IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) e-ISSN: 2319-2380, p-ISSN: 2319-2372. Volume 8, Issue 3 Ver. I (Mar. 2015), PP 16-18 www.iosrjournals.org DOI: 10.9790/2380-08311618 www.iosrjournals.org 16 | Page Nested Reverse Transcriptase-PCR (NRT-PCR) Assay for detection of Classical Swine Fever Virus P. Thakuria 1 , S. Sarma 2 , D. K. Sarma 3 , D. J. Kalita 4 , K. Sharma 5 , R. Sharma 6 and P. Roychoudhury 7 1,2and 4 Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Guwahati-781022, Assam, India 5 and 6 Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati- 781022, Assam, India. 3 Director, NRC on Pig, ICAR, Rani, Guwahati-781031, Assam, India 7 Central Agricultural University, Mizoram, India Abstract :Classical Swine Fever(CSF) or Hog Cholera is one of the most feared and devastating disease of pigs. The disease has become main threat to pig industry in counrties with a dense pig population and is known to cause more deaths in pigs as compared to many other infectious agents. The disease is endemic in many parts of India including Assam. The virus belongs to genus Pestivirus and family Flaviviridae and has close antigenic similarity with the two other members of this genus ie. Border disease virus (BDV) and Bovine viral diarrhea virus (BVDV). Out of these CSFV induces severe illness in young piglets and thereby cause severe economic loss in pig industry.It is important to differentiate CSF from other pestiviruses. nRT-PCR is a suitable approach for screening of suspected cases of disease and is now accepted by many countries and the European Union. Among several genes tested E2 is found to be more effective for genotyping of CSFV isolates. The envelope glycoprotein E2 is highly conserved and CSFV specific.The present investigation was conducted to detect CSFV from Tissue culture fluid using Nested Reverse Transcriptase- Ploymerase Chain Reaction (nRT-PCR) assay by amplifying the CSFV specific E2 gene fragment. Keywords: CSFV, E2 glycoprotein, Flaviviridae, Hog cholera Virus, Pestivirus, nRT-PCR. I. Introduction A major portion of the national economy of India is contributed by pig industry. Keeping the pace with other part of India, the North Eastern region of India specially Assam has also made a significant progress in pig industry in the recent years. A vast majority of the population being of tribal origin, pig rearing and consumption of pork have been traditionally popular in this region. In recent times the popularity of pork among the non-tribal particularly in urban areas is also increasing steadily. Among various infectious agents associated with diseases of pig is the virus. Out of these viral agents, Classical Swine Fever Virus (CSFV)is responsible for the most devastating disease that causes stillbirth, abortion, persistent infection [1] .Also compared to any other infectious disease CSF causes more number of deaths in pigs. Therefore, it is a cause of fear or threat to pig industry. The etiological agent of CSF is the Classical Swine Fever Virus (CSFV), a member of the genus Pestivirus, of the family Flaviviridae. The genome of the CSFV is single stranded RNA. The RNA is positive sense, the number of nucleotides in the RNA is approximately 12300bp and GC content is 46%.The CSFV measures approximately 40±3 nm in diameter and the inner core or nucleocapsid alone is about 29±3nm.The virus has an envelope with glycosylated membrane proteins and icosahedral symmetry [2].The virus has a non- translated region at either end (5´ NTR and 3´ NTR), encompassing a single open reading frame encoding a large protein that is cleaved into smaller fragments. The genes encoding the structural proteins (Cprotein, Erns, E1 and E2) are found towards the 5´ end of the genome, and include the major envelope glycoprotein gene E2.The genes encoding non-structural proteins (N pro , P 7 , NS2, NS3, NS4A, NS4B, NS5A and NS5B) are located mainly in the 3´ two thirds of the genome, and include the polymerase gene NS5B .The glycoprotein E2 is most immunogenic protein and neutralizing antibodies are directed to it during CSFV infection [3, 4]. For detection of CSF viral nucleic acid, the highly conserved E2 gene fragment of CSFV (Lowing et al., 1996) was used in the present study. Specific primers were used to amplify E2 gene fragment of CSFV isolates from Assam using nRT-PCR.