key: cord-0033222-t45lhp7l authors: Malik, Yashpal Singh title: Abstracts of the Papers (Oral/Poster) Presented During the XXI National Conference of Indian Virological Society (IVS) on “Immunobiology and Management of Viral Diseases in 21st Century”, Held from 8–10 November, 2012, at Indian Veterinary Research Institute (IVRI) Mukteswar Campus, Mukteswar, Nainital 263138, Uttarakhand date: 2013-04-17 journal: Indian J Virol DOI: 10.1007/s13337-013-0132-5 sha: 4d525eb3d4138cda7d335312b1651342215f09ef doc_id: 33222 cord_uid: t45lhp7l nan Specific types of high risk Human Papillomavirus (HR-HPV) types, particularly the HPV type 16 and HPV 18 are known to cause cervical cancer. The expression of two viral oncogenes E6 and E7 responsible for tumorigenic transformation, is mainly dependent on specific host cell transcription factor, Activator Protein 1 (AP-1) which acts as a signaling epicenter for cervical cancer. Although recently two prophylactic HPV vaccines have been developed, there is no therapeutic molecule available for the treatment of cervical lesions. We demonstrate that Curcumin (Diferuloylmethane), a yellow pigment, used in traditional medicines and present in the dietary spice turmeric (Curcuma longa), can selectively down regulate HPV 18 transcription as well as AP-1 binding activity. Curcumin can also reverse the expression dynamics of c-fos and fra-1 in tumorigenic HeLa cells by mimicking their expression pattern in normal cells. But the bioavailability of curcumin is extremely poor due to its hydrophobicity, rapid metabolism and lack of specificity in targeting cancer cells. Therefore, a folic acidcurcumin conjugate for mutation has been developed by attaching one molecule of curcumin with two molecules of folic acid (Cur-2FA) to make curcumin hydrophilic, enhance its bioavailability and to target only cancer cells which specifically express high level of folate receptors. The effect of both native curcumin and Cur-2FA on HPV and AP-1 has been analyzed in HPV positive cervical cancer and HPV negative breast cancer cell lines, by band shift assay, confocal microscopy, flow cytometry and western blotting including their cytotoxic potential and targeted cellular uptake of curcumin. In comparison to native curcumin, curcumin-2FA has been found to be at least two times more effective in inducing down regulation of HPV transcription, AP-1 activity and expression of its components particularly c-fos and fra-1. Curcumin-2FA was found to be more potent than curcumin in inducing apoptosis inhibits tumor cell proliferation and Cur-2FA conjugate was specifically taken up by cancer cells. In mice, curcumin-2FA was more bioavailable and had a longer half-life than curcumin. We demonstrate that this nontoxic low molecular weight curcumin-2Folic acid conjugate formulation specifically targets cancer cells which overexpress high affinity folate receptor and enhanced cellular uptake facilitating increased bioavailability and targeted delivery to cancer cells. Director, National Institute of Virology, Pune Concerned with emergence of many new diseases during the last decade the World health Assembly adopted International Health Regulations in 2005. Accordingly, each country is having obligation to monitor infectious diseases of international concern by developing minimum capacity for surveillance and diagnosis. Awareness has also increased in communities and vague diagnosis like viral fever or viral aetiology is no more acceptable to patients. Amongst infectious diseases viral diseases are the most important but capacity for surveillance and diagnosis is limited in most of the countries. Therefore, it is a major challenge to countries to develop virology to meet national and international expectations. The major viral outbreaks in India during last decade include Nipah virus, SARS, Chandipura, Avian Influenza, Chikungunya and Crimean Congo haemorrhagic fever. Nipah virus outbreak in Siliguri in 2001 exposed ill preparedness of national laboratories to provide timely diagnosis and lack of infection control practices in hospitals. The disease created unparalleled scare to health care workers. SARS posed extra ordinary pressure on health system due to peculiar nature of spread and resultant monitoring of airport and sea ports to detect cases, contact tracing and quarantine of suspected patients in infectious disease hospitals. Discovery of Chandipura as a causative agent of encephalitis in central India was a masterpiece investigation. Involvement of virus has been proved year after year and is fully established. Being disease of low socioeconomic strata there is little interest to commercialize the diagnostics and vaccines developed by the institute. This brings important issue of governmental policy for neglected important diseases of specific populations in the country. Avian influenza is caused by highly pathogenic Influenza H5N1 virus. This virus kills almost 100% infected chickens and causes 40-90% mortality in humans in different countries. The virus became greatest worry to public health and also to the food economy. Influenza viruses have notorious history of world wide spread in shortest possible time and with widespread occurrence of this virus in many countries it was considered as the prime candidate for the pandemic. Unprecedented pandemic preparedness plans were prepared by countries causing huge financial burden on health systems around the world. Chikungunya outbreak after 34 year took everyone by surprise. The main vector Aedes aegypti, which was earlier designated as urban mosquito has developed strong foothold in rural areas also. As a result both urban and rural communities experienced massive outbreak. To add to problem the Aedes albopictus also played important role in some ecological settings in places like Kerala. Finally in 2009, H1N1 pandemic appeared to test all the claims of our preparedness to contain infectious diseases. The virus appeared in Mexico/USA and within a month travelled to most part of the word once again to prove that spread of this virus remains unmatched. The virus caused millions of the cases and many deaths causing panic in public health system. It was real life time experience to be part of the team responsible for management and control of the pandemic. Quick identification and fantastic public health response to Crimean Congo Haemorrhagic Fever outbreak in Gujarat was reassuring and satisfactory and greatly appreciated. The highly fatal and obnoxious disease is a real threat to health care workers particularly young doctors and nurses in Gujarat and Rajasthan states of the country. Aetiology of many outbreaks like encephalitis in Gorakhpur and other parts of eastern UP remained elusive. Similarly, several outbreaks of haemorrhagic manifestations are also unidentified. The epidemics and pandemics provided tremendous pressure on public health system of the country and posed unique challenge to laboratories and scientists of the country. On one hand progressive evolution in public health preparedness and response has been reassuring and satisfactory but on the other hand lot more is expected from government and scientists to meet future s challenges. Newer diagnostics, standard and uniform protocols, quality control and assurance, vaccines and antivirals need to be developed. Hospital records and infection control practices need to be modernized. Infectious disease hospitals are in pathetic state and need review and fresh investment by governments. Citrus originated in South-east Asia including N.E. India. Many of the important virus diseases now recognized in citrus appear to have originated in India and the orient, and to have moved with citrus as it was carried to other parts of the world through traders. Economic importance of citrus viruses has been realized world over. Therefore, for the first time delegates from 11 countries participated in a conference of citrus virus diseases in Riverside, California in 1957. At this meeting in California, an organization now known as 'International Organization of Citrus Virologists' (IOCV) was established. IOCV has organized 18 conferences since its inception in 1957. These conferences are held once in three years in different citrus growing countries. The 12th IOCV conference was held in India at Indian Agricultural Research Institute, New Delhi in 1992. To avoid introduction of viruses through the movement of citrus germplasm, the International Organization of Citrus Virologist, (IOCV), FAO, and IBPGR jointly published FAO/IBPGR Technical Guidelines for the Safe Movement of Citrus Germplasm and the I was a member of this team. Citrus psorosis was the 1st virus disease reported in 1933 in California and budwood certification was started against it in 1938. Many important discoveries were made between 1950 to 1975 on virus chemistry and structure, Identification of new pathogens like mycoplasma-like organisms and viroids and discovery of density gradient centrifugation which helped the purification of viruses. In India, Citrus was grown as seedling trees. Horticultural techniques like budding and grafting started by 1920. Since then grafted plants were raised in various parts of India. Decline or dieback started in sweet orange on rough lemon and became a baffling problem by 1960. Dieback was attributed to various causes like Fungi, physiological disorders, deficiency, and soil etc. After the discovery of greening disease in India, dieback was suggested to be caused by Greening ? fungi like Diplodia, Fusarium etc. In India, the researches on citrus viruses were restricted only to symptomatology, transmission, vector, and virus-vector relationship by 1980. The research work on virus and virus-like diseases was strengthen after the establishment of the Advanced Centre for Plant Virology at IARI, New Delhi in 1984 with the assistance of UNDP of FAO and establishment of a National Research Centre for citrus (NRCC) at Nagpur in 1986 by ICAR. In citrus, more than 40 virus and virus-like diseases have been described from most citrus growing countries but in India, 21 virus and virus like diseases are known to occur. Some of the economically important diseases were reported and worked out by us. Important among these are: Indian citrus ringspotvirus (Mandarivirus), Citrus yellow mosaic virus (Badnavirus), Citrus yellow vein clearing virus, Pummelo yellow mosaic rhabdovirus, Citrus yellow corky vein viroids. After 1990, reliable serological and molecular diagnostics were developed for detection of viruses in citrus and many viruses infecting citrus were sequenced. Details of these events will be discussed. Both DNA and RNA viruses cause disease in livestock and poultry. RNA viruses are more prone to mutations and hence the diseases caused by RNA viruses, such as Foot and mouth disease, Bluetongue, African horse sickness, rabies are difficult to control. Carrier status and reservoirs of viruses pose further problem in this regard. Bats and wildlife are known as carriers/reservoirs of many viral diseases. There are instances where the virus can be maintained and excreted by the healthy individual for a long period as has been reported for rabies virus in dog saliva, herpes and lentiviruses in animals and man. For bluetongue virus cattle serve as reservoirs and for malignant catarrhal fever virus sheep act as reservoir. Pigs are known to amplify FMD and influenza viruses. Since in developing countries, sheep, goats, cattle, buffaloes and pigs often graze or live together in the company of humans, these close contacts increase the chances of spread and maintenance of existing viral diseases as well appearance of new viral diseases. Immunosuppression by herpes and retroviruses further complicate the epidemiology of animal viral diseases. As a result of this, non-existent forgotten diseases like Tuberculosis have reemerged. Prions responsible for Mad cow disease in cattle and Scrapie in sheep have belied the notion that an infectious agent need to have nucleic acid. Origin of viruses and place of viruses in total micro biome still remains less understood, particularly when there are viruses of animals, birds, fishes, lower animals, bacteria, mycoplasma, fungi, etc. Changing life style, societal development, climate change has made visible impact on the occurrence of new viral diseases. In recent decades, occurrence of new viral diseases has gone up and now a new disease appears almost every year or alternate years, 75% of which are zoonotic in nature. Similarly, about 63% of the known 1465 existing infectious diseases are zoonotic. Besides causing diseases, there are some positive aspects of viruses. For example, the Newcastle disease virus has been shown beneficial application in resolving tumors in humans due to its oncolytic properties. Bacteriophages have been shown to have the potential of using for prevention/cure of mastitis caused by Staphylococci and Streptococci. Rinderpest, also known as Cattle Plague, is a viral disease of cattle, buffalo, sheep, goats, pigs and other wild ruminant species. It is difficult to trace back to the time when the disease was recorded for the first time, it is believed that the disease originated from Asia and it ravaged the livestock in Asia and Europe from time immemorial. Though the disease in India was first recorded in Assam during 1752, it must have been prevalent through centuries. Rinderpest has been linked with the development of modern veterinary science globally. This devastating disease, in the absence of any known treatment or vaccine used to cripple the economy as it resulted in high morbidity and mortality in bovines, adversely affecting livestock and agricultural production leading to poverty of rural masses. The mortality could be as high as 80% or more. Efforts to counteract Rinderpest have inspired many developments in the history of veterinary science including the creation of first veterinary school at Lyon in France in 1762. It also triggered the establishment of public veterinary services in Europe and creation of Indian civil veterinary services in India in 1891. Considering the high morbidity/mortality and enormous economic losses due to Rinderpest, the Govt. of India in 1868 appointed ''The Cattle Plague Commission'' to study the problem and suggest remedial measures. Ever since the establishment of Imperial Bacteriological Laboratory (IBL) at Pune in 1889 on the recommendation of this Commission, priority was given for conducting research on Rinderpest. Sustained research work started after shifting of the IBL to Mukteshwar (Indian Veterinary Research Institute) in 1893. The research work laid emphasis on diagnosis and vaccine development to control/prevent the disease in domestic livestock. Though the efforts to effectively control Rinderpest were initiated very early in 20th century, major thrust came after 1950s. The presence of Rinderpest in India was confirmed by Cattle Plague Commission in 1871. It was reported that hundreds of thousands cattle died every year. Agricultural activity like ploughing of fields and transportation of agricultural products were drastically affected. On the recommendation of Cattle Plague Commission the Imperial Bacteriological laboratory (now known as Indian Veterinary Research Institute (IVRI) was established at Pune on 8th December, 1889 for undertaking researches on Rinderpest and other infectious diseases of livestock and poultry. Because of the huge losses and socio-economic implications of rinderpest, the Government of India invited Prof. Robert Koch and his team to Mukteshwar in 1897 to provide his scientific inputs to control Rinderpest. Prof. Robert Koch and his team along with Dr Alfred Lingard, the then Head of the Laboratory, conducted experiments on the use of bile from infected animals as vaccine with inconclusive results. However, Prof. Koch confirmed that the disease prevalent in India and the one he was working in South Africa were similar. In 1899-Anti-Rinderpest serum was developed at IVRI, Mukteshwar and used as a method of passive immunization. The protection conferred was for about 14 days only. Thereafter, serum simultaneous method of vaccination was standardized at Mukteshwar and shown to be superior to the Anti-Rinderpest serum. While the antiserum provided immunity for about a week only, in serum simultaneous method, involving the use of live virus along with the serum, there was a risk for the spread of the disease to the susceptible in contact animals. This method remained in vogue till the epoch making discovery of Dr. J.T. Edwards for successfully adopting and fixing the Rinderpest virus in heterologous host (goat) made a great breakthrough for the control of Rinderpest in India and abroad and also paved the way for the development of other attenuated viral vaccines which provided lifelong immunity. The serum simultaneous method of vaccination was replaced by goat tissue Rinderpest virus (GTRV) vaccine in 1931 for its use among plain cattle of India. Further refinement in the development of goat tissue Rinderpest virus vaccine (GTV) was responsible for launching the National Rinderpest eradication program (NREP) in India in 1954. National Rinderpest Eradication program of India provided impetus to the FAO initiative for installing South Asia Rinderpest Eradication campaign (SAREC). Systematic vaccination campaigns were conducted in North, West, Central and East India during 1956-1964 . Subsequently, Tissue culture Rinderpest (TCRP) vaccine was introduced as it was safe in all breeds of cattle including exotic European breeds and their crossbreds in which GTV was considered as hot vaccine. This mass vaccination provided sustainability to livestock development programs, especially the cross breeding program in cattle with high milk yielding exotic breeds. Systematic vaccination decreased the incidence of the disease incidence but did not eliminate the infection in the country. It was with this concern that National Program on Rinderpest Eradication (NPRE) was initiated in 1992 with the financial support from European Union. Under this program, based on epidemiological history of Rinderpest, the country was demarcated into four specific zones (A, B, C and D) for vaccination and follow up action to eradicate Rinderpest. The campaign was finally successful with focused vaccination and zoo sanitary measures with audit on sero monitoring system. About 1600 million doses of vaccination against Rinderpest were carried out between the years 1956-1998. The last outbreak of Rinderpest occurred in 1995 in India in Tamil Nadu, and vaccination was stopped in 2000. India's sero-surveillance exercise covering 480 million livestock population and involving 86,000 veterinarians and para veterinary staff seems to be the largest of its kind in the world. Though India had a long campaign to conquer Rinderpest, it provided historic evidence to the world community that the stamping out policy as was adopted in Europe and other countries could be avoided. In India, because of the socio religious considerations, slaughtering of animals is considered inhumane and sentiments of Indian population are against the cow slaughter policy. This ethical approach to eradicate rinderpest in India seems to be the greatest achievement in the world history for eradication of a viral disease of food animals. Finally India achieved the freedom both from Rinderpest disease and virus infection in 2004 and 2006, respectively. Rinderpest eradication could be considered a revolution. The FAO of United nations stated that Rinderpest eradication was instrumental in enabling the green revolution in India. Rinderpest control provided assured draft power for crop production before the introduction and popularization of mechanization of crop farming. Non availability of animal draft power in pre-independence and post-independence period till 1980 resulted in declined planted area leading to smaller harvest. There was enhanced productivity of cattle through cross breeding and artificial breeding. India gained additional food production due to Rinderpest control/eradication from 1965 to 1998 which added up to 289 billion US dollars. This is the greatest contribution of veterinary scientists to crop science and dairy development program in India after Independence. Eradication of Rinderpest mainly benefitted livestock keepers and enhanced food and nutritional security. Above all there has been argumentation in both meat and milk production. The milk production increased 2.99 times more from the year 1955 to 1995 and 4.796 times by 2006. The meat production increased to 17.99 times from the year 1959 to 1995. Impact of Rinderpest control on values in million dollars milk and meat has been substantial. There has been 102.06 times increase in income from milk and 193.96 times more from meat from 1950-1951 to 2005-2006 . Increase in terms of tons for milk production was 72719 (000 tones) from . Increase in terms of meat production was 2671 (000 tons) from . Value in respect of milk is 15563.56 million US dollars and meat comes to 435011 million dollars between the years 1950 to 1996. Rinderpest eradication has also contributed towards conservation of biodiversity as well as species that might be threatened will have one less problem to face. The expenditure on livestock health care programs has also Abstracts 101 reduced substantially due to the eradication of Rinderpest. Subsequent to global eradication of Rinderpest as declared by FAO on 28th June, 2011, future generation should be in preparedness for rapid and unequivocal diagnosis in an emergency situation if Rinderpest re-emerges. Updated laboratory facilities for prompt detection of virus and trained manpower in selected places need to be defined and biological material of Rinderpest of value, like vaccine strains, virulent viruses and antisera are capsulated under bio containment facilities with periodical check up by the eminent FAO experts/ national consultants in various countries. The major viral disease reported in India by IVRI, Mukteshwar after Rinderpest was Ranikhet disease of poultry in the year [1927] [1928] . Efforts made to attenuate the virus by serial passage in the developing chicken embryos succeeded in 1942 in the form of a modified live virus R2B which provided satisfactory immunity but did not cause disease. However, the R2B strain was hot in imported breeds of poultry. On similar lines attenuated vaccine was developed for Fowl pox by serially passaging the field virus in embryonated chicken embryos. Attenuation of wild virus for vaccine development has also been attempted in primary cell cultures, established cell lines and mice for several diseases including sheep pox, goat pox, orf, camel pox, rabies, African horse sickness. With the introduction of imported high yielding exotic germplasm and their crossbreds, the problem of foot and mouth disease (FMD) took gigantic proportions threatening the dairy industry. Series of killed vaccines against FMD were developed using different chemicals for inactivation of the cell culture grown virus for large scale vaccine production, both stationary cultures in large flasks and suspension cultures in fermenters have been developed. Presently BEI inactivated, aluminum hydroxide oil adjuvanted trivalent vaccine, having virus types OA and Asia1 is in vogue. Rabbits and chicken embryos have also been used for attenuation of viruses for producing lapinised and avianised vaccines. Alternative passages in cell culture and chicken embryos and unnatural route of infection have also been used for vaccine development. In some diseases, such as ILT, Newcastle disease, Marek's disease, naturally occurring mild/ attenuated viruses have been used successfully. Attenuation of vaccine candidate strains of viruses is both time consuming and uncertain method based on hit and trial method. African Horse Sickness, a vector born viral disease of equines which is endemic in South Africa, hit India in 1960-1961 for the first time through Rajasthan border. IVRI, Indian Army and National Institute of Virology, Pune jointly work together on war footing to contain the infection and finally succeeded in eliminating clinical cases by 1965 following strict regulations under the law on infectious diseases for collection of samples for diagnosis and disease investigation, destruction and proper disposal of affected animals. The virus isolated was found to be Type IX. Diagnostics along with mouse brain tissue vaccine were developed successfully. India is free from AHS and OIE has given negative status. However, we need to have preparedness for prompt diagnosis of the infection, in case the disease struck again as AHS episodes outside South Africa have occurred after about 30-35 years. Efforts should be directed to prioritize the existing, emerging and exotic viral diseases of livestock and poultry considering their economic, zoonotic and epizootiological characteristics, and threat perceptions for undertaking appropriate R&D and policy interventions. The diagnostic tests should qualify to OIE requirements which require harmonization, validation and accreditation of all the diagnostic tests in place as a mandatory requirement for health certification for export and import of livestock and livestock products, semen and embryos. Unlike bacterial diseases, viral diseases are difficult to control due to their rapid spread and lack of effective and affordable antiviral drugs. Rapid, specific and precise diagnosis is of utmost importance for timely and effective control of viral diseases. The diagnostic test should be sensitive, specific and at the same time cost effective. Where ever possible, recombinant antigen and monoclonal antibodies should be used in ELISA and other diagnostic assays. Only OIE recommended tests for various diseases, should be used for trade certification purposes. DIVA test along with marker vaccine should be developed for all those diseases where vaccination policy is in vogue, for differentiating the infected animals from the diseased ones. A DIVA has already been developed for FMD at Project Directorate on Foot & Mouth Disease, Mukteswar. An array of diagnostic tests from conventional AGPT, neutralization, CFT, HA-HI and agglutination to the second and third generation series tests, including IFT, IPT, IEM, ELISA, Dot-ELISA, Dip-Strip, EIAs, RIAs, PCR, PCR-ELISAs, LAMP, RT-PCR, Nested PCR, Real Time PCR, Multiplex PCR, nucleic acid hybridization, gene sequencing are now available and many more are under development. Signal (light, electronic) amplification and detection methods are the most exciting ones among these. Now there are tests having sensitivity of detecting as less as 10 virus particles. New innovation in molecular diagnostics include automated PCR, Real-time computerized PCR analysis, second generation PCR kits, DNA amplification finger printing, DNA Chips and Bio Sensors, lab-on-a-chip, micro arrays, nucleic acid sequence based amplification (NASBA), ligase chain reaction (LCR), DNA probe test based on strand displacement amplification (SDA), self-sustained sequence replication reaction (3SR), application of quantum dots and molecular imaging for the diagnosis of respiratory viruses and surface enhanced Raman spectroscopy (SERS) using laser and nanotechnology. From field application point of view, development of Pen-side diagnostic tests is the need of hour as there is limitation of laboratory based diagnosis in rural areas where most of the livestock is available. Work is in progress at IVRI and TANUVS for developing Pen-side diagnostic tests for viral diseases of poultry and other animals. Ideal vaccines should be potent, capable of imparting lasting immunity, having adequate protective immunogenic mass without other unwanted extraneous antigens, easy to administer, safe for all category of animals including pregnant ones and capable of providing immunity in young animals and after in-ovo vaccination of poultry. The vaccine should have good keeping quality at refrigeration temperature as well as at ambient temperature. Development of recombinant bivalent and multivalent vaccines should receive priority of researchers as these vaccines will reduce the cost of vaccination as well as stress to the animals. Immunomodulators including adjuvants and cytokines should be used to enhance the immune response of killed vaccines. The dose of the vaccine should be as small as possible (about 1 to 2 ml for large animals). The vaccine should be able to induce adequate humoral as well as cellular immune responses. The vaccines should be updated periodically to accommodate the antigenic variations in the pathogen. Innovations are also required for devising easier administration of vaccines, such as patch vaccination, pressure vaccination, and vaccine administration through drinking water and feed pallets. This will need to develop thermo tolerant live virus vaccines TANUVAS has developed a thermo stable vaccine against Newcastle disease in feed pellet. Research is in progress at IVRI and other institutions for developing virus like particle (VLPs) vaccines against FMD and Blue tongue viruses devoid of viral genome, through reverse genetics. These vaccines will not have the possibility of reverting back to virulence which is common apprehension against the use of live modified viral vaccines. Modern biology and genetic engineering approaches have made it possible to develop safer and potent vaccines in the form of DNA vaccines, peptide and synthetic vaccines. Understanding of immune mechanism against viral diseases in various host species will help in developing better vaccines utilizing the knowledge of cytokines, histocompatibility complex, interferon ant interference, s-RNAi, TLRs and marker assisted selection for disease resistance breeding. We need to switch over from first and second generation vaccines to third generation vaccines on case to case basis. Antigenic and genetic analysis of highly pathogenic H5N1 viruses from Indian outbreaks from 2006 to 2010 was carried out for selection of hemagglutinin (HA) gene donor vaccine candidate. On the basis of 3-D antigenic cartography A/chicken/West Bengal/80995/2008 H5N1 virus was found to be the best fit as the HA gene donor virus. For development of reverse genetics based non-pathogenic H5 vaccine strain, the basic amino acid cleavage site RRRKKR*GLF (major genetic character responsible for highly pathogenic nature of H5N1 viruses) in the HA gene of the selected H5N1 strain was modified to IETR*GLF by site directed mutagenesis using reverse-complimentary primers. The mutation in the pHH21-HA plasmid was confirmed by nucleotide sequencing. Using the mutated HA gene in the reverse genetics system of type A influenza virus (WSN/33), a recombinant H5N2 virus was generated from cloned gene segments of influenza virus as a non-pathogenic vaccine candidate for developing DIVA marker vaccine against H5N1 in poultry. Non-structural protein 1 (NS1) of influenza A viruses counteracts the host immune response against the influenza viruses by not only inhibiting the nuclear export and maturation of host cell messenger RNA (mRNA), but by also blocking the dsRNA-activated protein kinase (PKR) mediated inhibition of viral RNA (vRNA) translation. Down regulation of NS1 gene expression in the host cell may be a potent antiviral strategy to provide protection against the influenza virus infection. We observed the effect of siRNAs, synthesized against NS1 gene, on the inhibition of virus replication in Balb/c mice. The potency of siRNAs was assessed by plaque assay, real time RT-PCR, western blotting and histopathological analysis in mouse lung samples. We also assessed the cytokine levels in the Bronchioalveolar lavage fluid (BALF) of mice by ELISA. The protective effect of siRNAs was also evaluated by performing animal survival assay. When siRNA was administered in Balb/c mice, 92% reduction in the levels of NS1 gene expression in mice lungs was observed. A significant reduction in the lung virus titers and bronchial inflammation was also detected in the presence of siRNA as compared to the untreated virus control. A decrease in IFN-c & TNF-a and an increase in IFN-a1, IFN-b & IL-1b was observed in BALF samples of siRNA treated-virus infected mice. The siRNA effectively protected the mice from lethal influenza A virus challenge over a 21 day period. The study was validated by the use of selectively disabled mutants of each set of siRNA. Our findings suggest that siRNA targeted against NS1 gene of influenza A virus can provide considerable protection to the virus infected host cells and may be used as potential candidates for nucleic acid based antiviral therapy for prevention of influenza A virus infection. Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata-700010 (WB); Email: chawlam70@gmail.com Virus infection brings about cell death as a consequence of activation of the host cellular defense mechanism. Since early onset of cell death is detrimental to virus replication, viruses recruit certain proteins to activate cellular survival for counteracting apoptosis. Cell lysates were prepared from A549 and 293T cells infected with InfA/PR8 strain at increasing time points after infection followed by immunoblotting. Role of Matrix protein was assessed by expressing M1 protein in pcDNA6 and using M1 specific siRNA. During influenza A infection viral protein M1 (Matrix 1) activates survival genes at early infection periods but enhance the effects of apoptotic inducers late in the infection. At early infection periods M1 localizes mainly to nucleus and this translocation is cellular kinase mediated phosphorylation dependent. In the nucleus, M1 physically interacts with death domain-associated protein 6 (Daxx) which is a transcriptional repressor of survival genes, especially Birc2, Birc3, Birc5, c-flip, XIAP. Daxx interacts and inactivates RelB (NF-jB) transcription factor and recruits DNA methyltransferases, mainly Dnmt1 and Dnmt3a, to methylate RelB responsive promoters and thereby silence gene expression. Thus, M1 prevents Daxx's repressional function during infection thereby exerting survival role. On the other hand during late infection phases M1 interacts with stress-activated heat shock protein 70 (Hsp70) and shows pro-apoptotic function. Apart from its chaperonine activity it exerts anti-apoptotic function by binding to apoptosis protease-activating factor 1 (Apaf-1) thereby disrupting apoptosome formation. M1/Hsp70 complex formation results in reduced interaction between Hsp70 and Apaf-1 leading to procaspase-9 activation induced by cytochrome c and ATP. Influenza viruses modulate cellular innate immune response such as apoptosis by dual function of its matrix protein different stages of infection. Influenza A (H1N1) pdm09 was the first influenza pandemic of the 21st century. Though the overall global case fatality rate of the 2009 pandemic H1N1 appears to be low (less than 0.5%), the fatality rate in India was relatively higher (0.86%). Elucidating the viral determinants of disease severity is important for developing better prevention and treatment strategies. Nasal/throat swabs were collected from patients presenting with Influenza like Illness (ILI) and were tested for the presence of (H1N1) pdm09 virus by real-time PCR (CDC Protocol). The positive cases were categorized into mild, moderate or severe group based on the clinical presentation of the patient (n = 5 per group). Virus isolation was done using MDCK cell line. The haemagglutinin gene of the isolates was amplified by one step RT PCR using WHO-CDC primers. The expected size products were purified using USB Exosap-IT purification kit and subjected to DNA sequencing using Big Dye terminator V 3.1 cycle sequencing ready reaction kit. The obtained sequences were analysed using BioEd-itv7.0.9. The viral haemagglutinin gene sequences have been analysed for signature mutations in the three categories of patients with clinical symptoms. Live bird markets in Eastern India comprise of chicken and ducks sold in layered cages. Ducks are known reservoirs of various subtypes of avian influenza virus (AIV) and the chances of interspecies transmission and persistence of AIV in the environments is much higher under these conditions. In India, so far H9N2 and H4N6 subtype of low pathogenic AIV have been isolated from ducks and one more subtype of H11N1 was reported from a migratory bird. Here, we are reporting isolation and characterization of H11N9 subtype of AIV from a duck in the State of Jharkhand from a live bird market for the first time. In November 2011, cloacal and tracheal swab samples were collected from apparently healthy chickens and ducks in a live bird market located at Jamshedpur, Jharkhand. One of the cloacal samples collected from a nondescript duck was positive for AIV by NP gene based RT-PCR. Further subtyping by RT-PCR revealed that the virus belonged to H11N9 subtype. Subsequently, the virus was isolated by inoculation into 9-11 day old embryonated specific pathogen free chicken eggs. The subtype of the virus was confirmed as H11 by hemagglutination inhibition test with reference serum. Sequencing of hemagglutinin (HA), neuraminidase (NA) and non-structural gene (NS) was done and analysis indicated 97% homology of HA and NA genes and 99% homology with those of A/mallard/Czech Republic/13438-29K/2010 (H11N9) virus. This study emphasizes the need for targeted surveillance of live bird markets to understand the various subtypes of AIV that are circulating among Indian poultry. The recent emergence of a novel swine origin human influenza A virus poses a serious global health threat. The control of this pandemic swine flu by vaccination has become more difficult due to rapid mutation. In absence of effective vaccine, the control is mainly by treating the patient with antiviral drugs especially oseltamivir (Tamiflu). However, the recent 2009 pandemic H1N1 viruses have been found resistant to oseltamivir. This has necessitated to look for more effective and safe alternate antiviral drugs. The anti-flu property of Tulsi (Ocimum Sanctum) has been discovered by medical experts across the world quite recently. The present study describes the assessment of antiviral and immunomodulatory potential of ethanolic extract of tulsi leaves against the pandemic 2009 swine flu H1N1 virus. The antiviral activity was demonstrated through in vitro inhibitory potential in MDCK in terms of virus inhibition assay as indicated through CPE, cell viability, hemagglutination, immunofluorescence, real-time reverse transcription-PCR. The immunomodulatory potential of ethanolic extract of Ocimum was also assessed in Balb/c mice through haematological parameters, mRNA expression of immuno-modulating cytokines & TLR respectively. Further phytochemical characterization of ethanolic extract was accomplished by TLC, HPLC to identify the active constituents responsible for antiviral activity. The virus infectivity was suppressed by ethanolic extract of tulsi leaves in both dose and MOI dependent manner. The time point kinetics of virus inhibition assays indicated that the post treatment was more effective than pretreatment followed by simultaneous treatment. The in-vivo immunomodulatory activity in Balb/c mice revealed significant increment in the population of defensive W.B.C. through enhanced proliferation of monocytes and neutrophils. The release of immunomodulating cytokines (IL-4, IL-10, IL-15) & TLR-9 was significantly higher compared to control mice. In conclusion, the present study clearly indicated the immunomodulatory as well antiviral potential of tulsi leaves that can offer promising option for supplemental strategy to currently available antiinfluenza therapies. morbidity and mortality worldwide. The objective of this study was to compare the infectivity rates, antigenic variations and the serum cross-protection to other subtypes of influenza A virus among children of defense personnel in Delhi region, from July 2011 to March 2012. The study comprised of 208 patients presenting ILI. All the samples were screened for influenza virus by real-time RT-PCR and the blood samples of virus positive patients were collected to check cross-reactivity of antibody in serum. Of the 208 screened samples, 39 (18.8%) patients were found to be infected with influenza A virus. The PCR typing and sub-typing revealed 3 samples to be positive for H1N1-2009, 13 for seasonal H1N1, 19 for H3N2 and 4 samples were positive for both H3N2 and sH1N1. HAI was performed to analyze the cross reactivity of antibodies against various subtypes of influenza A viruses. It was observed that the previously infected seasonal H1 patients had a good level of cross-reactivity against H3 stain, but the H1N1-2009 infected patients were found to have fair titre of crossreactivity to sH1N1 strain but drastically less titre against H3 strain. The study revealed that the seasonal H3N2 (48.71%). infection was found to be highest as compared to seasonal H1N1 (33.33%) followed by H1N1-2009 (7.69%). The co-infection of sH1N1 & H3N2 (10.25%) was also observed among the infected patients. The antibody raised by H1N1-2009 gave significant cross-protection against seasonal H1N1 as compared to H3N2. Swine Origin Influenza A virus belonging to the Orthomyxoviridae family is an enveloped virus with segmented negative sense RNA genome surrounded by helical symmetry shell. Current influenza vaccines protect against homologous viruses but are less effective against antigenic variants and provide little protection against a different subtype. New vaccine strategies are therefore needed that can both accelerate production and provide broader spectrum of protection. Subunit vaccines like recombinant HA protein offers an alternative over conventional vaccine strategies that could save several months of manufacturing time. In contrast to conventional approaches there is no need for live influenza virus or large quantities of eggs, and subunit vaccines could be deployed earlier in the pandemic for effective reduction of morbidity and mortality. Moreover, it is also economical to produce these vaccines capable of inducing antibody that can neutralize the circulating strain of influenza virus. As it is very important to produce the antigenic protein in its native soluble form, prokaryotic system like bacteria may not be ideal for making this vaccine protein. With this background, it is practically important to have an alternate heterologous system that can make pandemic influenza HA protein and also can overcome the limitations associated with already reported systems. As Pichia offers several advantages including rapid and economical bulk production of recombinant proteins, an effective influenza HA subunit vaccine can be made with minimal notice for pandemic variants using Pichiapastoris. In this study, we have successfully developed the semi-continuous fermentation strategy for bulk production of H1N1-HA recombinant protein using Pichia system. Different fermentation parameters like Dissolved oxygen/aeration, Agitation, pH, Glycerol feed, methanol induction have been optimized for better expression of recombinant HA protein. The expressed proteins have been purified using Ion Exchange chromatography and the authenticity of the purified protein was confirmed using Immunoblotting technique. Influenza is a vaccine preventable viral infection that can occasionally cause severe or fatal disease, especially in the elderly, the very young and those with underlying illness. Influenza activity in India is monitored by National Influenza Center at National Institute of Virology (NIV). Clinical samples of suspected patients with influenza like illness were collected from outdoor patient department at local hospitals in Pune (September 2011 (September -2012 . Real time RT PCR was used to detect seasonal and pandemic influenza. Virus LIMS software was used for data management. During the study period, total 1307 clinical samples were tested for influenza virus and 10% samples (82-type B, 56-Pdm, 1-H3) were positive for influenza. Type B circulation was observed throughout the study period (except June 2012), however peak activity (32.92%) was observed in October 2011. A spurt in pandemic A (H1N1) cases was observed from February-April with 51.78% positivity and increased activity was observed from August to September during the rainy season in 2012. Pandemic A (H1N1) positivity was highest (46.42%) in age group 15-35 years while lowest (8.9%) in age group above 55. In case of type B, highest positivity (41.46%) was observed in the 5-15 age group while lowest (1.2%) in age group above 55. Overall Male to female ratio for influenza positivity is 1 The increasing understanding of the regulatory mechanisms involved in the pathogenesis of influenza is opening up opportunities for new therapeutic intervention. Since currently available treatment options against these viruses are limited owing to genetic drifts, there is a need for development of alternative therapies. DNAzymes (Dz), derived by in vitro selection processes, is one such discovery that has potential for selective gene silencing; thus we aimed to target the M2 gene of influenza A virus to down-regulate its replication in MDCK cells. Several 10-23 DNAzymes were designed and analyzed for their ability to specifically cleave the M2 gene of influenza A virus. The Dz that worked best was further standardized with MgCl 2 gradient to achieve the best results. The same concentrations of Dz were also transfected with the whole virus (Influenza A/PR/8/34) to study the inhibition of replication. RT-PCR and Realtime RT-PCR assays followed by western blot analysis were performed to detect the inhibition of the expression of M2 gene. We DNA vaccination represents a unique strategy to overcome the limitations of immunization with conventional vaccines which is restricted by the high variability of influenza viruses. We evaluated the protective efficacy of a plasmid DNA, encoding an evolutionarily conserved epitope of viral matrix protein, against the influenza A virus infection. It was found that the mice immunized via the intramuscular route elicited immune response to the peptide encoded by the plasmid DNA, with enhanced level of Th1 cytokines viz. IL-12 and IFNc production in the stimulated splenocyte supernatant. The T lymphocytes in the spleen of immunized mice significantly lysed the virus infected MDCK cells. A significant decrease in virus replication was also observed in the lungs of immunized mice and 63% of the mice were protected against the lethal challenge of influenza A viruses. These findings suggest that the plasmid DNA expressing a single matrix epitope may serve as a promising vaccine candidate to provide effective immunity in susceptible population. One of the most important vector borne diseases of ruminants worldwide is caused by bluetongue virus (BTV), an orbivirus of the Reoviridae family. The virus genome consists of 10 segments of dsRNA which code for 7 structural and 4 non-structural proteins. The outer capsid proteins VP2 (encoded by genome seg-2) and VP5 (encoded by genome seg-6) are the most important proteins responsible for serotype specificity, virus neutralization and haemagglutination. VP5 protein also enhances the protective neutralizing activity of VP2 protein inducing higher serotype specific antibody titre than the VP2 alone. The nucleotide sequence and the phylogenetic analysis of these genes can provide a rapid alternative approach for characterization of circulating strains, and for future molecular epidemiological investigations around the world. Out of the twenty-six BTV serotypes found worldwide, 22 were reported from different states of India. These include serotype 21 which was recently isolated from Andhra Pradesh, and was involved in severe outbreak of bluetongue in Indian native sheep. BTV21 (KMNO-7) and BTV16 were circulating at the same time. This co-circulation, along with the fact that the virus genome is segmented, provides an opportunity for these two isolates of different serotypes to simultaneously infect the same animal, and even the same cell or a same vector with the potential for generation of reassortant viruses. Present study was undertaken to conduct full length nucleotide sequencing of genome seg-2 and seg-6 of two Indian isolates VJW-64 (BTV16) and KMNO-7 (BTV21) which would lead to efficient characterization of the virus, eventually helping in selection of predominant candidate vaccine strains. For the purpose of sequencing, genome seg-2 and seg-6 were divided into several overlapping gene fragments and primer pairs specific to every such fragment were designed. Consensus full length gene sequences thus generated were submitted to GenBank. Nucleotide sequence homology analysis of genome seg-2 and seg-6 of these two Indian isolates revealed an inter-serotypic variation of 29% to 60.1% on the basis of vp2 gene. isolate of BTV16, which is much more than it shows with any isolate of BTV21. KMNO-7 (BTV21) significantly diverged from original strain of BTV21, and is a reassortant strain having acquired seg-6 from an isolate of BTV16. It was probably because of this reassortment that BTV21 was involved in a severe outbreak of bluetongue in Indian native sheep. In-silico restriction enzyme analysis revealed certain restriction sites which were specific to the Indian isolates of BTV16 (VJW-64) and BTV21 (KMNO-7). In this study full length sequencing of genome seg-2 and seg-6 of Indian isolate VJW-64 (BTV16) was carried out for the first time in India and that of KMNO-7 isolate (BTV21) for the 2nd time in the world. This study in itself is an important initiative towards developing a database of full length genome seg-2 and seg-6 nucleotide sequences of Indian isolates. It also provides some useful insights into the epidemiology of the bluetongue disease in India and undermines serotyping on genome seg-6 basis. An immunoaffinity chromatography for purification of biologically active (infective) bluetongue virus (BTV) has been optimized using polyclonal antibody to core particle of BTV. Anti-core antibody was produced in guinea pig which was further purified by ammonium sulfate precipitation. Immunoaffinity columns were prepared by noncovalent binding of anti-core antibody to cyanogen bromide-activated protein-A Sepharose beads. BTV-infected cell culture supernatant was added to this column, virus was captured by specific antibody, column was washed with buffer and then virus was eluted with a buffer consisting of 4 M MgCl 2 and 75 mM HEPES, pH 6.5. The infectivity of eluted BTV was tested on the cell culture. BTV purified by this method retained the antigenicity and infectivity as determined by sandwich ELISA and infectivity assay on BHK-21 cell respectively. There are many reports of occurrence of mixed infection of BTV with other viruses like peste des petitsruminants virus, capripox viruses, orf virus etc. Most of the times, conventional procedure of BTV isolation directly on cell culture from mixedly infected tissue or blood samples leads to isolation of non-BTV. This method will help in selective capture and enrichment of BTV from mixed population of viruses for efficient isolation on cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. India is enzootic for bluetongue and 21 different serotypes of Bluetongue viruses (BTV) have been reported based on virus isolation and seroprevalence. Bluetongue virus serotype-1 (BTV-1) is most prevalent in the north-western and southern states of India and a good number of viruses have been isolated from different hosts for the past more than 25 years. A study was conducted to understand the antigenic and genetic variations amongst these isolates by VP2 gene sequencing and cross-neutralization. Full length VP2 genes of 20 BTV-1 isolates were sequenced and sequence analyses revealed presence of two distinct geographical clusters of viruses confined to north-western and southern regions of India. Phylogenetically all Indian BTV-1 isolates are very closely related to the Australian isolates and therefore the viruses are 'eastern topotype' of BTV. The viruses of both north-western and southern cluster showed more than 97% sequence identity at nucleotide and amino acid level. In spite of close identity, cross-neutralization studies amongst the Indian BTV-1 isolates revealed co-existence of distinct neutralization variants. One neutralization resistant variant was found amongst the southern cluster of viruses in geographically restricted area of Andhra Pradesh. Antigenic variation was also observed amongst the north-western isolates but no distinct neutralization resistant variant was found. Cross neutralization data suggest that there are at least two distinct VP2 phenotypic variants existing in India and therefore selection of suitable vaccine candidate for BTV-1 should be done very critically to ensure maximum protection against the all the existing neutralization variants. Ruminants of Maharashtra Central Military veterinary laboratory (CMVL) of Indian Army still remains the only laboratory to achieve this rare feat since the year 2009 when accreditation was awarded for the first time to any animal disease diagnostic laboratory in India. Majority of the animal disease diagnostic laboratories in India are working under the Government or Government aided self financed institutions and performing the dual task of diagnostics and research on animal pathogens. While the consensus is growing by every day to establish international standard in the diagnostic task in the Government owned laboratories, however it's still a matter of debate to bring research work under this ambit due to obvious reasons. Even as in many developed countries worldwide quality systems have been adopted successfully in many key sectors including the research laboratories, research institutes in India are now showing an interest in setting up a system. Establishing the laboratory quality management system as per international standard ISO 17025:2005 is certainly an opportunity for them not only to improve their performance but also to reach a quality label. The success of CMVL in this regard can be taken as role model which is actively engaged in meeting diagnostic as well as research obligations to meet growing needs of Army apart from collaborating research with leading centers' of excellence in the country. The experiences of CMVL are discussed in detail in this paper to show that research institutes can immensely benefit from an international quality standard adjusted and adapted to their peculiar role and needs. In recent times Avian Reovirus (ARV) is associated with poor growth, increased feed conversion ratio and mortality in broilers. The present study was undertaken to characterize nine field isolates suspected for proventriculitis/tenosynovitis syndrome of ARV by isolation in SPF eggs, RNA-PAGE, RT-PCR and RFLP. The virus isolates and ARV vaccine (ARVv) taken as virus control were propagated in nine day old specific pathogen free chicken embryos by chorio-allantoic membrane (CAM) route. In eight out of nine samples the chicken embryos showing haemorrhages and yellowish green foci on the liver and death within 64-98 h were considered specific for ARV. RNA was extracted from the CAM of the above isolates by TRIZOL method and subjected to RNA-PAGE after heat treatment at 60°C in water bath for before loading in the wells. Out of nine isolates, 8 isolates and ARVv control showed profile of 10 segments consisting of large (L1, L2, L3), medium (M1, M2, M3) and small (S1, S2, S3, S4). The S1 and S3 genes of ARV isolates were amplified using gene specific Reo1F, Reo2R and Reo3F, Reo4R primers for S1 gene and P1F, P2R, P3R primers for S3 gene. PCR product sizes of 980 bp, 810 bp and 672 bp, 548 bp respectively were obtained that was specific for S1 and S3 genes of ARV in six of the eight isolates. On RE digestion of the 548 bp product of the S3 gene with enzyme Rsa I, 3 fragments of *180, *250 and *400 bp were observed in 3 of 6 samples and ARVv whereas product digests of *180 to *400 bp were obtained in other 3 samples. Based on the variations of the RE digest profiles the latter 3 isolates were designated as field isolates when compared with that of ARVv. To conclude, RNA-PAGE, RT-PCR and RFLP can be employed for detection of ARV and depending upon the similarities and dissimilarities in their RFLP profile the isolates can be identified as either vaccine or field strains of ARV. Buffalopox is an emerging contagious viral zoonosis of domestic buffaloes (Bubalus bubalis) which also infects cattle and humans. The disease is caused by buffalopox virus (BPXV)-a close variant of vaccinia virus (VACV) being recognised as an occupational zoonosis due to the naïve population against orthopoxviruses. Lack of information on host tropism of BPXV, led us to analyse the host range serpin 1 (SPI-1) gene of BPXVs isolated from outbreaks (2010 & 2011) in buffaloes, cattle and human in Maharashtra and Uttar Pradesh. The encoded protein of this gene is expressed in the early stages of infection and acts as anti-apoptosis factor in vaccinia virus. The virus was isolated in Vero cells from infected scabs collected from animals and humans and the extracted viral DNA was subjected to PCR amplification of serpin 1 gene. The amplicons were cloned in pTZ57R/T vector and sequenced commercially. An open reading frame (ORF) nucleotide sequence homology search was carried out using the NCBI BLAST. The nucleotide (nt) and deduced amino acid (aa) sequences were aligned using the CLUS-TAL W program and phylogenetic trees were constructed using neighbour-joining method of MEGA5 software. Comparative sequence analysis was done to elucidate variations among BPXV isolates from buffaloes, cattle and human as well as to determine the evolutionary relationship among OPXVs. Sequence analysis revealed that BPXV isolates (buffalo, cattle and human) shared maximum homology (99% at nt and 98.5% at aa level) among themselves as well as with VACV. Furthermore, phylogenetic analysis exhibited closest homology with VACV isolates followed by RPXV, CPXV, HSPV, Cantagalo virus, MPXV and VARV. The high degree of sequence similarity and close evolutionary relationship with VACV and other poxviruses indicates the conserved nature of the gene among Orthopoxviruses which could play a role similar to VACV in viral pathogenesis. This is the first report of genetic analysis of anti-apoptosis gene of buffalopox virus which will be useful in elucidating the host antiviral response. Infectious bronchitis virus (IBV) a threat to the domestic chicken causes acute and highly contagious respiratory disease. IBV is a member of the genus Coronavirus. The genome which is 27.6 kb genome in size is non-segmented, positive sense, single stranded RNA. All coronaviruses maintain a set of essential genes, including those that encode the polymerase (Pol), spike (S), small membrane (E), membrane (M), and nucleocapsid (N) proteins, in the order 5'-Pol-S-E-M-N-3'. The M glycoprotein which is partially exposed at the surface of the virion is a major Type II integral membrane protein and is essential for the production of coronavirus-like particles. Duck viral enteritis, which is caused by duck enteritis virus (DEV), is an acute, contagious and lethal disease. DEV is currently classified to belong to the alphaherpesvirinae subfamily of the herpesviridae family. The DNA polymerase, a product of UL30, is of central importance for successful viral replication in alpha herpes virinae group of viruses. The viral enzyme is involved in the initiating events of gene amplification processes. The UL30 protein is highly conserved among herpesviruses at the amino acid sequence level. This is one of the important protein for the replication of virus in the host cell. The DNA polymerase of herpes virus shows a significant similarity with that of eukaryotes. The DNA sequences encoding UL30 was identified from the genomic DNA of DEV. The amplification of the partial gene was carried out, using self designed primers in a polymerase chain reaction. A 1596 bp partial gene product was cloned into pTZ57R/T cloning vector and the UL30 gene was sequenced. The recombinant UL30 protein was expressed by subcloning the UL30 gene from a pET bacterial expression system after induction with 1 mM IPTG. The expressed recombinant protein obtained as a fusion protein with histidine tag was purified by affinity chromatography. The purified recombinant protein was confirmed by SDS-PAGE and western blot analysis using anti DEV sera which detected an 80 kDa protein band in the blot. The recombinant protein was further dialysed and concentrated using Amicon filters and the total yield of the protein was found to be 120 lg/ml. several sheep and goat pox outbreaks have been confirmed using a combination of diagnostic tools (clinical, autopsy and molecular) in districts of Mandi, Kangra, Shimla, Kinnaur, Kullu and La-haul&Spiti. The rapid increase in temporal and spatial distribution of pox outbreaks points to the fact that disease is emerging or remerging in the state. Since capripox virus is capable of persisting in the environment for long and able to spread fast, and also the fact that the diseases are highly contagious in nature it has become mandatory to take up blanket vaccination programme to induce herd immunity so that losses are prevented. Canine Parvo Virus type 2 (CPV-2) is responsible for acute gastroenteritis in pups, with a high rate of morality. CPV is prone to genetic evolution and over the three decades it has undergone several mutations there by resulting in emergence of different strains like CPVtype 2a, 2b, New-2a, New-2b and 2c. In March 2012, an outbreak of acute gastroenteritis occurred in pups of an organised canine breeding kennel located at Meerut, India. The breeding kennel has been stringently following the recommended vaccination schedule against CPV both in pups and in adult canines. During Mar-May 2012, diagnosis of CPV infection was first confirmed by commercial immunochromatographic (IC) test-kit and later confirmed by polymerase chain reaction (PCR). In the present study, isolates were genetically characterized by partial amplification of 611 bp of the gene encoding for capsid protein VP2. These sequences were then aligned and compared with sequences of 03 commercial vaccine stains and with reference CPV strains. Total of 21 faecal samples of pups suspected of CPV were collected during the current outbreak of which, 17 samples were found positive by IC test-kit and 19 samples were found positive by PCR. All the PCR positive samples were subjected for virus isolation in Madin-Darby canine kidney (MDCK) cell line from which 12 isolates were obtained. On the basis of the sequence alignment, these isolates were characterized as New CPV-2a. In all the isolates, at 297 position of VP2 gene, Alanine (Ala) was present instead of Serine (Ser-Ala), while, at 496 position' Asparagine (Asn) was present. At 325 position of VP2 gene, all the isolates showed a point mutation i.e. presence of Isoleucine (Ile) instead of Tyrosine (Tyr). CPV strains present in three different commercial vaccines were also analysed and were characterized as CPV type 2. On the basis of geographic pattern, it has been observed that CPVtype 2b variants are predominant in Northern India while CPV-type 2a variants are prevalent in Southern and Central India. This is the first report establishing the prevalence of New CPV-2a from the Northern India. Further, it is inferred that CPV vaccine strains of most of the commercial vaccines are different from the circulating CPV outbreak strains. This difference raises the concern over the CPV strains contained in most of the commercially available products in India. Peste des petits ruminants is an acute, febrile and infectious viral disease of goats and sheep, characterized by mucopurulent nasal and occular discharges, necrotising and erosive stomatitis, pneumonia and inflammation of gastro-intestinal tract leading to severe diarrhea. The disease is caused by a Morbillivirus of the family Paramyxoviridae. The present study was aimed to study the incidence of Peste des petitsruminants virus (PPRV) in goats of selected areas of Gujarat by sandwich ELISA and to derive estimates of overall, locationwise, agewise and sexwise incidence and samplewise positivity rates. In addition, the study also involved standardization and application of novel Nucleocapsid (N) gene based RT-PCR, for diagnosis of PPR. A total of 46 clinical samples from 31 goats suspected of PPRV from selected areas of Gujarat were tested by sandwich-ELISA, of which 23 animals were found positive yielding an overall incidence rate of 74.19%. All the 46 clinical samples, a reference vaccine virus (Sungri isolate) and a blood sample from apparently healthy goat were processed for RNA extraction using TRI Reagent Ò . PCR amplification was done using in silico designed N-gene specific primer pair N1-N2. Reference vaccine virus as well as fifteen (32.61%) of the 46 clinical samples, including fourteen blood samples and one nasal swab, produced the desired amplion of approximately 463 bp with primer pair N1-N2. Factors Classical swine fever (CSF), caused by CSF virus, is a highly contagious disease of domestic pigs and wild boar having profound socio-economic impact on piggery husbandry in developing countries like India. CSF is endemic disease in our country and numbers of outbreaks occur every year in many states of the country. Among the three regions of the CSFV genome, targeted for classifying the isolates into different genotypes for deducing the molecular epidemiological information, 190 nucleotide (nt) long major immunogenic protein E2 gene sequence is routinely considered. In order to genetically assort the circulating CSFV isolates in the country, in the present study, we aligned 190 nt of E2 protein of 31 Indian isolates of CSFV belonging to the state of Assam, Andaman and Nicobar Islands, Haryana, Kerala, Meghalaya, Mizoram, Utt-arKhand, Uttar Pradesh, West Bengal, using ClaustalW and phylogenetic tree was constructed using MEGA 5. The phylogenetic grouping using neighbor joining method indicated that these Indian isolates belonged to three genotypic groups viz., genotype 1. Rabies is one of the most important zoonotic disease of the world which primarily depends on the vaccination to control. Despite of the long history and the remarkable progress in the knowledge, prevention and control of rabies still the disease has maintained its global distribution in animals as well as humans. In the vaccine production the rabies virus is grown on BHK-21 cells followed by further downstream process & finally blended as a vaccine. It is very essential that the growth of the virus is monitored as an in-process control. This can be done by a regular estimation of the infectivity titers. The infectivity titer of rabies can be indirectly measured by an in vivo method known as MICIT-mouse intracerebral inoculation test using weaned inbred mice (8-10 g) where the titre of virus is expressed in MID50/ml. In this method different dilutions of the test substance is inoculated intracerebrally in groups of 10 mice each and based on the specific mortality due to disease the median lethal dose in 50% of the population (MID50) is estimated using Reed-Muench formula. MICIT test is a time consuming and cumbersome with some inherent biological variations due to certain factors like age, body weight and sex of animal etc. Rabies infectivity can also be quantified using an in vitro method in BHK-21 cells known as Fluorescent antibody test (FAT) using FITC-labelled anti-rabies immunoglobulin, following acetone fixation. The stained cells were washed in buffer and read under bluelight fluorescence microscope to detect the characteristic green fluorescence associated with rabies antigen corpuscles. As the mice test is regarded as a gold standard the present study is undertaken to establish a correlation between the two methods to ensure the reliability of using the method as an estimate and eventually to replace the use of animals in MICIT method. A total of 21 samples of viral harvest from regular production lots of rabies virus were estimated for its respective titers by both the methods. All the samples were tested for its titre as per standard method of MICIT and FAT (OIE2000 Buffalopox is a highly contagious, zoonotic viral disease affecting buffaloes, cattle and humans and is being reported time-to-time from several states of India. The etiological agent, buffalopox virus (BPXV) is a close variant of vaccinia virus (VACV), the virus used for smallpox vaccination. Considering the zoonotic importance and increased incidence and severity of buffalopox infection in humans in the Indian sub-continent combined with vulnerable population lacking antibodies to smallpox and related orthopoxviruses, studies are warranted to develop recombinant antigen-based prophylactics and diagnostic assays for epidemiological investigations and appropriate control measures. The present study carried out to over-express the major immunogenic protein of BPXV, H3L (*35 kDa) in prokaryotic system and to evaluate for its diagnostic as well as prophylactic potential. This was the first attempt in the proteomic study of BPXV. In the present study, H3L gene of BPXV-Vij/96 was amplified (*840 bp), cloned into a pET32a expression vector and recombinant H3L over-expressed in E coli. Recombinant H3L fusion protein (*50 kDa) was purified under native condition using affinity chromatography and its specific immunoreactivity was confirmed using anti-orthopoxvirus sera (BPXV and CMLV) in Western blot. Recombinant H3L forms only monomers as analysed by native and denatured PAGE followed by Western blot. Recombinant H3L protein was found to be immunogenic in adult mice and guinea pigs following immunization along with adjuvants as revealed by ELISA and serum neutralization test assays. Further, passive protection studies carried out in suckling mice using hyperimmune sera (HIS) raised against recombinant H3L, showed its prophylactic ability by protecting against virulent BPXV. Purified recombinant H3L protein was used as an antigen in an indirect ELISA format and showed specific reactivity to buffalopox and camelpox sera. There was no cross reactivity with HIS or infected sera against orf, goatpox, sheeppox, PPR, FMD and bluetongue viruses. The study indicated that the recombinant H3L protein is a potential candidate/reagent in the development of prophylactics/diagnostics for buffalopox and camelpox infections. VP6 based RT-PCR assay were used to screen a total of 191 diarrhoeic faecal samples from bovine calves (138 from cattle calves and 53 from buffalo calves) collected from different regions of India for the presence of RV and the VP6 based RT-PCR detected 14.1% samples positive for RV compared to 11.5% by RNA-PAGE screening. For the rapid and sensitive diagnostics, aSYBR Green based Real-Time PCR assay was developed with the self-designed primers targeting the conserved region of the NSP4 gene with an amplicon size of 130 bp. For optimization, standard curve was constructed using ten-fold serially diluted plasmid containing the NSP4 gene insert of 130 bp. The standard curve parameters determined were in the optimum range; the slope was -3.336 with a high regression coefficient (R 2 ) of 0.997. The PCR efficiency was 99.4%. A Ct value of less than 35 and a Tm value of 78.35°C (± 1.0°C) were considered as positive. This newly developed qPCR was found 100% specific as no amplification from related enteric viruses was seen. The qPCR assay was compared with available diagnostic assays on 80 clinical samples chosen randomly and its sensitivity in comparison to RNA-PAGE and RT-PCR assay was 4.02 and 3.28 times higher, indicative of suitability of this assay for screening of the samples for bovine RVAs in future. Further validation by screening sufficient more number of field samples is essential for its use at large scale in molecular epidemiological studies of RVs in India. RNA interference mediated through small interfering RNA (siRNA) mediates degradation of gene transcript in sequence dependent manner. This potential of siRNA has been exploited against many viruses by inhibiting the expression of viral genes that are crucial to viral pathogenesis and virus replication. In this study, the potential of RNAi has been evaluated as antiviral agent against rabies. The siRNAs targeting rabies virus (RV) glycoprotein (G) gene and nucleoprotein (N) gene was designed and evaluated using plasmid co-transfection approach in HEK-293 cells. The potent siRNAs as single and multiple form were delivered using lentiviral vector system. Lentiviruses viz., Lenti-G7, Lenti-N and Lenti-G7 N expressing siRNA targeting RV-genes were constructed. To evaluate the effect of siRNA delivery using lentivirus system, three homogenous cell lines namely, BHK-G7, BHK-N and BHK-G7 N constitutively expressing siRNA were prepared and antirabies effect was analysed by challenging these cell lines with RV-PV-11 strain. Forty eight hours post-infection, the reduction in G gene and N gene transcript were quantified and compared with respective controls using real-time PCR. There was significant reduction in RV-G and N transcripts in all the cell lines. Further, the effect of siRNA on RV multiplication was evaluated in RV challenged cell lines by direct florescent antibody technique (dFAT). There was significant reduction in RV multiplication in all the siRNA expressing cell lines compared to control. To evaluate the anti-rabies effect of siRNAs delivered using lentivirus in vivo in mice, the mice were treated intracerebrally with 5 9 10 5 transduction units of Lenti-G7, Lenti-N and LentiG7N. Five days post-lentivirus treatment the mice were challenged with 20 LD50 of RV-CVS strain using intra-masseter route. The challenged mice were observed for 14 days for rabies specific symptoms and death. There was 66.6% protection in mice treated with Lenti-G7. The mice treated with either Lenti-N or Lenti-G7 N showed complete protection. The control untreated mice died within 9 days post-challenge showing lethal nature of challenge virus. These observations demonstrated the potential of lentiviral delivered siRNAs in inhibition of rabies virus multiplication. This holds the potential of siRNAs as antiviral agent against rabies. Bluetongue (BT) is an infectious, non contagious arthropod borne viral disease of wild and domestic ruminants especially sheep which inflicts major losses on subsistence sheep farmers in southern India. Affected sheep may have erosions and ulcerations on the mucous membranes, dyspnea, lameness and inflammation of the coronary band. The disease is caused by bluetongue virus (BTV) the type species of the genus Orbivirus and belongs to family Reoviridae, transmitted between their ruminant hosts by certain haematophagous Culicoides biting midge. It is OIE list 'A' multispecies disease. Till 2008, twenty four distinct serotypes of BTV (BTV-1 to BTV-24) have been isolated and characterised worldwide. Recently, BTV-25 has been reported from Switzerland and BTV-26 from Kuwait. The present study was carried out with the objectives to identify and characterize the culicodes species procured from southern India. A total of sixty seven Culicoides DNA samples extracted using a nondestructive DNA extraction method were amplified using mitochondrial gene specific primers. An amplicon of 523 bp was obtained in all the amplified samples. All these positive samples were further targeted for sequencing PCR and the products were purified and dissolved in Hi Di formamide. The samples were sequenced in the DNA analyzer ABI PRISM 3100. The sequencing data obtained was analyzed using computer softwares. A total 48 (71.64%) samples were identified as C. oxystoma, 6 (8.95%) as C. reconditus, 4 (5.97%) as C. pseudopalidipennis, 3 (4.47%) each of as C. Schultzei and C. imicola, 2 (2.98%) as C. peregrines and one (1.49%) as C. kubenesis, respectively. The nucleotide sequence based analysis revealed that the majority of the Culicoides species belonged C. oxystoma and showed their prevalence in India earlier also. The blast analysis revealed that the Indian isolates of C. Oxystoma were 90-99% similar with Japanese and Israel isolates. The literature on sequences related to C. oxystoma is very scanty. The only sequences belonging to C. oxystoma have been of Japanese and Israel origin. The phylogenetic analysis revealed huge genomic diversity among Indian sequences as all the Indian isolates formed more than eight clusters distantly related to Japanese and Israel sequences. The sequences of Israel and Japanese origin grouped together to form one separate cluster. Out of 48 Indian sequences, only one showed closeness to Japanese sequence. The rest of all of the Indian sequences grouped in five major clusters showing identity with each other. However, seven of the Indian sequences depicted distant identity with other Indian sequences and very distantly placed from Israel and Japanese sequences. The observations from the present study indicated that the world literature on C. oxystoma sequences is scanty and the information generated in the present study could be useful in molecular characterization and determining the association of this vector with spread of BTV virus depending upon the movement of this vector in various part of the country. The sequencing data generated in this study could form the basis for developing database of C. oxystoma in the country. Bluetongue virus (BTV) is a prototype species of the genus Orbivirus within the family Reoviridae which causes Bluetongue disease (BT) in domestic as well as wild ruminants. BTV is non-enveloped virus having 10 segmented dsRNA genome. Segmented 6 encode VP5 protein along with VP2 protein gives serotype specificity to the virus. A pair of vp5 gene specific designed primer generating an amplicon size of 823 bp of Indian isolate of BTV9 was designed. The segment 6 of an unknown Indian isolate of BTV was amplified using this primer, sequenced and analyzed. Nucleotide sequence analysis revealed that Indian BTV9 serotype showed more than 97% identity with other Indian and European BTV9 isolate and more than 90% with Japanese isolates. However, only 68.8% identity was found with South African isolates. More than 97% identity was observed based on deduced amino acids sequences with Indian, Japanese and European isolates and only 73.4% with South African BTV9 isolates. The phylogenetic study based on vp5 gene nucleotide and deduced amino acid sequences revealed that the BTV9 isolate in present study formed a major cluster including European and other Indian isolates of BTV9 indicated the western origin of this BTV9 isolate. The in silico restriction enzyme analysis (REA) with AflIII, BtrI and HindII showed a common pattern of restriction sites in Indian and European isolates at 1152, 1153 and 591 and lack of any of these restriction sites in African and other Japanese isolates further confirm the western origin of BTV9 Indian isolate. Further, the lack of BsmAI restriction site in Indian BTV9 isolate could differentiate it from European, Australian, Japanese and South African isolates. Within a nucleotype the nucleotide and deduced amino acid sequence identities reported were [76% and [86%, respectively. The BTV9 isolate used in present study showed maximum 76-80.2% nucleotide and 88-93.2% amino acids sequence identity with nucleotype 'B' serotypes (serotype 3, 5, 6, 13, 14, 16 and 21) . This placed all the Indian BTV9 isolates in nucleotype 'B'. However, the BTV9 isolates from South Africa had shown maximum 76.2-77.5% nucleotide and 87.5-89.4% amino acid identity with nucleotype 'C' serotypes (serotypes 1, 2 and 23). This placed South African BTV9 isolates in nucleotype 'C'. Hence the BTV9 of Indian origin in the current study indicated high probability of reassortment in segment 6 which differentiates it from BTV9 of South African isolates. These observations further suggest occurrence of BTV 9 isolates at global level that can be assigned two different nucleotypes B and C. In the autumn of 2011, blood samples (n = 51) were collected from goats in Pithoragarh area of Uttarakhand (India). About 64% of these samples were tested positive for BTV antigen (by a sandwich ELISA) including a number of samples being strong positive. From a strong antigen-positive blood sample, a BTV was isolated (named as PTG-13) on cell culture, which was confirmed as BTV-1 by RT-PCR coupled with partial sequencing of genome segment-2. The cytopathic effects, typical of BTV, appeared as early as on 18 h post infection and virus titer was measured at third passage on BHK-21 cells which was found to be (10 8.7 TCID 50 /ml). The neutralization behavior of PTG-13 was studied by virus neutralization with hyperimmune sera (HIS) prepared against a panel of eleven BTV-1 isolates collected from different parts of the country over a period of more than two decades. PTG-13 was completely neutralized between 1:16 and 1:32 dilution of HIS against two of the south Indian BTV-1 isolates. HIS against six north Indian isolates neutralized the PTG-13 virus at 1:4 dilution. However, HIS against three of the south Indian isolates could not neutralize the PTG-13 virus. One-way neutralization data suggest that PTG-13 has closer antigenic relation with two south Indian isolates than with the north Indian isolates. Neutralization resistance of PTG-13 with HIS against three south Indian isolates suggests that PTG-13 could be a VP2 phenotypic variant of BTV-1 co-existing in southern India. Detailed genetic analyses and crossneutralization of the Indian BTV-1 isolates will provide better understanding of the level of genetic and antigenic relatedness between the isolates. The disease is subclinical in cattle and goats, and these animals can act as reservoir hosts for the virus. In the autumn of 2011, blood samples (n = 51) were collected from goats in Pithoragarh area of Uttarakhand (India). About 64% of these samples were tested positive for BTV antigen (by a sandwich ELISA) including a number of samples being strong positive. From a strong antigen-positive blood sample, a BTV was isolated (named as PTG-13) on cell culture, which was confirmed as BTV-1 by RT-PCR and partial sequencing of genome segment-2. The goat plasma samples were found to contain high titer of neutralizing antibody against BTV-23, however, the virus could not be isolated. Interestingly, no neutralizing antibody was detected against PTG-13 or other BTV-1 isolate, which suggests that sampling was done probably before the development of neutralizing antibody against PTG-13 virus in the host. Isolation of BTV-1 (PTG-13) and presence of BTV-23 neutralizing antibody in blood samples indicate that goats were naturally infected with BTV-1 and 23. The dual infection in goats with two BTV serotypes has potential for intertypic genetic reassortment leading to evolution of more virulent virus strain. Rotaviruses are a major cause of acute gastroenteritis in the case of several mammalian and avian species. According to WHO, the rate of infection and fatality due to rotavirus is more in developing countries. Rotavirus causes runting and stunting syndrome is a major cause of great economic impact among the poultry rearers. The virus belongs to the family Reoviridae and has a double stranded RNA as its genome. The virion is non-enveloped and genome is distributed among 11 segments of dsRNA. The outer layer of the virus is made up of VP4 and VP7 proteins and intermediate layer made up of VP6. The VP6 proteins are group specific protein which helps to classify rotavirus into different groups. Electrophoretic pattern of rotaviruses on poly acrylamide is also specific and according to all these seven groups of virus (A-G) are identified till now. Among these groups group A rotaviruses affect both mammals and birds and groups D, F, G are identified only from the avian species. During the current study samples collected from enteritis cases in poultry from foot hills of the Uttarakhand were screened for the presences of different groups of rotaviruses. Enteric samples were collected as fecal samples, intestinal contents, peeled off intestinal mucosa etc. all the samples were processed and a 10% PBS suspension of the samples were made. RNA was isolated using standard TRIZOL method. Initial screening was done using conventional RNA-PAGE. The cDNAs were prepared from the isolated RNA using standard procedures and group specific VP6 gene based RT-PCR was used for detection of group A and D rotaviruses. Out of the seventy samples screened 21 were found positive for group A rotavirus and 6 were found positive for group D rotavirus. One of the samples was having mixed infection of both group A and D. The results of present study confirms circulation of avian group A and D rotavirus in this region and warrants further studies to characterize the viruses in depth so as to develop a good vaccine for preventing the infection in poultry. Canine parvovirus-2 (CPV-2), of the genus Parvovirus of family Parvoviridae, is an important pathogen of dogs which causes acute enteritis, myocarditis and lymphopenia, especially in pups. CPV-2 induced pathogenesis in animals is attributed to apoptosis in infected cells. Caspases and p53 play an important role in mediating apoptosis. In this study we confirmed that the CPV-2 induced apoptosis of MDCK cells is mediated by extrinsic, intrinsic/mitochondrial and, endoplasmic reticulum stress mediated pathways and is p53 dependent. MDCK cells infected with CPV-2 at 30-40% confluency with 0.01 m.o.i. were harvested at 24 h, 48 h and 72 h post infection. The total RNA was isolated from the harvested cells and the cDNA synthesized using random hexamer primers and MMLV-RT. Real time PCR was carried out to evaluate the expression of caspase-3 (Effector caspase), Caspase-8 (initiator caspase in extrinsic pathway), Caspase-9 (initiator caspase in intrinsic pathway), Caspase-12 (initiator caspase in endoplasmic reticulum stress mediated pathway) and p53. The expression of the above caspases and p53 was found to be upregulated at all time points in comparison to control(s) and increased with time, indicating the involvement of all these caspases. The expression of caspase 3, caspase 8, caspase 9, caspase 12 and p53 at 72 h post infection was found to 9.80 ± 0.85; 5.10 ± 0.55; 3.98 ± 0.55; 2.82 ± 0.30 and 4.27 ± 0.62, folds higher than control(s), respectively. The real time results were also confirmed by IFAT which showed bright fluorescence as compared to controls with caspase-3, 8, 9, 12 at 48 h post infection. The efflux of cytochrome C and increase in percentage of caspase-9 positive cells detected by flowcytometry at all time points with respect to control further confirmed the involvement of intrinsic pathway. Viral metagenomics, coupled with high-throughput sequencing and bio-informatic analysis of mammoth genomic libraries, has recently emerged as a potential tool to characterize the unculturable viruses from the environment as well as animal samples. Animal disease outbreaks which seem to be multi-factorial and complex in nature due to involvement of multiple microbes, with genetic variations at species, subspecies, strains level coupled with non-isolation of etiological agent, pose greater challenge to the researchers. In such scenario, metagenomic approach provides a novel way to unravel the mysteries of disease outbreak as well as better understanding of microbial characteristics, host-pathogen interaction and insights into design and development of novel diagnostics/therapeutics. In India, although, the realm of viral metagenomics is at infancy, the approach seems to grow exponentially in the days ahead as the infrastructure and technique employed become largely applicable in most veterinary research laboratories with a greater focus to safeguard the health scenario of livestock population. Avian reoviruses (ARVs) are the cause of an important emerging disease of the commercial poultry birds in India. Serological screening has revealed its wide spread distribution in poultry population and there is a report about the high economic losses due to stunting syndrome caused by ARVs on several broiler farms in southern parts of the country. The ARV isolate used in the present study was obtained from the Avian Diseases Section, Division of Pathology, IVRI, Izatnagar. In the present study, rB encoding gene of ARV field strain was amplified, cloned into pTZ57R/T vector, sequenced and finally expressed. Phylogenetic analysis of rB encoding gene showed nearly complete homology with American, Canadian and Chinese isolates except Taiwanian isolates of ARV, muscovy duck reovirus strain 89026 and two turkey reovirus strains NC98 and Tx99. The presence of a large no of antigenic regions in rB protein was determined using PROTEAN programme of DNA STAR software. The rB encoding gene was successfully expressed from a pET bacterial expression system. The recombinant protein was confirmed by SDS-PAGE and western blot analysis using hyperimmune sera against ARV raised in rabbit. This protein can be further purified and can be used in rB ELISA for diagnosis of ARV infection and to better predict the immune status of dam for successful vaccination strategy. Infectious bronchitis (IB) is an economically important disease of poultry and it affects chickens of all ages causing respiratory and nephrotic syndromes in broilers, reduced egg production in layers and breeders. Rapid diagnosis and immune status determination are critical to controlling outbreaks of infectious bronchitis virus (IBV). The nucleocapsid (N) protein, a major structural protein of IBV, is the preferred protein for use in development of group specific serologic assays. It has highly conserved sequences, which share 91 to 96.5% identity among various strains, is produced abundantly during infection, and has high immunogenicity, readily inducing antibodies as well as cytotoxic T lymphocyte immunity in chickens. In the present study, propagation of an Indian isolate of IBV was done by chorioallantoic method in 10 days-old specific pathogen free embryonated chicken eggs. Characteristic curling and dwarfing of embryos was noticed in all embryos at sixth day post inoculation. Viral RNA was isolated from allantoic fluid by TRIzol method and transcribed into cDNA by gene specific primers. About 1.2 kb nucleocapsid gene was amplified by RT-PCR. The amplified product was cloned and the nucleotide sequence of the N gene was determined. The Indian IBV isolate exhibited more than 90% homology with the M41 vaccine strain. Efforts are now being made to clone and express the N protein in prokaryotic expression system for development of recombinant N protein based ELISA which will be useful for detecting IBV specific antibody. A recombinant UL30 antigen-based single serum dilution enzyme linked immuno sorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively. From the start of 21st century, researchers are interested in the field of nanotechnology. Nanotechnology based diagnostics and therapeutics have started budding through scientific research. Nanobiosensors are one among them, used for detection of chemicals or biological materials. Nanometer refers to a unit which is one billionth of a meter. Biosensors are analytical devices incorporating a biological material which is intimately associated with a physicochemical transducer or transducing microsystem. Nanobiosensors are known for their high sensitivity, specificity, rapidity and portability. There are various types of nanobiosensors based on various principles namely, electrochemical biosensors, voltammetric and amperometric sensors, impedance sensors, optical fiber based sensors, surface plasmon resonance based biosensors, quartz crystal microbalance and atomic force microscopy based nanobiosensors. Nanobiosensors have been designed for diagnosis of dreadful human viral diseases like HIV, hepatitis B, Hepatitis C, Ebola virus etc. Nanobiosensors have also been designed for diagnosis of animal diseases like avian influenza virus, infectious bovine rhinotracheitis, rabies, bovine leukemia virus. In future, nanobiosensors may replace the current viral disease diagnostics as they are time consuming and expensive. and age groups and is a major cause of direct economical loss to the cattle industry. The disease is widely distributed around the world and is endemic in India, with outbreaks reported from almost all the states. Once the infection is established, the virus remains in latency and persists lifelong. It is the most common viral pathogen found in bovine semen and is one of the major hurdle in implementation of government run breeding and artificial insemination (AI) programmes. To avoid risk of spreading infection, regular testing of semen at semen collection centers is necessary. Therefore, a rapid, sensitive and user-friendly test that can be adapted at semen collection centers for routine screening IBR virus in bovine semen is always in demand. A LAMP based visual detection assay was developed for detection of IBR viral genome. The reaction includes three pairs of primers specific to IBR viral genome which gives unique specificity to the assay. The test was carried out using a simple heat block. The developed assay could detect as low as 10 fg of viral DNA per reaction, which was 10-times more sensitive when compared to conventional PCR. Further, the developed assay was adapted for rapid and sensitive detection of the IBR virus in bovine semen in a user-friendly manner by differentiating positive and negative reaction by visual colour development. The whole LAMP assay, including DNA isolation, isothermal amplification and visualization of results with naked eye could be completed in 90 min. The assay had an analytical sensitivity of 0.215 TCID 50 or 0.4 infective virus particles per reaction when spiked into IBRV negative semen. The LAMP assay was validated for specificity, sensitivity and repeatability using spiked bovine semen. The test has been evaluated on 198 semen samples collected from bulls to detect IBR virus. The rapidity and ease of performing the test makes it an attractive option to be employed in government run IBR control programme as well as breeding and artificial insemination programmes. The use of chemo and radiotherapy for treatment of cancer is limited due to genotoxic side effects on healthy cells, involvement of antiapoptotic signal transduction pathways that prevent cell death and requirement of functional p53 for induction of apoptosis in cancerous cells. Efforts are beings made worldwide towards the development of new anticancer therapies as an alternative to chemotherapy. Viral gene therapy is one of the most potent therapeutics that is being ventured into worldwide. Canine parvo Virus-2 (CPV-2) is one of the viruses with an inherent oncolytic property. The Non-Structural protein-1 (NS1) protein of CPV-2 plays a major role in parvoviral cytotoxicity and pathogenicity in permissive cells. The oncolytic potential of CPV2-NS1 was established in vitro. Prior to taking up the in vivo studies, the present study was undertaken, to clone Canine Parvovirus Ns1 gene and reporter gene GFP in eukaryotic expression vector pVIVO2mcs and characterize the double construct in mammalian cells. The genes were successfully cloned in pVIVO2mcs and characterized for their expression as demonstrated by fluorescence microscopy and immunofluorescence. This characterized double gene construct will be used to evaluate the oncolytic potential of CPV-2 NS1 in experimentally induced in vivo tumour model. Having successfully eradicated Rinderpest, greater thrust is being made to untangle mysteries related to Foot-and-Mouth Disease (FMD). FMD is caused by Foot-and-Mouth Disease virus (FMDV), an Apthovirus under family Picornaviridae. FMD causes critical economic losses to small or large animal holders. Investigation on cytokine responses after natural Foot-and-Mouth disease outbreak and vaccination will reveal significant information. In the present investigation, blood samples from 7 adult Holstein-Friesian crossbred breeding bulls (Bos taurus 9 Bos indicus) of Germplasm center, Indian Veterinary Research Institute (IVRI), Izatnagar and 4 vaccinated Holstein-Friesian crossbred cattle were collected in RNAprotect Ò Animal blood tubes (Qiagen, Germany). To immunize cattle (cowsof Dairy Section, IVRI, Mukteshwar and young calves at experimental animal shed of PD-FMD) trivalent inactivated vaccine (FMDV; Type 'O', Type A' and Type 'Asia 1') was used. After incubation at room temperature for 2 h, these RNAprotect Ò Animal blood tubes were stored at -80°C. Purified total RNA was extracted as per manufacturer's instructions (RNeasy Ò protect Animal blood kit, Qiagen, Germany). One blood sample of Mithun (Bos gaurus) showing clinical FMDV infection was also processed. Cytokine, IL-2 mRNA expression level in the total RNA was detected in one-step real-time polymerase chain reaction (PCR) (7500 real-time PCR, AB Applied Biosystems, USA) using comparative CT (DD) type of experiment (QuantiTect Ò SYBR Ò Green RT-PCR kit, Qiagen, Germany). Melt curve analysis was performed. Expression levels of IL-2 mRNA were depicted in the form of Relative Quantitation (RQ) values vs Target/Sample. Odd sample of Mithun showed lower level of IL-2 mRNA expression. Increased IL-2 mRNA expression levels were found in FMDV infected breeding bulls than the vaccinated cattle. NDV, an avian virus is classified in the genus Avulavirus of Paramyxoviridae family. It causes economically significant disease in birds of various species. The NDV genome consists of six genes arranged in the order 3 0 -NP-P-M-F-HN-L-5 0 which encodes for six structural and two non-structural proteins. The two additional non structural proteins, V and W are formed by the RNA editing process during P gene transcription. In this study, V and W genes of NDV amplified and cloned in eukaryotic expression vector pcDNA3.1 (?), were assessed for their apoptotic potential in human cervical cancer cell line (HeLa). The HeLa cells at around 70% confluency were transfected with pcDNA.ndv.v and pcDNA.ndv.w. The transfected HeLa cells were harvested at 24 h, 48 h and 72 h post transfection and analyzed for induction of apoptosis by DNA fragmentation, PI staining and Annexin v binding assays. No inter-nucleosomal cleavage was observed in the transfected cells even at 72 h post transfection indicating that these two genes did not play role in Abstracts 123 induction of apoptosis by NDV. It was further confirmed the study of phosphatidylserine translocation in transfected cells using Annexin V binding by flow cytometry. The pro-apoptotic role of these genes (proteins) were also studied by staining transfected cells with propium iodide where flowcytometric analysis revealed slight decrease in sub-G1 peak (hypodiploid cell population) as compared with even vehicle and vector control, indicating that this protein is not involved in causing apoptosis rather it has anti-apoptotic role. In conclusion our study indicated that V and W lacks pro-apoptotic behaviour and seems to be play antiapoptotic role in HeLa cells. Vaccination is considered as the cheapest and best alternative to control the incidence of the disease and its ultimate eradication. Sheeppox live attenuated vaccines are highly immunogenic but their usefulness is limited because they stimulate a pock reaction and/or lead to the death of some of the vaccinated animals. Therefore, there is a need for the development and selection of efficacious and safe attenuated live indigenous vaccine to combat the disease. In the present study, sheeppox vaccine made from indigenous sheeppox virus-Srinagar, 38/00 (SPPV-Srin) attenuated in Vero cells at Division of Virology, IVRI, Mukteswar was compared in terms of their safety, potency, sero-conversion and protective dose with the commercial in-use vaccines, Roumanian Fanar (SPPV-RF), a foreign strain and Ranipet (SPPV-R), an indigenous strain adapted in primary lamb testes cells and calf thyroid cells respectively. Identity of SPPV vaccine strains were confirmed by PCR, PCR-RFLP and sequencing. Animal experiments were carried out in sero-negative sheep as per the OIE recommendations. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was found to be ''hot''; not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed delayed hyper sensitivity reaction and resisted virulent SPPV challenge, while control animals developed disease. SPPV could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine by PCR and real time PCR. All vaccines were found potent and the PD 50 was highest for SPPV (Srin and R) followed by RF. The immunized animals were sero-converted following vaccination with sustained antibody responses after challenge was assessed by ELISA and serum neutralization test. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines, in addition to better safety. Thus, there is potential benefit in use of indigenous SPPV-Srin vaccine for control and eradication of sheeppox in India. Newcastle disease virus is an infectious disease of poultry which causes severe economic losses in domestic poultry. New generation vaccines are currently in demand to avert deficiencies of vaccines currently in market. Among these, rational design of vaccine candidate is a promising approach. Multi-epitopic DNA vaccine construct containing fusion and haemagglutinin epitopes were designed with additional elements like endoplasmic reticulum secretory signal sequence, poly histidine and gene optimization to improve immunogenicity. Mycobacterium tuberculosis HSP70 was used as a genetic adjuvant to increase innate and adaptive immune responses. Transient expression of the construct was confirmed with an immunofluorescent assay in Vero cells. The constructs were injected subcutaneously into 15 days-old specific pathogen free chickens. Immune responses were studied with HI, ELISA, LTT and FACS assays. Protection was assessed by challenging with a virulent virus. HSP mediated multi epitopes improved the immune responses and conferred protection against Newcastle disease. The results indicate that a rationally designed DNA vaccine construct can improve vaccine strategies involving nucleic acid immunizations. Use of medicinal plant/herb extracts for viral infection has raised future of phytoantiviral therapeutic agent. Jatropha curcas Linn is a multipurpose, drought resistance, perennial plant belonging to Euphorbiaceae family. The present study was undertaken to observe the effect of Jatropha curcas leaf extract on replication kinetics of infectious bursal disease virus (IBDV) and also the mechanism of antiviral action was explored. Chick embryo fibroblast (CEF) adapted IBDV was treated with 50% methanolic extract of Jatropha curcas leaves and the viral replication kinetics was assessed by virus titration (TCID50), reverse transcription polymerase chain reaction (RT-PCR) and quantitative real time PCR. The antiviral action of extract was determined by observing the effect of extract on virion binding to host cell, RNA entry and damage to nucleic acid. The results indicated that 50% methanolic extract of Jatropha curcas leaves was found to inhibit viral replication possibly through direct antiviral effect of one or more of the constituents of the extract. The mechanism by which the extract of J. curcas inhibits viral replication was explored. The results showed that the extract inhibits viral RNA entry into host cell, when added simultaneously at the time of infection, whereas no effect on virion attachment to host cell as well as no apparent damage to RNA genome was observed. Thus these experiments demonstrated that antiviral activity of J. curcas leaves extract, the activity may be due to direct action of one or more of phytochemical present in extract which in part inhibiting viral RNA entry into host cell. Among the infectious agents associated with gastroenteritis, rotaviruses (RVs) are the major ones, while picobirnaviruses (PBVs) are emerging viruses affecting number of mammalian and avian species. The present study describes the development of a diagnostic assay for the detection of bovine PBVs. A new RT-PCR assay was developed with the self-designed primers targeting RdRp gene (segment-2) with detection limit of 4.45 9 10 2 copies/ll of plasmid having specific 272 bps amplicon. This RT-PCR diagnostic assay was used to screen a total of 228 faecal samples collected from diarrhoeic bovine calves (176 from cattle and 52 from buffaloes) from different regions of the Indiaand the assay detected 5.26% samples for PBVs compared to 2.63% by RNA-PAGE. Further genotyping of PBVs presented the predominance of genogroup I while one sample, the first of its kind in the world revealed mixed infection of genogroup I and II in bovines. The sequence analysis of positive amplicons of bovine PBVs unveiled the highly diverse nature irrespective of their place of isolation. Grippingly, three clones from a single sample showed significant sequence divergence. These findings advocated the circulation of more than one strain of PBVs within a single host. Since the inadequate availability of PBVs sequences in public genome databases, our bovine PBVs isolates makes a separate cluster on phylogenetic analysis and some of the isolates displayed higher sequence homology with human and porcine isolates showing probability of interspecies transmission of the virus. A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid, specific and highly sensitive detection of Capripox virus (CaPV) genome. The assay was optimized using purified viral genomic DNA and the reliable amplification was observed at 63°C for 60 min using a set of four primers targeting DNA polymerase gene of CaPV. The amplified LAMP products was identified by gel electrophoresis and direct observation by naked-eye for presence of turbidity and colorimetric detection of the amplified products following the addition of SYBR Green I and Hydroxy Naphthol Blue (HNB) dyes. The specificity of the LAMP assay was assessed by amplifying 9 different strains of CaPV (5 sheep pox and 4 goat pox viruses) isolated in different geographical areas. No cross-reactivity with other related viruses, including orf virus (ORFV), buffalpox virus (BPXV) and camelpox virus (CMLV), was detected. The detection limit of LAMP is found to be 10 copies/ll of standard plasmid and 10 fold higher to standard gel based PCR (100 copies/ll). The assay was evaluated for detection of CaPV in field clinical samples (n = 200) and cell culture isolates (n = 9) and compared with conventional PCR and TaqMan probe based QPCR. As a field diagnostic test, the stability of LAMP reagents including primers and enzyme after exposure at 37°C is checked and found to be stable for 5 days without affecting diagnostic efficacy of the assay. The study proves that the LAMP assay is a simple and cost-effective on-site diagnostic tool for rapid clinical diagnosis of capripox in sheep and goats suitable in resource limited field laboratories. . In majority of the virus-serum regimens site 3 was found to be substituted in the populations. Variants could be identified with substitutions at site 2 or 3 only but site 1 variants were always accompanied by substitutions elsewhere on the capsid. Changes observed in VP3 region were always associated with substitutions at VP1 suggesting a minor role for such residues which act in synergy with other sites towards the neutralization escape phenotype. Presence of substitutions at the same locations as identified in this study in the Indian field isolates supports the importance of these sites. When the P1 sequence dataset of serotype A Indian field outbreak viruses were aligned, VP1 143 position revealed as many as 5 amino acids (T, R, G, A, V). Interestingly, a positively charged residue 'R' was seen exclusively in genotype 16 and 10, while genotype 18 including the vaccine strain showed T 143 . When the P50 genetic variants along with its parent population (P0) were subjected to two dimensional microneutralization test and plaque reduction neutralization (PRN) assay using the BCS, a considerable reduction in the SN 50 titre (*0.47 log 10 titre) and PRN 70 titre (*0.32 log 10 titre) was noticed. Hence, it is presumed that amino acid substitution at VP1 Polymorphism of bovine leukocyte antigen (BoLA) DRB3 gene is being intensively investigated for potential association with economically important diseases of cattle. Accordingly, we investigated the association of DRB3 Exon 2 polymorphism as evidenced by the variation in the binding pockets with variability in immune response to inactivated trivalent (O, A and Asia1) Foot and Mouth Disease virus (FMDV) vaccine in a closed population of crossbred cattle. Antibody titer of C1.8 was set as the cut off value to distinguish the protected (C1.8) and unprotected (\1.8) animals. Eleven different alleles of over 3% frequency were detected in the population. We found that DRB3 alleles *0201, *0801 and *1501 always ranked high for protective immune response whereas alleles *0701, *1103 and *1101 consistently ranked low for unprotected immune response for all the three serotypes. Rank correlation of DRB3 alleles among the three serotypes was positive, high in magnitude and statistically significant (p \ 0.05). Logistic regression analysis revealed that odds of protection from the vaccine were highest for all the three serotypes if allele *1501 was present and strengthened the results of allele ranking. Predicted amino acid substitution in the peptide binding pockets revealed that all the important sites had high Wu-Kabat index. Similarly, specific residues in pockets were crucial for immune response to FMD vaccine. There were specific substitutions in un-protected alleles such as absence of acidic amino acids substituted by basic amino acid at b71, presence of non-polar cysteine or basic histidine at b30 and presence of polar tyrosine at b37. From the observations, we hypothesize that the substitutions lead to unique conformational changes in the protein products of the studied alleles that would associate with the protective or unprotective antibody response to FMDV vaccine. The knowledge has potential implications in future selection programs if integrated with the complete BoLA haplotype details and production traits of the herd. Foot and mouth disease virus serotype Asia1 accounts for about 12% of the FMD outbreaks recorded in India. Five serotype Asia1 isolates collected from different place were analyzed at capsid coding region. For three isolates (PD509/2010, PD18/2011 and PD155/2011), sequences were generated directly from clinical materials. For two isolates (PD508/2010 and IND327/2009), sequences were generated from cell culture supernatants. The integrin binding ligand 'RGD' tripeptide motif was conserved in all the five isolates. The leucine residue at position ?4 and alanine at position ?2 were 100% conserved in all isolates compared. But the leucine at ?1 position revealed frequent variations. Methionine was found conserved in all the four isolates except PD155/2010 in which it was L. R56 in VP3 and R135 in VP2 thought to be critical for heparan sulfate binding were 100% conserved in all the isolates. High degree of conservation of these residues indicates that this virus can utilize cell surface heparan sulfate to gain entry into the cells. The S73 euroasiatic signature in VP4, residues predicted to be important in 1A-1B cleavage and histidine residues at positions 21, 87, 145, 157 and 174 in VP2 that mediate H-bonding at 1B/1C interphase were conserved. The cleavage sites for FMDV type Asia1 isolates from this analysis are ;GAG for L/P1, ALLA;DKK(R)T for 1A/1B,PSKE;GIVP for 1B/ 1C, ARR(Q)E(Q);TTT(A)T for 1C/1D, PEKQ; for 1D/2A. The residues critical for antigenic sites were fully conserved. At least one residue in VP2 (56) and six amino acid sites in VP1 (35, 45, 47, 119, 132 AND 169) was found to be under positive selection. In similarity plot and boot scan analysis, no evidence of recombination was detected. In Maximum Likelihood tree all the isolates were clustered in Lineage C which has been in circulation in India since 2005. No discrepancy in topology between VP1 and P1 was observed. Camelpox is the contagious skin disease of camelids caused by camelpox virus (CMLV) and the disease causes economic impact in terms of morbidity, mortality, loss of weight and reduced milk yield. Both inactivated and live attenuated vaccines are available in few countries. Considering the disadvantages with inactivated vaccine, a live vaccine is the best choice as a long-term solution towards control of camelpox. With the right earnest, a Vero cell attenuated live vaccine from an Indian isolate of CMLV (named CMLV-1) has been developed recently at IVRI, Mukteswar and it was found safe, potent and efficacious. But a study with respect to thermo stability has not been undertaken. Here a research was carried out on evaluation of thermo stability (with and without extrinsic stabilizers) of the vaccine virus. Though the pox viruses are thermo-stable in general, almost reports on the stability of camelpox vaccine virus are found to be nil. On evaluation of intrinsic thermo-stability, the current indigenous isolate was found to have an expiry period of only 9 days and 4 days at 37°C and 45°C, respectively. Hence, a study was carried out to analyse the stability of CMLV vaccine with extrinsic stabilizer combinations to use it on the field for a long period. A total of three stabilizers were used for freeze-drying the vaccine and stability of both freeze-dried and reconstituted vaccine was tested at different temperatures. It revealed that the camelpox vaccine lyophilized with TAA stabilizer (Trehalose dehydrate with amino acids and divalent cations) appeared relatively superior with an expiry period of 44 months at 4°C, 215 days at 25°C, 22 days at 37°C and 20 days at 45°C. Among the three stabilizers, BUGS stabilizer appeared relatively inferior to others at all temperatures. Further, among four diluents viz. PBS, 0.85% NaCl, distilled water and 1 M MgSO 4 used for reconstitution of the vaccine, PBS appeared better followed by 0.85% NaCl for reconstitution of the vaccine. Bacteriophages are viruses which infect bacteria. Even though phages were recognized anti-bacterial agents in the 1920s, the advent of broad spectrum antibiotics overwhelmed it. But with the start of development of antimicrobial resistance and destruction of commensal microbes by broad spectrum antibiotics, it is time to close the chapter of ''antibiotics'' and start a new era with bacteriophages. Compared with antibiotics, phages are able to replicate and make themselves available in the target site, able to mutate and counteract bacterial mutations, very specific towards target species and very low concentrations are necessary for action. In veterinary field, phages are used against bovine mastitis, enteric infections in calves and poultry. A high, but comparable percentage of NSP antibody prevalence {(44/ 125 (35.20%) and 122/399 (30.57%)} was found at both time points 6 months apart indicating an exposure of animals to FMD virus before first collection. In past there had been procurement of sheep into the farm from local sources. The serum samples were also tested in liquid phase blocking ELISA (LPB ELISA) to assess the level of structural protein (SP) antibody titre against FMDV serotypes O, A and Asia 1. The overall herd immunity (log 10 titer of C1.8 for all three serotypes) in the farm was found to be 0% (0/125) and 29.32% (117/399) for the samples collected at two different points of time as mentioned. The farm was under the practice of 'once annual' vaccination against FMD, which was done in the month of May, 2011. Declining herd immunity as evident from low SP antibody titre might have made the farm susceptible to FMDV exposure. Hence, a 'biannual' vaccination schedule may be more suitable to build up optimum level of protective antibody against FMD. Present investigation was conducted on eight Karan-Fries bulls maintained at A.B. Complex, NDRI, Karnal, India from October, 2011 to December, 2011 to study the effect of FMD vaccination on semen quality in KF bulls. A total of 48 ejaculates were taken before vaccination while a total of 112 ejaculates collected after vaccination to study the effect of vaccination stress on semen quality parameters. There was significant increase in rectal temperature during post vaccination up to one week. FMD vaccination had no significant (p = 0.101) effect on ejaculate volume and CMA 3 positive spermatozoa, which represents chromatin integrity. After FMD vaccination there was significant decrease in mass activity, sperm motility, live spermatozoa, sperm concentration, HOST positive and acrosomal integrity, which represents the structural defect in spermatozoa during epididymal maturation stage. After three weeks post-vaccination more than 50% bulls have produced satisfactory acceptable fresh semen quality. There was no correlation between antibody titre to different serotype FMD vaccine with fresh. When the bulls are classified into sire bred HF (n = 3, Group I) and sire half bred (n = 5, Group II) on the basis of exotic genetic level the data revealed that vaccination had more detrimental effect on fresh and frozen semen of Group I comparison to Group II. The testosterone concentration was higher in all the bulls during 1st week after vaccination compared to the pre-vaccination period, which may be due to stress immediately after vaccination and reduced to normal level after one week. No significant correlation was found between testosterone concentrations and semen characteristics. Many RNA viruses, viz., viral hemorrhagic septicemia virus (VHSV), grass carp reovirus (GCRV) and infectious hematopoietic necrosis virus (IHNV) infect a wide variety of fish species, and their ds (double stranded) RNAs are expected to be recognized by TLR3. We investigated molecular interaction between viral dsRNA and TLR3 inrohu fish (Labeorohita) following full-length TLR3-cDNA cloning by rapid amplification of cDNA ends (RACE). The domain architecture in rohu TLR3 (rTLR3) was analyzed by SMART, Pfam and SignalP3.0, and the leucine rich repeat regions (LRR) were manually identified by locating ''LxxLxLxxNxL'' motifs. The 3D-structure of TLR3 was created by homology modeling and molecular dynamics (MD) simulation was carried out in GROMACS 4.0.3 program. In rTLR3, poly I: C (synthetic dsRNA) binding regions were predicted in AutoDock4.0 and GOLD4.1, and the dsRNA of GCRV, VHSV and IHNV binding regions were identified by HADDOCK. Analysis of dsRNA-mediated TLR3-signaling cascade was carried out by intravenous injection of poly I: Cinrohu fingerlings, and quantitative real-time PCR analysis of type I IFN, Mx, TNF-a and IL-1b genes expressions. The full-length rTLR3-cDNA comprised of 873 amino acid (aa) residues with a signal peptide of 22 aa. The mature rTLR3 comprised of ectodomain (ECD), trans-membrane (TM) domain, and TIR domain with 706, 23 and 117 aa respectively. The horseshoe-shaped ECD of rTLR3 consisted of 27 LRRs, and structurally resembled human TLR3-ECD. In rLTR3-ECD, the peptides at LRR 4-6, 13-14 and 20-22 were predicted as poly I: C binding sites, and LRR 8-15 and LRR17-24 were identified as GCRV, VHSV and IHNV-dsRNA binding sites. Injection of polyI:Cactivated TLR3-signaling resulting in significant (p \ 0.05) up-regulation type I IFN, Mx,TNF-a and IL-1b genes expressions. These data together highlight the conserved function of TLR3 in recognizing viral infections and innate immunity from lower to higher eukaryotes. Role of innate immune system is crucial in the non specific first line antiviral defence in fish, which co-ordinate with adaptive immunity for the control of infection. Betanodavirus infection in fish is a very serious neuropathological viral disease causing large scale mortalities in marine fishes. We have developed cell lines from marine finfish Amphiprionsebae (Clown fish) and used for finding out the effect of viral infection in the expression of type I interferon and interferon stimulated genes following pre-treatment with different synthetic TLR ligands such as poly I:C and CpG ODNs. Among the different genes investigated (Type I Interferon (IFN), ISGs (Interferon stimulated genes) such as Mx, ISG-15, IRF-3 and viperin), interferon stimulation was more or less uniform in all the treatments, while virus infection and poly I:Ctransfection had higher immune gene expression compared to CpG ODN. Induction of ISG-15 was higher in poly I:C while virus infection alone had higher expression of viperin (Vig-1). Poly I:C and CpG pre-treated cells upon virus infection showed high induction of all except the ISG-15 genes tested in poly I:C treatment with highest expression of 25.36 fold for Vig-1 gene followed by 11.94 fold for ISG-15. CpG pretreatment also showed similar upward trend of gene expression (13.59 fold) for Vig-1 gene and 12.75 fold increase for ISG-15 gene. Other immune genes exhibited lesser induction compared to Vig-1 and ISG-15. The study indicated that poly I:C and CpG ODNs could induce increased innate immune response in clownfish against betanodavirus. The increased expression of immune genes would help enhance the antiviral defence in live fish and could act as potential immunostimulatory compounds and also as adjuvants in vaccination trials. Aquatic Animal Health and Environment Division, Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, Chennai-600 028; Email: kpjithendran@yahoo.com Viral nervous necrosis (VNN, also known as viral encephalopathy and retinopathy-VER) is one of the emerging viral diseases of finfishes caused by Betanodavirus belonging to the family Nodaviridae. Betanodavirusis one of the two genera making up the family Nodaviridae infecting fish along with Alphanodavirus infecting insects. This RNA virus is icosahedral (25-34 nm) infects a large range of host species, at least 40 species of marine and freshwater fish world-wide and the known host range, continues to expand as new species of fish are used for aquaculture. Of further interest is the potential of wild fish to become sub-clinical carriers as viruscontaminated water spreads from aquaculture enterprises into the marine environment particularly for those countries with large mariculture industries. In India, the virus has been reported from Asian seabass and freshwater ornamentals. A study revealed that 18.5% (n = 243) of the fish samples from wild, hatchery and farmed samples of fish species from different geographical locations were positive for the virus of the genotype RGNNV. The cultured fish species (seabass, mullet and milkfish etc.) revealed 8% infection in the form of sub-clinical infection. Characterization such as the morphology, growth study on cell line, temperature sensitivity, EM, partial nucleotide sequencing etc. has been undertaken and will be discussed. Iridovirus is a large double-stranded DNA virus with icosahedral symmetry, and ranges in size from 120-200 nm in diameter. Family Iridoviridae comprises three piscine iridoviruses, including the genera Lymphocystivirus, Ranavirus, and Megalocytivirus, all of which are known causative agent of fish iridoviral diseases. Among them, Ranavirus and Megalocytivirus are always associated with mass mortalities. The genus Ranavirus is having a broad host and geographic range and it causes Epizootic haematopoietic necrosis in fishes. Another group of emerging iridoviral disease, Red sea bream iridoviral disease causes considerable mortality in both farmed as well cultured marine fish of around 30 species. The most characteristic histopathological change in target organs viz., liver, spleen and kidney include the presence of basophilic, hypertrophied cells, often in large numbers. The iridoviral diseases are mainly diagnosed by gross lesions, histopathology, immunohisto-chemistry, PCR and virus isolation in cell culture. Lymphocystis is a common, chronic and benign infection caused by an iridovirus (Lymphocystivirus) affecting many species of teleosts world wide including India. They results in uniquely hypertrophied paraffin like nodules in the skin and fins. Mixed infections of iridovirus and betanodavirus are also in literature and found in Indian waters also. Co-infection of two different species of viruses affecting same fish host from sub-clinically affected seabass and milk fish under culture has been observed rarely. However, persistent and latent infections is the feature in field until adverse environmental conditions, such as overcrowding, temperature or poor water quality trigger the onset of disease in aquaculture sector. Mourilyan virus (MoV), a virus which is closely related to Bunyaviruses was first detected in Penaeusmonodon shrimp from Eastern Australia. MoV is an enveloped negative-sense ssRNA virus containing four genome segments which includes the L RNA segment encoding the RdRp, the M segment encoding two transmembrane glycoproteins (G1 and G2), and two small segments (S1 and S2) the nucleoprotein (N) and a small non-structural protein (NSs2). In the present study, we confirm for the first time the natural prevalence of MoV in India. The shrimp (P. monodon) from the culture ponds located at East coast of India were found positive for MoV using gene specific primers by RT-PCR. The shrimp collected from these farm appeared healthy with no visible signs of any disease. The sequence analysis of the G2 virion envelope glycoprotein gene PCR product of MoV showed close similarity to the Australian isolate of MoV. The viral gene isolated from P. monodon exhibited 76% nucleotide and 81% amino acid sequence identity on comparison. The amino acid sequence obtained for Indian MoV isolate revealed 39 amino acid replacement on comparison with Australian MoV isolate. Virus presence could be detected in various shrimp tissues such as gills, gut, hepatopancreas, muscle and haemocytes. However, due to the sequence variations present in the Indian isolate of MoV, it may be necessary to design internal primers based on the G2 virion envelope glycoprotein gene sequence specific to Indian isolate of MoV to detect low level of MoV infection in shrimp. Induction of innate immune pathways is critical for early host defense, but there is limited understanding of how teleost fishes recognize pathogen molecules and activate these pathways. Toll-like receptors (TLRs) have emerged as crucial sensors of invading microbes through recognition of pathogen-associated molecular patterns (PAMPs) in viruses, bacteria, fungi and protozoa. TLR3 is known to recognize double stranded RNA in humans, mice, pigs and fishes, which is a viral PAMP. In addition it is induced by bacterial as well as single stranded RNA viruses in fishes, indicating its role in recognition of bacteria and certain viruses. Bariliusbendelisis, is a coldwater fish that inhabits the streams and rivers and is a member of familyCyprinidae. In order to find out the homology between the TLR3 of this fish with other members of Cyprinidae, we designed a set of primers and amplified a fragment of around 1 kb. This amplicon was cloned in pTZ57R/T vector and sequenced. Analysis of the sequence revealed that it was 85% identical with TLR3 of Daniorario. Further to characterise this gene, the complete nucleotide sequence has to be worked out. This would enable the characterization of its agonists, its expression analysis before and after induction. The outcomes of the study will reveal the possible role of TLR3 in viral recognition in Bariliusbendelisis. Computational biology is an important tool in deciphering the structure of a target protein when X-ray structure is unknown. Infectious hematopoietic necrosis virus (IHNV) causes a serious disease in salmonids fish like trout and salmon. The glycoprotein of this virus plays an important role in attachment and fusion. However the X-ray structure of this glycoprotein is not yet known. Study shows that glycoproteins of viruses are involved in entry and eliciting protective immunity. Therefore, the understanding the structure besides function of this proteins is important in order to find the specific sites on viral proteins which binds to precise receptors on cell surface. Thein silico 3D model can help to understand the structure of this glycoprotein so that we could get more information about its active sites. The physiochemical characteristics of these glycoproteins were determined to predict protein structural and functional classes and performance. Using ExPASy program, the signal peptide and transmembrane regions of the proteins were identified. The structure was obtained through homology modelling using MODELLER 9.11 and SWISS-MODEL. Model optimization, quality assessment and visualization was done using combination of several in silico approaches like GROMOS96 10 force field in Swiss-Pdb Viewer, What IF Web Interface, ERRAT. Antimicrobial Peptide Genes of Golden Mahseer (Tor putitora) Directorate of Coldwater Fisheries Research Bhimtal, Infectious diseases are the bottleneck in development of aquaculture industries worldwide. Innate immune system is the major host defence system of fish and antimicrobial peptides (AMPs) are the unique defence peptides of this defence system. They are around 30 residues in length and cationic in nature. AMPs have broad spectrum activity and are generated within few minutes of microbial infections. As the AMPs are generated within the host, these natural products act as alternative endogenous antibiotics to combat infectious diseases. Conventional antibiotics are associated with development of antibiotics resistant pathogens and side effects but it has been observed that AMPs are not associated with such limitations. Therefore, AMPs can be used as alternatives to antibiotics in aquaculture for the treatment of infectious diseases as they have no such limitations. AMPs have immunomodulatory and anti-inflammatory activities. In response to viral infections, AMPs are known to stimulate the expression of TLR3 thus generate antiviral state in fish. Massive neuronal destruction in a Japanese encephalitis (JE) infected individual results in neuro-cognitive malfunction. This could be caused by direct hit of neurons or alterations in the infected host cell physiology. The present study was undertaken to investigate JE virus mediated alteration in functional gene expression profiles in mouse neuroblastoma cells (Neuro2A). Microarray analysis was performed on total RNA extracted from JEV infected Neuro2a cells. TUNEL assay and Real-time PCR were employed to validate microarray results. An upregulation of 660 and downregulation of 949 genes was observed in JEV infected Neuro2a cells in the microarray analysis. Genes involved in apoptosis, oncogenesis, metabolism, neurodegeneration and immunological functions were found to be differentially expressed. Pro-apoptotic genes (p53, VEGF, Gadd45) were upregulated while anti-apoptotic gene bcl-2 was downregulated. Similarly, alteration of expression of genes associated with neurodegeneration was observed. Upregulation of Hsp5, Enah, Btg2, Rassf and Apo e while downregulation of Casp6, Aplp1, Atp1a, Rtn4 and Nde1 noticed was validated in terms of fold change using threshold cycle number obtained from real-time PCR. TUNEL assay, DNA fragmentation and Real time PCR confirmed apoptosis in infected cells. Downregulation of expression and subsequent functional pathway of genes associated with neuronal regulation may result in improper neurogenesis, synaptogenesis, prevent neuritic growth and promote neurodegeneration. Global expansion of CHIKV endemic areas in recent years can be ascribed to increasing vector population, adaptability of virus to new vector and new geographical areas. Here, we developed a SYBR Green I based quantitative real time RT-PCR assay and applied for vector surveillance and to study the virus vector interactions. In the present study, SYBR Green I based quantitative real time RT-PCR assay was developed and validated with field caught mosquitoes. The detection efficiency of this assay was confirmed in mosquito pools of different pool sizes. Cross reactivity studies were done with Abstracts 129 related alphaviruses and flaviviruses. This assay was further adopted to assess the replication kinetics of CHIKV at different anatomical sites in Ae. aegypti at different days of post infection (pi). The detection efficiency of the assay was impervious to mosquitoes of different pool sizes. Vector surveillance has resulted in detection of CHIKV RNA in Ae. aegypti pools, confirming its vectorial potential for CHIKV in northern India. The assessment of the assay was further carried out by studying the competence of Indian Aedes aegypti for CHIKV, which revealed a cent percent infection rate and dissemination rate with 60% transmission rate. The replication kinetics of CHIKV in different anatomical sites of Aedes aegypti revealed the highest mean titre of CHIKV RNA at day 6 pi in midgut and at day 10 pi in saliva, legs and wings. The implementation of the assay in detecting lower viral load makes it a remarkable tool for surveillance of virus activity in mosquitoes with higher sensitivity and specificity. The identification of infected mosquito is considered as a potential indicator of an impending outbreak. Since, there is a paucity of vaccine or antiviral drugs for chikungunya virus, early and sensitive detection will help in proper management and will arrest spread of virus among human population. Flaviviruses are a useful model for studying the evolution of vectorborne diseases as their evolutionary characteristics are believed to have been determined through a combination of constraints imposed by the arthropod vector and the vertebrate hosts. It is not fully known which genes of these viruses have evolved for adaptability in different hosts and what is the extent of selection pressure. In this study, we determined the evolutionary timescales of West Nile (WN) viruses and compared all the genes of WNVs to identify patterns of selection pressure. The analyses was carried out using full genomes, E-gene as well as structural and non-structural regions separately, considering 39 representative sequences of all WNV lineages, 1 to 5. Molecular clock analysis was done using the Bayesian Markov Chain Monte Carlo (MCMC) method implemented in BEAST and selection pressure analysis was performed by using the methods available in the Datamonkey web server. The rate of nucleotide substitution (*6-8 9 10 -4 subs/site/year) and mean root age of all WNV lineages was comparable in case of E-gene, structural and non-structural region. Based on the E-gene, the estimate for the mean age of dominant lineage 1a and lineage 5 which includes Indian isolates (1957, 1980) was found to be 1911 and 1927 respectively. No evident selection pressure was noted in the structural region while selection pressure was noted in the non structural region at NS3-249 and NS4A-85. Mapping of known T-cell epitopes on the NS3 structure showed that the positively selected site NS3-249P/T/A was not associated with the known epitopes. However, it has been reported that this site is responsible for avian virulence in WNV. Overall, molecular clock as well as selection pressure analyses collectively showed that the WNV evolution is constrained by purifying selection. Similar studies in the context of additional Indian isolates need to be carried out in future. Entomological studies were conducted during an outbreak caused by Chikungunya virus at Jamshedpur city, Jharkhand during 2011. Breeding of Aedes aegypti mosquitoes was encountered in all the affected areas. Drinking water containers were comparatively free from mosquito breeding. Immature stages of the vector were mainly found in non potable water containers kept outdoors. Large number of larvae and pupae were observed in those containers which have been dysfunctional and stacked in the backyards of houses. Presence of Ae. aegyptipupae in the indoor and outdoor containers, high density of adult female mosquitoes and the crowded housing settlements seems to have added to the risk of virus transmission and spread. Numerous tyre vulcanizing shops on the streets have added to the Aeaegypti breeding spots where mosquito breeding and biting activity was observed. Adult mosquito catch from Jamshedpur was processed in the laboratory for virus isolation in C6/36 cell line, but virus could not be isolated. A colony of Aedes aegypti population from Jamshedpur was established in the laboratory and F1 generation adult female mosquitoes were infected with chikungunya virus by membrane feeding technique. After an incubation period of 8 days, the mosquitoes were screened by immunofluorescent antibody test. Percent positivity was found to be 58%. Insecticide susceptibility of Jamshedpur the mosquitoes was also carried out by dose-response assay on 3rd-4th stage larvae. Insecticide tolerance was observed in Jamshedpur strain to DDT, malathion and delta-methrin when compared with Pune strain of Ae. aegypti (Probit analysis). Approx 1-2 fold increase in LD50 values was observed in Jamshedpur strain as compared to Pune strain. Hepatitis C virus (HCV) infection is a serious global health problem that affects 180 million people worldwide and it is estimated that 3-4 million people are newly infected each year. HCV infection is an emerging threat and result in chronic liver disease that often progresses to liver cirrhosis and hepatocellular carcinoma. HCV is a small enveloped virus belonging to the Hepacivirus genus in the Flaviviridae family. It possesses a positive-sense, single-stranded RNA genome encoding a polyprotein that is processed by cellular and viral proteases into 10 different proteins, including structural proteins (core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B NS5A, and NS5B). Based on nucleotide sequences, HCV is grouped into six genotypes and more than 80 subtypes so far, that differ from each other by 31 to 33%. HCV-1b is the most common genotype in the worldwide while HCV-3 is the most prevalent in Indian subcontinent. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, pegylated interferon a in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. So, the prevention through vaccination remains the only method for its control. One possible vaccination strategy that can be used against viral infection is to produce artificial virus-like particles (VLPs). These VLPs, corresponding to empty virions, are therefore, non-pathogenic and generally highly immunogenic, the major two characteristics required for the development of an effective vaccine. In this approach, in order to develop a vaccine against HCV, the gene encoding the small envelope protein or the non-structural proteins is cloned into a BAC-EBV virus vector and is expressed in epithelial cells. (2006) (2007) (2008) , multiple serotypes were detected. A total of 46 (33 typeable and 13 nontypeable) EV positive fecal specimens from this study were inoculated in RD cell culture for virus isolation. Culture isolates were confirmed for the presence of EVs by RT-PCR using 5 0 NCR specific primers and genotyped on the basis of VP1 or VP1-2A gene sequences. Phylogenetic analysis of these sequences was carried out using Kimura-2 parameter algorithm and Neighbourjoining method, MEGA 4.0 software. Twenty two of 33 (66.6%) typeable and 3 of 13 (23.1%) nontypeable culture isolates showed cytopathic effect and tested positive for EV RNA while 22 (20 typeable and 2 nontypeable) of 25 (88%) EV positive isolates were genogrouped and genotyped. HEV-B (86.3%) {Echo-13, Echo-6, Echo-7, Echo-1, CB-2, HEV-B, Echo-27, Echo-29, Echo-11, CA-9, EV-75} was found to be predominant genogroup that was followed by HEV-A (4.5%) {EV-90} and HEV-C (4.5%) {CA-22}. Among the serotypes isolated in study, echovirus-13 was found to be predominant. Isolation of novel serotypes EV-75 and EV-90 and dual EV types (Echo-14/EV-76) from a single specimen was also achieved in this study. HCV Infection is an evolving public health problem globally, infecting approximately 3% of the world population and placing approximately 170 million people at risk for developing chronic liver disease. The natural history of HCV infection is characterized by an often clinically unapparent acute phase, followed by chronic infection in over half of those infected. Epidemiological assessment of the magnitude of the problem is essential for public health planning for preventing the HCV infection. The present study was aimed to know the prevalence and yearly trend of HCV infection among blood donors of Kumaon region of Uttarakhand. Blood donation records over 5 years (Oct 2007 to Sept 2012)from blood bank of Soben Singh Jeena Base hospital, Haldwani were reviewed retrospectively for seroprevalence and yearly trend of HCV infection. Total prevalence of HCV infection was 0.64%, higher rates were found among Sikh (3.28%), followed by Muslim (1.8%) and Hindu population (0.31%) respectively. Maximum antiHCV prevalence was seen in Udham Singh Nagar District (3.25%) where as prevalence in Nainital District was only 0.23%. Statistically significant higher seroprevalence was seen in replacement donors in comparison to voluntary donors. Increasing yearly trend of HCV infection was seen from 2007 to 2012 among blood donors. The study highlights the need for more stringent screening in blood donors. The replacement donors should be discouraged. All this is not possible without increased public awareness of the magnitude and implications of this infection and its mode of spread. Health authorities have to include hepatitis C on their radar as a disease which can result in significant morbidity and mortality in the years to come. HCV positive donors should be informed about their disease, counselled and referred to hepatologist, and permanently deferred for future donations. On the outset of West Nile virus (WNV) isolation from patient serum in Kerala, a study was carried out to determine vector competence of local population of Culexquinque fasciatus in comparison to laboratory strain (Pune). Growth kinetics of WNV in the two populations demonstrated an identical growth pattern though the virus intake by the Kerala strain was low compared to Pune strain. Maximum virus yield in Kerala and Pune populations was observed on 17th day post infection (PI) yielding 7.15 and 7.48 log 10 PFU/ml respectively. Both the strains replicated the virus to [5 log 10 PFU/ml within 5 days of feeding on the virus blood mixture and maintained the titer in ascending order throughout the study period. A rapid increase in virus titer from 2.15 log 10 on 1st day PI to 5.36 log 10 PFU/ ml on 5th day PI was observed in the Kerala strain while the Pune strain demonstrated a gradual increase. Virus dissemination to legs was observed on 7th day PI in both the strains. Presence of WNV in saliva, which is a marker for vector competence, was detected on 7th day PI yielding 2.67 log 10 and 2.15 log 10 PFU/ml by the Kerala and Pune strains respectively. The high replication potential of WNV by the mosquitoes, rapid dissemination salivary glands, ability to feed on humans and birds and its abundance makes the Kerala strain of Cxquinque fasciatus a competent vector for the transmission of WNV in Kerala. Epidemics of dengue virus (DENV) have been recorded from almost all over India at different times since 1940s. Evolutionary dynamics of both DENV-1/2 in India have been restricted to analyses based on the Envelope (E) gene sequences and have shown a change of genotypes over the time period. The establishment of the Cosmopolitan genotype was noted in India in both cases since the 1980s. It is thus important to investigate the role of selection pressure in the different genes of the DEN viruses of this genotype. Whole genomes of representative DENV-1 isolates (n = 128) and DENV-2 (n = 138) as well as a sub-dataset including only the Cosmopolitan genotype were used in this study. For the E gene, larger datasets (n = 267/289 respectively for DENV-1/-2) were used. The selection pressure analysis was performed by using the SLAC, FEL and REL methods available at the Datamonkey web server. Selection pressure analyses of DENV-1 sequences, showed one positively selected codon site in the capsid protein (112) and two positively selected sites in the nonstructural proteins at NS1-94, NS3-288. For DENV-2, one positively selected site (360) in the E-protein and 4 sites in the non-structural proteins at NS1-714, 715, NS2A-433 and NS3-255 were noted. Residue 360 was also the only positively selected site within the Cosmopolitan genotype of DENV-2. This site is found to be located near the domain I/III interface involved during conformational changes of the E-protein in association with virus entry and the putative receptor-binding loop in domain III. Overall, It was noted that DENV-1/2 evolution process is mainly constrained by purifying selection. The positively selected site in the E-protein of DENV-2 may have a role in membrane fusion efficiency and virus-cell interactions. NS3 that has been reported to be the most immunogenic antigen in dengue, showed evidence of positively selection for both DENV-1/2 and thus needs further investigation. The Dengue non structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The NS1 gene was amplified from cell culture infected viral RNA using reverse transcription polymerase chain reaction. The amplified gene was further cloned into pET28a (?) vector under the control of T7 RNA polymerase promoter. In order to increase the purity level of the recombinant protein during purification, the transgene was engineered to carry 6X-Histidine tags at both N-terminal and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli rosetta gamicells. The selected bacterial transformant known to express the recombinant NS1 protein was further used to optimize the expression conditions viz IPTG concentration, media type, temperature and harvest time. With optimized conditions, a good expression of NS1 was noticed as early as 0.5 h of post induction. The size of the expressed protein was found to be *43 KDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The recombinant protein was found to be partly soluble and major portion was found in inclusion bodies. The NS1 protein was purified from inclusion bodies, refolded and dialyzed against PBS. Presence of double fusion tag facilitated the recombinant NS1 protein to withstand stringent purification steps that resulted in improved purity. Acute Encephalitis Syndrome (AES) is defined as clinically, as a person of any age, at any time of year with the acute onset of fever and a change in mental status. Encephalitis is a significant cause of morbidity and mortality in children each year in Uttar Pradesh. The cause of encephalitis varies depending on the season, the area of the country, and the exposure of the child. Viruses are the leading cause of encephalitis. These include enteroviruses, Japanese Encephalitis virus, West Nile virus, Herpes simplex virus, Measles and Mumps. In this study we screened more than 300 Cerebrospinal fluid (CSF) samples from AES cases admitted in BRD Medical College, Gorakhpur during June to September 2012. For screening large no of CSF samples 24 well cell culture plate method was adopted. All the samples were passaged in Rhabdomyosarcoma (RD) cell line for minimum of three passages. Cytopathic effect (CPE) was observed for 5-7 days in each passage and recorded. All the tissue culture fluid was subjected to PCR for Enteroviruses, Parecho, Flavi, and Herpes virus. CPE positive samples were also screened with antigen capture ELISA and IFA for enterovirus. PCR negative and CPE positive samples were further subjected for electron microscopy for identification. The outcome of this study will help in knowing the cause of disease. The significance of these findings will also be helpful in future for deciding proper line of treatment and in planning the control strategy of AES cases. (DEN-1, 2, 3 & 4) each capable of producing a wide spectrum of signs and symptoms varying from sub clinical disease to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), that characterize dengue infection. The timely diagnosis of dengue is essential not only to prevent the complications such as dengue hemorrhagic fever and dengue shock syndrome but also to prevent the spread and containment of the disease. This study was conducted in Virology laboratory, Department of Microbiology, Maulana Azad Medical College New Delhi. 400 patients with acute febrile illness of clinically suspected dengue cases attending the outpatient department, emergency or wards of Lok Nayak Hospitals from January to September 2012 were included in the study. Serum samples from these patients were tested for both IgM ELISA (NIV PUNE) and NS1 antigen ELISA (Pan Bio) by commercially available kits as per the manufacturer's instructions. Out of the 400 patient samples tested; 54 were positive for either NS1 antigen or IgM antibody by Capture ELISA. Dengue NS1 antigen capture ELISA gave an overall positivity rate of 66.6% (36/54) and IgM ELISA gave an overall positivity rate of 46.29% (25/54). Only NS1 antigen can be used to test during the first four days of fever. IgM ELISA begins to show positive by fifth day of illness and gradually its positivity increases. The sensitivity of IgM antibody detection by capture ELISA is insufficient in the early phase of the infection and it would be beneficial to identify early cases so as to prevent its complications. Therefore NS1 antigen assay along with IgM antibody detection assays is a useful tool for early detection of dengue infection. Dengue an emerging arboviral disease because of its rapidly increasing incidence and expanding geographic range causes frequent outbreaks in tropical and subtropical regions with significant morbidity and mortality. It is classified antigenically into four serotypes; since these serotypes vary in their virulence their detection and analysis of spatial and temporal transition are essential. Sera were collected from 400 patients of clinically suspected dengue cases attending the out/inpatient department of Lok Nayak Hospital, Delhi from July 2012 to September 2012. Dengue infection was confirmed by serology, NS-1 antigen and RT-PCR. Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 400 samples tested for dengue virus RNA, 48 (12%) were found to be positive. Multiple dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 and DENV-1 being the predominant serotype. This study indicates hyperendemicity of dengue in the region with the presence of multiple serotypes and high rates of co-infection and local genomic evolution of the viral strains involved in this outbreak which emphasize the need to prevent and control dengue infection. However the association of concurrent infections with severe forms of disease (DHF/DSS) needs further studies as different serotypes vary in their virulence and this should be a point of concern for health care agencies. Health authorities should consider strengthening surveillance for dengue infection, given the potential for future outbreaks with increased severity. respectively and none were found positive for JE and forty two (28%) patients were positive for MTB by real time PCR. While 2 (4%) each were positive for JE & Dengue IgM ELISA respectively. None of the samples were found positive for WN and CHIKV IgM. Herpes group of viruses and MTB were found to be the commonest causes of meningoencephalitis in Jaipur. Rajasthan is supposed to be JE nonendemic area but 2 patients were found to be positive for JE IgM is a significant finding but its important to rule out cross reacting antibodies. However steps should be taken to carry out active surveillance for presence of JE in our area as it causes outbreaks and epidemics with high mortality but is a vaccine preventable disease. Transmission Dynamics of Japanese Encephalitis Virus in Assam J. Borah, P. Dutta, S. A. Khan, P. Chowdhury, R. Topno and J. Mahanta Entomology and Filariasis Section (Arbovirology Group), RMRC, ICMR, NE Region (Assam) The incidence of Japanese encephalitis (JE) in recent times has shown an increasing trend in India and has become a major public health problem. There is no treatment for JE. The present study aims at identifying the temporal transmission pattern of JE virus (JEV) infection by tracking the sentinel pigs, mosquito vectors and human JE incidence in Assam. This information will provide baseline data to design and implement JE prevention programmes in endemic areas of the country. The suspected human cases were confirmed by ELISA and Viral neutralization test. JE antibody seroconversion in sentinel pigs was detected by Haemagglutination inhibition test. Per man hour density and minimum infection rate in collected adult mosquitoes were determined by standard formula. Data were analysed by using SPSS software. Sentinel pig seroconversions were significantly associated with human cases 4 weeks before their occurrence; highly correlated during the same time and till 2 weeks before case occurrence and remained significantly correlated up to 2 weeks after human case occurrence. JEV was detected in the same month in pigs and mosquitoes; peaks of pig seroconversion were preceded by JEV infection peaks in vectors by 1-2 months. Kaplan-Meier analysis indicated that detection of JEV positive mosquitoes was significantly associated with the median time to occurrence of seroconversion in pigs. This study reveals the temporal relationship of transmission of JE in an Indian setting which may depict identical scenarios in other pig-rearing areas of South East Asia with a similar ecological niche and potential mosquito vectors. This will not only help in predicting JEV activity in a particular JE endemic area but also accelerate the initiation of timely vector control measures and animal vaccination programmes to reduce the risk of JEV infection. This will also alert the local health authority and the administrator for management of any future JE outbreak in areas under their jurisdiction. Flaviviruses has been documented in experimental models. A study was conducted to evaluate 8 antibiotics (Tetracycline and Aminoglycoside group compounds) against JEV strain P20778 in Baby Hamster Kidney 21 (BHK21) cell line. CC 50 for drugs were calculated based on Optical density (OD) readings by MTT assay at 490 nm. Antiviral activity of drugs at non toxic concentrations was evaluated by Cytopathic Effect Inhibition (CPEI) assay. Virus yield reduction assay (VYRA) was done to compare infectious drug titers of infected cultures treated at different concentrations. Effect of drugs upon kinetics of JEV induced pathogenicity was studied. Plaque reduction assay at non cytotoxic concentration of drugs were carried out. CC 50 of drugs ranged from 7-520 lg/ml as assessed by MTT assay. CPEI assay with non toxic concentration of drugs showed that all eight antibiotic compounds showed antiviral efficacy inhibiting JEV induced CPE. VYRA revealed decline of virus titer (TCID 50 ) at varied drug concentrations. One drug induced decline in plaques numbers with increase of the drug concentration. It can be concluded that at non toxic concentration, antibiotics have inhibited JEV induced typical CPE, reduction in TCID 50 of drug treated compounds and reduction in JEV induced plaques treated with antibiotics. These observations showed that the antibiotics (Tetracyclines and Aminoglycosides groups) affect JEV induced pathogenicity in in vitro. However, mode of drug efficacy is to be studied, the study is in progress. Department of Microbiology, University of Delhi South Campus, New Delhi, Email: rkaul@south.du.ac.in to be associated with a large number of human cancers like Burkitt's lymphoma, nasopharyngeal cancer, Hodgkin's disease as well as a number of B cell lymphomas in ADDS patients and transplant associated immunoblastic lymphomas, and also with invasive breast cancer. The in vitro infection of B cells with EBV gives rise to lymphoblastiod cell lines (LCLs), which expresses a subset of 12 latent viral transcripts. Chronic inflammation at various steps is associated closely with progression of tumorigenesis including cellular transformation, survival, proliferation, invasion and angiogenesis leading to cancer. Various stimuli like cytokines, hormones, and mitogens can cause induction of Cyclooxygenase-2 (Cox-2). Cox-2 have been reported to be expressed in EBV associated cancer. EBV transformed lymphoblastiod cell lines were used as model for latent infection. LPS was used to up-regulate COX-2 expression and EBV was detected in cell free supernatant using PCR targeting Bam W region of EBV genome. We found that inducing LCLs with LPS, unregulated Cox-2 expression, which is also responsible for inducing Lytic reactivation of EBV in latently infected cells. Cox-2 is a multifunctional moderator protein and is capable of inducing lytic reactivation of EBV in latently infected cells. Cox-2 mediated pathway play a critical role in regulation of EBV life cycle and EBV mediated malignancies. Our studies suggest a direct link between Cox-2 upregulation, chronic inflammation and EBV associated malignancies. Acute gastroenteritis is one of the most frequent cause of infectious disease, worldwide and norovirus (NoV) has been found to be most common cause of viral gastroenteritis. A 6-year (2005-2010) NoV surveillance study was conducted in the city of Pune, India to find out trends and temporal variations in the circulation of NoV strains. A total of 1424 fecal specimens collected from children (B 5 year) with acute gastroenteritis were tested by RT-PCR for detection and characterization of NoV by using primers targeting RdRp and VP1 (capsid) regions. PCR products were sequenced and analyzed phylogenetically. NoV positivity was 7.8% (111/1424) and varied between 5.3-11.6% in 6 years of study. The rate of infection was higher (55.8%) in children with B1 In recent years the rate of food borne outbreaks increases throughout the world. It is estimated that there are between 81 million cases of food-borne illness each year and approximately 50% of these cases are associated with meat and poultry. Microbial contamination of raw meat and meat products has been the predominant cause of food related illness. Major food borne pathogens are E.coli, and Salmonella which are the common flora associated with the meat products. Customers concern towards the use of chemical preservatives has increased and hence biological preservative is opted to be the best Phage based therapies have a great potential for a variety of antibacterial application. Bacteriophages are the viruses that are capable of infecting bacteria and lyse the cells. Phages have been proved to be safe for consumption and FDA approvals have also been given for the application of phages for bioprocessing of food materials. Based on the above objective an attempt has been made to isolate Bacteriophages against major food borne pathogens. Salmonella and E.coli cultures were isolated from the fresh and packed meat products. A total of 10 salmonella cultures and 5 E.coli cultures were isolated. Salmonella cultures were identified by using specific primers invA139 (5 0 -GT GAAATTATCGCCACGTTCGGGCAA-3 0 ) and invA141 (5 0 -TCAT CGCACCGTCAAAGGAACC-3 0 ). Bacteriophages were isolated from the meat samples by soft agar overlay method. A total of 6 Bacteriophages were isolated. Among the six phages BPMR2 phage showed ambivalent activity against the test strains of Salmonella and E.coli. Stability of the phage against wide range of pH and chlorine was checked. The phage BPMR2 was capable of surviving in pH range between 4-9. But at pH 2 the phage activity cannot be detected. Chlorine inhibited the phage activity completely. Significance of the current work is that Bacteriophages are mostly very specific to their host, but here bacteriophage BPMR2 is capable of lysing two food borne pathogens, Salmonella and E.coli which makes it an ideal agent that can be used in bioprocessing of the meat products. 187) were included in the study. HBoV was detected in faecal samples by PCR amplification of VP1/VP2 region and positive amplicons were characterized by sequencing. Phylogenetic analysis was performed using MEGA 5 computational tool. HBoV was detected in 21/372 (5.6%) test samples but none of the control samples. Co-infection with Rotavirus was observed in 3 (14.3%) cases. Nucleotide sequence analysis of partial VP1/ VP2 region of the 21 strains illustrated the predominance of HBoV1 (57.1%). HBoV2, HBoV3 and HBoV4 were detected at 19.1%, 9.5% and 14.3%, respectively. Phylogenetic analysis showed that the study strains shared 92-99% nucleotide identity with their respective prototype strains. HBoV positivity was observed in children B1 year of age throughout the year, with peak HBoV activity occurring in July and December. Severity assessment of AGE revealed moderate infection in 7 and severe in 14 of the HBoV positive cases. The study reports detection of HBoV in paediatric population with AGE for the first time in India and circulation of all 4 genotypes in the study region. Although our study corroborates association of HBoVs with AGE, further epidemiological studies are warranted to establish a causal relationship between HBoV and AGE. Viral Hepatitis E, an important disease of public health concern, conventionally relies on anti-HEV IgM serology. In an outbreak situation, collection of sample by venipuncture for laboratory confirmation is often difficult. Thus testing the specimens of dried blood spots (DBS) on filter papers can proof to be an easy, simple and reliable alternative. The present study was designed to evaluate the applicability of anti-HEV IgM detection from DBS samples and to determine the stability of anti-HEV IgM detection at varied time interval and at various storage temperatures. Paired blood and DBS sample were collected from 44 jaundice patients and 8 healthy controls during an outbreak of hepatitis E. These samples were tested for anti-HEV IgM antibodies using commercially available ELISA kit. The DBS were tested by the HEV IgM ELISA kit with in-house modification accommodating varying disc diameter, incubation time of disc elution and sample diluents. Out of 44 samples, 21 samples were stored at 4°C and 37°C and tested for a period of 210 days and 65 days respectively. Phosphate Buffer saline with 0.5% fetal bovine serum was found to be the best sample diluents. The optimum diameter and incubation time for elution was found to be 6 mm and 4 h respectively. Three cut offs (CO 1 : kit cut off, CO 2 : mean of negative controls above 3SD and Camelpox and buffalopox are considered as emerging and reemerging viral diseases. In this study, Loop-mediated isothermal amplification (LAMP) assay targeting A-type inclusion body protein gene (ATI) of orthopox viruses (OPV) namely buffalopox virus (BPXV) and camelpox viruses (CMLV), for specific, sensitive and rapid detection of both of these zoonotic viruses. The assay was optimized using purified viral genomic DNA from density gradient purified CMLV and BPXV vaccine viruses and standard reagents namely LAMP primers, Betaine, MgSO 4 and BstDNA polymerase and it resulted in reliable specific amplification at 63°C for 45 min. The amplified LAMP products was identified by agarose gel electrophoresis and subsequent direct visualization under UV light or observation by naked-eye for presence of turbidity and color change following the addition of SYBR Green I and Hydroxyl naphthol blue (HNB) dyes. The analytical specificity of the LAMP and conventional PCR assays was evaluated using other related poxviruses (Goatpox, Sheeppox, and Orf virus), which revealed that, specific amplification only for OPVs. LAMP assay had shown 100 fold higher sensitivity compared to conventional PCR when tested using purified viral DNA and standard plasmid construct. Further, the developed assay was evaluated using cell culture isolates {CMLV (n = 10) and BPXV (n = 12)} and clinical samples (n = 100) of animals and humans. These results prove that the developed OPV-LAMP is a simple and cost-effective diagnostic tool for rapid, highly sensitive and specific detection of OPVs from clinical samples of both animals and humans without the need of high precision tools like PCR/real-time PCR cyclers in field diagnostic laboratories. Hepatitis C virus (HCV), is the one of the major causes of parenterally transmitted non-A and B hepatitis, affecting more than 170 million people worldwide. HCV genotypes have shown geographical linkages and the genotyping is known to play a critical role in the outcome of the HCV infection, progression of disease, and response to interferon therapy. In India, approximately 1.8% to 2.5% of the population is infected with HCV and at present there is not a single report on the confirmation of detection, and genotypic distribution of HCV in Uttarakhand region of North India. We describe here the detection and distribution pattern of prevalent genotypes of Hepatitis C virus (HCV) in Uttarakhand state of India. In the study, 18 serum samples from healthy blood donors and 21 serum samples from chronic hepatitis patient, were screened for anti-HCV antibodies by using 4th Generation TRI-DOT Immunoassay followed by 5 0 UTR region PCR-restriction fragment length polymorphism (RFLP), sequencing and phylogenetic analysis. Twelve out of eighteen healthy blood donors noted a very high prevalence of anti-HCV antibodies (66.67%) and all the 21 patients suffering from chronic hepatitis were detected positive for both anti-HCV antibodies and PCR assay. The restriction patterns obtained clearly indicated the presence of genotype 3 in all the clinical samples but patterns were found to be flawed when subtyping was considered. The sequence based HCV genotyping and subtyping revealed predominance of HCV genotype 3a (77.8%) followed by 3b (22.2%) in the state. Phylogenetic analysis clustered seven HCV-3a and three HCV-3b Uttarakhand isolates with the Canadian HCV-3a and 3b isolates. HCV genotype 3a remains the dominant genotype in Northern India but also indicates circulation of 3b type in Uttarakhand state. The close association between Indian and Canadian isolates may suggest the origin of current circulating strains in Uttarakhand region back to Canada but needs more elaborative investigations to confirm this assumption. Stringent screening of blood collected from blood donors is in high demand in India. This study is aimed to obtain new insight into the inhibitory effect of EBNA3C during the process of TAp63a induced apoptosis in B cell. Human B cells and EBV transformed lymphoblastoid cells were cultured in vitro. Expression vectors containing EBNA3C, p63 isoforms and their truncations were transfected in these cell lines and further assays were performed. Apoptosis were detected by annexin-V staining using flowcytometry. Mitochondrial membrane potential assay was performed by DiOC6 staining and cytochrome c release by western blot. Interaction between EBNA3C and p63 isoforms were checked by immunoprecipitation and co-immunoprecipitation. Downstream signaling of extrinsic and intrinsic pathways of apoptosis was traced by using ligand, inhibitors and siRNA studies. TAp63a expression in B cells induces apoptosis which involves activation of caspases and death receptors. TAp63a also induces mitochondria mediated apoptotic pathway. EBNA3C expression reduces the rate of apoptosis in these cells by interfering the downstream targets of TAp63a. TAp63a induces and recruits genes that play roles in different steps of apoptosis program which is inhibited by EBNA3C. These findings explain one possible mechanism by which EBV blocks the p63 mediated apoptosis especially in EBV transformed LCLs. Email: anukumar74@gmail.com, anukumar@icmr.org.in Chandipura virus produces encephalitis/encephalopathy in naturally infected young children and experimentally infected young susceptible mice. Neuro-tropism is major feature in many viral infections. Neurotropic viruses use nervous tissues for replication and also take a route to central nervous system (CNS). This study was undertaken to find out the neuro-invasive behaviour of Chandipura virus in young susceptible mice. Young mice less than 14 days old are susceptible to Chandipura virus infection and adults are refractive. The susceptible mice were infected with virus via foot pad injection. Different nervous tissues including sciatic, spinal cord, brain and other tissues were collected at 24 h intervals up to 72 h post infection (PI). The tissues were processed for virus quantification and histology. Adult mice were infected through different routes including intra-cerebral route to determine the requirement of nervous tissues for virus replication. In virus infected susceptible mice it was observed that time dependent increase in viral RNA copies in different nervous tissues. Other tissues like spleen, lung, kidney etc. the peak virus titre was noticed at 24 h PI and it was later declined. Perivascular cuffing and other signs of inflammation were noticed in lower, upper spinal cord and brain. TUNEL positive nuclei in brain indicated that cell death and/or damage in the CNS. Infected susceptible mice also showed gross neurological symptoms like hemiplegia and/or paraplegia of hind limb, circling movement, eye lesions etc. In adult mice, the virus reached the brain through intranasal and intra-cerebral route of inoculation. However the pathogenesis noticed only in intra-cerebrally inoculated mice. No pathogenesis noticed in adult mice through all other routes employed in this study. From this study, we concluded that in young susceptible mice the virus might migrate through the nervous tissues and finally reach the CNS. However in adult mice, the nerve tissues neither pick up nor transport the virus to CNS. The pathogenesis in young mice might be due to the virus replication induced damages in the nervous system. The results are preliminary and a detailed study is necessary to confirm of mechanism of axonal transport in Chandipura virus infection. Hepatitis E virus (HEV) infection is endemic in India. The disease manifestation ranges from self limiting acute viral hepatitis (AVH) to life threatening acute liver failure (ALF). The clinical course of the disease, possibly, thought to be immune-mediated. Toll-like Receptors (TLRs), a class of evolutionary-conserved protein, plays a key role in sensing pathogen associated molecular patterns (PAMPs). Downstream signaling of TLRs modulates the production of cytokines and plays a crucial role in determining the course of immune response and disease pathogenesis. Thus, the present study was aimed at elucidating the role of TLRs and their cytokine modulation in the immunopathogenesis of HEV. Anti-coagulated blood was collected from 50 AVH-HEV, 30 ALF-HEV patients, and 50 apparently healthy-controls (HC). PBMCs were separated using Ficoll-Hypaque. One part was processed for RNA extraction (unstimulated) another cultured in RPMI-1640 and pulsed (Stimulated) with recombinant HEV-ORF2 protein (452-617 a.a). Geneexpression levels of TLR (2, 3, 4, 7 and 8) were checked in unstimulated and stimulated group using semi-quantitative Real-time-PCR. Lymphocyte proliferation-index was estimated using Colorimetric-MTT assay. Cytokine levels were checked in the Serum and culture-supernatant using Cytokine-Bead-Array. TLR3 silencing experiments were performed in the PBMCs of representative HEV patients. Post silencing cytokine levels were estimated. TLR3 geneexpression in AVH was significantly higher than ALF (215 Vs 21 fold-increase; (p \ 0.0001). Proliferation-Index of AVH was significantly higher than ALF (3.25 ± 0.876 Vs 1.748 ± 0.253; p \ 0.008). Significantly higher amount of IFN-c detected in the culture-supernatant of AVH Vs ALF (292.3 ± 88 pg/ml Vs 3.235 ± 0.8 pg/ml; p \ 0.0001). IFN-c: IL-4 ratio in the AVH was 89.76 and in ALF was 0.589. Circulating TGF-b levels were significantly elevated in patients as compared to HC (HC: 57.40 ± 27.48 ng/ml, AVH: 322.8 ± 212.2 ng/ml, ALF: 334.5 ± 203.8 ng/ ml). After silencing TLR-3 using specific siRNA significant decrease in IFN-c in the PBMC culture-supernatant observed (without-siRNA 25.57 pg/ml Vs with siRNA 4.30 pg/ml; p = 0.0349). A lower level of TLR3 gene-expression followed by minimal levels of INF-c production leads to a low proliferation index which contributes to a marked Th2-shift in ALF-patients. On the other hand, AVH-patients demonstrated robust Th1-type of immune-response mediated by TLR3 pathway, thus providing a strong pointer towards involvement of TLR3 in the immunopathogenesis of HEV infection and can be a potential therapeutic target for antiviral agents. Cassava (Manihot esculenta Crantz, Family. Euphorbiaceae) is the third largest source of dietary carbohydrates in the world. Cassava mosaic disease, very common in the cassava crop, is caused by the geminiviruses Indian cassava mosaic virus (ICMV) and Sri lankan cassava mosaic virus (SLCMV) in India. This investigation describes an SLCMV DNA cloned from CMD-affected cassava, using Rolling Circle Amplification (RCA). Its sequence analysis and infectivity on Nicotiana benthamiana, using biolistic inoculation is described. Infected materials were collected from Attur, Tamil Nadu. RCA technique was used to clone geminiviral DNAs. Total DNA was extracted from symptomatic cassava samples and used as a template for the RCA. A 2.7 kb cloned fragment was obtained in pTZ57R vector (Fermentas) and sequenced. Sequences were analysed by using the BLAST programme of the NCBI server. For infectivity analysis of the cloned fragment of viral segment was released from the vector backbone of the recombinant plasmid by digestion with Pst I restriction enzyme [MBI Fermentas], self-ligated, amplified by RCA and was used directly for the analysis of infectivity on 30-35 days old Nicotiana benthamiana plants. The sequence resembled DNA-A of begomoviruses and was found to be having the highest sequence identity (99%) to SLCMV. The name SLCMV-Attur is proposed for the isolate. Biolistically inoculated Nicotiana benthamiana showed symptoms of leaf rolling and deformation after 30 dpi. The results of this study comparing the sequence variations and phylogenetic analysis with full-length begomoviral sequences reported from plants belonging to various taxonomic families from Indian subcontinent and rest of the world indicated that a strong relationship exists between begomoviruses infecting plants belonging to the same family and their geographic location. Biolistic inoculation results gave conclusive evidence that the cloned DNA is infectious in Nicotiana benthamiana plants. In a survey conducted on the diversity of begomoviruses infecting tomato in Northern India, nearly 30% of the tomato leaf samples had mixed infection of two bipartite begomoviruses, ToLCNDV and ToLCPalV. The plants showed brilliant yellow blotches, yellow mottling and severe leaf curl symptoms. To study the interaction between the two bipartite begomoviruses, the genomic components of ToLCPalV and ToLCNDV were cloned and partial tandem repeat constructs of DNA A and DNA B components were made in the Tiplasmid derivative pBIN19. The DNA A and DNA B components of ToLCNDV and ToLCPalV were agroinoculated to Nicotiana benthamiana, tomato and cucurbitaceous hosts to study the interaction between the components. Agroinoculation of ToLCPalV alone produced downward leaf curling, mild yellowing, leaf curling in N. benthamiana and tomato at 10 days post inoculation (DPI). The ToLCPalV constructs were readily infectious on bottle gourd and cucumber produced yellowing, downward leaf curling and yellow blotches. Contrastingly, ToLCNDV components induced vein clearing, yellow mottling and severe leaf curl symptoms in N. benthamiana and tomato; the ToLCNDV components were not able to induce any symptoms in cucurbitaceous hosts. When agroinoculations were done by exchanging the components (DNA A of ToLCNDV with DNA B of ToLCPalV) on tomato, characteristically severe symptom expression, downward leaf curling, brilliant yellow blotches in leaf lamina, reduction of leaf lamina were seen in the plants inoculated with ToLCNDV A and ToLCPalV B. No such severe symptoms were seen in plants inoculated with combination of ToLCPal A and ToLCNDV B. Mixed inoculation with all four components led to severe symptoms expression though dominated by ToLCPal B mediated symptoms. Semi-quantitative PCR analysis of viral replicative forms clearly showed that all four components replicate efficiently on par with each other that the viral DNA components attained 500 ng/100 mg of leaf at 15DPI. The vector whitefly picked up the virion particles from the plants inoculated with homologous, heterologous and mixed combinations transmitted it to 85% of test plants. The acquiring and transmission of all the four components efficiently by the vector, indicates how association of two viruses may get established and perpetuated by subsequent transmission to healthy plants. Importance of these results in the context of resistance breaking and epidemics due to emergence of new variants is discussed. Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute (IARI), New Delhi-110012; Email: leafcurl@rediffmail.com Cucumber green mottle mosaic virus (CGMMV), a member of genus Tobamovirus, infects cucurbits worldwide. The present study was undertaken to use the CGMMV genome for useful purposes. The complete genome of an isolate of CGMMV from Delhi characterized previously was used to design an a vector for transient expression of protein in plants. The genome of CGMMV was cloned in two parts; the one contain 5 0 backbone that included the 5 0 untranslated region (UTR), replicase protein and movement protein genes and the other part contained coat protein gene and 3 0 UTR. Two modular constructs, 5 0 module and 3 0 module were developed by adding an intron (IN) sequence and recombination sequences (RS) to the 3 0 end of the 5 0 backbone and multiple cloning sites, IN and RS to the 5 0 end of 3 0 backbone. When the mixture of 5 0 , 3 0 and integrase constructs were agroinfiltrated in Nicotiana benthamiana, recombination of the 5 0 and 3 0 genome was evident by RT-PCR and formation of virus particles was observed in electron microscope. To examine the ability of the vector system to express heterologous protein, beta-glucuronidase (GUS) gene was cloned in the MCS of 3 0 module. When a mixture of 5 0 module, 3 0 GUS module and an integrase gene constructs was agroinfiltrated to N. benthamiana and cucumber leaves, GUS expression was detected at 10-12 days post infiltration. As CGMMV produces mild symptoms, the vector based on CGMMV will be potentially useful to produce pharmaceutical proteins in cucurbits. is a major limiting factor for papaya and cucurbit cultivation worldwide. The present study was undertaken to clone and express the genes encoding antibody to the recombinant coat protein (rCP) and to assess the engineered monoclonal antibody (mAb) for the detection of PRSV. A 33 kDa rCP gene of PRSV was expressed in E. coli and purified rCP of about 50 lg was used to immunize the mouse through intraparitoneal route and subsequently three booster doses were given using 25 lg of the protein. The serum was drawn at 45 days post immunization showed high reactivity with PRSV infected leaf samples as well as with the purified rCP in ELISA. The spleen was isolated from the rabbit in order to purify mRNA to clone the antibody variable region genes (VH and VL) using universal degenerate primers. The VH and VL genes were 351 and 360 nucleotides long respectively, which contained the framework regions and complimentary determining regions and belong to the family IgG1 and kappa chain, respectively. The VH and VL genes were used to develop the expression constructs in pET28a(?) vector and 14 kDa protein was obtained in E. coli. The amount of purified VH and VL proteins was 3-4 mg/liter of bacterial culture. The crude sonicated pellet as well as purified VH and VL proteins were used as an engineered mAb to detect PRSV in the infected tissues and showed high binding affinity with purified protein in dot immunobinding assay. The present study successfully generated engineered mAb fragments to PRSV in E.coli. This is the first report of engineered mAb to PRSV. The approach may be useful to produce diagnostic mAb against other plant viruses. Chilli (Capsicum annum) is an important spice crop cultivated throughout Tamil Nadu. Leaf Curl disease of chilli has emerged as serious problem in the Perambalur district, the major chilli growing area of Tamil Nadu. During 2010-2011, very high disease incidence (up to 100% of plants) was observed in farmers field in Esanai, Anugur, Konnaripalayam, Narnamangalam villages. The characteristics field symptoms were upward curling, puckering and reduced size of leaves. Severely affected plants were stunted and produced small and no fruits. Electron microscope examination of field samples revealed few, typical geminate particles isolated by Honda et al., method. The presence of begomovirus was confirmed by polymerase chain reactions (PCR) using the replication protein primers ChiLCVFP 5 0 -ATGAAATA TGAACARCCG-3 0 and ChiLCVRP 5 0 -CCATCCRAACATTCAGG Email: rabinr1956@gmail.com Cassava in Tamil Nadu is cultivated as a tuberous root crop and its roots are the major source of dietary and industrial starch serving as input for the major starch and sago industries. Tamil Nadu has an area of 95,000 ha (40% of the total area under cassava in India, 2006) and 60% of cassava produced is utilized industrially to produce starch, sago and other value added products. Since, cassava is mainly propagated vegetatively, it is particularly prone to viral infections, which tends to build up in successive cycles of propagation (Calvert and Thresh, 2002) . In Tamil Nadu, the cassava mosaic disease has been reported to be caused by Sri Lankan cassava mosaic virus (Dutt et al., 2005 and Rajinimala et al., 2006) . Crop loss data due to infection with Cassava Mosaic Disease have been reported from many countries including India (Fauquet and Fargette, 1990) . The average yield loss caused by cassava mosaic disease was estimated to be 50% (Fauquet and Fargette, 1990) . From India Malathi et al. (1985) has reported that the disease causes a yield loss of 17-88%. Cursory perusal of the literature revealed that many studies have been attempted to quantify yield losses due to CMD infection but information on qualitative loss is limited. Hence, we attempted to study both the quantitative and qualitative loss due to SLCMV infection in cassava variety CO2. This study was conducted in an area of 50 cents in total (Virus indexed healthy plants in an area of 29 cents and SLCMV infected plants in an area of 21 cents). The yield obtained from infected plants was 969 kg when compared to healthy plants yield 3392 kg. Further the quality of the tubers was also analyzed and it was found that the starch content of tubers collected from healthy tubers was 28% whereas it was 21.5% in infected tubers. The protein content of tubers from healthy plants was 0.44% and it was 0.39% in virus infected tubers. Our studies indicate that infection with Sri Lankan cassava mosaic disease can reduce the starch content of the tubers to the tune of 7%. Since, Tamil Nadu has more than 800 cassava based industries (which depends mainly on the cassava starch), this study has significance to the economy of Tamil Nadu and farmers. The coat protein (CP) gene of five Indian Garlic common latent virus (GarCLV) isolates was determined. Length of the CP gene was found to be 960 bp, encoding a protein of 319 amino acids. Comparative nucleic acid sequence analyses revealed a 4.3% divergence among the Indian isolates with an overall 11.9% diversity among all available isolates from around the world. (Or), Aurangabad (Au), Coimbatore (Co), Jalna (Jal), Vijayawada (Vij), Jalgaon (Jalg) and Varanasi (Var)]. Thereafter, cloning and sequence analysis of full-length viral DNA and associated betasatellites was done from selected four and six samples respectively. The results indicated that CP sequences fell into two groups each showing more than 95% identity within the group but less than 80% between the groups. One group showed highest identity to BYVMV CP sequences, while other to Mesta Yellow Vein Mosaic Virus CP sequences. The cloned betasatellites shared more than 95% similarity to betasatellites associated with BY-VMD. The recombination analysis of the clones was performed by using Recombination Detection Program (RDP) RDP analysis could detect an event of possible recombination between BYVMV and MeYVMV in one of the full-length isolate. This report indicates strongly that BYVMD in India is associated with at least two viruses, BYVMV and MeYVMV. It also indicated that recombination is quite widespread in begomoviruses affecting okra. Sequence analysis of cloned betasatellites from seven locations indicated that there was high sequence identity (90% or \90%) among all the cloned molecules. Cherry necrotic rusty mottle virus is a graft transmissible, unassigned member in the family Betaflexiviridae. CNRMV infects sweet cherry (Prunus avium) and has been reported in North America, Europe and Japan. Diseased plants show brown angular necrotic spots, rusty chlorotic areas, shot holes of the leaves, blisters, gum pockets and general necrosis of the bark. In India, sweet cherry is grown mainly in the North-Western states of Jammu and Kashmir (J&K), Himachal Pradesh (H.P.) and in hilly regions of Uttarakhand (U.K.). This perennial crop is usually propagated by grafting and, therefore, is easily infected with viruses when contaminated material is used as a propagation source. Therefore, it is desirable to devise diagnostic protocols for CNRMV in quarantine and certification programs. To understand the health status of Cherry orchards and to determine the incidence of the virus in India, a survey was conducted in the months of May and September in Srinagar, Pulwama, Shopian, Sopore and Ganderbal regions of J&K and Rohru, Narkanda and Bhutti regions of H.P. For the preliminary detection of CNRMV, symptomatic leaf and twig samples were collected and RT-PCR was carried out. The incidence of CNRMV was found to be 7% and 66% from J&K and H.P., respectively. In order to characterize the virus at the molecular level, the CP gene and TGB were amplified by RT-PCR using specific primers. The partial genome sequence of JK10 isolate consists of 2203 nucleotides. Comparison of the sequence with the already submitted complete CNRMV sequences revealed a similar genetic organization with the ORF 2, ORF 2a, ORF 3, ORF 4, ORF 5 and ORF5a in similar positions and 3 0 UTR region of almost identical size. The sequence of JK10 isolate showed 91% and 84% similarity to the type isolate of CNRMV from Germany (AF237816) at the nucleotide and amino acid level, respectively. Phylogenetic analysis based on JK10 isolate coat protein gene with selected members of the family Betaflexiviridae showed that the isolate is most closely related to CNRMV flowering cherry isolate, FC4 from Japan (EU188438) sharing 94% and 96% identity at nt and aa levels respectively. and Cucumismelo (Muskmelon) hosts was identified on the basis of nucleocapsid (N) protein gene characteristics. The partial N gene of GBNV from muskmelon, chrysanthemum and dahlia of 477 nucleotide long encoding 159 amino acids amplified using species specific forward and degenerate reverse primers. Comparative sequence analyses of GBNV revealed 93-100% and 94-100% identities at nucleotide and amino acid level respectively with the corresponding region of GBNV N gene from different hosts. Similarly, in phylogenetic relationships, the N gene sequences in this study clustered with the GBNV sequences from other hosts used for comparisons, this forms the first evidence of occurrence of GBNV in ornamental and cucurbitaceous crop in India. Gladiolus cultivars are of various colors, sizes, shapes and flowering time which make it a high choice for commercially cultivation. The commercial production of gladiolus is being hampered by infection of several plant viruses deteriorating the quality of blooms. Approximately 200 gladiolus cultivars are being maintained and grown in CSIR-NBRI, Lucknow for cut-flowers and annual flower show. The leaf mosaic, color breaking and deformation of flowers, and corm distortion symptoms were observed in many gladiolus plants of different cultivars. The natural occurrence of potyvirus was detected byRT-PCR tests in Vink's Glory, Aldebaran and Sylvia cultivars. The elimination of potyvirus was attempted by in vitro culturing in combined with chemotherapy of cormel explants from infected gladiolus of cvs. Vink's Glory, Aldebaran and Sylvia. The infected cormels were surface sterilized and cultured in MS media supplemented with 1.0 mg/L BAP, 0.5 mg/L IAA and 2 mg/L 2,4 D growth hormones and various concentrations of virazole (30, 40 and 50 mg/ L). The successful regeneration was achieved in MS media supplemented with 30 mg/Lvirazole. At 40 mg/L concentration of virazole, corms survived but there was no growth, however, 50 mg/L concentration was found lethal. Three randomly chosen regenerated shoot lets of Sylvia cultivar were tested by RT-PCR to confirm the potyvirus and two/three plants were found to be virus-free. The virus-free shootlets are being multiplied by in vitro propagation to obtain more virus-free plants. Elimination of potyvirus infection from gladiolus and development of the virus-free culture using chemotherapy would be useful for the floriculture industry for the mass propagation and quality production of blooms. , were used as template for PCR amplification using CaYMV forward and reverse primer pair spanning the most conserved region of RT/RNaseH of CaYMV. During agarose gel electrophoresis, PCR amplified product showed the expected *550 bp band in many diseased samples, but no amplification in asymptomatic sample. Two independent PCR amplified products were gel purified and got sequenced. The sequence data of 565 nucleotides obtained were analyzed to resolve any ambiguity and submitted to GenBank under the accessions: JX228965 and JX228966. Gerbera (Gerbera jamesonii) belongs to the family Asteraceae and is commonly known as African Daisy. It is popularly used as decorative garden and container plants, besides sold mostly as cut-flower sticks. Gerbera has been cultivated almost in all parts of country, yet more commercially in Karnataka, Maharashtra and North-Eastern states including Uttar Pradesh. Gerbera cultivation has a major setback in the commercial production because of the several plant viruses which deteriorate the commercial quality of flowers. One of the commercially important cultivar of gerbera cv. Zingaroo was found to be infected by severe chlorotic mosaic, floret and flower deformation disease. The natural occurrence of Cucumber mosaic virus (CMV) associated with the disease of G. jamesonii has been detected by western blot immunoassay using CMV antiserum (PVAS242a) and RT-PCR tests employing coat protein gene specific primers. In present study, the elimination of CMV from infected gerbera has been attempted by in vitro culturing of floral buds and pedicel explants. The basal MS medium supplemented with combination of plant growth hormones (1.0 mg/L BAP and 0.5 mg/LIAA) was optimized for gerbera regeneration. The regenerated shootlets were first tested by RT-PCR using CMV-CP specific primers for presence or absence of CMV. One of the randomly selected shootlet was found to be free from CMV and further multiplied using their proliferation shoots in regeneration medium additionally supplemented with Adenine sulfate (Ads, 0.5 mg/L) growth hormone. A total of 15 regenerated shootlets were rooted in rooting medium (MS medium supplemented with 0.5 mg/LIAA and 35.0 mg/L Ads hormones. The rooted plantlets were re-confirmed by RT-PCR, acclimatized and established in glasshouse. Elimination of CMV infection from gerbera and development of virus-free gerbera plants would be helpful to provide healthy planting material to gerbera growers and for improvement of better quality production of gerbera in the country. Peanut mottle virus (PeMoV), a serologically distinct Potyvirus (family Potyviridae)is one of the major seed borne virus affecting groundnut and soybean all over the world. The symptom of the virus in peanut is mild and thus its occurrence is often unnoticed in the field. PeMoV occurring in India has not been characterized at molecular level. In the present study, the coat protein (CP) gene of PeMoV isolate from peanut in Andhra Pradesh was cloned, sequenced and used to express in E.coli. Polyclonal antiserum (PAb) to the core recombinant CP of PeMoV was generated and used in serological diagnostic tests. The full length sequence of PeMoV CP (JX088125) gene showed 99% similarity with the PeMoV Isolate (DQ868539) from Israel. The core CP gene (540 bp) was cloned into pET28a(?) expression vector and the recombinant CP was expressed as a fusion protein containing N and C terminal hexa-histidine tag. A high titre PAb produced against the recombinant CP efficiently detected PeMoV in the infected peanut leaf samples. The PAb was used in ELISA to test 1641 peanut samples during 2010-2012 from several fields near Hyderabad, which showed 12% incidence of PeMoV. This is the first report of immunodiagnosis of PeMoV using polyclonal antiserum against bacterial expressed viral antigen. In the present study, we have developed a full length nucleocapsid protein gene and a core NP gene transgenic constructs from an isolate of WBNV characterized from northern India. To achieve optimal conditions for regeneration of watermelon cv. Sugar Baby, six types of explants (immature cotyledon, mature distal cotyledon, mature proximal cotyledon, distal hypocotyls, proximal hypocotyls, basal epicotyls) were evaluated on MS medium supplemented with different concentrations and combinations of hormones. The results showed that proximal immature cotyledons cultured in MS ?BAP (2 mg/l) ? IAA (0.1 mg/l) achieved the highest rate (76%) of regeneration. Agrobacterium tumefaciens strain EHA 105 carrying the construct in a binary vector pBI121, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency. The optimal conditions for transformation of watermelon were evaluated and 14.2% of GUS transformed plants were obtained. The optimised protocol was then adapted to transform watermelon plants using both the transgene constructs of WBNV. However, a very low rate (0.9%) of transformation using WBNV based transgenes was achieved. Technology, Palampur. Eight sweet cherry (Prunus avium) and five sour cherry (P. cerasus) leaf samples were collected and tested for five viruses that are known to infect Prunus species by reverse transcription-polymerase reaction (RT-PCR). Viruses that were analyzed included Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Cherry necrotic rusty mottle virus (CNRMV), Cherry virus A (CVA) and Prunus necrotic ringspot virus (PNRSV). Only CVA was detected from all sour cherry and five sweet cherry plants by RT-PCR assay with a new set of primers CVA5839U/CVAL specific to a 1539-bp fragment of the CVA replicase gene and 3 0 UTR. To confirm RT-PCR results, CVA amplification products were sequenced (accession no. FN669549). Sequence analysis of the 1539 bp by BLAST search in GenBank showed 91% similarity to the type isolate from Germany (Accession no. NC_003689) and 99% similarity to the Indian isolate (Accession no. FN691959) of CVA at nucleotide level. To the best of our knowledge, this is the first report of CVA from sour cherry in India. During the survey of Asteraceous plants in Gorakhpur district of North-Eastern Uttar Pradesh a severe yellow mosaic and vein clearing symptoms were recorded on Ageratum conyzoide. The symptomatic leaves were analyzed through PCR assays with specific primers (TLCV); detect positive *800 bp amplicons. The PCR amplified product was directly sequenced and sequence was submitted in GenBank with accession no. GQ412352. Sequence similarity and phylogenetic analysis confirmed the presence of begomovirus Ageratum enation virus (AEV) on Ageratum conyzoides. In current scenario, AEV is widely distributed throughout Asia. The frequency with which new strains of AEV are appearing in several agricultural crops and non-crop species which indicates that the virus species has more virulent strains in field and pose a serious threat to cultivated and non cultivated crop species. In last 5 years its number is increasing very fast. So far many strains have been reported from a variety of plants. It indicates that in coming years Ageratum enation virus will pose serious constraint next to Tomato leaf curl New Delhi virus to agricultural crops due to its more virulent strains with broad host range. The widespread occurrence of AEV on various crop and non crops species, suggests us to formulate strict control strategy measure to check further spread of this virus in new locality. Existence of begomovirus has been increased at the alarming level in the India and it has been reported on various crops by different workers from the different regions of India. Therefore an immediate attention is required to check the further spread of this begomovirus in nature. During survey in North-Eastern of Uttar Pradesh, severe chlorosis, crinkling and curling symptoms were noticed on leaves of Catharanthus roseus plant. The virus was partially purified from the symptomatic leaves by differential centrifugation. Electron microscopy of the partially purified preparation revealed the 760 nm 9 14 nm filamentous flexuous particles. The causal virus was found transmitted through mechanical transmission to healthy Catharanthus roseus plants as well as through Aphis gossypii, A. nerii, A. craccivora, Brevicoryne brassicae, Myzus percicae, and Macrosiphoniella sanbormii. Immuno-diagnostic studies (DAC-ELISA and DIBA) showed positive serological relationships of the causal virus with polyclonal antiserum of Watermelon mosaic virus (WMV). The RT-PCR assays amplified fragment of *330 bp with coat protein specific primer of WMV. The particle morphology, serology and RT-PCR results confirmed that the virus associated with chlorotic leaves of Catharanthus roseus in the present study is Watermelon mosaic virus. The virus infection also found to deform the palisade cells in shape and size and number of chloroplast were reduced in tissue of infected leaves under histopathological observation. Cowpea mild mottle virus (CPMMV), a member of genus Carlavirus (family Betaflexiviridae) transmitted by whitefly (Bemisia tabaci), contains long flexuous particle, positive sense ssRNA of about 8.12 kb. It is an emerging as a threat to cultivation of field crops in tropical and subtropical regions of Africa and Asia. During 2011-2012 soybean crops grown at Indian Agricultural Research Institute (IARI), New Delhi, experimental field was found to be severely affected by mosaic disease with disease incidence of 18.6-71.2%. Infected soybean produced symptoms like mosaic, mottling, leaf deformation and stunting. The virus could infect soybean, French bean, mungbean, urdbean, cowpea, tobacco, fenugreek and asparagus bean through mechanically sap inoculation. Seed transmission of virus was ranged from 0.62 to 14.2%, depending on soybean cultivars. Under electron microscopy a long flexuous particle measuring 620-650 9 12-15 nm in size from symptomatic soybean leaves was observed. Carlavirus specific primer detected the associated virus with mosaic disease of soybean as a member of Carlavirus amplifying 930 nt fragment of the viral genome and this virus was further confirmed as a strain of CPMMV using another specific primer pair amplifying 216 nt fragment. One soybean isolate from Delhi designated as CPMMV-D1 was characterised by sequencing of 1289 nt fragment of 3 0 end of the genome covering CP (867 nt), nucleic acid binding protein (NABP) gene (303 nt) and 3 0 UTR (120 nt). Sequence analysis showed that the present isolate shared 68-73% nt identity with other isolates available in GenBank. In phylogenetic analysis, CPMMV-D1 placed separately in phylogenetic tree. The present isolate differed from previously reported two Indian groundnut isolates CPMMV-S and CPMMV-M, sharing 73% nt identity. Earlier it was presumed that Soybean mosaic virus (SMV) is the causal agent of mosaic disease in soybean under Delhi condition but present study ruled out the SMV-etiology and determined CPMMV-etiology of mosaic disease in soybean under Delhi condition. This is the first report of identification and molecular characterization of CPMMV infecting soybean in India. Most or all of the flexible filamentous plant viruses share a common protein fold and helical chemistry and are used widely in nanobiotechnology as various engineering platforms. Little is known about the structure of flexible filamentous plant virus coat proteins compared to rigid rod tobamoviruses and icozahedral plant viruses. Mandarivirusis a monotypic filamentous plant virus genus in the family Alphaflexiviridae with Indian citrus ringspot virus (ICRSV) as type species. ICRSV contains a genome of 7560 base positive sense single stranded RNA coding six different proteins. Coat protein (CP), the only structural protein of this virus is a 325 residue peptide of 34 kDa that self assembles into a flexible helical particle of around 650 nm in length. An attempt was made to construct the 3D model structure of ICRSV CP through threading the templates available in protein databank using iterative threading assembly refinement server which is an integrated platform for automated protein structure and function prediction. Secondary structure of the ICRSV CP has 101 helix and 224 coils with a confidence score ranging from 6-9 on an average. The predicted 3D structure of ICRSV CP has a TM score of 0.33 ± 0.11. This TM score is in the range of 0.17 to 0.5, which indicate the predicted 3D model cover a satisfactory level of topology with the native structure. The structural analogue of the 3D model of ICRSV CP was found to be crystal structure of CP of Papaya mosaic virus with a TM score *0.5. Six out of the top 10 alignments of the 3D model of ICRSV CP with a normalized Z score of C2 reported from the following threading programs MUSTER, PROSPECT2, HHSEARCH, SP3, PPA-I, HHSEARCH I and SPARKS were with the crystal structure of CP of Papaya mosaic virus, a flexible filamentous virus of Potexvirus genus. Potexvirus is the closest relative of Mandarivirus and CP of both viruses show homology at the aminoacid level in sequence analysis. 3D model of the ICRSV CP generated was verified with structure validation programme PRO-CHECK and Ramachandran plot of the model showed 96% of the aminoacid residues in allowed region with 83.3% in most favoured region. This implicates that the model generated is of moderately good quality. The structure of CP generated may serve as a model for new experimental advancement with Mandarivirus coat protein as a platform for engineering nanoparticles for vaccine production and peptide expression like its closest relative Potexvirus. Coat protein (CP) gene of Garlic common latent virus (GarCLV) was over-expressed in Escherichia coli strain BL21 expression system as 40 kDa fusion protein bearing Histidine tag (6His) at its both N and C terminals. The purified protein reacted positively in western blotting with anti GarCLV polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbit. Antisera to GarCLV (titre 1:2000) detected the virus by direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) in GarCLV positive garlic sample. Further, the specific reactivity of the antisera was confirmed through western blotting and immunosorbent electron microscopy (ISEM). Antiserum developed was successfully utilized for detection of the GarCLV infection in 46 out of 48 different garlic accessions. The immunoreagents developed will be useful for the virus indexing in garlic tissue culture programme and quarantine certification programme as well. (RT-PCR) assay was developed for simultaneous detection of these two viruses with an internal control (NADH dehydrogenase subunit 5 gene) to minimize the risk of getting false negative results. Specific primers were designed against coat protein gene of both the viruses and reported sequences of internal control were used in this study. Uniplex RT-PCR assay was standardized for the detection of these viruses (ACLSV &ApMV) individually. Later the standardized RT-PCR conditions were used for duplex RT-PCR analysis with an internal control wherein the amplicons appeared as faint bands. Therefore, RT-PCR conditions were again optimized by modifying the composition of PCR mix i.e., buffer, dNTPs, primer and Taq DNA polymerase and by minor change in the PCR cycling conditions. This optimized PCR mix and PCR conditions showed sharp expected size of amplicons in duplex RT-PCR with respect to two viruses and multiplex RT-PCR with respect to internal control. The results were confirmed by sequencing the amplified product and then by BLAST analysis. Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both the viruses in field infected apple plants. It gave clear bands of desired sizes along with an internal control. The duplex RT-PCR assay has detection sensitivity as that of uniplex RT-PCR assay for respective viruses. So, the duplex RT-PCR provides a simple, rapid, sensitive and convenient way for simultaneous detection of ACLSV &ApMV by reducing the time and cost of the consumables. Mycovirus research is an emerging and potential field of virus research but is still in its infancy, especially in Indian context. In the present study 18 isolates of different fungi were screened for the presence of virus like particles (VLP's). Negative staining and electron microscopy indexed 7 Chrysosporium sps., Candida albicans, Trichophyton mentagrophytes VLP's elimination was attempted through hyphal tipping and thermotherapy. Immediate heat treatment was successful in eliminating VLP's from C. pseudomerdarium. Similarly, C. albicans was treated with cycloheximide at 10, 20, 50, 100, 200 lg/ml concentration. VLP's were eliminated at 50 lg/ml. Partial purification of VLP's of C. pseudomerdarium and C. albicans was done. Extraction of nucleic acid directly from mycelium gave band in both the isolates through Agarose Gel Electrophoresis. Nature of nucleic acid was ascertained by RNAse and DNAse treatment. Presence of band in low salt (0.01 SSC) RNAse test of C. pseudomerdarium led to conclusion that nucleic acid can be dsRNA. High salt and DNAse treatment did not reveal any band in any of the isolates. Studies of fungal virulence and hypovirulence can increase our understanding of molecular mechanism influencing expression of virulence in plant pathogens and expand potential of fungal virulence as unique mechanism of action for biological control. Molecular Characterization of Coat