Introduction
In India, the Foot and Mouth Disease (FMD) is endemic and occurs in all parts of the country throughout the year. The serotypes prevalent in India are O, Asia 1, A and C. Out of these four serotypes, type-O accounts for more than 85% of the outbreaks followed by types Asia 1 and A. Type C is the least prevalent and has not been recorded since 1995. Though the morbidity rate due to FMD is high, the mortality rate is low. The disease situation is complicated by several factors and the notable ones among them are, (i) a large population of susceptible animals (more than 470 million), (ii) no systematic vaccination and (iii) unrestricted movement of animals within the country. The economic losses caused by the disease are mainly due to loss in milk production and reduction in the working ability of work animals. The direct loss due to this in India is estimated to be more than Rs. 3500-5000 crores per year. In addition to this, the milk and milk products, meat and hide are not accepted by countries free from the disease causing reduction in the export potential of livestock industry in our country.

The work carried out by the All India Co-ordinated Research Project (AICRP) on FMD of Indian Council of Agricultural Research (ICAR) during the last 3 decades has helped in identifying the prevalence of the disease, distribution of virus types, development of quality diagnostics for the precise typing and antigenic analysis and in knowing the molecular epidemiology of the disease.

Molecular epidemiology of FMD
Molecular epidemiology is dependent on the availability of molecular (nucleotide or amino acid) sequences of infectious agents. It has helped to a great extent in tracing the route and spread of any infectious disease in the past few years, which was not completely possible by conventional serological methods. The determination of FMDV nucleotide sequence and phylogenetic analysis is more precise and is now a definite technique used world over for characterizing the individual outbreak strains. This study helps in pinpointing the origin, spread, relatedness and distribution pattern of the different field strains in very clear terms. The basic technology i.e., reverse transcription, polymerase chain reaction and cycle sequencing, is used for obtaining the nucleotide sequence of the FMDV field isolates. The nucleotide sequence data thus generated from the outbreak strains are compared with each other, the vaccine virus strain(s) used in the country, and with the sequences available from the Databank (GenBank). The sequences are aligned, and the results are expressed in the form of a tree (Phylogenetic tree), showing evolutionary relationships between the strains compared, in various specialized computer programs (OMIGATM 2.0, EpiSeq 2.1, PHYLIP). A phylogenetic tree was constructed using the UPGMA method as implemented in the computer program Neighbor and dendrogram plotted using the program DRAWGRAM both from the PHYLIP 3.5c phylogeny package. The UPGMA method constructs a tree by successive (agglomerative) clustering using an average-linkage method of clustering. Finally the dendrograms against each serotype were plotted using the Freelance Graphics program. In the studies pertaining to the molecular epidemiology of FMDV, it has been accepted that isolates showing >15% nucleotide divergence form a distinct genotype, and those showing <5% nucleotide difference are closely related and could be from viruses closely related temporally or from the same outbreak. 

The results are interpreted as the genetic relatedness/divergence among the isolates from the phylogenetic tree. The evolutionary related sequences (or outbreaks caused by single or related strains) cluster at a particular place in the phylogenetic tree compared to the distantly related (divergent) strains either from the same or distantly separated geographical locations. This study is undertaken by comparing the nucleotide sequence of the whole capsid protein (VP1-4) encoding genes or the highly immunodominant capsid protein (VP1) gene of FMDV. 

Phylogenetic analysis of type O isolates
Foot and Mouth disease virus serotype-O is the most prevalent serotype all over the world wherever the disease is prevalent. In India also this is the most prevalent serotype and accounts for more than 85% of the total recorded outbreaks. There are seven geographically distinct genotypes (designated as TOPOTYPES) worldover in the FMDV serotype O. The detailed genetic analysis carried out on nearly 300 Indian field isolates representing the last 15 years showed that they are genetically heterogeneous in nature but all of them belong to a single genotype (designated as Middle-East-South Asia [ME-SA] topotype) with genetic inter-relationship of 0-12%. Within single genotype the isolates could be further subdivided into 16 sub-genotypes.

In order to see how the Indian isolates grouped with those from Bangladesh, Middle East, and countries of Far East Asia, we constructed dendrograms from the published sequences from the above countries. In the dendrogram (Fig.1) all the Indian isolates from India, Bangladesh, Saudi Arabia, Egypt and Turkey formed one genotype. The second genotype was formed by isolates from Far East Asia (China, Hong Kong, Taiwan) and the third one by European isolates. 

From the sequencing studies following conclusions could be drawn

1. Group 1 is the dominant genetic group responsible for the majority of outbreaks caused by FMDV type O in India. This group is widespread in the country and members have no species barrier. Members of this group are also responsible for outbreaks in Indian Sub-continent and the Middle East.

Figure - 1

(Dendrogram showing genetic relationship between FMDV type O strains)

2. Group 3 and 4 are mainly confined to the states of southern India.
3. Within a given state and in a given period FMDV isolates belonging to different genetic group are responsible for field outbreaks. 
4. As the field isolates are not very divergent from the vaccine strain INDR2/75, the present vaccine confers protection against them.

During the year 2000-2001 new outbreaks were recorded in several countries of the world (Japan, South Korea, Russia, South Africa, United Kingdom, Ireland, Netherlands and France) due to type O virus belonging to this ME-SA topotype and the dominant strain which caused all these recent outbreaks has been designated as PAN-ASIAN strain. The detailed sequence analysis of the Indian field strains of type O also revealed that majority of the outbreaks in the country during the last 3 years belong to this genetic group (Pan-Asian). This group also contained some older isolates recovered from the states of Gujarat, Maharashtra, Orissa and Punjab. The isolate IND231/88 recovered from Gujarat in the year 1982 is the oldest member in this group indicating their presence in the country as early as 1982. None of the older isolates (isolated before1994) from southern India, grouped with the above strains, suggesting the possibility of their entry to this region only recently. 

Interestingly, the year 2001 also encountered emergence of a new genetic group which is about 8% divergent from the Pan-Asian group. Preliminary studies indicated that members of this group are responsible for outbreaks in Karnataka, Gujarat, Punjab, Haryana, and Uttar Pradesh. An isolate from Gujarat seems to be the ancestor for this genetic group. This group is responsible for the majority of recent outbreaks in Karnataka. However, both Pan-Asian strain and the new genetic group caused the recent outbreaks in Haryana and Punjab. Sequencing studies also indicated the presence of other genetic groups in the country, but they were/are responsible for sporadic outbreaks.

The amino acid sequence corresponding to the whole VP1 was aligned and analysed. The analysis indicated that the neutralising antigenic sites (1, 3 and 5) on the isolates are conserved and the present vaccine strain would be able to confer protection against them (1,6).

Phylogenetic analysis of type A isolates
Till date 1D gene sequence of 120 isolates (40 partial and 80 complete) recovered from 12 different states of the country have been determined. During the past three years, the partial sequences have been compared with each other and the international strains available in the nucleotide databank and found to be more divergent at the nucleotide level compared to serotypes O and Asia 1 isolates from India. The work was extended to sequence the complete VP1 protein gene of the virus, as it has been argued over the years that more the number of sites compared better and more precise are the results. The complete VP1 protein gene sequences of 80 type A isolates were compared with each other and the vaccine strains (IND 17/77 and IND 490/97 [WBN 17/82]) and the international reference strain A22 IRAQ 24/64. In the phylogenetic tree, drawn using the UPGMA method, the isolates were distributed into three major genotypes (designated as I to III). The genotype I consisted of isolates recovered before 1990 including A22 Iraq 24/64 and one Indian vaccine strain IND 17/77. It comprises of 6 strains, and distributed in 5 different genetic groups. The genotypes II and III seem to co-circulate in the country and are distributed in atleast 12 different states in India. It is to be noted from the dendrogram (Fig.2) that the genotypes I and II share a common ancestor, and genotype III is a recent divergent genotype from these two genotypes. It is important to mention that none of the isolates from 1999 and 2000 outbreaks grouped at any point of time in the phylogenetic tree. The genotypes II and III are made up of 4 and 5 genetic groups respectively, which further support the genetic heterogeneity of the virus in our country. The oldest isolate in the genotype III is IND 21/90, which has approximately 12% (average) nucleotide divergent from the rest of the isolates in the group. In the dendrogram drawn with partial nucleotide sequence of VP1 protein gene, the isolate IND 21/90 was grouped with the isolates from genotype I and II, indicating the evolutionary relationship with this group of virus. Furthermore, IND 21/90 shares some of the amino acids (Arginine at 143 of VP1) with genotypes I and II, and is different (Threonine at 143 of VP1) from most of the isolates in genotype III. 

Virus isolates recovered over a period of 12 years and distributed within a large number of genetic groups reflected the selection and amplification of diverse mutant populations from the quasi species pools of FMD virus in the field. The rapid selection of divergent strains in the field situations could be due to insufficient immunity in animals.

The alignment of the deduced amino acid (a.a.) sequences of 45 FMDV type A strains shows, the amino acid replacements were not distributed along the entire length of the selected VP1 sequence (158-213 a.a. region). The isolates also did not show any uniform pattern of amino acid replacements as per the place of isolation (state-wise distribution) of the isolates. The nucleotide substitution along the sequences compared resulted in amino acid replacement at 16 different positions. There are 2 major areas (VP1 165-171 and 191-198 amino acid regions) on the protein where amino acid replacements are observed. One (VP1 165-171 amino acid region) of these areas is immunodeterminant in case of type A viruses. The residue VP1-168 is located on a distinct loop structure on the protein. In both the Indian (IND17/77 and NDDB-A) vaccine strains there is Arginine (R), whereas in A22-Iraq 24/64 it is Glutamine (Q) at VP1-168. This region in A10-Holland is identified to contain a minor antigenic site. The field isolate IND145/87 seems to be very closely related to IND17/77 genetically as well as antigenically. (3,4,6).

Figure 2

(Dendrogram showing genetic relationship between type A isolates)

Phylogenetic analysis of type Asia1 isolates
The sequence analysis of more than 120 (42 partial and 78 complete VP1 sequences) field isolates of Asia1 collected from different states in the country were sequenced in the VP1 region and, were compared with the isolates whose sequence was generated earlier. (Fig.3). The sequence of two vaccine strains (IND 63/72 and IND 491/97[WBN 117/85]) and one isolate each from China, Pakistan and Israel was used in the comparison. When Asia1 FMDV sequences were grouped according to the criterion followed for poliovirus (viruses with <15% nucleotide divergence), they were segregated in to 4 different genotypes. The Asia 1 strain from Pakistan (PAK 1/54), Israel (Shamir/89) and China (China) formed one genotype each, and all Indian isolates including the two vaccine viruses (IND 63/72 and IND 491/97) formed a single genotype. 

Isolates collected in the recent years form the larger group and are responsible for most of the outbreaks and distributed at least in nine different states in the northern and the southern parts of the country. Further, the prevalence of viruses from more than one genetic lineage is seen in the same state/region within a period of one year. The above observations are to be examined in the light of the fact that there is no restriction on animal movement in the country, except those imposed by natural barriers. When outbreaks occur year round and in all parts of the country, unrestricted animal movement could introduce new strains into areas where they were not quite prevalent, and these new strains could dominate over strains already existing in the area. The small percentages of vaccinated animals also play a minor, nevertheless important role in the emergence of dominant viral strains in a region. This is also evident where a clear epidemiological relationship could be established between the vaccine virus (IND 63/72) and some of the field isolates. This could either be due to inadvertent release of vaccine virus in the field or incomplete inactivation of the virus during vaccine preparation. Based on these findings the following conclusions could be drawn:

1. Sequencing and phylogenetic analysis of Asia1 viruses distributed them into four different genotypes: The prototype Asia 1 virus (PAK 1/54), one isolate each from Israel (Shamir/89) and China formed three individual genotypes. Another genotype was formed of the viruses from India, which comprised of different genetic groups. 

2. A high degree of genetic heterogeneity was observed among the Indian isolates and their distribution in the country probably reflects the lack of restriction on animal movement.

3. Although a high degree of genetic heterogeneity was observed among Indian isolates, sequence comparison of so many isolates from India showed the existence of a single genotype in 15 years.

Figure 3

(Dendrogram showing genetic relationship between type Asia 1 isolates) 

4. Isolates of recent years are distinct and different from the isolates collected in the late eighties and early nineties.

Comparison of the deduced amino acid sequences of the field isolates in relation to the vaccine virus showed a non-synonymous difference of 14-27 residues out of 409 non-synonymous positions. This clearly showed that whatever non-synonymous changes are there in the field isolates they are not getting fixed whereas, synonymous changes are responsible for nucleotide divergence in the population. The nucleotide substitutes/site/year was approximately 1.32 X 10-2. 

The nucleotide and deduced amino acid sequence in the 130-160 region (codons) of VP1 polypeptide of sixty-five field isolates of foot-and mouth disease (FMD) virus serotype Asia1 was determined and the consensus/majority residues were deduced. Comparison of the amino acid sequences among the field isolates revealed conservation of residues at 130-132, 134-136, 142-144, 148 and 156-160. Variation was observed among the field isolates at rest of the amino acid residues between 133 to 138 and 148 to 154 regions which were unique for Asia1 virus and are presumably responsible for the distinct antigenic nature of this serotype. The two stretches of amino acid TQPT (Thr-Gln-Pro-Thr neutral) groups associated with VSNR (Val-Ser-Asn-Arg alkaline) tetrapeptides instead of ETTS (Glu-Thr-Thr-Ser acidic), and L (M/S) GQ (Leu-Met/Ser-Gly-Gln less alkaline) tetrapeptides were observed in many isolates. The list of distribution seems to suggest that at least two clusters of Asia 1 isolates were parental viruses mutating under natural conditions and diverging step by step.

The amino acid sequence alignment shows that substitutions are scattered along the entire length of the sequence. There are areas where the frequency of substitution is higher. Three such regions i.e., the G-H loop (position 140-160), the B-C loop, (position 40-60), and the C-terminus of VP1 (position 210-211). One extra amino acid was found at position VP1 44 in majority of the viruses (1,2,5,6). 

References

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