R. Venkataramanan

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.

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.

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.

Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge

Vaccination is a well accepted strategy for control of foot-and-mouth disease (FMD) in endemic countries. Currently, chemically inactivated virus antigens are used for preparation of FMD vaccine. To develop a non-infectious and safe recombinant vaccine, we expressed structural polypeptide of FMDV (O/IND/R2/75) using baculovirus expression system. We show that inclusion of mutated viral 3C protease in frame with the polypeptide (P1-2A), enhanced the yield of structural proteins. The structural proteins retained antigenicity and assembled into empty virus-like particles (VLPs). Immunization of guinea pigs with purified fractions of the VLPs resulted in humoral and cell mediated immune response by 4 weeks. The VLPs elicited comparable humoral immune response and relatively higher cell mediated immune response, when compared to conventional vaccine in guinea pigs. Further, up to 70% of the VLP immunized guinea pigs were protected against challenge with homologous guinea pig adapted virus. Our results highlight the application of recombinant FMDV VLPs in FMD vaccination.

Antigenic features of foot-and-mouth disease virus serotype Asia1 as revealed by monoclonal antibodies and neutralization-escape mutants

Neutralizable antigenic sites/epitopes of serotype Asial foot-and-mouth disease virus (strain IND63/72) were identified using monoclonal antibodies (mabs) and their neutralization-escape mutants. Relative affinity/reactivity of the mabs for viral (both native and trypsin-cleaved) and subviral antigens in enzyme-linked immunosorbent assay (ELISA) showed dominance of trypsin-sensitive and conformation-dependent neutralizable antigenic sites. Characterization of neutralization escape mutants identified at least four independent trypsin-sensitive neutralizable antigenic sites on Asial FMD virus. One site was identified by mabs B3, 1A, 24, 2A, 40 and 63, second site by mabs 34 and 81, third site by mab 72 and fourth site by mab 89. The reaction profile of the mabs with selected field isolates in ELISA identified four different neutralization epitopes within the site B3/1A/24/2A/40/63.

Synthesis and antibacterial activity of a novel series of acylides active against community acquired respiratory pathogens.

A novel series of acylides 4 were designed to overcome antibacterial resistance and evaluated for in vitro and in vivo activity. This series of acylides was designed from clarithromycin by changing the substitution on the desosamine nitrogen, followed by conversion to 3-O-acyl and 11,12-carbamate. These compounds showed significantly potent antibacterial activity against not only Gram-positive pathogens, including macrolide-lincosamide-streptogramin B (MLS(B))-resistant and efflux-resistant strains, but also Gram-negative pathogens such as Haemophilus influenzae. These acylides also showed better activity against telithromycin resistant Streptococcus pneumoniae strains.

Serotype C foot-and-mouth disease virus isolates from india belong to a separate so far not described lineage

Complete 1D gene sequences of 13 Indian foot-and-mouth disease virus (FMDV) type C field isolates and a vaccine strain (C-Bombay/64) were determined. All the field isolates showed a greater genetic homogeneity (95-100%) among themselves and were 19.7-21.2% divergent from the vaccine strain. In the phylogenetic analysis, the Indian field isolates formed a separate lineage (lineage VII) different from the previously identified six lineages (lineage I-VI) in type C FMDV [J. Virol. 66 (1992) 3557]. The vaccine strain was grouped with European lineage (lineage II). Comparison of the deduced amino acid sequences of antigenic sites A and C of field isolates showed no significant variation from the vaccine strain. One-way serological relationship determined in ELISA showed antigenic closeness of the field isolates with C-Bombay/64.

Nucleotide Sequence of the Structural Protein-Encoding Region of Foot-and-Mouth Disease Virus A22-India

Nucleotide sequence of the structural protein-encoding region of foot-and-mouth disease virus (FMDV) A22-India 17/77 was determined using non-radioisotopic technique. Comparison of nucleotide and deduced amino acid sequence with A22-Iraq 24/64 revealed 175 synonymous (silent) and 42 non-synonymous nucleotide changes resulting in 34 amino acid substitutions along the capsid proteins (VP1–VP4). Out of the 4 structural proteins VP4 is highly conserved. The highly variable and immunodominant protein VP1 showed 47% of the total amino acid substitutions. VP2 and VP3 contain 38.2% and 14.7% of the amino acid substitutions, respectively. The VP1-based phylogenetic analysis of 18 different type A viruses including A22-India 17/77 divided them in to two broad genetic groups (Asian and European/South American), and each group is further subdivided in to two separate genotypes. A22-India 17/77, A22-Iraq 24/64 and A22-Azerbaijan/65 formed one genotype and the 4 Chinese strains formed a separate genotype in the Asian group of viruses. In the European/ South American group, A-Argentina/87 represents one genotype and the remaining 10 strains formed the second genotype in this group.