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Archives of Virology

, Volume 160, Issue 11, pp 2749–2761 | Cite as

Development of novel antibodies against non-structural proteins nsP1, nsP3 and nsP4 of chikungunya virus: potential use in basic research

  • Sameer Kumar
  • Prabhudutta Mamidi
  • Abhishek Kumar
  • Itishree Basantray
  • Umarani Bramha
  • Anshuman Dixit
  • Prasanta Kumar Maiti
  • Sujay Singh
  • Amol Ratnakar Suryawanshi
  • Subhasis Chattopadhyay
  • Soma ChattopadhyayEmail author
Original Article

Abstract

Chikungunya virus (CHIKV) has reemerged recently as an important pathogen, causing several large epidemics worldwide. This necessitates the development of better reagents to understand its biology and to establish effective and safe control measures. The present study describes the development and characterization of polyclonal antibodies (pAbs) against synthetic peptides of CHIKV non-structural proteins (nsPs; nsP1, nsP3 and nsP4). The reactivity of these pAbs was demonstrated by ELISA and Western blot. Additionally, in vitro infection studies in a mammalian system confirmed that these pAbs are highly sensitive and specific for CHIKV nsPs, as these proteins were detected very early during viral replication. Homology analysis of the selected epitope sequences revealed that they are conserved among all of the CHIKV strains of different genotypes, while comparison with other alphavirus sequences showed that none of them are 100 % identical to the epitope sequences (except Onyong-nyong and Igbo Ora viruses, which show 100 % identity to the nsP4 epitope). Interestingly, two different forms of CHIKV nsP1 and three different forms of nsP3 were detected in Western blot analysis during infection; however, further experimental investigations are required to confirm their identity. Also, the use of these antibodies demonstrated faster and enhanced expression profiles of all CHIKV nsPs in 2006 Indian outbreak strains when compared to the CHIKV prototype strain, suggesting the epidemic potential of the 2006 isolate. Accordingly, it can be suggested that the pAbs reported in this study can be used as sensitive and specific tools for experimental investigations of CHIKV replication and infection.

Keywords

Vero Cell Semliki Forest Virus Chikungunya Virus CHIKV Infection nsP4 Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank Dr. Manmohan Parida for kindly providing the virus strains (S-27 and DRDE-06), CHIKV polyclonal antibody, and Vero cell lines. This work was supported by Department of Biotechnology, Ministry of Science and Technology, Govt. of India vide grant no. BT/PR13118/GBD/27/186/2009, BT/PR15173/GBD/27/356/2011 and by Council of Scientific and Industrial Research (CSIR), Ministry of Science and Technology, Govt. of India vide Project No. 37(1542)/12/EMR-II).

References

  1. 1.
    Strauss EG, Strauss JH (1986) Structure and replication of the alphavirus genome. The Togaviridae and Flaviviridae. Plenum Press, New YorkGoogle Scholar
  2. 2.
    Robinson MC (1955) An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952–53. I. Clinical features. Trans R Soc Trop Med Hyg 49(1):28–32CrossRefPubMedGoogle Scholar
  3. 3.
    Powers AM, Brault AC, Tesh RB, Weaver SC (2000) Re-emergence of Chikungunya and O’nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. J Gen Virol 81(Pt 2):471–479CrossRefPubMedGoogle Scholar
  4. 4.
    Thaikruea L, Charearnsook O, Reanphumkarnkit S, Dissomboon P, Phonjan R, Ratchbud S, Kounsang Y, Buranapiyawong D (1997) Chikungunya in Thailand: a re-emerging disease? Southeast Asian J Trop Med Public Health 28(2):359–364PubMedGoogle Scholar
  5. 5.
    Diallo M, Thonnon J, Traore-Lamizana M, Fontenille D (1999) Vectors of Chikungunya virus in Senegal: current data and transmission cycles. Am J Trop Med Hyg 60(2):281–286PubMedGoogle Scholar
  6. 6.
    Lahariya C, Pradhan SK (2006) Emergence of chikungunya virus in Indian subcontinent after 32 years: a review. J Vector Borne Dis 43(4):151–160PubMedGoogle Scholar
  7. 7.
    Staples JE, Fischer M (2014) Chikungunya virus in the Americas—what a vectorborne pathogen can do. N Engl J Med 371(10):887–889. doi: 10.1056/NEJMp1407698 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Arankalle VA, Shrivastava S, Cherian S, Gunjikar RS, Walimbe AM, Jadhav SM, Sudeep AB, Mishra AC (2007) Genetic divergence of Chikungunya viruses in India (1963–2006) with special reference to the 2005–2006 explosive epidemic. J Gen Virol 88(Pt 7):1967–1976. doi: 10.1099/vir.0.82714-0 CrossRefPubMedGoogle Scholar
  9. 9.
    CDC (2012) Chikungunya outbreak—Cambodia, February–March 2012. MMWR Morb Mortal Wkly Rep 61:737–740Google Scholar
  10. 10.
    Moyen N, Thiberville SD, Pastorino B, Nougairede A, Thirion L, Mombouli JV, Dimi Y, Leparc-Goffart I, Capobianchi MR, Lepfoundzou AD, de Lamballerie X (2014) First reported chikungunya Fever outbreak in the republic of congo, 2011. PLoS ONE 9(12):e115938. doi: 10.1371/journal.pone.0115938 PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Nkoghe D, Kassa RF, Caron M, Grard G, Mombo I, Bikie B, Paupy C, Becquart P, Bisvigou U, Leroy EM (2012) Clinical forms of chikungunya in Gabon, 2010. PLoS Neglect Trop Dis 6(2):e1517. doi: 10.1371/journal.pntd.0001517 CrossRefGoogle Scholar
  12. 12.
    Wu D, Wu J, Zhang Q, Zhong H, Ke C, Deng X, Guan D, Li H, Zhang Y, Zhou H, He J, Li L (2010) Yang X (2012) Chikungunya outbreak in Guangdong Province, China. Emerg Infect Dis 18(3):493–495. doi: 10.3201/eid1803.110034 CrossRefGoogle Scholar
  13. 13.
    Angelini R, Finarelli AC, Angelini P, Po C, Petropulacos K, Macini P, Fiorentini C, Fortuna C, Venturi G, Romi R, Majori G, Nicoletti L, Rezza G, Cassone A (2007) An outbreak of chikungunya fever in the province of Ravenna, Italy. Euro Surveill 12(9):E070906.1PubMedGoogle Scholar
  14. 14.
    Rianthavorn P, Prianantathavorn K, Wuttirattanakowit N, Theamboonlers A, Poovorawan Y (2010) An outbreak of chikungunya in southern Thailand from 2008 to 2009 caused by African strains with A226V mutation. Int J Infect Dis 14(Suppl 3):e161–e165. doi: 10.1016/j.ijid.2010.01.001 CrossRefPubMedGoogle Scholar
  15. 15.
    Staikowsky F, Le Roux K, Schuffenecker I, Laurent P, Grivard P, Develay A, Michault A (2008) Retrospective survey of Chikungunya disease in Reunion Island hospital staff. Epidemiol Infect 136(2):196–206. doi: 10.1017/S0950268807008424 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Van Bortel W, Dorleans F, Rosine J, Blateau A, Rousset D, Matheus S, Leparc-Goffart I, Flusin O, Prat C, Cesaire R, Najioullah F, Ardillon V, Balleydier E, Carvalho L, Lemaitre A, Noel H, Servas V, Six C, Zurbaran M, Leon L, Guinard A, van den Kerkhof J, Henry M, Fanoy E, Braks M, Reimerink J, Swaan C, Georges R, Brooks L, Freedman J, Sudre B, Zeller H (2014) Chikungunya outbreak in the Caribbean region, December 2013 to March 2014, and the significance for Europe. Euro Surveill 19(13)Google Scholar
  17. 17.
    Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, Lavenir R, Pardigon N, Reynes JM, Pettinelli F, Biscornet L, Diancourt L, Michel S, Duquerroy S, Guigon G, Frenkiel MP, Brehin AC, Cubito N, Despres P, Kunst F, Rey FA, Zeller H, Brisse S (2006) Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Med 3(7):e263. doi: 10.1371/journal.pmed.0030263 PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Higashi N, Matsumoto A, Tabata K, Nagatomo Y (1967) Electron microscope study of development of Chikungunya virus in green monkey kidney stable (VERO) cells. Virology 33(1):55–69CrossRefPubMedGoogle Scholar
  19. 19.
    Powers AM, Brault AC, Shirako Y, Strauss EG, Kang W, Strauss JH, Weaver SC (2001) Evolutionary relationships and systematics of the alphaviruses. J Virol 75(21):10118–10131. doi: 10.1128/JVI.75.21.10118-10131.2001 PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Simizu B, Yamamoto K, Hashimoto K, Ogata T (1984) Structural proteins of Chikungunya virus. J Virol 51(1):254–258PubMedCentralPubMedGoogle Scholar
  21. 21.
    Strauss JH, Strauss EG (1994) The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58(3):491–562PubMedCentralPubMedGoogle Scholar
  22. 22.
    Jose J, Snyder JE, Kuhn RJ (2009) A structural and functional perspective of alphavirus replication and assembly. Future Microbiol 4(7):837–856. doi: 10.2217/fmb.09.59 PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Rheme C, Ehrengruber MU, Grandgirard D (2005) Alphaviral cytotoxicity and its implication in vector development. Exp Physiol 90(1):45–52. doi: 10.1113/expphysiol.2004.028142 CrossRefPubMedGoogle Scholar
  24. 24.
    Wang YF, Sawicki SG, Sawicki DL (1991) Sindbis virus nsP1 functions in negative-strand RNA synthesis. J Virol 65(2):985–988PubMedCentralPubMedGoogle Scholar
  25. 25.
    Ahola T, Kaariainen L (1995) Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci USA 92(2):507–511PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Laakkonen P, Hyvonen M, Peranen J, Kaariainen L (1994) Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli. J Virol 68(11):7418–7425PubMedCentralPubMedGoogle Scholar
  27. 27.
    Laakkonen P, Auvinen P, Kujala P, Kaariainen L (1998) Alphavirus replicase protein NSP1 induces filopodia and rearrangement of actin filaments. J Virol 72(12):10265–10269PubMedCentralPubMedGoogle Scholar
  28. 28.
    Pastorino BA, Peyrefitte CN, Almeras L, Grandadam M, Rolland D, Tolou HJ, Bessaud M (2008) Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus. Virus Res 131(2):293–298. doi: 10.1016/j.virusres.2007.09.009 CrossRefPubMedGoogle Scholar
  29. 29.
    Karpe YA, Aher PP, Lole KS (2011) NTPase and 5’-RNA triphosphatase activities of Chikungunya virus nsP2 protein. PLoS ONE 6(7):e22336. doi: 10.1371/journal.pone.0022336 PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Hardy WR, Hahn YS, de Groot RJ, Strauss EG, Strauss JH (1990) Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus. Virology 177(1):199–208CrossRefPubMedGoogle Scholar
  31. 31.
    Sawicki SG, Sawicki DL, Kaariainen L, Keranen S (1981) A Sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis. Virology 115(1):161–172CrossRefPubMedGoogle Scholar
  32. 32.
    Shirako Y, Strauss JH (1998) Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase. J Virol 72(3):2310–2315PubMedCentralPubMedGoogle Scholar
  33. 33.
    Sawicki D, Barkhimer DB, Sawicki SG, Rice CM, Schlesinger S (1990) Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4. Virology 174(1):43–52CrossRefPubMedGoogle Scholar
  34. 34.
    Wang YF, Sawicki SG, Sawicki DL (1994) Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA. J Virol 68(10):6466–6475PubMedCentralPubMedGoogle Scholar
  35. 35.
    Chattopadhyay S, Kumar A, Mamidi P, Nayak TK, Das I, Chhatai J, Basantray I, Bramha U, Maiti PK, Singh S, Suryawanshi AR, Chattopadhyay S (2014) Development and characterization of monoclonal antibody against non-structural protein-2 of Chikungunya virus and its application. J Virol Methods 199:86–94. doi: 10.1016/j.jviromet.2014.01.008 CrossRefPubMedGoogle Scholar
  36. 36.
    Das I, Basantray I, Mamidi P, Nayak TK, B M P, Chattopadhyay S, Chattopadhyay S (2014) Heat shock protein 90 positively regulates Chikungunya virus replication by stabilizing viral non-structural protein nsP2 during infection. PLoS ONE 9(6):e100531. doi: 10.1371/journal.pone.0100531 PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Kumar A, Mamidi P, Das I, Nayak TK, Kumar S, Chhatai J, Chattopadhyay S, Suryawanshi AR, Chattopadhyay S (2014) A novel 2006 Indian outbreak strain of Chikungunya virus exhibits different pattern of infection as compared to prototype strain. PLoS ONE 9(1):e85714. doi: 10.1371/journal.pone.0085714 PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Chattopadhyay S, Weller SK (2006) DNA binding activity of the herpes simplex virus type 1 origin binding protein, UL9, can be modulated by sequences in the N terminus: correlation between transdominance and DNA binding. J Virol 80(9):4491–4500. doi: 10.1128/JVI.80.9.4491-4500.2006 PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Kam YW, Lee WW, Simarmata D, Le Grand R, Tolou H, Merits A, Roques P, Ng LF (2014) Unique epitopes recognized by antibodies induced in Chikungunya virus-infected non-human primates: implications for the study of immunopathology and vaccine development. PLoS ONE 9(4):e95647. doi: 10.1371/journal.pone.0095647 PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Scholte FE, Tas A, Martina BE, Cordioli P, Narayanan K, Makino S, Snijder EJ, van Hemert MJ (2013) Characterization of synthetic Chikungunya viruses based on the consensus sequence of recent E1-226V isolates. PLoS ONE 8(8):e71047. doi: 10.1371/journal.pone.0071047 PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Laakkonen P, Ahola T, Kaariainen L (1996) The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme. J Biol Chem 271(45):28567–28571CrossRefPubMedGoogle Scholar
  42. 42.
    Peranen J, Laakkonen P, Hyvonen M, Kaariainen L (1995) The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles. Virology 208(2):610–620. doi: 10.1006/viro.1995.1192 CrossRefPubMedGoogle Scholar
  43. 43.
    Li GP, La Starza MW, Hardy WR, Strauss JH, Rice CM (1990) Phosphorylation of Sindbis virus nsP3 in vivo and in vitro. Virology 179(1):416–427CrossRefPubMedGoogle Scholar
  44. 44.
    Peranen J, Takkinen K, Kalkkinen N, Kaariainen L (1988) Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein. J Gen Virol 69(Pt 9):2165–2178CrossRefPubMedGoogle Scholar
  45. 45.
    Vihinen H, Saarinen J (2000) Phosphorylation site analysis of Semliki forest virus nonstructural protein 3. J Biol Chem 275(36):27775–27783. doi: 10.1074/jbc.M002195200 PubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Sameer Kumar
    • 1
  • Prabhudutta Mamidi
    • 1
  • Abhishek Kumar
    • 1
  • Itishree Basantray
    • 1
  • Umarani Bramha
    • 1
  • Anshuman Dixit
    • 1
  • Prasanta Kumar Maiti
    • 2
  • Sujay Singh
    • 2
  • Amol Ratnakar Suryawanshi
    • 1
  • Subhasis Chattopadhyay
    • 3
  • Soma Chattopadhyay
    • 1
    Email author
  1. 1.Infectious Disease BiologyInstitute of Life SciencesBhubaneswarIndia
  2. 2.Imgenex India Pvt. Ltd.BhubaneswarIndia
  3. 3.School of Biological SciencesNational Institute of Science Education and ResearchBhubaneswarIndia

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