, India Received 5 December 2004; received in revised form 2 March 2005; accepted 2 March 2005 Available online 25 April 2005

Abstract Recent reports indicated presence of two antigenic and genetic groups (genotypes VI and VII) of foot-and-mouth disease virus (FMDV) type A in India and are divergent from the vaccine strains. In order to choose suitable eld isolate as candidate vaccine strain, anti-sera against representative isolates from both the genotypes and two in-use vaccine strains are tested in neutralization assay. Two candidate vaccine strains from both the genotypes are selected with close antigenic match to the eld isolates. From the result it is evident that IND 81/00 (genotypes VII), gave a better antigenic coverage (antigenic relationship (r)-value >0.40 with 79% of isolates of 20022003) than IND 258/99 (genotype VI; r-value > 0.40 with 42% of 20022003 isolates). Phylogenetic analysis based on P1 genomic region placed all the recent isolates (20012003) into genotype VII, with emergence of a new variant virus (VIIbVP359 del) having amino acid deletion at an antigenically critical residue (VP359 ), indicating a major evolutionary jump probably due to immune selection. Though very limited in its extent, this data indicates an apparent dominance of genotype VII over genotype VI and underscores the need to continue further molecular epidemiological investigations to substantiate this nding. 2005 Elsevier B.V. All rights reserved.
Keywords: Foot-and-mouth disease virus; Serotype A; Antigenic analysis; Phylogenetic analysis; Variant virus

1. Introduction Foot-and-mouth disease (FMD) is an extremely contagious viral disease of various artiodactylae animals including cattle, buffaloes, pigs, sheep and goats, and many wild animals. The etiological agent of the disease, foot-and-mouth disease virus (FMDV), is a non-enveloped icosahedral virus of genus Aphthovirus, family Picornaviridae with a singlestranded and positive-sense RNA (Racaniello, 2001). Sixty copies each of the four structural proteins, VP1VP4, and the newly synthesized genomic RNA joins together to form the complete infective virion. Structural proteins VP1VP3 forms outer surface of the virion, while VP4 is entirely internal (Acharya et al., 1989). The three surface exposed

Corresponding author. Tel.: +91 5942 286122; fax: +91 5942 286307. E-mail address: tosh64@email.com (C. Tosh).

capsid proteins carry the virus neutralizing antigenic sites (reviewed in Mateu, 1995). At the antigenic level, FMDV isolates sampled worldwide have been classied into seven distinct serotypes, named serotypes O, A, C, Asia 1 and SAT 13 with multiple subtypes within each serotype (Pereira, 1977). Of the three serotypes (O, A and Asia 1) prevalent in India in recent years, type A is antigenically and genetically more diverse than others. Based on the VP1 gene sequence analysis, FMDV type A exists as 10 genotypes, IX, globally (Tosh et al., 2002). In India, four genotypes have been identied, and in recent years genotypes VI and VII are predominantly co-circulating. Preliminary serological studies using serum neutralization test and sandwich ELISA indicated divergence of few representative isolates of both the genotypes from one of the currently used vaccine strains, IND 17/77 (Anon, 19982001). More elaborate studies of representative serotype A isolates collected over a period

0168-1702/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2005.03.021

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of nine years (19932001) showed a poor inter-genotypic antigenic coverage among the genotype VI and VII isolates (Tosh et al., 2003; Mittal et al., 2005), suggesting the requirement of two strains, one from each genotype, in the vaccine for better efcacy. In such a scenario, a hunt for a strain that shows a close antigenic relationship with isolates from both the genotypes is highly pertinent as it may be used as a potential vaccine strain. Moreover, high endemicity of FMD in India entails molecular epidemiological studies to be continued occasionally for representative isolates across the country to obtain additional information on FMDV evolution. In this study, we report the antigenic and genetic analyses of FMDV type A eld isolates between 1999 and 2003 in order to select a suitable vaccine strain that could provide antigenic coverage to both the groups of viruses (genotypes VI and VII) co-circulating in India.

Table 1 History of the eld isolates used in the study Virus isolate IND 17/77 IND 490/97 IND 106/99 IND 233/99 IND 237/99 IND 252/99 IND 258/99 IND 314/99 IND 317/99 IND 6/00 IND 8/00 IND 40/00 IND 81/00 IND 173/00 IND 174/00 IND 225/00 IND 240/00 IND 331/00 IND 338/00 IND 66/01 IND 68/01 IND 433/01 IND 460/02 IND 461/02 IND 24/03 IND 25/03 IND 30/03 IND 144/03 IND 145/03 IND 153/03 IND 154/03 IND 155/03 IND 161/03 IND 163/03 IND 206/03 IND 209/03 IND 233/03 IND 247/03 IND 270/03 IND 279/03 IND 281/03 Isolation date 00.00.1977 00.00.1982 00.00.1999 00.00.1999 23.03.1999 27.01.1999 12.02.1999 23.07.1999 03.08.1999 20.11.1999 06.11.1999 29.12.1999 29.01.2000 23.03.2000 00.00.2000 17.05.2000 16.05.2000 13.06.2000 26.08.2000 19.09.2000 26.11.2000 00.12.2001 17.12.2002 20.12.2002 25.11.2002 28.11.2002 18.12.2002 28.01.2003 10.01.2003 27.01.2003 00.02.2003 00.00.2003 11.01.2003 29.01.2003 00.00.2002 00.00.2002 00.00.2003 00.00.2002 00.00.2003 15.03.2003 16.03.2003 Place of outbreak Tamilnadu West Bengal Hisar, Haryana Hisar, Haryana Churu, Rajasthan Bhavnagar, Gujarat Bhavnagar, Gujarat Goalpara, Assam Hailakandi, Assam Kurnool, Andhra Pradesh Tinsukia, Assam Bangalore, Karnataka Bangalore, Karnataka Bangalore, Karnataka Hessarghat, Karnataka Bangalore, Karnataka Sonitpur, Assam Assam Assam Darrang, Assam Mizoram Ludhiana, Punjab Mathura, Uttar Pradesh Mathura, Uttar Pradesh Khanapara, Assam Kamrup, Assam Shillong, Meghalaya Mathura, Uttar Pradesh Mathura, Uttar Pradesh Kodagu, Karnataka Bareilly, Uttar Pradesh Moradabad, Uttar Pradesh CampJial, Bihar Mahuabagh, Bihar Sonipat, Haryana Rohtak, Haryana Karnal, Haryana Jhajjar, Haryana Dang, Gujarat Saharanpur, Uttar Pradesh Muzaffar Nagar, Uttar Pradesh Host Bovine Bovine Buffalo Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Buffalo Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Buffalo Bovine Bovine Bovine NA NA Bovine Buffalo Bovine Buffalo NA Buffalo Buffalo

2. Materials and methods 2.1. Viruses Thirty-nine FMDV type A eld isolates collected between 1999 and 2003 and two vaccine strains [IND 17/77 (isolated in 1977) and IND 490/97 (isolated in 1982)] in India were used for antigenic analysis. The histories of the FMDV eld isolates, including host species, year of isolation and geographical distribution are described in Table 1. The virus isolates represent 56 passages in monolayer cultures of BHK21 cells and are obtained from the National foot-and-mouth disease virus repository maintained in this laboratory. Nine isolates collected during 20012003 were sequenced at the capsid-coding (P1) region. 2.2. Animal sera BHK-21 cell monolayer propagated FMDVs were puried to get complete virus (146S) particles and the concentration of 146S particles was estimated as previously described (Wagner et al., 1970; Doel and Baccarini, 1981). Puried 146S particles were inactivated with binary-ethyleneimine (BEI) as per Bahnemann (1975). Rabbit immune sera against BEI-inactivated 146S particles were obtained as previously described (Tosh et al., 2003). 2.3. Neutralization test In order to evaluate the antigenic diversity in the eld isolates, two-dimensional micro-neutralization test (2D-MNT) was carried out using standard method (Rweyemamu et al., 1978), with rabbit polyclonal anti-sera raised against reference viruses. BHK-21 cells were used as the indicator system in neutralization test. The end point titre of the serum against homologous and heterologous viruses was calculated as the reciprocal of the last dilution of serum that neutralizes 100 TCID50 in 50% of the wells. One-way antigenic relation-

NA, not available.

ships (r-value) of the eld isolates relative to the reference strains was calculated, which is expressed as the ratio between heterologous/homologous serum titre. The criteria of Samuel et al., 1990 were applied for interpreting the antigenic relationships; an r-value range of 0.401.00 indicates that the existing vaccine strain provides enough protection; while in the range of 0.200.39 indicates a need for a more potent vaccine. However, r-values below 0.20 stipulate the necessity for a new vaccine strain. 2.4. Viral RNA extraction, polymerase chain reaction and sequencing Viral RNA was extracted from 460 l of infected cell culture supernatant using RNeasy mini Kit (Qiagen) according to the manufacturers protocol, and eluted

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with 50 l of nuclease free water. RNA was reversetranscribed using AMV RT (Promega) and negativesense primer NK61 (5 -GACATGTCCTCCTGCATCTG-3 ; Knowles and Samuel, 1995). The P1 region of the viral genome was amplied using primers NK61 and L463R (virus sense, 5 -ACCTCCRACGGGTGGTACGC; George, 2000) with Hotstar Taq (Qiagen). Polymerase chain reaction (PCR) was carried out on a thermal block (Hybaid) with the following thermal proles: 1 cycle at 95 C (15 min); 35 cycles at 95 C (30 s), 50 C (30 s) and 72 C (3.5 min) and 1 cycle at 72 C (10 min). PCR products were eluted from 1% agarose gel using QIAquick gel extraction kit (Qiagen). Sequencing reactions (fmol kit, Promega) were carried out on puried PCR products using Cy-5 labeled primers NK72 (5 -GAAGGGCCCAGGGTTGGACTC; Knowles and Samuel, 1995), CTL-A20, CTL-A9 (5 -TCTCCAGGTCAGAGAAGTAGTACG and 5 -CTCAGGCGTGTCCGGCGGT, respectively; Tosh et al., 2000), DH1 and DH6 (5 -AACAACTACTACATGCA and 5 -TTGTTCTGAGTGTTGGTTGTGTG, respectively; Sabarinath, 2001) and analyzed on an automated sequencer (ALF Express system II, Pharmacia Biotech). 2.5. Phylogenetic analysis Nine P1 sequences determined in this study were aligned with the 40 published Indian FMDV type A sequences (Tosh et al., 2003; Mittal et al., 2005) and 2 exotic A22 reference strains [A22 Iraq 24/64 (Bolwell et al., 1989) and A22/550 Azerbaijan/65 (GenBank accession number: X74812)] using the CLUSTAL X 1.8 (Thompson et al., 1997). The aligned sequences were used to construct the phylogenetic tree by the neighbor-joining method (Saitou and Nei, 1987), using MEGA 2.1 (Kumar et al., 2001). Evolutionary distances were calculated using two-parameter model of Kimura (1980). The reliability of the branching orders was estimated by bootstrapping (1000 replicates). The tree was rooted using FMDV serotype O strains [O1/Kaufbeuren (GenBank accession no: X00871), O1/Campos (AJ320488) and O2/Brescia (M55287)] as outgroup. Percentage similarities/differences were estimated using Megalign program from Lasergene package (DNASTAR Inc., USA).

3. Results 3.1. Antigenic relationships Antigenic analysis of the eld isolates in relation to the vaccine strain(s) is signicant for testing the appropriateness of the existing vaccine strain as well as for the selection of new vaccine strain(s), if required. One-way antigenic relationships (r-values) of FMDV type A eld isolates against the two vaccine strains [IND 17/77 (genotype IV) and IND 490/97 (genotype VI)] and the eight eld isolates, representing genotypes VI (IND 233/99, IND 237/99, IND 258/99 and

IND 68/01) and VII (IND 6/00, IND 40/00, IND 81/00 and IND 173/00) are shown in Table 2. Of the 29 isolates analyzed against the vaccine strains, none showed an r-value in the range of 0.401.00 with IND 490/97 while one isolate had an r-value in 0.401.00 ranges with IND 17/77 (Table 2a). In case of IND 490/97, 93% (n = 27) of the isolates had their r-values < 0.20 indicating the need for a new vaccine strain. Furthermore, 52% of the isolates had an r-value of less than 0.20 against IND 17/77 while 45% of them demonstrated r-values between 0.20 and 0.39. Among the representatives of genotypes VI, only 24% (n = 7) of the isolates showed an r-value in the range of 0.401.00 against IND 233/99 and IND 237/99, while 38% were in this range against IND 258/99. However, none of the isolates had an r-value > 0.40 with IND 68/01, another representative of genotype VI. The r-values in the range of 0.00.19 were found for 17 (59%), 10 (34%), 8 (28%) and 22 (76%) isolates with IND 233/99, IND 237/99, IND 258/99 and IND 68/01, respectively, implying that the genotype VI isolates are showing a poor antigenic relationships with the recent isolates (Table 2a). Of the four (IND 6/00, IND 40/00, IND 81/00 and IND 173/00) representative isolates of genotype VII under study, IND 6/00 and IND 173/00 behaved poorly with 76 and 72% of the recent eld isolates, respectively, showing r-values < 0.20 (Table 2a). However, 76% of the isolates showed an r-value of >0.40 with IND 81/00, indicating a wide antigenic spectrum of IND 81/00. Only two isolates showed an r-value below 0.20, while ve isolates had r-values in the range of 0.200.39 for IND 81/00. Antiserum to IND 40/00 showed poor antigenic relationship (r-values < 0.20) with 41% of the isolates under study. Moreover, 14% of the recent isolates revealed an r-value between 0.20 and 0.39, while r-values in the range of 0.401.00 were observed in case of 45% of the isolates. In order to have an extensive antigenic comparison of IND 40/00 and IND 81/00, the candidate vaccine strains of genotype VII with broader antigenic reactivity among the four analyzed, the study was further extended to include a total of 40 eld isolates sampled between 1999 and 2003 (Table 2a and b). As compared to IND 40/00, IND 81/00 demonstrated signicantly wide antigenic coverage with 63% (n = 25) of the isolates showing r-values above 0.40, 22% (n = 9) in the range of 0.200.39 and only 15% (n = 6) of the viruses under study below 0.20. The corresponding values for IND 40/00 were 40, 30 and 30%, respectively. Moreover, for more recent isolates (20022003; Table 1), IND 81/00 had antigenic relationship values in the range of 0.401.00 for 79% of the isolates with respect to 37% for IND 40/00. Intra-genotypic antigenic coverage of IND 81/00 (86% of genotype VII isolates studied had an r-value > 0.40) was also found to be better than that of IND 40/00 (50% had an r-value of >0.40). However, there was no signicant difference in the inter-genotypic antigenic coverage of IND 40/00 and IND 81/00 with genotype VI isolates (r-value > 0.40 for 3 and 4 isolates, respectively, out of 14).

R.K. Jangra et al. / Virus Research 112 (2005) 5259 Table 2 One-way antigenic relationship (r-value) of FMDV type A eld isolates in 2D-MNT Genotype Virus (a) Anti-146S rabbit sera against IND 17/77a IV VI IND 17/77 IND 490/97 IND 233/99 IND 237/99 IND 258/99 IND 68/01 IND 6/00 IND 40/00 IND 81/00 IND 173/00 IND 433/01 IND 460/02 IND 24/03 IND 30/03 IND 144/03 IND 153/03 IND 154/03 IND 163/03 IND 270/03 IND 461/02 IND 25/03 IND 145/03 IND 155/03 IND 161/03 IND 206/03 IND 209/03 IND 233/03 IND 247/03 IND 279/03 IND 281/03 Virus 1.00* 0.22 <0.2 <0.2 0.25 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 0.25 <0.2 0.25 0.23 0.25 0.64 <0.2 <0.2 <0.2 0.32 0.22 <0.2 0.24 0.22 0.24 0.32 0.32 IND 490/97a <0.20 1.00 <0.2 <0.2 <0.2 <0.2 0.25 0.33 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 IND 233/99b <0.2 0.38 1.00 0.79 0.75 1.00 0.43 0.38 0.58 0.21 <0.2 <0.2 <0.2 <0.2 <0.2 0.37 <0.2 <0.2 <0.2 <0.2 <0.2 0.50 0.50 <0.2 <0.2 <0.2 <0.2 <0.2 0.25 <0.2 IND 237/99b <0.2 <0.2 0.71 1.00 1.00 1.00 <0.2 <0.2 0.28 <0.2 0.25 0.25 0.24 0.65 <0.2 0.32 <0.2 <0.2 0.32 0.35 0.47 0.24 0.35 <0.2 0.25 0.50 <0.2 0.60 0.25 0.25 IND 258/99b <0.2 <0.2 1.00 1.00 1.00 0.52 <0.2 <0.2 <0.2 <0.2 0.25 0.25 <0.2 0.34 0.34 0.70 0.24 0.29 0.20 0.25 0.24 0.50 0.72 <0.2 0.50 0.25 0.65 0.53 1.00 0.68 IND 68/01b <0.2 <0.2 0.25 0.25 0.25 1.00 <0.2 <0.2 <0.2 <0.2 0.35 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 0.25 0.36 0.38 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 IND 6/00c <0.2 0.74 <0.2 <0.2 <0.2 <0.2 1.00 1.00 0.50 0.50 <0.2 0.50 <0.2 0.36 0.50 0.28 <0.2 0.25 0.25 <0.2 <0.2 0.25 <0.2 0.25 <0.2 <0.2 <0.2 <0.2 1.00 0.50 IND 40/00c 0.67 0.33 0.50 0.23 0.50 <0.2 0.92 1.00 0.61 0.96 <0.2 <0.2 <0.2 0.25 0.50 <0.2 <0.2 <0.2 0.98 <0.2 <0.2 <0.2 <0.2 0.66 0.51 0.36 <0.2 0.48 0.66 1.00 IND 81/00c <0.2 0.35 0.72 0.50 0.50 0.38 0.73 1.00 1.00 1.00 0.70 0.74 0.55 1.00 1.00 0.50 <0.2 0.34 0.65 1.00 0.47 1.00 0.74 0.95 0.55 0.57 0.38 0.52 0.36 0.54

55

IND 173/00c 0.25 0.50 0.25 <0.2 <0.2 <0.2 0.70 0.50 0.50 1.00 <0.2 <0.2 0.25 <0.2 <0.2 <0.2 <0.2 <0.2 0.26 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2

VII

Not known

Genotype

(b) Anti-146S rabbit sera against IND 237/99b IND 258/99b 0.50 0.68 0.50 0.77 0.25 0.72 <0.2 0.73 0.74 ND ND IND 40/00c 0.23 0.46 0.26 0.23 0.23 0.24 0.23 0.23 0.23 0.60 0.92 IND 81/00c 0.33 0.51 <0.2 <0.2 0.34 <0.2 <0.2 0.33 0.33 0.53 0.66

VI

IND 106/99 IND 252/99 IND 314/99 IND 317/99 IND 8/00d IND 240/00 IND 331/00 IND 338/00 IND 66/01 IND 174/00 IND 225/00

0.77 0.50 1.00 0.77 1.00 0.25 <0.2 0.77 1.00 ND ND

VII

Neutralization data for the isolates IND 17/77, IND 490/97, IND 233/99, IND 40/00, IND 237/99, IND 258/99, IND 68/01, IND 6/00, IND 81/00 and IND 173/00 with rabbit anti-sera raised against IND 17/77, IND 490/97, IND 233/99 and IND 40/00 were from the previous studies (Tosh et al., 2003; Mittal et al., 2005). Genotype designation of the eld isolates is shown in roman numerals (dened as in Fig. 1). a Anti-146S rabbit sera against vaccine strains. b Anti-146S rabbit sera against representatives of genotypes VI isolates. c Anti-146S rabbit sera against genotype VII isolates. d Genotyping is based on VP1 gene (Mittal, 2003). r-value, ND, not determined.

Studies with two representative candidate vaccine strains (IND 237/99 and IND 258/99) of genotype VI, which showed comparatively better antigenic coverage among the four analyzed, were further stretched to include a total of 38 iso-

lates. Both the candidate vaccine strains demonstrated good antigenic relationship with isolates of genotype VI as 77% (10 out of 13) of the isolates studied had their r-values in the range of 0.401.00 (Table 2). However, both IND 237/99 and

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IND 258/99 had very poor inter-genotypic antigenic coverage as for each virus only 1 of the 13 isolates studied had their r-values above 0.40. Further, 59% (10 out of 17) of the isolates of year 19992001 revealed good antigenic related-

ness with both IND 237/99 and IND 258/99. On the contrary, IND 258/99 behaved better than IND 237/99 as 42% (8 out of 19) of the isolates of year 20022003 demonstrated a good antigenic relationship (r-values > 0.40) as compared to 21%

Fig. 1. Neighbor-joining tree showing the phylogenetic relationship of FMDV type A isolates. The tree was rooted using FMDV type O strains (O1/Kaufbeuren [GenBank accession no: X00871], O1/Campos [AJ320488] and O2/Brescia [M55287]). Bootstrap support values above 70% out of 1000 replicates are shown near the major nodes. Horizontal branch lengths are drawn to scale. Isolates sequenced in this study are underlined. The genotypes/sub-genotypes are shown in roman numerals. The variant virus isolates are shaded.

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(4 out of 19) for IND 237/99. To sum up, 18 out of 38 isolates under study demonstrated good antigenic match with IND 258/99 as compared to 14 out of 38 isolates for IND 237/99. Thus, in comparison to IND 237/99, IND 258/99 displayed a marginally better antigenic coverage of the eld isolates in the present study. 3.2. P1 sequence analysis In the phylogenetic tree (Fig. 1), all the eld isolates were clustered into previously identied two major genotypes VI and VII (Tosh et al., 2002, 2003) with high condence values (100% out of 1000 bootstrap replicates). An interesting observation was that eight of the nine newly sequenced isolates lacked a codon at nucleotide positions 10841086 [corresponding to VP359 position of IND 17/77 (AF204108)]. These eight isolates grouped closely together forming a distinct cluster within the previously identied sub-genotype VIIb, and bootstrap value of 100% supported this cluster (shaded in Fig. 1); we have named this cluster as sub-genotype VIIbVP359 del to differentiate from the main group. They showed very close genetic relationship with 97.698.8% nucleotide identity among themselves and 91.293.7% identity to other members of sub-genotype VIIb. Another isolate, IND 433/01 (sub-genotype VIId in Fig. 1), although shared branching with VIIc, formed a distinct group (98% bootstrap support) with 91.292% nucleotide identity to VIIc isolates. Alignment of nucleotide and deduced amino acid sequences of P1 region revealed common substitutions that are unique to the eight variant isolates (VIIbVP359 del). These differences were found at 10 synonymous positions (nt 156 A G, 315 A T, 450 C T, 546 A C, 831 T C, 840 A C, 915 C T, 1350 G A, 1371 C T and 1887 C A) compared to IND 17/77 that were not shared with all other Indian isolates analyzed. Similarly, ve deduced amino acid residues were unique to VIIbVP359 del at positions VP2 86 (D N), 88 (T A) and 138 (Y H), VP1 3 (T S) and 168 (R Q).

4. Discussion In this report, we present antigenic analysis of a total of 39 eld isolates sampled between 1999 and 2003 with the vaccine strains and eight eld isolates (IND 233/99, IND 237/99, IND 258/99, IND 68/01 representing genotype VI and IND 6/00, IND 40/00, IND 81/00, IND 173/00 representing genotype VII) in 2D-MNT. Moreover, P1 sequences of nine recent type A isolates (20012003) representing six Indian states were determined and compared with the published sequences to track the epidemiological changes over time. One-way antigenic relationships of the eld isolates were rst analyzed in relation to the reference/vaccine strains. An r-value of <0.20 indicates a signicant serological variation

and applying this criteria it is seen that the majority (93%) of the eld isolates are signicantly different from the vaccine strain IND 490/97 (Table 2a). Similarly, antigenic diversity of the eld isolates from IND 17/77 (another vaccine strain) was also observed; r-value of 00.19 and 0.200.39 was observed with 52 and 45% of the eld isolates, respectively, indicating major antigenic diversity of the eld isolates from the two vaccine strains. This nding supports the recent reports (Tosh et al., 2003; Mittal et al., 2005) on antigenic shift of the Indian type A eld isolates from the current vaccine strains. Of the four genotype VI-specic sera tested (Table 2a), two (IND 233/99 and IND 68/01) gave very poor reactivity, i.e. 024% of the isolates showed an r-value > 0.40, while 2438% of the isolates gave homologous reactivity with IND 237/99 and IND 258/99. Similarly, of the four genotype VII-specic sera tested, two (IND 6/00 and IND 173/00) gave antigenic relationship of >0.40 with 014% of the isolates, whereas 4576% of the isolates were well covered antigenically by IND 40/00 and IND 81/00 (Table 2a). Two candidate vaccine strains each from genotypes VI and VII with close antigenic match to the eld isolates were selected and tested with more number of isolates (3840 isolates) sampled between 1999 and 2003 in 2D-MNT. From the result (Table 2), it is evident that 4063% of the isolates gave an r-value of >0.4 against IND 40/00 and IND 81/00 (genotypes VII), whereas 3747% of the isolates showed an r-value > 0.40 with IND 237/99 and IND 258/99 (genotypes VI). Of the 19 recent eld isolates (sampled during 20022003), 37 and 79% showed homologous reactivity against IND 40/00 and IND 81/00, respectively, indicating antigenic superiority of IND 81/00 over IND 40/00 (Table 2a). But only 21 and 42% of the above isolates gave an r-value > 0.40 with IND 237/99 and IND 258/99, respectively. In general, the led isolates studied could broadly be divided into two major antigenic groups with very poor cross reactivity. To further characterize the eld isolates, nucleotide sequences of the P1 region for a subset of nine randomly selected isolates were generated and compared with each other and the published Indian isolates (Tosh et al., 2003; Mittal et al., 2005). The most striking feature of the results presented is the detection of eight isolates that lacked a codon at VP359 position, which was found to be antigenically critical in FMDV type A10 (Thomas et al., 1988). Furthermore, we detected positive Darwinian selection at VP359 position in FMDV type A (Tosh et al., 2003; Mittal et al., 2005). Deletion of this critical residue in the isolates suggests immune evasion of the variant virus by changing the epitope structure and it may be due to immune pressure. In the remaining one isolate, IND 433/01, no deletion was detected with a total of 2211 nucleotides. In the phylogenetic tree, all the newly sequenced isolates clustered with genotype VII and none grouped with genotype VI (Fig. 1). Of the nine isolates, eight grouped with subgenotype VIIb and the remaining one (IND 433/01) grouped separately in the tree (sub-genotype VIId). The eight isolates

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that grouped within sub-genotype VIIb show 97.698.8% nucleotide identity to one another and 91.293.7% identity to other members of sub-genotype VIIb, and we have named this new cluster as sub-genotype VIIbVP359 del. Analysis of nucleotide and deduced amino acid sequences of P1 region revealed common substitutions that are unique to the eight variant isolates. The differences were at 10 synonymous positions (nt 156 A G, 315 A T, 450 C T, 546 A C, 831 TC, 840 A C, 915 C T, 1350 G A, 1371 C T and 1887 C A) compared to IND 17/77, and were not shared with any other Indian isolates analyzed. Similarly, ve amino acid residues, i.e. VP2 86 (D N), 88 (T A) and 138 (Y H), VP1 3 (T S) and 168 (R Q) were common to this group. From the result, it appears that sub-genotype VIIb was the major source of virus for the recent outbreaks in India, although one isolate belonging to a new sub-genotype, i.e. VIId (IND 433/01) was also identied (Fig. 1). This novel genetic cluster (VIIbVP359 del) was observed as early as November 2002 from the state of Assam (IND 24/03) and by February 2003 it has spread into ve other states including Gujarat at the western and Karnataka at the southern parts of India. This widespread distribution of the current genetic group VIIbVP359 del of FMDV type A implies unrestricted animal movement in the country, which has been demonstrated previously (Pattnaik et al., 1998; Hemadri et al., 2000; Tosh et al., 2002). More or less the FMDV type A status in other regions of the country will be similar to this observation as evidenced indirectly from the close antigenic relationships of the eld isolates (sampled during 20022003) with IND 81/00. Overall, the grouping pattern of the isolates in the tree is of temporal nature (i.e. by year of isolation) rather than geographical isolation. In summary, FMDV type A isolates studied are highly divergent antigenically and genetically from the two vaccine strains. Moreover, none of the genotype-specic candidate vaccine strains gave a complete antigenic coverage to all the isolates studied. Hence, the recent eld isolates could broadly be divided into two antigenic groups with poor cross reactivity among themselves. IND 81/00 (genotype VII candidate vaccine strain) gave a better antigenic coverage (r-value > 0.40 with 79% of 20022003 isolates) compared to IND 258/99 (genotype VI candidate vaccine strain; r-value > 0.40 with 42% of 20022003 isolates). However, obvious disparities in epitope recognition by the immune system across different species entail assessment of credibility of IND 81/00 as candidate vaccine strain in homologous host system. Majority of the recent isolates belonged to genotype VII and none of them was found to group with genotype VI. The molecular epidemiological analysis reveals the emergence of a variant virus with deletion at an antigenically critical residue. This deletion is an indicative of major evolutionary jump, which could be due to immune selection. The probable progenitor of the variant virus was seen rst time from the state of Assam and spread to ve other states (Meghalaya, Bihar, Uttar Pradesh, Gujarat and Karnataka) in the country within

a period of 4 months as revealed from the data. Though very limited in its extent, this data indicates an apparent dominance of genotype VII isolates over that of genotype VI and underscores the need to continue further molecular epidemiological investigations to substantiate this nding. Acknowledgements We thank two anonymous referees for their comments. We are thankful to Indian Council of Agricultural Research for providing necessary facilities to carry out this work. R.K.J. thanks the Indian Veterinary Research Institute (Bareilly, Uttar Pradesh) for his M.V.Sc. fellowship. References
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