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Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of livestock, primarily affecting cattle, buffalo and pigs. FMD virus serotypes O, A and Asia1 are prevalent in India and systematic efforts are on to control and eventually eradicate the disease from the country. FMD epidemiology is complex due to factors like co-circulation, extinction, emergence and re-emergence of genotypes/lineages within the three serotypes, animal movement, diverse farm practices and large number of susceptible livestock in the country. Systematic vaccination, prompt diagnosis, strict biosecurity measures, and regular monitoring of vaccinal immunity and surveillance of virus circulation are indispensible features for the effective implementation of the control measures. Availability of suitable companion diagnostic tests is very important in this endeavour. In this review, the diagnostic assays developed and validated in India and their contribution in FMD control programme is presented.

Foot-and-mouth disease (FMD) virus serotype O Ind2001 lineage within the Middle East-South Asia topotype is the major cause of recent FMD incidences in India. A sub-lineage of Ind2001 caused severe outbreaks in the southern region of the country during 2013 and also reported for the first time from Libya. In this study, we conducted a detailed evolutionary analysis of Ind2001 lineage. Phylogenetic analysis of Ind2001 lineage based on maximum likelihood method revealed two major splits and three sub-lineages. The mean nucleotide substitution rate for this lineage was calculated to be 6.338×10(-3)substitutions/site/year (s/s/y), which is similar to those of PanAsian sub-lineages. Evolutionary time scale analysis indicated that the Ind2001 lineage might have originated in 1989. The sub-lineage Ind2001d that caused 2013 outbreaks seems to be relatively more divergent genetically from other Ind2001 sub-lineages. Seven codons in the VP1 region of Ind2001 were found to be under positive selection. Four out of 24 recent Ind2001 strains tested in 2D-MNT had antigenic relationship value of <0.3 with the serotype O vaccine strain indicating intra-epidemic antigenic diversity. Amino acid substitutions found in these minor variants with reference to antigenic diversity have been discussed. The dominance of antigenically homologous strains indicates absence of vaccine immunity in the majority of the affected hosts. Taken together, the evolution of Ind2001 lineage deviates from the strict molecular clock and a typical lineage evolutionary dynamics characterized by periodic emergence and re-emergence of Ind2001 and PanAsia lineage have been observed in respect of serotype O., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease virus (FMDV) serotype Asia1 was first reported in India in 1951, where three major genetic lineages (B, C and D) of this serotype have been described until now. In this study, the capsid protein coding region of serotype Asia1 viruses (n = 99) from India were analyzed, giving importance to the viruses circulating since 2007. All of the isolates (n = 50) recovered during 2007-2013 were found to group within the re-emerging cluster of lineage C (designated as sublineage C(R)). The evolutionary rate of sublineage C(R) was estimated to be slightly higher than that of the serotype as a whole, and the time of the most recent common ancestor for this cluster was estimated to be approximately 2001. In comparison to the older isolates of lineage C (1993-2001), the re-emerging viruses showed variation at eight amino acid positions, including substitutions at the antigenically critical residues VP279 and VP2131. However, no direct correlation was found between sequence variations and antigenic relationships. The number of codons under positive selection and the nature of the selection pressure varied widely among the structural proteins, implying a heterogeneous pattern of evolution in serotype Asia1. While episodic diversifying selection appears to play a major role in shaping the evolution of VP1 and VP3, selection pressure acting on codons of VP2 is largely pervasive. Further, episodic positive selection appears to be responsible for the early diversification of lineage C. Recombination events identified in the structural protein coding region indicates its probable role in adaptive evolution of serotype Asia1 viruses.

The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Three of the seven serotypes of foot-and-mouth disease (FMD) virus are prevailing in India. A massive vaccination campaign is on to control and eradicate the disease from the country. However, FMD vaccines provide short term immunity, hence regular assessment of antibody level in the vaccinated herds is indispensible for the success of the control programme. The antibodies are quantitatively estimated, either by virus neutralization test or by end-point dilution liquid-phase-blocking ELISA (LPBE). Millions of cattle and buffalo in the country are now systematically vaccinated, and thousands of serum samples are routinely screened in the country for estimation of herd immunity against FMDV serotypes O, A and Asia1. Testing such a large number of serum samples within limited a period of time by the conventional end point dilution method of LPBE requires lots of man power, and biological reagents. A more economical high throughput single dilution LPBE (SdLPBE) assay was optimized and validated for quantitative estimation of antibody levels against the three FMD virus serotypes. The assay was thoroughly validated against LPBE method before adopting it for country-wide use. The biological reagents used in the assay were prepared in thermo-stable form to enable transportation to the field level FMD diagnostic laboratories., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

The complete nucleotide sequence of a new lineage of foot and mouth disease virus (FMDV) serotype O was determined. The lineage designated as Ind2011 first appeared during 2011 in the Southern region of India. Excluding the poly C tract and poly A tail, the genome of Ind2011 ranged from 8,169 to 8,172 nucleotides. Variation in the genome length was due to insertions/deletions in LF-UTR. The lineage had a higher sequence identity with lineage PanAsia-1 at P1 and P2 regions, and with lineage PanAsia-2 at P3 and L regions. Phylogenetically, the isolates were placed closely to both PanAsia-1 and 2 lineages, and appear to be a novel variant of the PanAsian lineage.

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. In India, the disease is endemic in nature and is controlled primarily by prophylactic bi-annual mass vaccination. In this control programme, liquid-phase blocking ELISA (LPBE) is being used widely for post vaccination seromonitoring. In order to develop an alternative assay to LPBE, the recombinant capsid polyprotein (rP1) of FMD virus (FMDV) serotype O was expressed in Escherichia coli and used as an antigen for the detection of antibodies to FMDV. The capsid polyprotein of FMDV serotype O could be expressed successfully as a recombinant 6xHis-SUMO tagged protein in soluble form. In a Western blot assay, the rP1 protein reacted strongly with anti-FMDV serotype O guinea pig and bovine serum. Further, in this study, an rP1 protein-based solid phase competitive ELISA (rP1-SPCE) was developed and evaluated with a set of serum samples representing the various epidemiological situation of the country. The performance of the rP1-SPCE was compared with the in-house LPBE, and overall, an excellent agreement (kappa = 0.95) was observed between the two tests. This report demonstrates that the recombinant capsid polyprotein-based ELISA has the potential to be an easy-to-perform, safe alternative to the conventional LPBE for the quantitative detection of antibodies to FMDV serotype O., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study involves evaluation of antioxidant potential of Crocus sativus and its main constituents, safranal (SFN) and crocin (CRO), in bronchial epithelial cells, followed antiinflammatory potential of the active constituent safranal, in a murine model of asthma. To investigate the antioxidizing potential of Crocus sativus and its main constituents in bronchial epithelial cells, the stress was induced in these cells by a combination of different cytokines that resulted in an increase in nitric oxide production (NO), induced nitric oxide synthase (iNOS) levels, peroxynitrite ion generation, and cytochrome c release. Treatment with saffron and its constituents safranal and crocin resulted in a decrease of NO, iNOS levels, peroxynitrite ion generation, and prevented cytochrome c release. However, safranal significantly reduced oxidative stress in bronchial epithelial cells via iNOS reduction besides preventing apoptosis in these cells. In the murine model of asthma study, antiinflammatory role of safranal was characterized by increased airway hyper-responsiveness, airway cellular infiltration, and epithelial cell injury. Safranal pretreatment to these allergically inflamed mice lead to a significant decrease in airway hyper-responsiveness and airway cellular infiltration to the lungs. It also reduced iNOS production, bronchial epithelial cell apoptosis, and Th2 type cytokine production in the lungs., (Copyright © 2015 John Wiley & Sons, Ltd.)

In recent times, majority of the foot-and-mouth disease (FMD) outbreaks in India are caused by serotype O Ind2001 lineage. The lineage has diverged into four sub-lineages (Ind2001a, b, c and d). We report here the genetic and antigenic analyses of nine Ind2001d isolates that caused outbreaks during April 2013-March 2014 in India. The length of the genomes of outbreak viruses varied between 8153 and 8181 nucleotides without any insertion or deletion in the coding region. Of the nine isolates analyzed antigenically against the currently used Indian vaccine strain INDR2/1975, eight showed good cross serological match (>0.3) indicating optimal antigenic coverage by the vaccine strain. An unprecedented deletion of 22 nucleotides between position 57 and 78 was observed in the 3' untranslated region of one of the isolates without compromising the virus viability, which imply that partial distortion in SL2 of 3'UTR may not have influence on virus viability at least under in-vitro conditions. Recently the Ind2001 lineage has been reported from several countries including Libya and spread of this lineage across a wide geographical area needs to be monitored carefully to avoid any future pandemic., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 βE-βF loop, VP1 C-terminus and VP3 β-B knob) surrounding the fivefold symmetry axis (VP1 βF-βG loop) and at other discrete sites on the capsid (VP3 βG-βH loop, VP1 C-terminus, VP2 βC strand and VP1 βG-βH loop). A lysine insertion in the VP1 βF-βG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 βG-βH loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 βB-βC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production., (© 2015 The Authors.)

Field outbreak strains of foot-and-mouth disease virus (FMDV) infect host cells through certain Arg-Gly-Asp (RGD) dependent integrin family of cellular receptors. In contrast, FMDV adapted in non-host cell cultures are reported to acquire the ability to infect cells via heparin sulphate (HS) or other unidentified cell surface molecules. It has been reported that during the serial passage of FMDV serotype A in BHK-21 cell culture, VP2 E131K (E2131K) substitution was fixed within the heparin sulphate binding site. The fixation of positively charged residue at position VP2 131 of serotype A is considered to associate with the ability to utilise alternative receptor. In this study, an infectious full-length cDNA clone for Indian FMDV vaccine strain A IND 40/2000 was constructed. Through site-directed mutagenesis on the cDNA clone, recombinant virus containing positive charged amino acid residue at position VP2 131 was rescued. The recombinant mutated virus was shown to have specific and strong affinity for HS and demonstrated an enhanced infectivity in BHK-21 cell line. The introduction of lysine residue at VP2 131 position that allows cell culture adaptation of FMDV serotype A could be exploited for the generation of vaccine seed stocks with improved growth properties in BHK-21 cell line., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Foot-and-mouth disease (FMD) is a highly infectious disease of transboundary importance. Routine biannual vaccination along with surveillance activities is seen as the principal to control FMD in India. Non-structural protein (NSP) based immunoassays are the test of choice for the differentiation between infected and vaccinated population. In this study, 3D protein of FMD virus was expressed in Escherichia coli and an indirect ELISA (I-ELISA) was developed to detect 3D-antibodies in the infected bovines. 3D I-ELISA demonstrated comparable diagnostic sensitivity (97.6%) but lower specificity (80.8%) as compared to the in-house r3AB3 I-ELISA. However, the specificity values varied significantly for naïve and vaccinated samples and were observed to be 98.42% and 76.93%, respectively. A moderate degree of concordance (88.5%) was observed between the overall results of two ELISAs. 3D I-ELISA displayed a considerably lower specificity in uninfected vaccinated samples, thereby suggesting against its application for serosurveillance in intensively vaccinated population. However by virtue of its high diagnostic sensitivity and longer duration of persistence of 3D-antibody post-infection, 3D I-ELISA could be adopted for seroepidemiological investigations in regions not practicing vaccination and could be extended to susceptible species which are generally not covered by vaccination., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Ursolic acid (1), a natural pentacyclic triterpenic acid, afforded a variety of ring-C transformed products (5-11) when treated with N-bromosuccinimide under the influence of a range of protective groups and solvents. The synthesized compounds have been evaluated for cytotoxic activity against prostate PC 3, leukemia THP 1, cervical Hela and lung A-549 cancer cell lines. Among the tested analogs, compounds 5, 8, 9 and 10 showed potent activity against PC 3, THP 1 and Hela cell lines. Especially, compound 10 showed enhanced activity against the Hela cell line than the parent compound. Compounds 5, 8 and 9 showed comparable activities against PC 3 and THP 1 cell lines.

Differentiation of Foot-and-Mouth Disease infected from vaccinated animals is essential for effective implementation of vaccination based control programme. Detection of antibodies against 3ABC non-structural protein of FMD virus by immunodiagnostic assays provides reliable indication of FMD infection. Sero-monitoring of FMD in the large country like India is a big task where thousands of serum samples are annually screened. Currently, monoclonal or polyclonal antibodies are widely used in these immunodiagnostic assays. Considering the large population of livestock in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody against 3B region of 3ABC poly-protein was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. Two formats of enzyme immunoassays (sandwich and competitive ELISAs) were optimized using scFv with objective to differentiate FMD infected among the vaccinated population. The assays were statistically validated by testing 2150 serum samples. Diagnostic sensitivity/specificity of sandwich and competitive ELISAs were determined by ROC method as 92.2%/95.5% and 89.5%/93.5%, respectively. This study demonstrated that scFv is a suitable alternate for immunodiagnosis of FMD on large scale., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Foot-and-mouth disease is a highly infectious and contagious disease of livestock animals with transboundary and economical importance. Animals in the endemic settings are regularly vaccinated in addition to intensive surveillance for control of the disease. Under intensive vaccination, detection of infected animals among the vaccinated population is essential to monitor the infection and to track down the virus movement. Sero-surveillance and retrospective disease diagnosis is performed primarily by detecting antibodies against non-structural proteins (NSPs) of FMD virus which are usually absent in the inactivated vaccine formulations. The study was conducted with an objective to compare simultaneously performance of six NSP ELISAs in detecting infected animals in the areas covered under intensive vaccination, and to assess their fit-for-purpose attribute for sero-surveillance of FMD in India. A panel of bovine serum samples consisting of samples collected from infected with FMDV, vaccinated and naive animals were constituted. In addition, samples collected at random from areas having varied FMD situation and vaccination coverage were tested simultaneously by the six NSP ELISAs to compare their performances. The four indigenous assays showed varying degrees of correlation with the two commercial kits. The study validated that, in all the groups of samples, the indigenous assays were equally sensitive and specific as the two commercial kits. Among all the six assays, PrioCheck and in-house 3ABC I-ELISAs showed maximum sensitivity for detection of infected animals, whereas 3AB3 I-ELISA and 3ABC C-ELISA showed maximum specificity. The study concluded that the in-house available assays are equally capable as the commercially available kits for differentiation of infected animals under intensive vaccination and identifies the 3AB3 I-ELISA with optimum sensitivity and specificity for the purpose of sero-surveillance in India., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Detection of antibodies to nonstructural proteins (NSP) of foot-and-mouth disease virus is the preferred diagnostic method to differentiate infected from vaccinated animals. In India, an endemic region practising preventive biannual vaccination, 3AB3 indirect ELISA (r3AB3 I-ELISA) has been employed as the primary screening test for serosurveillance. However, because of the variability observed in the immune response to the NSPs, the likelihood of detecting or confirming an infected animal is increased if an antibody profile against multiple NSPs is considered for diagnosis. In this study, all three copies of NSP 3B were expressed in a prokaryotic system to develop an indirect ELISA (r3B I-ELISA). At the decided cutoff of 40 percent positivity, the diagnostic sensitivity and specificity of the r3B I-ELISA were estimated to be 92.1% (95% CI: 89.0-94.5) and 98.1% (95% CI: 96.9-98.8), respectively, as compared to 97.04% and 95.04% for r3AB3 I-ELISA. Although r3B I-ELISA displayed lower sensitivity compared to the screening assay, which could possibly be attributed to additional relevant B-cell epitopes in the carboxy-terminal half of the 3A protein, the former achieved considerably higher specificity on repeatedly vaccinated animals. NSP antibodies could be detected from 10 to as late as 998 days postinfection in experimental calves. Substantial agreement in the test results (90.6%) was found between the two ELISAs. The r3B I-ELISA, when used in conjunction with the r3AB3 I-ELISA as an integrated system, can potentially augment the efficiency and confidence of detection of infected herds against the backdrop of intensive vaccination.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of transboundary importance. In India, since the launch of the FMD control programme, there has been a substantial increase in the vaccinated bovine population. In this scenario, there is a need for additional locally developed non-structural protein (NSP)-based immnoassays for efficient identification of FMD virus (FMDV)-infected animals in the vaccinated population. The 2B NSP of FMDV, lacking the transmembrane domain (Δ2B), was expressed successfully in a prokaryotic system, and an indirect ELISA (I-ELISA) was developed and validated in this study. The diagnostic sensitivity and specificity of the Δ2B I-ELISA were found to be 95.3 % and 94.6 %, respectively. In experimentally infected cattle, the assay could consistently detect Δ2B-NSP-specific antibodies from 10 to approximately 400 days postinfection. The assay was further validated with bovine serum samples collected randomly from different parts of the country. The performance of the Δ2B I-ELISA was compared with the in-house r3AB3 I-ELISA, and the overall concordance in test results was found to be 86.49 %. The Δ2B I-ELISA could be useful as a screening or confirmatory assay in the surveillance of FMD irrespective of vaccination.

There is emerging evidence that stem cells can rejuvenate damaged cells by mitochondrial transfer. Earlier studies show that epithelial mitochondrial dysfunction is critical in asthma pathogenesis. Here we show for the first time that Miro1, a mitochondrial Rho-GTPase, regulates intercellular mitochondrial movement from mesenchymal stem cells (MSC) to epithelial cells (EC). We demonstrate that overexpression of Miro1 in MSC (MSCmiro(Hi)) leads to enhanced mitochondrial transfer and rescue of epithelial injury, while Miro1 knockdown (MSCmiro(Lo)) leads to loss of efficacy. Treatment with MSCmiro(Hi) was associated with greater therapeutic efficacy, when compared to control MSC, in mouse models of rotenone (Rot) induced airway injury and allergic airway inflammation (AAI). Notably, airway hyperresponsiveness and remodeling were reversed by MSCmiro(Hi) in three separate allergen-induced asthma models. In a human in vitro system, MSCmiro(Hi) reversed mitochondrial dysfunction in bronchial epithelial cells treated with pro-inflammatory supernatant of IL-13-induced macrophages. Anti-inflammatory MSC products like NO, TGF-β, IL-10 and PGE2, were unchanged by Miro1 overexpression, excluding non-specific paracrine effects. In summary, Miro1 overexpression leads to increased stem cell repair.

Idiopathic pulmonary fibrosis (IPF) is a fatal disorder resulting from the progressive remodeling of lungs, with no known effective treatment. Although transforming growth factor (TGF)-β has a well-established role in lung fibrosis, clinical experience with neutralizing antibodies to TGF-β has been disappointing, and strategies to directly suppress TGF-β1 secretion are needed. In this study we used a combination of in silico, in vitro, and in vivo approaches to identify microRNAs involved in TGF-β1 regulation and to validate the role of miR-326 in pulmonary fibrosis.We show that hsa-miR-326 regulates TGF-β1 expression and that hsa-miR-326 levels are inversely correlated to TGF-β1 protein levels in multiple human cell lines. The increase in TGF-β1 expression during the progression of bleomycin-induced lung fibrosis in mice was associated with loss of mmu-miR-326. Restoration of mmu-miR-326 levels by intranasal delivery of miR-326 mimics was sufficient to inhibit TGF-β1 expression and attenuate the fibrotic response. Moreover, human IPF lung specimens had markedly diminished miR-326 expression as compared with nonfibrotic lungs. Additional targets of miR-326 controlling TGF-β signaling and fibrosis-related pathways were identified, and miR-326 was found to down-regulate profibrotic genes, such as Ets1, Smad3, and matrix metalloproteinase 9, whereas it up-regulates antifibrotic genes, such as Smad7. Our results suggest for the first time that miR-326 plays a key role in regulating TGF-β1 expression and other profibrotic genes and could be useful in developing better therapeutic strategies for alleviating lung fibrosis.

In this study, the Indian foot-and-mouth disease virus (FMDV) vaccine strain (A IND 40/2000) was passaged under homologous bovine convalescent serum (BCS) pressure to gain insight into the evolutionary dynamics of the antigenic sites. A considerable drop in the neutralization titres of the BCS for the isolated variants as compared to the parental population in either virus neutralization or plaque reduction neutralization test was observed. T143K substitution preceding the integrin binding 'RGD'-motif in the βG-βH loop of VP1 was found to be selected consistently and exclusively under immune pressure. By virtue of its location within an immunodominant site, sequence heterogeneity observed in the field viruses and residues already mapped in the neutralizing monoclonal antibody resistant mutants, position 143 in VP1 was predicted to be a critical residue of an important neutralizing epitope in serotype A FMDV. Using next-generation sequencing approach, the gradual overtaking of the originally dominating major variant by a minor one under a selective environment could be demonstrated. In the control passage regimen, VP2 E131K substitution was fixed within the heparan sulfate binding pocket probably as a result of adaptation to use alternative cellular receptors. But at the same time, these substitutions arising under selection forces other than immune pressure changed the antigenic behaviour of the virus inadvertently., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, an RNA transfection was used to rescue infectious foot-and-mouth disease (FMD) virus from clinical samples in BHK-21 cell line for diagnosis of FMD. Tissue samples (n=190) were subjected to FMD virus isolation by conventional cell culture and also by RNA transfection. FMD virus was isolated from 62% of the clinical samples by RNA transfection, whereas virus was isolated only from 16% of the clinical samples in conventional cell culture method, suggesting better performance of the RNA transfection. Virus was rescued from 67% and 10% of ELISA negative but multiplex PCR positive samples by RNA transfection and conventional cell culture, respectively. The efficiency of transfection was studied on clinical samples subjected to temperature as high as 37°C and varying pH (pH 4-9). Except up to 1 week of storage at 4°C at pH 7.5, virus isolation was not possible by cell culture. Virus was rescued by transfection from samples stored at 4°C for any of the applied pH up to 4 weeks, and when stored at 37°C virus could be rescued up to 4 weeks at pH 7.5 suggesting the fitness of transfection to isolate virus from clinical samples stored under inappropriate conditions. The sequence data and antigenic relationships with the vaccine strains, between virus rescued by transfection and conventional cell culture, were comparable. The RNA transfection will help to increase the efficiency of virus isolation, diagnosis and molecular epidemiological studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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. However, repeated attempts to recover viable virus from the RNA transcripts with a G320-T point mutation introduced in the short fragment of the 5' untranslated region failed. The possible destabilizing effect of such a mutation on the predicted long stem-loop structure at the 5'-end of the genome and its implications for viral genome replication are discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Serological investigation to detect foot-and-mouth disease (FMD) virus circulation in the domestic small ruminant population of India was conducted. A total of 4407 and 4035 serum samples from sheep and goats, respectively were collected at random covering majority of the states across the country during 2010-2012. These samples were analyzed for antibodies against the non-structural proteins (NSP) of FMD virus in an indirect 3AB NSP ELISA and against the structural proteins (SP) in a liquid phase blocking (LPB) ELISA. A total of 20.35% sheep and 13.60% goats were found to be positive for 3AB NSP antibodies providing a serological evidence of extensive viral activity. In LPB ELISA, only 4.54% sheep and 6.27% goats were found to have protective antibody (log10 titre of ≥1.8) against all three serotype strains in the vaccine, which correlates with "no or sparse vaccination" scenario in these species in the country. Hence, to check silent amplification and dissemination of virus in a mixed farming set up, small ruminants may be brought under the ambit of routine vaccination and surveillance programmes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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. The amplified RT-LAMP products were identified by agarose gel electrophoresis with ethidium bromide staining or observation by naked eye for the presence of turbidity and colour change following the addition of hydroxyl naphthol blue (HNB). The specificity of the assay was demonstrated by the absence of amplification of genome extracted from other viruses or cellular origin. With respect to analytical sensitivity the developed RT-LAMP assay was found more sensitive than routinely used multiplex PCR (mPCR). Further, the assay was evaluated with RNA extracted from cell cultured isolates (n = 50), tongue epithelial samples (n = 150) and semen samples from infected bulls (n = 13). In conclusion, RT-LAMP with HNB dye was shown to be simple, specific and sensitive assay for rapid diagnosis of FMDV infection. Further, the assay has the potential for field deployment and use for rapid FMDV surveillance in India.

Foot and mouth disease virus (FMDV) serotype Asia1 was first identified in India in 1951 and since then causing significant proportion of FMD outbreaks in the country. In this paper genetic analysis of 219 isolates from India collected over a period of 48 years is described. Bayesian approach was used to estimate the date of divergence and evolutionary rate. Phylogenetic analysis indicated the circulation of three lineages (B, C and D) of which lineage B formed one genotype (I) which was prevalent during 1964-2000. Genotype II constituted by lineage C and D has been in circulation since 1979 till date. We observed dramatic form of clade turnover in serotype Asia1 in India. The time scale analysis indicated that the most recent common ancestors for Indian Asia1 strains existed around 77 years ago. The evolutionary rate of serotype Asia1 viruses (genotype II) from India was estimated at 5.871×10(-3) substitutions per site, per year. We observed several connections in our phylogeographic analysis indicating intense flow of virus among states. The antigenically critical sites were frequently substituted and positive selection was evident at many sites. Maximum likelihood analysis suggested that the strains circulating in the country since 2005 were different from the genetic groups (I-VII) identified earlier and designated here as Group VIII., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease (FMD) is a transboundary animal disease caused by foot-and-mouth disease virus. In India, systematic preventive vaccination using inactivated trivalent (O, A and Asia 1) vaccine is the strategy being adopted to control FMD. The use of non-structural protein (NSP)-contaminated inactivated vaccine raises concerns over differentiation of infected and vaccinated animals (DIVA) by NSP based immunoassays. However, 2C being a membrane associated protein usually remain absent in vaccine formulations and thus, anti-2C response is one of the most reliable indicator of the FMDV infection. In this study, 34 amino acids from N-terminus of 2C protein were removed to eliminate membrane-binding amphipathic helicase activity for the expression of recombinant protein in soluble form. Truncated 2C (2Ct) was utilized for development of an indirect ELISA (I-ELISA) for bovine and the developed 2Ct I-ELISA was validated using a panel constituting of serum of naïve, vaccinated and infected animals. The assay was compared with the in-house r3AB3 I-ELISA and the overall concordance was 85.31%. The diagnostic sensitivity and specificity of the 2Ct I-ELISA were 92.9% and 94.0%, respectively. The apparent prevalence of anti-2C antibodies for random bovine samples tested by the developed assay was 23.7%. The developed ELISA will help in augmenting the sensitivity of detection if used in combination with r3AB3 I-ELISA for sero-surveillance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease virus (FMDV) is constantly evolving under neutralizing antibody pressure in either naturally infected or vaccinated animals. This study was carried out to understand the dynamics of evolution of antigenic sites. Neutralizing antibody-resistant populations of three strains of FMDV serotype O (INDR2/1975, IND120/2002 and IND271/2001) were isolated by serial propagation in BHK-21 cells in the presence of sub-neutralizing level of bovine vaccinal sera (BVS). In the partial neutralization escape variants, fixation of aa substitutions were observed at critical residues of all established antigenic sites of serotype O {144 of VP1 (site 1), 45 and 48 of VP1 (site 3), 72 and 134 of VP2 (Site 2)} except site 4 and 5. In majority of the variant populations, site 3 was found to be substituted and therefore immunodominance may not be associated with a particular site, rather it appears to be a virus strain and infected host specific affair. Substitutions were also observed in proximity to the identified residues {41 and 51 (βB-βC loop), 133, 140 and 143 (βG-βH loop), 201, 204 and 209 (C terminus) of VP1, 71 and 75 (βB-βC loop), 131 (βE-αB region), 174 and 179 (βG-βH loop) and 219 (C terminus) of VP3} within antigenic sites of serotype O or other serotypes which could be significant in terms of neutralizing antibody binding and immune escape. Presence of similar residues in the Indian field viruses as selected in the variants supports the importance of these sites in antigenic diversification of serotype O FMD virus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In India, emergence of Ind2001 lineage of foot-and-mouth disease virus (FMDV) serotype O was recorded in the year 2001. After causing sporadic incidences, the Ind2001 lineage that re-surged in 2008 out-competed PanAsia from the field during 2009 and continued its dominance during 2010 and 2011 as well. The lineage has diversified in due course of time, leading to two sub-lineages (Ind2001a and Ind2001b). The sub-lineage Ind2001a include isolates collected during 2001-2002 and sub-lineage Ind2001b is constituted largely by isolates collected during 2008-2012. The nucleotide substitution rate of sub-lineage Ind2001b was estimated at 6.58×10⁻³ substitutions/site/year. The most stable PanAsia lineage is restricted only to few outbreaks. During 2011, emergence of a new genetic group with >9% nucleotide divergence from rest of the lineages circulating in the country was detected and named as lineage Ind2011. Two specific amino acid substitutions at positions VP1-36 (F) and VP2-133 (T) were observed in the Ind2011 lineage. The new lineage at present is restricted only to southern states of the country. It is uncertain whether the emergence was triggered by immune pressure or due to a bottleneck in transmission or selected for higher fitness value. Six sites (4, 68, 83, 135, 138 and 209) in VP1 protein were identified to undergo episodic diversifying selection in serotype O field isolates. Both emerging and re-emerging lineages had appropriate antigenic match with currently used vaccine strain, INDR2/1975. Irrespective of genetic variability, the field isolates showed remarkable conservation at antigenically critical residues that might contribute to the observed antigenic stability. With the emergence of a new genetic group after a span of 10 years, the overall epidemiological scenario in the region is expected to change in the coming years., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease (FMD) is endemic in India and causes severe economic loss. Status of FMD in the country for five fiscal years is presented. Outbreaks were more in number in 2007-2008 than 2010-2011. Three serotypes of FMD virus (O, A and Asia1) are prevalent. Serotype O was responsible for 80% of the confirmed outbreaks/cases, whereas Asia1 and A caused 12% and 8%, respectively. Geographical region-wise assessment indicated varying prevalence rate in different regions viz; 43% in Eastern region, 31.5% in Southern region, 11.6% in North-eastern region, 5% Central region, 4.4% Western region and 4% in Northern region. Highest number of outbreaks/cases was recorded in the month of September and lowest in June. Emergence and re-emergence of different genotypes/lineages within the serotypes were evident in real-time investigation carried out from time to time. Continues antigenic divergence in serotype A resulted in change in the vaccine strain in 2009. As on date, all genetic diversity within the serotypes is well tolerated by the vaccine strains. Unrestricted animal movements in the country play a major role in the spread of FMD., (© 2012 Blackwell Verlag GmbH.)

The objective of this study was to investigate the endometrial expression of pro-inflammatory cytokines (IL1β, IL6, IL8 and TNFα) along with TLR4 and CD14 in normal and endometritic buffaloes. The genitalia were collected in the abattoir and divided into three groups as normal (gr. I=12), clinical endometritis (CE, gr. II=12) based on positive color reaction to white side test of uterine discharge and sub-clinical endometritis (SCE, gr. III=12) based on endometrial cytology (presence of ≥5% PMNs) and histopathology. The equal numbers of genitalia were grouped into follicular and luteal stage in each group. Endometrial tissue scrapings were used for total RNA extraction and cDNA was transcribed and amplified by Real time PCR. The results showed several fold higher expression of all cytokine transcripts in CE (gr. II), whereas significant up-regulation of CD14 (1 to 2-fold), IL6 (15 to 36-fold), IL8 (8 to 14-fold) and TNFα (10 to 11-fold) mRNA was observed in SCE. This indicates that the evaluation of expression patterns of certain cytokines gene holds promise to diagnose the severity and degree of uterine inflammation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dynamic variations in mitochondrial shape have been related to function. However, tools to automatically classify and enumerate mitochondrial shapes are lacking, as are systematic studies exploring the relationship of such shapes to mitochondrial stress. Here we show that during increased generation of mitochondrial reactive oxygen species (mtROS), mitochondria change their shape from tubular to donut or blob forms, which can be computationally quantified. Imaging of cells treated with rotenone or antimycin, showed time and dose-dependent conversion of tubular forms to donut-shaped mitochondria followed by appearance of blob forms. Time-lapse images showed reversible transitions from tubular to donut shapes and unidirectional transitions between donut and blob shapes. Blobs were the predominant sources of mtROS and appeared to be related to mitochondrial-calcium influx. Mitochondrial shape change could be prevented by either pretreatment with antioxidants like N-acetyl cysteine or inhibition of the mitochondrial calcium uniporter. This work represents a novel approach towards relating mitochondrial shape to function, through integration of cellular markers and a novel shape classification algorithm.

The phylogenetic analysis of VP1 sequences of the 39 type O foot and mouth virus (FMDV) isolates collected from different regions of India during the year of 2001-12 revealed that all isolates belonged to the Middle East - South Asia (ME-SA) topotype. Based on the amount of divergence among the isolates, the viruses were further classified into three distinct lineages namely Ind 2001, PanAsia and PanAsia-2 as well as a minor, unnamed group. Ind 2001 lineage viruses accounted for most of the current type O outbreaks. At the nucleotide level these isolates showed a divergence of 2% to 14% with an average sequence variation of ~9.9%. The serological spectrum of the current vaccine strain was studied by using bovine vaccinate serum (BVS) raised against O/IND/R2/75. All the current field isolates (n=24) were homologous ('r' value 0.4 to 1.0) to the vaccine strain. Examination of the amino acid sequences for selection pressure revealed the positive selection at amino acid sites 13 and 45., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Differentiation of infected from vaccinated animals (DIVA) is essential for effective control of foot-and-mouth disease (FMD) by vaccination. The antibody response against FMD viral non-structural proteins (NSPs) has been used widely for this purpose. Among all the NSPs, the 3ABC polyprotein has been recognized as the most appropriate indicator for DIVA. In this study, mutated full-length 3ABC polyprotein was expressed in a prokaryotic system and monoclonal antibody against the recombinant protein was developed. A competitive ELISA (C-ELISA) for DIVA was standardized for different species of livestock animals using recombinant 3ABC and monoclonal antibodies. The diagnostic sensitivity and specificity of the assay were estimated by testing a panel of known serum samples consisting of sera from naive, vaccinated and infected animals as 86.9% with 66.4-97.2 (95%) confidence interval and 97% with 89.6-99.6 (95%) confidence interval respectively at 40% inhibition cut-off. The assay was validated further by testing sera from different livestock species collected at random from different parts of the country. The assay will provide a common method for testing sera from different species of livestock and wild animals. The C-ELISA is a sensitive and specific DIVA assay for FMD and can be used as a method for FMD control programme with vaccination., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease virus serotype O accounts for around 80 % of the outbreaks in India. Although Indian serotype O isolates belongs to the ME-SA topotype, circulation of different lineages has been noted. After its emergence in the year 2001, the 'Ind2001' lineage outcompeted the PanAsia lineage in causing serotype O outbreaks in the year 2009. Three isolates had an amino acid deletion at position 139 in the VP1 coding region and grouped with the 'Ind2001' lineage. The currently used Indian vaccine strain of serotype O covers all of the field isolates antigenically.

Emergence of genetically and antigenically divergent lineages/genotypes and poor intergenotypic antigenic coverage is a major concern in serotype A foot-and-mouth-disease virus (FMDV) in India. In 2009, to cover antigenic diversity emerged in serotype A virus field isolates, IND40/2000 was selected as the new vaccine strain for incorporation in the trivalent FMD vaccine formulation used in India. Although current vaccine strain (IND40/2000) covers most isolates antigenically, a few VP3(59)-deletion group isolates showed low r-value in routine vaccine matching exercise. The VP3(59)-deletion group within genotype 18 emerged first in late part of 2002 and in 2007 causing outbreaks along with non-deletion isolates of the same genotype. In case of emergence or re-emergence of more antigenically divergent isolates in future, a need for a new vaccine candidate to cover maximum isolates of both deletion and non-deletion group may arise. Four alternate candidate vaccine strains (IND281/2003, IND195/2007, IND360/2007 and IND123/2008) were selected based on set criteria and antigenic relationships with field isolates sampled between 2002 and 2009 were analyzed using a micro-neutralization test. Phylogenetic analysis based on capsid region of serotype A isolates revealed existence of two broad distinct clusters (VP3(59)-deletion and non-deletion group) within genotype 18. The VP3(59)-deletion group has diversified genetically with time giving rise to three different sub-lineages (clade18a, 18b and 18c). The present study indicates that the virus candidates IND281/2003 (VP3(59)-deletion group) and IND195/2007 (non-deletion group) can be used as an adjunct or alternative strain to currently used vaccine strain IND40/2000 in case of emergence of more antigenically divergent isolates in future., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Oxygen-sensing prolyl-hydroxylase (PHD)-2 negatively regulates hypoxia-inducible factor (HIF)1-α and suppresses the hypoxic response. Hypoxia signaling is thought to be proinflammatory but also attenuates cellular injury and apoptosis. Although increased hypoxic response has been noted in asthma, its functional relevance is unknown. The objectives of this study were to dissect the mechanisms and role of the hypoxic response in asthma pathophysiology. Experimental studies were conducted in mice using acute and chronic allergic models of asthma. The hypoxic response in allergically inflamed lungs was modulated by using pharmacologic PHD inhibitors (ethyl-3-4-dihydroxybenzoic acid [DHB], 1-10 mg/kg) or siRNA-mediated genetic knockdowns. Increased hypoxia response led to exacerbation of the asthma phenotype, with HIF-1α knockdown being beneficial. Chronically inflamed lungs from mice treated with 10 mg/kg DHB showed diffuse up-regulation of the hypoxia response, severe airway remodeling, and inflammation. Fatal asphyxiation during methacholine challenge was noted. However, bronchial epithelium restricted up-regulation of the hypoxia response seen with low-dose DHB (1 mg/kg) reduced epithelial injury and attenuated the asthmatic phenotype. Up-regulation of the hypoxia response was associated with increased expression of CX3CR1, a lymphocyte survival factor, and increased inflammatory cell infiltrate. This study shows that an exaggerated hypoxia response may contribute to airway inflammation, remodeling, and the development of asthma. However, the hypoxia response may also be protective of epithelial apoptosis at lower levels, and the net effects of modulating the hypoxia response may vary based on the context.

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. Coinfection of BHK-21 cells demonstrated abrupt loss in the genotype VI viral load with commensurate increase in the load of genotype VII as measured by the genotype differentiating ELISA, RT-PCR and real-time RT-PCR. The superinfection dynamics was shaped by temporal spacing of infection, where the invading genotype VII took more number of passages than coinfection to eventually overtake the resident genotype VI. It was speculated that such superior replicative fitness of genotype VII could have been a possible factor for the ultimate dominance of genotype VII in nature., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 99.1% and 96.4%, respectively. This assay could detect antibodies to 3AB (3AB-Ab) from 10 to as late as 900 days post-infection in cattle infected experimentally. The "in-house" assay demonstrated higher sensitivity than a commercial 3ABC ELISA kit particularly with samples obtained from the late stages of infection. Transient post-vaccinal 3AB-Ab response could be detected in one of the three commercial vaccines during the six-month vaccination regimen, which emphasizes the fact that for a DIVA-compatible diagnostic strategy to be a realistic option, all vaccines need to be quality checked for the NSP content., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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 VP3(59)-deletion and non-deletion group within genotypes VII. The VP3(59)-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.

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine., (Copyright © 2011 AlphaMed Press.)

Nardostachys jatamansi DC. is a highly reputed Medhya and Nootropic (Learning and Memory) Ayurvedic medicinal plant. Its use as herbal medicine singly and as an ingredient of multi-herbal formulations is fast increasing. In order to authenticate and evaluate it quantitatively, its standardization is highly warranted with respect to a reliable marker. In this connection a rapid and highly sensitive UPLC-QTOF MS method has been developed. The analysis was carried out on an Acquity BEH C(18) column with gradient elution of methanol-water and 3 mm ammonium acetate using QTOF mass detector in negative ionization mode. The method was validated over a concentration range of 9.76-156.25 ng/mL nardin. The calibration curve is linear with the correlation coefficient (r) and coefficient of determination (R(2)) were 0.9997 and 0.9995 respectively. The LOD and LOQ were 3.050 and 9.277 ng/mL respectively. The recovery of nardin in the range 96.36-111.13% achieved from spiked samples was consistent and reproducible. The inter-day and intra-day assay precision of the analytes over the entire concentration range was less than 5%. The developed method required only 4 min for chromatography to authenticate and quantify the marker, viz. nardin in N. jatamansi samples, in addition to the sample preparation time., (Copyright © 2010 John Wiley & Sons, Ltd.)

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. These genotypes correlated with distinct evolutionary lineages in the maximum-likelihood phylogeny. During the last decade, ten genotypes have been in circulation the world over and it was evident that no type A strain has transgressed the continental barriers during this period. A single genotype (genotype 18) within the Asia topotype has been circulating in India with neither any incursion nor any long distance movement of virus out of the country during the last ten years, although close genetic and epidemiological links between viruses from Bhutan and India were revealed.

Extreme antigenic and genetic heterogeneity of serotype A foot-and-mouth disease virus (FMDV) population has resulted in change of vaccine strains in India twice in the last decade. In such a situation, complete characterization of the vaccine strains is imperative. With regard to the frequent outbreaks of this disease, FMDV field strains are also of interest. Therefore three vaccine strains and two field strains of type A FMDV from India were completely sequenced and the obtained sequences were subjected to sequence and phylogenetic analyses. Based on the complete coding region, all the Indian strains clustered in the Asia topotype and exhibited a more than 11% nt divergence from the other Asian strains. The 5'-UTR of some Indian strains revealed block deletions of 43 and 86 nt corresponding to the pseudoknot region. Amino acids S44 in VP2 and F164 in VP1 were found to be the exclusive signatures for the Asia topotype. The vaccine strains differed at 65 aa positions in the capsid region, 13 of them antigenically critical. Variability at such positions is likely to affect the antigenic profile of these strains. Complete genome sequences of the vaccine strains presented here could serve as the reference for any comparative genomics in future.

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. The emergence of this group warrants rapid and accurate detection to facilitate early planning and implementation of an effective control policy. A rapid multiplex PCR assay was developed for detection of the dominating VP3(59)-deletion group with 100% sensitivity and specificity, even before generating sequence data and confirmatory phylogenetic analysis. This development is important for surveillance of FMD in India., (Copyright © 2010 Elsevier B.V. All rights reserved.)

India is endemic for foot-and-mouth disease (FMD), and goats constitute the second largest susceptible population of domestic livestock. FMD surveillance and control strategies in the country largely ignore small ruminants, known to be critical in the epidemiology of the disease. Here, serological investigations were carried out to generate estimates of antibody prevalence in goats of Orissa state to both non-structural (NSP-Ab) and structural proteins (SP-Ab) of FMD. The apparent overall NSP-Ab and SP-Ab seroprevalences were 38% and 20.7%, respectively, which signifies a very high level of FMD virus circulation in the goat population despite the lack of clinical signs in this species. The apparent prevalence of NSP-Ab and SP-Ab was positively correlated in the sampling areas. Interestingly, the values found for NSP-Ab prevalence were almost consistently higher than those found for SP-Ab prevalence. This could have been attributable to either issues related to sensitivity and specificity of the test systems employed or differences in the post-infection kinetics of NSP- and SP-Ab. The pattern that emerged from SP-Ab analysis indicated goats being infected with all three prevalent serotypes (O, A and Asia 1) and reinforces the concept that non-vaccinated goats can be exploited as tracer animals for detecting serotypes involved in outbreaks. The results underscore the requirement to bring caprine species under comprehensive surveillance and vaccination campaigns to check silent amplification, excretion and transmission of the virus., (© 2010 Blackwell Verlag GmbH.)

Background & Objectives: With the emergence of a new reassortant influenza A H1N1 virus that caused the 2009 pandemic it was felt necessary that pigs should be closely monitored for early detection of any influenza virus infection. Therefore, we investigated disease outbreaks with clinical history suggestive for swine influenza reported to our laboratory by owners of affected pig farms in Uttar Pradesh., Methods: Detection of swine influenza A virus (SIV) was attempted by isolation in embryonated chicken eggs. Presence of virus was detected by haemagglutination (HA) test and RT-PCR for amplification of different gene segments, cloning and sequencing. BLAST analysis of sequence data, phylogenetic analysis and mutation analysis based on HA, NA and matrix genes was done., Results: SIV could be isolated from one farm and all eight gene segments amplified by RT-PCR. BLAST analysis of partial nucleotide sequences and phylogenetic analysis using nucleotide sequence of HA (601 nt), NA (671 nt) and M (1031 nt) genes indicated close genetic relationship of the Indian swine isolate (A/Sw/UP-India-IVRI01/2009) with human pandemic 2009 (H1N1). The HA gene showed close relationship with the viruses of "North American Swine" lineage, whereas the NA and M genes clustered with the viruses of "Eurasian Swine" lineage, indicating a novel HA-NA reassortant. The remaining of 5 genes (NP, PA, PB1, PB2 and NS) belonged to "North American Swine" lineage., Interpretation & Conclusions: This is perhaps the first report describing swine influenza among Indian pigs caused by an influenza A H1N1 virus sharing close homology with the human pandemic (H1N1) 2009 virus. Further reassortment with circulating influenza viruses must be closely monitored.

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. In the present report, the complete VP1 genomic region of 41 FMDV Asia1 field isolates collected between 2003 and 2008 was sequenced. Phylogenetic analysis revealed reemergence of lineage C since 2005 following exclusive dominance of lineage D in the period between 2002 and 2004. At many positions lineage specific signature residues were identified. The antigenic relationship of the field isolates with the currently used vaccine strain IND63/72 was also determined, which reflects antigenic stability of serotype Asia1 in-spite of genetic heterogeneity., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

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. The VP3(59)-deletion group maintained its phylogenetic distinctness even at the LF-5' UTR. Eighteen lineage specific signatures detected here support independent evolutionary paths for the lineages. Extensive deletions of 45 and 89 nucleotides corresponding to the pseudoknot region were noticed. Conservation pattern in the 'A(253)AACA' motif in the cre/bus stem-loop indicates the importance of first three 'A' residues in VPg uridylylation. Of the three polypyrimidine tract binding protein (PTB) binding sites mapped on the internal ribosome entry site (IRES), the pyrimidine tract (Py tract) in the loop of domain 2 was found to be maximally conserved and it might be the major PTB binding site. Strikingly, a deletion group lineage specific transversion was noticed in the Py tract at the 3' end of IRES without significantly affecting its in vitro infectious titer. Hence, we presume that for efficient cap-independent viral translation, either a minimum number of pyrimidine residues rather than a complete Py tract or a Py tract tolerating transversions only at specific locations and a core motif 'CUUU' within the Py tract is essential.

The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. Here we analyzed the 3A region of serotype A virus in view of the emergence of a variant group in India with an amino acid deletion at an antigenically critical position of capsid protein, VP3. The 3A region exhibited extreme variability with 38% of the amino acid positions showing substitutions and the C-terminal third (127-151) region was most flexible. Genotype inclusive grouping of type A foot-and-mouth disease virus as observed in 1D region based phylogeny was much less apparent at 3A region possibly due to independent evolution of nonstructural and structural protein coding regions. Akin to the 1D region, the VP3(59)-deletion group maintained its phylogenetic distinctness even at the 3A region and was found to be diverging with time. Twelve lineage specific signature amino acid residues, of which four were identified to be experiencing positive selection, indicates fixation of advantageous mutations in a lineage specific manner. Six positions, all located in the hypervariable C-terminal third, were identified to be under positive selection and were presumed to be imparting the virus certain advantage accounting for its adaptability to wide host spectrum and rapid dissemination. A significant change of Q(44)H was noted only in the older lineage (VIIb) of the deletion group at a position where Q(44)R mutation is associated with guinea pig adaptation. As this site has been detected to be under positive selection, such a lineage specific substitution is thought to have imparted certain temporary advantage to the virus during its possible adaptation in wild or some understudied domestic hosts and must not have seriously compromised fitness upon readaptation in bovines. A conserved hydrophobic transmembrane domain from position 59 to 76 could be predicted which possibly anchors 3A to intracellular membranes for successful interaction with RNA replication complex.

We investigated the molecular epidemiology of foot-and-mouth disease virus (FMDV) serotype Asia 1, which caused outbreaks of disease in Asia during 2003-2007. Since 2004, the region affected by outbreaks of this serotype has increased from disease-endemic countries in southern Asia (Afghanistan, India, Iran, Nepal, Pakistan) northward to encompass Kyrgyzstan, Tajikistan, Uzbekistan, several regions of the People's Republic of China, Mongolia, Eastern Russia, and North Korea. Phylogenetic analysis of complete virus capsid protein 1 (VP1) gene sequences demonstrated that the FMDV isolates responsible for these outbreaks belonged to 6 groups within the Asia 1 serotype. Some contemporary strains were genetically closely related to isolates collected historically from the region as far back as 25 years ago. Our analyses also indicated that some viruses have spread large distances between countries in Asia within a short time.