Comparative efficacy of virus like particle (VLP) vaccine of foot-and-mouth-disease virus (FMDV) type O adjuvanted with poly I:C or CpG in guinea pigs.

Foot-and-mouth disease (FMD) is one of the most contagious and economically important diseases of cloven-hoofed livestock. Currently used inactivated FMD vaccines have short lived immunity besides risk of handling live virus. We studied recombinant FMD virus like particles (VLPs) encoded by FMDV type O/IND/R2/75 polyprotein genes expressed in Sf9 cells and adjuvanted with CpG or Poly I:C in inducing protective immune response in guinea pigs.

Diagnostic Potential of Recombinant scFv Antibodies Generated Against Hemagglutinin Protein of Influenza A Virus.

Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA) has been a preferred target for generation of neutralizing-antibodies as potent therapeutic/diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment antibodies were constructed using the phage-display technology to aid in diagnosis and treatment of human influenza A virus infections.

Development and evaluation of a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) for rapid detection of foot and mouth disease virus in India.

A simple, rapid and sensitive diagnostic assay for Foot-and-mouth disease (FMD) is required for deployment in the field. In this study, development of Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) assay based on the 3D polymerase gene for specific and rapid detection FMD virus (FMDV) was carried out. The assay was optimised with viral RNA extracted from serotype O, A and Asia 1 FMDV vaccine strains, which resulted a reliable amplification at 65 °C for 60 min.

RNA structure disrupting G320-T transversion within the short fragment of the 5' untranslated region prevents rescue of infectious foot-and-mouth disease virus.

A full-length cDNA clone of an Indian foot-and-mouth disease virus strain, Asia 1 IND 491/1997 was assembled downstream of the T7 promoter in the pBluescript II SK (+) vector by sequential ligation of four PCR-generated subgenomic fragments. RNA transcribed from that construct were transfected into BHK-21 and LFBK cells to rescue infectious virus. The in vitro growth kinetics, plaque morphology, infectivity titer, antigenic profile and virulence characteristics in unweaned mice infected with the recombinant virus were comparable to those infected with the parental virus.

Recombinant non-structural polyprotein 3AB-based serodiagnostic strategy for FMD surveillance in bovines irrespective of vaccination.

In India, the proportion of bovines vaccinated against foot-and-mouth disease (FMD) is increasing since the implementation of the Government supported 'FMD Control Programme', and non-structural protein (NSP)-based serological assays for discriminating between antibodies induced by infection or vaccination (DIVA) could be useful. The FMD virus NSP 3AB was expressed in a prokaryotic system and an indirect ELISA (r3AB(3) I-ELISA) was developed and validated as a screening assay for detecting virus in vaccinated bovines. The diagnostic sensitivity of the assay was estimated to be 96%, while the diagnostic specificity varied between the naïve and vaccinates as 99.

Experimental evidence for competitive growth advantage of genotype VII over VI: implications for foot-and-mouth disease virus serotype A genotype turnover in nature.

In India, systematic genotype replacement has been observed for serotype A foot-and-mouth disease virus. After a decade of co-circulation of genotypes VI and VII, genotype VII emerged as the single dominant genotype since 2001. To derive possible explanations for such epochal evolution dynamics, in vitro intergenotype growth competition experiments involving both co- and superinfection regimes were conducted.

Phylogenetic structure of serotype A foot-and-mouth disease virus: global diversity and the Indian perspective.

Global epidemiological analysis is vital for implementing progressive regional foot-and-mouth disease control programmes. Here, we have generated VP1 region sequences for 55 Indian type A outbreak strains and have included complete VP1 sequences from 46 other countries to obtain a comprehensive global phylogeographical impression. A total of 26 regional genotypes within three continental topotypes, based on a 15% nucleotide divergence cut-off criterion, could be identified.

Multiplex PCR for rapid detection of serotype A foot-and-mouth disease virus variants with amino acid deletion at position 59 of the capsid protein VP3.

In India, there has been co-circulation, extinction and emergence of genotypes/lineages within serotype A foot-and-mouth disease (FMD) virus. At present an antigenically heterogeneous, unique lineage within genotype VII dominates the field outbreaks. This genetic cluster has amino acid deletion at position 59 of VP3 (VP3(59)-deletion group), considered to be critical antigenically.

Phylogenetic analysis of Indian serotype Asia1 foot-and-mouth-disease virus isolates revealed emergence and reemergence of different genetic lineages.

Foot-and-mouth-disease virus (FMDV) serotype Asia1 causes significant number of disease outbreaks in India. Indian Asia1 virus isolates were shown to be genetically heterogeneous and of the two lineages (lineage B and lineage C) described in India, lineage C caused majority of the outbreaks. Emergence of a novel divergent lineage (lineage D) within lineage C has been described in 2001.

Phylogenetic analysis of 3C protease (3C(pro)) coding region of Foot-and-mouth disease virus type A.

Nucleotide sequence analysis of the 3C protease (3C(pro)) region of Foot-and-mouth disease virus type A (FMDV-A) isolates from India has revealed incongruous phylogenetic grouping between 3C(pro) and VP1 region possibly due to the genetic recombination or independent evolution of non-structural and structural protein coding regions. Similar to the VP1 region, the emerging VP3(59)-deletion group maintained its genetic distinctiveness at 3C(pro) region and was found to be diverging with time. Two lineage specific signature aa residues were detected for the deletion group in proof of lineage specific drift or selection events.

Comparative analysis of the large fragment of the 5' untranslated region (LF-5' UTR) of serotype A foot-and-mouth disease virus field isolates from India.

India is endemic for foot-and-mouth disease (FMD) and in recent years a unique group within serotype A, carrying a codon deletion at an antigenically critical site in capsid protein VP3 has emerged (VP3(59)-deletion group). This tempted us to analyze the noncoding region, which is an under represented area, though critically associated with virus biology and pathogenesis. Analysis of the large fragment of 5' untranslated region (LF-5' UTR) of type A FMD virus revealed discrepancy in the overall tree topology between LF-5' UTR and 1D region possibly due to independent evolution of coding and noncoding regions.

Analysis of the leader proteinase (L(pro)) region of type A foot-and-mouth disease virus with due emphasis on phylogeny and evolution of the emerging VP3(59)-deletion lineage from India.

Genotype inclusive grouping of Indian type A isolates as observed in 1D region based phylogeny was distorted at complete L(pro) region, where the VP3(59)-deletion group lineages of genotype VII clustered away from both genotypes VII and VI, confirming its uniqueness and independent evolution of L(pro) and 1D region. Akin to the 1D region, this deletion group is gradually diverging genetically even at L region forming more number of lineages and inter-lineage distance at L region is considerably more than that for 1D region. The deletion group is restricted to India only as none of the exotic sequences clustered within this group.

Enhanced immune response of DNA vaccine (VP1-pCDNA) adsorbed on cationic PLG for foot and mouth disease in guinea pigs.

Foot and mouth disease (FMD) is the major constraint to international trade in livestock and animal products. Though conventional vaccine has shown to provide protection, it has several limitations, like short duration of immunity and poor cell mediated immune response compared to DNA vaccines, which are known to induce both cell mediated as well as humoral responses. The present work envisages the production of DNA vaccine construct with partial 1D gene (coding for VP1) of FMDV type 'A' and studied the efficacy of the vaccine coated on cationic PLGA micro-particles in guinea pigs.

Comparative genomics of serotype Asia 1 foot-and-mouth disease virus isolates from India sampled over the last two decades.

This study deals with a comparative analysis of complete genome sequences of twenty-one serotype Asia 1 foot-and-mouth disease (FMD) field viruses isolated over a period of two decades from India, two vaccine strains and seven exotic sequences. The Indian viruses could be grouped in to three distinct lineages at the entire coding region, evolving independently probably under differential selection pressure as evident from the lineage-specific signatures identified. This comparison revealed 80% of amino acids at the polyprotein region to be invariant.

Assessment of suitability of two serotype A candidate vaccine strains for inclusion in FMD vaccine in India.

The recent type A foot and mouth disease virus field isolates recovered in India are shown to be antigenically quite divergent from the in-use vaccine strain (IND 17/82), warranting the selection of a suitable vaccine strain which can cover this diversity in antigenic spectrum. In earlier studies employing neutralization test with anti-146S rabbit sera raised against eight candidate vaccine strains, IND 81/00 and IND 40/00 belonging to genotype VII were found to offer the best antigenic coverage. In order to assess the credibility of IND 81/00 and IND 40/00 as vaccine strains, 17 recent isolates received during 2005-2006 and representative isolates from older genotypes were subjected to two-dimensional micro-neutralization assay using bovine convalescent serum (against IND 81/00 and IND 40/00) and bovine vaccinate serum (against IND 40/00).

Genotype differentiating RT-PCR and sandwich ELISA: handy tools in epidemiological investigation of foot and mouth disease.

Years of molecular epidemiological surveillance has revealed co-circulation of two antigenically divergent genotypes of foot and mouth disease virus serotype A in India. Genotype differentiating RT-PCR and sandwich ELISA were developed as fast, cost-effective and user-friendly alternatives to 1D region based phylogeny for detection and differentiation of genotype VI and VII. The RT-PCR assay targeting 1D region was found to be more sensitive and authentic in distinguishing genotypes than sandwich ELISA.

Development and comparison of genome detection assays for the diagnosis of foot-and-mouth disease suspected clinical samples.

Detection of foot-and-mouth disease virus (FMDV) from clinical specimens by conventional sandwich enzyme-linked immunosorbent assay (ELISA) and virus isolation in cell culture is often compromised owing to limited sensitivity and inactivation during transit, respectively. A RT-PCR (oligoprobing) ELISA in both solid and aqueous phase hybridization formats targeting an across serotype conserved site at 3C-3D region was developed and its effectiveness was compared with that of the known targets at the IRES region. A non-isotopic RNA dot hybridization assay with colorimetric detection targeting both the IRES and the 3D region were also validated, which is capable of handling high throughput samples with ease.

Sequence analysis of the non-structural 3A and 3C protein-coding regions of foot-and-mouth disease virus serotype Asia1 field isolates from an endemic country.

A total of 18 foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates belonging to two different lineages (including the divergent group) as delineated earlier in VP1-based phylogeny were sequenced in the non-structural 3A and 3C protein-coding regions. The phylogenetic trees representing the regions coding for the non-structural proteins were very similar to that of the structural VP1 protein-coding region. Phylogenetic comparison at 3C region revealed clustering of Asia1 viruses with the isolates of serotypes O, A and C in the previously identified clade.

A novel genetic lineage differentiating RT-PCR as a useful tool in molecular epidemiology of foot-and-mouth disease in India.

Comparison of nucleotide sequences at the VP1 coding region of foot-and-mouth disease serotype Asia1 viruses from India has revealed two genetic lineages with emergence of a genetically divergent group in recent years. In this study a simple, fast, relatively costeffective multi-primer RT-PCR assay to differentiate genetic lineages of type Asia1 viruses was developed. Efforts were made in the design of novel lineage-specific primers and in optimization of the multi-primer assay protocol in conjunction with the use of the serotype specific primer for confirmation of serotype Asia1 virus.

Genetic comparison of large fragment of the 5'untranslated region among foot-and-mouth disease viruses with special reference to serotype Asia1.

Foot-and-mouth disease (FMD), the most economically important disease of cloven-hoofed animals, is endemic in India. Sequence analysis revealed that phylogenetic grouping of type Asia1 field isolates on the basis of the large fragment of the 5'untranslated region (5'LF-UTR) was quite similar to that based on the sequences of the capsid-coding (VP1) region of the same viruses. The existence of two distinct lineages of type Asia1 suggested by the study on the VP1 region was further supported by the detection of a difference in length and predicted secondary structure of the 5'LF-UTR between the two lineages.

Development and evaluation of a multiplex PCR for differentiation of foot-and-mouth disease virus strains native to India.

A multiplex PCR (mPCR) for the differentiation of Indian FMDV serotypes, O, A, Asia 1 and C was developed and evaluated on 142 clinical and 39 cell culture samples. On the latter samples both the tests worked well with 100% efficiency, whereas on clinical samples mPCR had better efficiency than ELISA. The test was found to be specific for FMDV.

Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome.

India is endemic for foot-and-mouth disease and it continues to be a major threat to the livestock industry despite vaccination programmes. In the present study, the ability of specific small interfering (si)RNAs directed against different genomic regions of foot-and-mouth disease virus (FMDV) to inhibit virus replication in BHK-21 cells was examined. For preliminary evaluation of possible siRNA-mediated FMDV inhibition, a cocktail of several unique populations of 12-30bp siRNAs were successfully produced corresponding to three target regions located at structural (VP3-VP1), non-structural (2A-2C), and non-structural-untranslated (3D-3'UTR) region of serotype Asia1.

Complete nucleotide sequence analysis of a vaccine strain and a field isolate of foot-and-mouth disease virus serotype Asia1 with an insertion in VP1 genomic region.

Complete nucleotide sequences except the poly (C) tract and poly (A) tail of a vaccine strain (IND 491/97) and an atypical field isolate (IND 321/01) of Foot-and-mouth disease virus (FMDV) serotype Asia1 are described. Amino acid (aa) sequence analysis of the VP1 protein of the field isolate revealed that the latter has 212 instead of 210 or 211 aa found in the so far available sequences of other FMDV isolates of Asia1 serotype. The insertion was localized in the hypervariable region of aa 130-160 of VP1 protein.