Divakar Hemadri

Phylogenetic analysis of serotype A foot-and-mouth disease virus isolated in India between 1977 and 2000.

Summary. The genetic diversity among the Indian serotype A Foot-and-mouth disease virus (FMDV) isolates sampled over a period of 24 years (1977–2000) was studied by sequencing the VP1 gene. In the phylogenetic tree, constructed from 83 Indian and 37 other available sequences, the FMDV type A isolates were distributed into 10 major genotypes (designated as I–X). The Indian isolates were distributed in 4 genotypes (I, IV, VI and VII), and co-circulation of at least 2 genotypes (VI and VII) in different states of the country in recent years is evident from the result. The study also revealed differential geographic distribution of genotypes, for example, some (genotypes I and VII) were recovered from large geographical areas, some time even across the continents, suggesting the spread of the viruses beyond continental barriers. Localization of genotypes restricting to a particular country (genotypes III and X) was also observed in the study. The genetic diversity in the field isolates has been discussed in relation to the amino acid substitutions at the known antigenic sites. This work provides valuable insights into the epidemiological situation of type A FMDV in India and necessary information in the selection of suitable vaccine strain(s), if required, for the National FMD control program.

Multiple origins of foot-and-mouth disease virus serotype Asia 1 outbreaks, 2003-2007.

Viruses in 6 genetic groups have caused recent outbreaks in Asia.Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence.

Emergence of a New Strain of Type O Foot-and-Mouth Disease Virus: Its Phylogenetic and Evolutionary Relationship with the PanAsia Pandemic Strain

In India, Foot-and-mouth disease virus (FMDV) serotype O has been associated with more than 75% of the outbreaks. Previous studies with this serotype have indicated that the viruses circulating in India belong to a single genotype. Recent (February 2001) FMD epidemics in Europe have focussed global attention on the source of the virus and have been traced to a strain, PanAsia (serotype O), which is present in India since 1990. In this study, to further characterize the isolates belonging to the PanAsian strain, we sequenced the complete VP1-encoding (1D) gene for 71 FMDV serotype O isolates from India recovered from the field outbreaks during the last 4 decades (1962–2001). All the isolates in the tree were distributed in to three major branches (designated as A, B and C); the branch C is further divided into four groups (I–IV), of which the group IV belongs to the PanAsia strain. Furthermore, we show that the PanAsia strain has been circulating endemically since 1982 (not 1990 as reported earlier) and has been the most dominant outbreak strain in the recent years and distributed at least in 17 states of the country. During the year 2001, another new group (group III) of virus with genetic divergence of 5.4–11.1% at nucleotide level from the PanAsia strain is found to co-circulate endemically, and is slowly replacing it. At amino acid level this strain differed from PanAsia strain at five amino acid positions in the VP1. Although these strains are divergent at nucleotide level, they maintained a good antigenic relationship with one of the vaccine strains (IND R2/75) widely used in the country. Given the ability of the PanAsia virus to persist, spread and to outcompete other strains, the present trend could be of serious concern as the newly emerging virus is replacing it. If this is true, then there is another equally divergent strain as PanAsia that may pose a serious threat to the global dairy and meat industries.

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1.

Summary. Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia 1 virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I–IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 × 10−2 synonymous substitutions per nucleotide per year.

Sequence and phylogenetic analysis of the L and VP1 genes of foot-and-mouth disease virus serotype Asia1

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, we determine the nucleotide (nt) sequence of the L (603 nt) and VP1 (633 nt) genes of 27 FMDV serotype Asia 1 isolates recovered from different outbreaks in India, and compared with each other and the vaccine strain, IND 63/72, used in the country. Independent phylogenetic analyses on both the aligned gene sequences identified two major lineages (designated A & B) in the Asia 1 isolates. Both L- and VP1-based trees were congruent with respect to the major branching pattern of the isolates. The lineage A is represented by the isolates of 1986-2000 including the vaccine strain IND 63/72, whereas, lineage B appeared to be dominant and responsible for most of the recent outbreaks. A correlation was observed between the clustering of the isolates in the phylogenetic tree and the amino acid changes at many of the positions in VP1 as well as in L protein. The annual rate of evolution in L and VP1 genes was found similar and estimated to be 4.0 x 10(-3) and 3.8 x 10(-3) substitutions per nucleotide, respectively. Our result, largely from the congruence in phylogenetic trees and the rate of evolution in both the genes, suggests the possibility for the use of L gene sequence in phylogenetic comparison of FMDV.

Genetic and antigenic analysis of two recently circulating genotypes of type A foot-and-mouth disease virus in India: evidence for positive selection in the capsid-coding genes

Summary. We have analyzed isolates of two recently circulating genotypes (genotypes VI and VII) of type A foot-and-mouth disease virus (FMDV) from India. Maximum-likelihood models provided support for the presence of positively selected sites in the capsid-coding (P1) region. Positive selection was detected at a number of amino acid positions behind a background of strong purifying selection. Among the positively selected sites, four were identified at known critical antigenic residues (VP2 79, VP3 59 and 70 and, VP1 83), suggesting that FMDVs are under pressure from the immune system. Two residues (VP2 134 and VP3 59) that are part of the heparan sulfate-binding pocket in subtype A22 FMDV are also inferred to be under positive selection. Antigenic divergence was observed between and within the genotypes in neutralization tests with sera raised against the representative isolates from genotypes VI and VII. The two vaccine strains showed one-way antigenic relationships (r value) of <0.2 with 64% of the isolates, whereas, with genotypes VI and VII an r value of >0.4 was observed with 24% and 64% of the isolates, respectively. No correlation could be deduced from the amino acid substitutions at specific critical residues and lower r values in the field isolates.

Phylogeny, genome evolution, and antigenic variability among endemic foot-and-mouth disease virus type A isolates from India

Summary.The capsid-coding (P1) and 3A regions of foot-and-mouth disease virus (FMDV) type A field isolates including two vaccine strains collected during 1977–2000 were analyzed. In the phylogenies, the isolates were distributed into two previously identified genotypes VI and VII, with multiple sub-genotypes that are temporally clustered. Comparison of the antigenic relationships of field isolates with the two vaccine strains (IND 17/77 and IND 490/97) and the reference strains of the genotypes VI (IND 233/99) and VII (IND 40/00) indicated two broad antigenic groups that correlate with the phylogenetic groupings (genotypes VI and VII), and are highly divergent from the vaccine strains. The maximum likelihood method of selection analysis identified a number of amino acid sites within the P1 region to be under weak positive selection. Some of the positively selected sites were mapped at/near the antigenically critical amino acid sites of the P1 region, indicating host immune pressure as one of the important driving force behind the observed genetic and antigenic diversity in FMDV. A small number of selected sites are located in the heparan sulphate-binding pocket of the virus, suggesting a fitness advantage for cell entry of the virus. No positive selection was detected in the 3A dataset.

The non-structural leader protein gene of foot-and-mouth disease virus is highly variable between serotypes.

Aphthoviruses are unique among picornaviruses in that they alone encode a functional L proteinase as the first component of the viral polyprotein. The L genes of a few Indian foot-and-mouth disease viruses were sequenced and compared with those available to study the extent of variation in this gene. Besides the two in-frame start codons present in all FMDV L genes, the Asia-I vaccine virus had an additional in-frame AUG (start) codon, at codon position 3. Amino acid sequence comparison revealed that 39.8% of positions were capable of accepting replacements, yet the residues of the catalytic dyad were totally conserved. Sequence comparison at the C-terminus of the protein indicated that K/R↓GAGQS is sufficient for L/P1 cleavage. Phylogenetic analysis based on the L gene sequences did not reveal any serotype-specific clustering. The probable implications of the observed high variability in this non-structural gene is briefly discussed.

Characterization of foot-and-mouth disease serotype Asia1 viruses grown in the presence of polyclonal antisera in serology and nucleotide sequence analysis

Summary.Foot-and-mouth disease viruses (FMDV) have a high rate of mutation and spontaneous mutants can be readily isolated in the laboratory. In this study, plaque purified FMDV Asia1 vaccine strains (IND 63/72 and IND 491/97) were passaged in-vitro in Baby Hamster Kidney-21 cell monolayers in the presence of sub-neutralizing levels of antiviral polyclonal sera (APS), raised in guinea pigs against the purified and inactivated whole virus particles of IND 63/72, IND 491/97 and IND 13/01. After serial passages under selective immune pressure, the viruses starts growing in the presence of undiluted sera and showed certain characteristics like an increased resistance to neutralization by APS and reduction in plaque counts on titration in plaque assay. Cross-neutralization of these viruses with above-mentioned APS revealed selection of three complete and one partial polyclonal antibody resistant (PAR) viruses based on the ‘r’ value in micro neutralization test. Alterations were detected at several amino acid residues in the structural protein-coding P1 region. Many of the residues inferred to be positively selected sites in other serotypes of this virus were also prone to substitution under immune selection pressure in Asia1 virus. The present work extends the finding that selection exerted by host antibody also plays a major role in the rapid evolution of FMDV Asia1, as observed in other serotypes.

Comparative complete genome analysis of Indian type A foot-and-mouth disease virus field isolates

Comparative complete genome analysis of 17 serotype A Indian field isolates representing different genotypes and sub-lineages is presented in this report. Overall 79% of amino acids were invariant in the coding region. Chunk deletion of nucleotide was observed in S and L fragment of 5′-UTR. More variability which is comparable to that of capsid coding region was found in L and 3A region. Functional motifs and residues critical for virus biology were conserved most. Polyprotein cleavage sites accepted few changes. Many sites were detected to be under positive selection in L, P1, 2C, 3A, 3C, and 3D region and of which some are functionally important and antigenically critical. Genotype/lineage specific signature residues could be identified which implies evolution under different selection pressure. Transmembrane domain could be predicted in 2B, 2C, 3A, and 3C proteins in agreement with their membrane binding properties. Phylogenetic analysis at complete coding region placed the isolates in genotype IV, VI, and VII and two broad clusters comprising VP359-deletion and non-deletion group within genotypes VII. The VP359-deletion group has diversified genetically with time giving rise to three lineages. Incongruence in tree topology observed for different non structural protein coding region and UTRs-based phylogeny indicate suspected recombination.