Cloning and expression of NS3 gene of Pakistani isolate type 2 dengue virus

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Introduction: Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines.

Material and Methods: Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors.

Results: The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells.

Conclusion: Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

1. Akram D.S., Igarashi A., Takasu T.: Dengue virus infection among children with undifferentiated fever in Karachi. Indian J Pediatr 1998, 65, 735–740.

2. Bartelma G., Padmanabhan R.: Expression, Purification, and Characterization of the RNA 5′-triphosphatase activity of dengue virus type 2 nonstructural protein 3. Virology 2002, 299, 122–132.

3. Bhatt S., Gething P.W., Brady O.J., Messina J.P., Farlow A.W., Hay S.I.: The global distribution and burden of dengue. Nature 2013, 496, 504–507.

4. Chan Y.C., Salahuddin N.I., Khan J., Tan H.C., Seah C.L.K., Chow V.T.K.: Dengue haemorrhagic fever outbreak in Karachi, Pakistan, 1994. Trans R Soc Trop Med Hyg 1995, 89, 619–620.

5. Clum S., Ebner K.E., Padmanabhan R.: Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B. J Biol Chem United States; 1997, 272, 30715–30723.

6. Das S., Pingle M.R., Muñoz-Jordán J., Rundell M.S., Rondini S., Golightly L.M.: Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay. J Clin Microbiol 2008, 46, 3276–3284.

7. Falgout B., Miller R.H., Lai C.J.: Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity. J Virol 1993, 67, 2034–2042.

8. Fatima Z., Idrees M., Bajwa M.A., Tahir Z., Ullah O., Ali M.: Serotype and genotype analysis of dengue virus by sequencing followed by phylogenetic analysis using samples from three mini outbreaks-2007–2009 in Pakistan. BMC Microbiol 2011, 11, 200.

9. Gibbons R.V., Vaughn D.W.: Dengue: an escalating problem. BMJ England 2002, 324, 1563–1566.

10. Gubler D.J.: Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998, 11, 480–496.

11. Jahan F.: Dengue fever (DF) in Pakistan. Asia Pac Fam Med 2011, 10, 1-4.

12. Keller T.H., Chen Y.L., Knox J.E., Lim S.P., Ma N.L., Patel S.J., Sampath A., Wang Q.Y., Yin Z., Vasudevan S.G.: Finding new medicines for flaviviral targets. Novartis Found Symp 2006, 277, 102–114.

13. Khan E., Hasan R., Mehraj V., Nasir A., Siddiqui J., Hewson R.: Co-circulations of two genotypes of dengue virus in 2006 out-break of dengue hemorrhagic fever in Karachi, Pakistan. J Clin Virol 2008, 43, 176–179.

14. Khan E., Kisat M., Khan N., Nasir A., Ayub S., Hasan R.: Demographic and clinical features of dengue fever in Pakistan from 2003–2007: A retrospective cross-sectional study. PLoS One 2010, 5, 1–7.

15. Leardkamolkarn V., Sirigulpanit W., Phurimsak C., Kumkate S., Himakoun L., Sripanidkulchai B.: The inhibitory actions of houttuynia cordata aqueous extract on dengue virus and dengue-infected cells. J Food Biochem 2012, 36, 86–92.

16. Lescar J., Luo D., Xu T., Sampath A., Lim S.P., Vasudevan S.G.: Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from Dengue virus as a target. Antiviral Res 2008, 5, 94–101.

17. Libraty D.H., Young P.R., Pickering D., Endy T.P., Kalayanarooj S., Rothman A.L.: High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002, 186, 1165–1168.

18. Lindenbach B.D., Prágai B.M., Montserret R., Beran R.K.F., Pyle A.M., Rice C.M.: The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. J Virol 2007, 81, 8905–8918.

19. Natarajan S.: NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus. Genet Mol Biol Ribeirão Preto SP Brazil 2010, 2, 214–219.

20. Rai M.A.: Epidemic: control of dengue fever in Pakistan. Nature 2011, 479, 40-41.

21. Sambrook J., Russell D.W.: The inoue method for preparation and transformation of competent E. coli “ultra-competent” cells.

22. Shum D., Smith J.L., Hirsch A.J., Bhinder B., Radu C., Stein D.A., Nelson J.A., Fruh K., Djaballah H.: High-content assay to identify inhibitors of dengue virus infection. Assay Drug Develop Technol 2010, 8, 553–570.

23. Wilder-Smith A., Ooi E.E., Vasudevan S.G., Gubler D.J.: Update on dengue: epidemiology, virus evolution, antiviral drugs, and vaccine development. Curr Infect Dis Rep 2010, 12, 157–164.

Journal of Veterinary Research

formerly Bulletin of the Veterinary Institute in Pulawy

Journal Information

IMPACT FACTOR Bull Vet Inst Pulawy 2016: 0.462

CiteScore 2016: 0.46

SCImago Journal Rank (SJR) 2015: 0.230
Source Normalized Impact per Paper (SNIP) 2015: 0.383


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