Jajati K. Mohapatra

Status of foot-and-mouth disease in India.

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.

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. 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.

Molecular typing of canine parvovirus strains circulating from 2008 to 2012 in an organized kennel in India reveals the possibility of vaccination failure

Canine parvovirus-2 (CPV-2), which emerged in 1978, is considered as the major viral enteric pathogen of the canine population. With the emergence of new antigenic variants and incidences of vaccine failure, CPV has become one of the dreaded diseases of the canines worldwide. The present study was undertaken in an organized kennel from North India to ascertain the molecular basis of the CPV outbreaks in the vaccinated dogs. 415 samples were collected over a 5year period (2008-2012). The outbreak of the disease was more severe in 2012 with high incidence of mortality in pups with pronounced clinical symptoms. Molecular typing based on the VP2 gene was carried out with the 11 isolates from different years and compared with the CPV prototype and the vaccine strains. All the isolates in the study were either new CPV-2a (2012 isolates) or new CPV-2b (2008 and 2011 isolates). There were amino acid mutations at the Tyr324Ile and at the Thr440Ala position in five isolates from 2012 indicating new CPV mutants spreading in India. The CPV vaccines used in the present study failed to generate protective antibody titer against heterogeneous CPV antigenic types. The findings were confirmed when the affected pups were treated with hyper-immune heterogeneous purified immunoglobulins against CPV in dogs of different antigenic types.

Evolutionary dynamics of foot-and-mouth disease virus O/ME-SA/Ind2001 lineage

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.

Surface EMG analysis and changes in gait following electrical stimulation of quadriceps femoris and tibialis anterior in children with spastic cerebral palsy

Purpose: To evaluate the clinical feasibility and effect of neuromuscular electrical stimulation (NMES) therapy of quadriceps femoris (QF) and tibialis anterior (TA) muscles on improving gait and functional outcomes in children with spastic cerebral palsy (CP). Method: Ten children with spastic diplegic/hemiplegic CP who were in the age group of 7 to 14 years recruited from a rehabilitation institute were randomly assigned either to a control group or a NMES group. Both groups obtained conventional physiotherapy and muscle strengthening exercises. The NMES group in addition received surface electrical stimulation to QF and TA muscles for four weeks duration. Results: The NMES group showed significant improvements as compared to the control group in walking speed (mean difference: 7.83 meters per min, 95% confidence interval: 3.13 to 12.53, p<;60;0.01) and cadence (mean difference: 23.33 steps per min, 95% confidence interval: 5.90 to 40.77, p<;60;0.01). The NMES group also showed significant reduction in physiological cost index of walking or PCI (mean difference: -1.32 beats per meter, 95% confidence interval: -1.83 to -0.80, p<;60;0.001) indicating greater energy-efficiency of walking. No significant changes were seen in EMG parameters. Conclusions: The findings of this study suggests that NMES therapy together with conventional physiotherapy more efficiently improves walking ability and functional outcomes as compared to conventional physiotherapy alone in children with spastic CP.

Emergence of a novel lineage genetically divergent from the predominant Ind2001 lineage of serotype O foot-and-mouth disease virus in India

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.

Serological evidence of foot-and-mouth disease virus infection in randomly surveyed goat population of Orissa, India.

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.

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

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

Immunodiagnosis of foot-and-mouth disease using mutated recombinant 3ABC polyprotein in a competitive ELISA

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.

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

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