A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

Characterization of humoral and cellular immune responses elicited by reduced doses of Brucella abortus S19 (calfhood) vaccine in cattle calves of India.

Brucella abortus S19 vaccine is a stable attenuated smooth strain, globally used as calfhood vaccine for the prevention of bovine brucellosis. Various agencies demonstrated different doses for vaccinating cattle and buffalo calves leading to ambiguity in selecting a suitable immune vaccine dose. The current study aimed at evaluating four graded doses of S19 vaccine to arrive at the dose which could produce comparable effectiveness as that of full dose prescribed by Indian Pharmacopeia among the Indian calves. Four vaccine doses of which the first dose consisted of full dose (40 × 109 CFU/dose) and the other three were 1/10th, 1/20th, 1/100th reduced doses along with control were tested. Each vaccine dose was administered to 13 cattle calves of 4-5 months of age maintained in separate groups. The blood samples were collected on 0 to 240 days post-vaccination (DPV) at the intervals of 0, 14, 28, 45, 60, 90, 150, 180 and 240 for assessment of vaccine-induced innate, humoral and cell-mediated immune responses. The sero-conversion of all vaccinated animals on DPV 45 and persistence of antibody till DPV 240 were noticed. No significant differences were observed in antibody response between animal groups that received full and 1/10th reduced doses. Innate and cell-mediated response by IL-6, TNF-α¸ IFN-γ, CD4+ and CD8+ cell counts showed dose-dependent responses with no significant difference between full dose and 1/10th reduced doses. The results suggest a possible one log reduction of full dose without compromising immune responses to aid larger vaccination coverage for creating herd immunity.

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India.

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

Chicken Feather Waste Valorization Into Nutritive Protein Hydrolysate: Role of Novel Thermostable Keratinase From Bacillus pacificus RSA27.

Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from Bacillus pacificus RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report K m and V max values of 5.69 mg/ml and 142.40 µg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and in silico structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp128, Leu162, Asn164, Ile166, and Val168) and a catalytic triad of Asp119, His151, and Ser308, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 µmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.

An integrated surveillance network for antimicrobial resistance, India.

OBJECTIVE: To assess the preparedness of veterinary laboratories in India to participate in an integrated antimicrobial resistance surveillance network and to address gaps in provision identified. METHODS: The Indian Council of Medical Research and the Indian Council of Agricultural Research collaborated: (i) to select eight nationally representative veterinary microbiology laboratories whose capacity for participating in an integrated antimicrobial resistance surveillance network would be assessed using a standardized tool; (ii) to identify gaps in provision from the assessment findings; and (iii) to develop a plan, and take the necessary steps to address these gaps in consultation with participating organizations. FINDINGS: The main gaps in provision identified were: (i) a lack of dedicated funding for antimicrobial resistance surveillance; (ii) the absence of standard guidelines for antimicrobial susceptibility testing; (iii) a shortage of reference strains for testing and quality assurance; and (iv) the absence of mechanisms for sharing data. We addressed these gaps by creating a veterinary standard operating procedure for antimicrobial susceptibility testing, by carrying out a validation exercise to identify problems with implementing the procedure and by conducting capacity-building workshops for veterinary laboratories. CONCLUSION: Antimicrobial resistance surveillance networks depend on the availability of accurate, quality-controlled testing. The challenges identified in creating an integrated surveillance network for India can be overcome by developing a comprehensive plan for improving laboratory capacity in human, veterinary and environmental sectors that is supported by the necessary funds. The study's findings may provide guidance for other low- and middle-income countries planning to develop a similar network.

Pathological and immunological characterization of bluetongue virus serotype 1 infection in type I interferons blocked immunocompetent adult mice.

Introduction: Wild-type adult mice with intact interferon (IFN) system were neither susceptible to bluetongue virus (BTV) infection nor showed signs of morbidity/mortality. Establishment of immunologically competent wild-type adult mouse model with type I IFNs blockade is necessary to assess the pathogenesis, immune responses and testing of BTV vaccines. Objectives: Present study aimed to establish and characterize BTV serotype 1 infection in immunocompetent adult mice with type I IFNs blockade at the time of infection by studying immune responses and sequential pathology. Methods: Adult mice were administered with anti-mouse IFN-α/ß receptor subunit-1 (IFNAR1) blocking antibody (Clone: MAR1-5A3) 24 h before and after BTV serotype 1 infection, and sacrificed at various time points. Sequential pathology, BTV localization by immunohistochemistry and quantification by qRT-PCR, immune cell kinetics and apoptosis by flow cytometry, and cytokines estimation by c-ELISA and qRT-PCR were studied. Results: IFNAR blocked-infected mice developed clinical signs and typical lesions of BT; whereas, isotype-infected control mice did not develop any disease. The IFNAR blocked-infected mice showed enlarged, edematous, and congested lymph nodes (LNs) and spleen, and vascular (congestion and hemorrhage) and pneumonic lesions in lungs. Histopathologically, marked lymphoid depletion with "starry-sky pattern" due to lymphocytes apoptosis was noticed in the LNs and spleen. BTV antigen was detected and quantified in lymphoid organs, lungs, and other organs at various time points. Initial leukopenia (increased CD4+/CD8+ T cells ratio) followed by leukocytosis (decreased CD4+/CD8+ T cells ratio) and significantly increased biochemical values were noticed in IFNAR blocked-infected mice. Increased apoptotic cells in PBMCs and tissues coincided with viral load and levels of different cytokines in blood, spleen and draining LNs and notably varied between time points in IFNAR blocked-infected mice. Conclusion: Present study is first to characterize BTV serotype 1 infection in immunocompetent adult mouse with type I IFNs blockade. The findings will be useful for studying pathogenesis and testing the efficacy of BTV vaccines.

Animal disease surveillance: Its importance & present status in India.

Animal disease surveillance encompasses systematic collection of long-term data on disease events, risk factors and other relevant parameters followed by analyzing the same with reference to temporal and spatial characteristics to arrive at a conclusion so that necessary preventive measures can be taken. In India, the animal disease surveillance is done through National Animal Disease Reporting System, which is a web-based information technology system for disease reporting from States and Union Territories with the aim to record, monitor livestock disease situation and to initiate the preventive and curative action in a swift manner during disease emergencies. National Animal Disease Referral Expert System is a dynamic geographic information system and remote sensing-enabled expert system that captures an incidence of 13 economically important livestock diseases from all over the country and also provides livestock disease forecasting. The laboratories under State and Central governments, several research institutes under the Indian Council of Agricultural Research and veterinary colleges are involved in livestock disease diagnosis including zoonotic diseases. An integrated surveillance system is necessary for early detection of emerging/zoonotic diseases in humans. This review provides information on disease reporting and surveillance systems in animal health sector and the need for One Health approach to improve and strengthen the zoonotic disease surveillance system in India.

Foot and Mouth Disease (FMD) incidence in cattle and buffaloes and its associated farm-level economic costs in endemic India.

The study investigated the important epidemiological parameters and farm-level economic costs of FMD incidence in cattle and buffaloes during 2013-14 to 2015-16 in various states of India. Multistage random sampling procedure was adopted for the primary survey and data was collected through face-to-face personal interview from 18,609 cattle and buffalo rearing farm households from 123 districts across twelve states and one Union Territory. Besides epidemiological parameters, different farm-level direct and indirect loss associated with FMD was assessed at disaggregated level (states) by employing deterministic mathematical models. Highest number of affected villages and disease incidence was observed in non- FMD control programme (FMD-CP) implemented Madhya Pradesh and Assam states, respectively whereas negligible incidence was in FMD-CP implemented Punjab state. The disease incidence was high during 2013-14 and declined during 2014-15 and 2015-16, respectively implied severe incidence scenario (2013-14) succeeded by moderate (2014-15) and mild (2015-16) scenarios. The crossbred and high productive animals were severely affected than local breeds whereas on sexwise and agewise comparison revealed higher incidence in females and adult animals. During severe incidence scenario, milk loss/animal ranged from USD 6.87-47.44, 18.42-125.88, 16.33-91.43, and 27.17-123.62; mortality loss/animal ranged from USD 32.61-804.27, 30.76-577.7, 65.36-502.2, and 188.04-413.7; distress sale loss/animal ranged from USD 3.22-188.63, 64.34-519.3, 214.47-341.8, and 209.11-450.3; and opportunity cost of labour/animal from USD 5.49-54.29, 5.49-67.78; 7.95-31.37 and 9.83-72.38 in indigenous cattle, crossbred cattle, local and improved buffalo, respectively. The estimated draught power loss/animal varied from USD 39.46-142.94 with least being in Madhya Pradesh and highest in Assam states whereas the median treatment cost/animal was USD 9.18 and USD 27.07 in indigenous cattle and upgraded buffaloes, respectively. The total farm-level economic loss projected due to FMD in cattle and buffaloes in India was USD 3159 million (INR 221,110 million), USD 270 million (INR 18,910 million) and USD 152 million (INR 10,610 million), respectively during the severe, moderate and mild incidence scenarios at 2015-16 constant prices. The loss varied across the states, and in severe incidence scenario, the country might lose USD 3.2 billion/year and hence, the bi-annual vaccination schedule need to be strictly implemented in all the states. Besides timely vaccination coverage, managing unabated animal movement, educating and motivating the farmers to vaccinate their animals might reduce the incidence and consequential losses to various stakeholders in endemic states like India.

An updated review on bluetongue virus: epidemiology, pathobiology, and advances in diagnosis and control with special reference to India.

Bluetongue (BT) is an economically important, non-contagious viral disease of domestic and wild ruminants. BT is caused by BT virus (BTV) and it belongs to the genus Orbivirus and family Reoviridae. BTV is transmitted by Culicoides midges and causes clinical disease in sheep, white-tailed deer, pronghorn antelope, bighorn sheep, and subclinical manifestation in cattle, goats and camelids. BT is a World Organization for Animal Health (OIE) listed multispecies disease and causes great socio-economic losses. To date, 28 serotypes of BTV have been reported worldwide and 23 serotypes have been reported from India. Transplacental transmission (TPT) and fetal abnormalities in ruminants had been reported with cell culture adopted live-attenuated vaccine strains of BTV. However, emergence of BTV-8 in Europe during 2006, confirmed TPT of wild-type/field strains of BTV. Diagnosis of BT is more important for control of disease and to ensure BTV-free trade of animals and their products. Reverse transcription polymerase chain reaction, agar gel immunodiffusion assay and competitive enzyme-linked immunosorbent assay are found to be sensitive and OIE recommended tests for diagnosis of BTV for international trade. Control measures include mass vaccination (most effective method), serological and entomological surveillance, forming restriction zones and sentinel programs. Major hindrances with control of BT in India are the presence of multiple BTV serotypes, high density of ruminant and vector populations. A pentavalent inactivated, adjuvanted vaccine is administered currently in India to control BT. Recombinant vaccines with DIVA strategies are urgently needed to combat this disease. This review is the first to summarise the seroprevalence of BTV in India for 40 years, economic impact and pathobiology.

Evaluation of the immune responses against reduced doses of Brucella abortus S19 (calfhood) vaccine in water buffaloes (Bubalus bubalis), India.

BACKGROUND: Brucella abortus S19 is the most widely used vaccine for the prevention of bovine brucellosis which remains the reference vaccine to which many other vaccine/s are compared. Considering the larger vaccination coverage by reduced dose of vaccine, the study aimed to compare reduced graded doses (1/10th, 1/20th and 1/100th) with standard dose of S19 vaccine (40 × 109CFU /dose) to determine the effective immunizing dose in water buffaloes. METHODS: A total of 25 female buffalo calves (Bubalus bubalis) in the age group of 4-5 months were equally grouped into five animals each in four test and one control groups and given with specified vaccine dose. The blood samples were collected on post vaccination days 14, 28, 45, 60, 90 and 120 for assessing innate (TNF-α and IL-12), humoral (IgG antibodies against Brucella LPS) and cell mediated immune responses (IFN-γ, CD4 + and CD8 + counts). RESULTS: The full dose, 1/10th and 1/20th reduced doses of S19 vaccine was capable of eliciting pathogen-specific antibody response, vaccine induced secretion of IL-12, TNF-α and IFN-γ with CD4 + and CD8 + effector T cell responses. Persistence of antibody and magnitude of immune responses were found dose dependent. CONCLUSION: Comparable immune responses were noticed with 1/10th reduced dose similar to standard dose. With this observation, decline of antibody titre will reduce the number of false positives and reduced dose of vaccine will facilitate larger vaccination coverage in the country.

Nanotherapeutics: An insight into healthcare and multi-dimensional applications in medical sector of the modern world.

In recent years nanotechnology has revolutionized the healthcare strategies and envisioned to have a tremendous impact to offer better health facilities. In this context, medical nanotechnology involves design, fabrication, regulation, and application of therapeutic drugs and devices having a size in nano-range (1-100 nm). Owing to the revolutionary implications in drug delivery and gene therapy, nanotherapeutics has gained increasing research interest in the current medical sector of the modern world. The areas which anticipate benefits from nano-based drug delivery systems are cancer, diabetes, infectious diseases, neurodegenerative diseases, blood disorders and orthopedic problems. The development of nanotherapeutics with multi-functionalities has considerable potential to fill the lacunae existing in the present therapeutic domain. Nanomedicines in the field of cancer management have enhanced permeability and retention of drugs thereby effectively targeting the affected tissues. Polymeric conjugates of asparaginase, polymeric micelles of paclitaxel have been recmended for various types of cancer treatment .The advancement of nano therapeutics and diagnostics can provide the improved effectiveness of the drug with less or no toxicity concerns. Similarly, diagnostic imaging is having potential future applications with newer imaging elements at nano level. The newly emerging field of nanorobotics can provide new directions in the field of healthcare. In this article, an attempt has been made to highlight the novel nanotherapeutic potentialities of polymeric nanoparticles, nanoemulsion, solid lipid nanoparticle, nanostructured lipid carriers, dendrimers, nanocapsules and nanosponges based approaches. The useful applications of these nano-medicines in the field of cancer, nutrition, and health have been discussed in details. Regulatory and safety concerns along with the commercial status of nanosystems have also been presented. In summary, a successful translation of emerging nanotherapeutics into commercial products may lead to an expansion of biomedical science. Towards the end of the review, future perspectives of this important field have been introduced briefly.

Molecular Modeling of Cloned Bacillus subtilis Keratinase and Its Insinuation in Psoriasis Treatment Using Docking Studies.

Present study demonstrated the expression of cloned Bacillus subtilis RSE163 keratinase gene and in silico binding affinities of deduced protein with psoriasis topical drugs for systemic absorption and permeation through skin. The ker gene expressed in E. coli showed significantly higher keratinase activity 450 ± 10.43 U representing 1342 bp nucleotides encoding 447 amino acids with molecular weight of 46 kDa. The modeled structure was validated using ramachandran's plot showing 305 residues (84.3%) in most favoured region. Docking studies using extra precision (XP) method of Glide showed optimum binding affinities with the drugs Acitretin (- 39.62 kcal/mol), Clobetasol propionate (- 37.90 kcal/mol), Fluticasone (- 38.53 kcal/mol), Desonide (- 32.23 kcal/mol), Anthralin (- 38.04 kcal/mol), Calcipotreine (- 21.55 kcal/mol) and Mometasone (- 28.40 kcal/mol) in comparison to other psoriasis drugs. The results can further be correlated with in vitro enzymatic experiments using keratinase as an effective drug mediator through skin to serve the unmet need of industries.

The Consensus from the Mycobacterium avium ssp. paratuberculosis (MAP) Conference 2017.

On March 24 and 25, 2017 researchers and clinicians from around the world met at Temple University in Philadelphia to discuss the current knowledge of Mycobacterium avium ssp. paratuberculosis (MAP) and its relationship to human disease. The conference was held because of shared concern that MAP is a zoonotic bacterium that poses a threat not only to animal health but also human health. In order to further study this problem, the conferees discussed ways to improve MAP diagnostic tests and discussed potential future anti-MAP clinical trials. The conference proceedings may be viewed on the www.Humanpara.org website. A summary of the salient work in this field is followed by recommendations from a majority of the conferees.

Concurrent Resolution of Chronic Diarrhea Likely Due to Crohn's Disease and Infection with Mycobacterium avium paratuberculosis.

Examination of samples of stool from a 61-year-old male patient, presenting with the clinical symptoms of Crohn's disease (CD), revealed massive shedding of acid fast bacilli with the morphology of Mycobacterium avium paratuberculosis (MAP), the causative agent of Johne's disease in cattle. MAP was cultured from the stool. Biotyping of the bacterium isolated from cultures of stool demonstrated, it was the Indian Bison biotype of MAP, the dominant biotype infecting livestock and humans in India. Based on this finding and because the patient was unresponsive to standard therapy used in India to treat patients with gastrointestinal inflammatory disorders, the patient was placed on a regimen of multi-antibiotic therapy, currently used to treat tuberculosis and CD. After 1 year of treatment, the patient's health was restored, concurrent with cessation of shedding of MAP in his stool. This patient is the first case shown to shed MAP from the stool who was cured of infection with antibiotics and who was concurrently cured of clinical signs of CD.

Pathology of Equine Influenza virus (H3N8) in Murine Model.

Equine influenza viruses (EIV)-H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.

Marker vaccine potential of foot-and-mouth disease virus with large deletion in the non-structural proteins 3A and 3B.

Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the traditional inactivated vaccines may sometimes contain traces of FMD viral (FMDV) non-structural protein (NSP), therefore, interfering with the NSP-based serological discrimination between infected and vaccinated animals. The availability of marker vaccine for differentiating FMD infected from vaccinated animals (DIVA) would be crucial for the control and subsequent eradication of FMD in India. In this study, we constructed a negative marker FMDV serotype O virus (vaccine strain O IND R2/1975), containing dual deletions of amino acid residues 93-143 and 10-37 in the non-structural proteins 3A and 3B, respectively through reverse genetics approach. The negative marker virus exhibited similar growth kinetics and plaque morphology in cell culture as compared to the wild type virus. In addition, we also developed and evaluated an indirect ELISA (I-ELISA) targeted to the deleted 3AB NSP region (truncated 3AB) which could be used as a companion differential diagnostic assay. The diagnostic sensitivity and specificity of the truncated 3AB I-ELISA were found to be 95.5% and 96%, respectively. The results from this study suggest that the availability negative marker virus and companion diagnostic assay could open a promising new avenue for the application of DIVA compatible marker vaccine for the control of FMD in India.

Megaprimer-mediated capsid swapping for the construction of custom-engineered chimeric foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the selection of appropriate candidate vaccine strain and its adaptation in cell culture to yield high titer of virus is a cumbersome process. An attractive approach to circumvent this tedious process is to replace the capsid coding sequence of an infectious full-genome length cDNA clone of a good vaccine strain with those of appropriate field strain, to produce custom-made chimeric FMD virus (FMDV). Nevertheless, the construction of chimeric virus can be difficult if the necessary endonuclease restriction sites are unavailable or unsuitable for swapping of the capsid sequence. Here we described an efficient method based on megaprimer-mediated capsid swapping for the construction of chimeric FMDV cDNA clones. Using FMDV vaccine strain A IND 40/2000 infectious clone (pA(40/2000)) as a donor plasmid, we exchanged the capsid sequence of pA(40/2000) with that of the viruses belonging to serotypes O (n = 5), A (n = 2), and Asia 1 (n = 2), and subsequently generated infectious FMDV from their respective chimeric cDNA clones. The chimeric viruses exhibited comparable infection kinetics, plaque phenotypes, antigenic profiles, and virion stability to the parental viruses. The results from this study suggest that megaprimer-based reverse genetics technology is useful for engineering chimeric vaccine strains for use in the control and prevention of FMD in endemic countries.

Capsid coding region diversity of re-emerging lineage C foot-and-mouth disease virus serotype Asia1 from India.

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.