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. 2017 Dec 21;32(6):511–519. doi: 10.1007/s12250-017-4059-7

Detection, isolation, and characterization of chikungunya viruses associated with the Pakistan outbreak of 2016–2017

Si-Qing Liu 1, Xiao Li 1,2, Ya-Nan Zhang 3, Ai-Li Gao 3, Cheng-Lin Deng 1, Jun-Hua Li 1, Shoukat Jehan 4, Nadia Jamil 4, Fei Deng 5, Hongping Wei 1, Bo Zhang 1,✉
PMCID: PMC6598907  PMID: 29285673

Abstract

The chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which has infected millions of people in Africa, Asia, Americas, and Europe since it reemerged in India and Indian Ocean regions in 2005–2006. Starting in the middle of November 2016, CHIKV has been widely spread, and more than 4,000 cases of infections in humans were confirmed in Pakistan. Here, we report the first isolation and characterization of CHIKV from the Pakistan outbreak. Eight CHIKV strains were newly isolated from human serum samples using a cell culture procedure. A full-length genome sequence and eight complete envelope (E1) sequences of CHIKV from Pakistan were obtained in this study. Alignment of the CHIKV E1 sequences revealed that the eight new CHIKV isolates were highly homogeneous, with only two nonsynonymous substitutions found at generally conserved sites (E99 and Q235). Based on the comparison of 342 E1 sequences, the two nonsynonymous mutations were located in well-recognized domains associated with viral functions such as the cell fusion and vector specificity, suggesting their potential functional importance. Phylogenetic analysis indicated that the CHIKV strains from Pakistan originated from CHIKV circulating in the Indian region. This study helps elucidate the epidemics of CHIKV in Pakistan and also provides a foundation for studies of evolution and expansion of CHIKV in South Asia.

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Keywords: chikungunya virus (CHIKV), Pakistan, virus isolation, clinical serum, molecular characterization

Acknowledgments

This work was supported by International Cooperation on key Technologies of Biosafety along the China–Pakistan Economic Corridor (153B42KYSB20170004), Major State Basic Research Development Program (2013FY 113500) and the National Natural Science Foundation of China (81572003 and 31600141). We are grateful to the Core Facility and Technical Support, Wuhan Institute of Virology, Wuhan Key Laboratory on Emerging Infectious Diseases and Biosafety, and Biosafety Level 3 Laboratory, Wuhan Institute of Virology for their helpful supports during the course of the work. We thank the administrator Hao Tang for his hard work in Biosafety Level 3 Laboratory.

References

  1. Chatterjee PK, Eng CH, Kielian M. Novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest virus fusion protein. J Virol. 2002;76:12712–12722. doi: 10.1128/JVI.76.24.12712-12722.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen R, Puri V, Fedorova N, Lin D, Hari KL, Jain R, Rodas JD, Das SR, Shabman RS, Weaver SC. Comprehensive genome- scale phylogenetic study provides new insights on the global expansion of chikungunya virus. J Virol. 2016;90:10600–10611. doi: 10.1128/JVI.01166-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Deng CL, Liu SQ, Zhang QY, Xu MY, Zhang HL, Gu DY, Shi L, He JA, Xiao GF, Zhang B. Isolation and characterization of zika virus imported to china using c6/36 mosquito cells. Virol Sin. 2016;31:176–179. doi: 10.1007/s12250-016-3778-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Deng CL, Liu SQ, Zhou DG, Xu LL, Li XD, Zhang PT, Li PH, Ye HQ, Wei HP, Yuan ZM, Qin CF, Zhang B. Development of neutralization assay using an egfp chikungunya virus. Viruses. 2016;8:181. doi: 10.3390/v8070181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeZure AD, Berkowitz NM, Graham BS, Ledgerwood JE. Whole-inactivated and virus-like particle vaccine strategies for chikungunya virus. J Infect Dis. 2016;214:S497–S499. doi: 10.1093/infdis/jiw352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Lescar J, Roussel A, Wien MW, Navaza J, Fuller SD, Wengler G, Wengler G, Rey FA. The fusion glycoprotein shell of semliki forest virus: An icosahedral assembly primed for fusogenic activation at endosomal ph. Cell. 2001;105:137–148. doi: 10.1016/S0092-8674(01)00303-8. [DOI] [PubMed] [Google Scholar]
  7. Levy-Mintz P, Kielian M. Mutagenesis of the putative fusion domain of the semliki forest virus spike protein. J Virol. 1991;65:4292–4300. doi: 10.1128/JVI.65.8.4292-4300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liu SQ, Zhang B. Zika virus: A flavivirus caused pandemics in latin america. Virol Sin. 2016;2:1–2. doi: 10.1007/s12250-016-3738-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Liu SQ, Li X, Deng CL, Yuan ZM, Zhang B. J Med Virol. 2017. Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of zika virus and chikungunya virus. [DOI] [PubMed] [Google Scholar]
  10. Martina BEE, Koraka P, Osterhaus ADME. Dengue virus pathogenesis: An integrated view. Clin Microbiol Rev. 2009;22:564–581. doi: 10.1128/CMR.00035-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ng LC, Hapuarachchi HC. Tracing the path of chikungunya virus—evolution and adaptation. Infect Genet Evol. 2010;10:876–885. doi: 10.1016/j.meegid.2010.07.012. [DOI] [PubMed] [Google Scholar]
  12. Rauf M, Fatima t Z, Manzoor S, Mehmood A, Bhatti S. Outbreak of chikungunya in pakistan. Lancet Infect Dis. 2017;17:258. doi: 10.1016/S1473-3099(17)30074-9. [DOI] [PubMed] [Google Scholar]
  13. Roussel A, Lescar J, Vaney MC, Wengler G, Wengler G, Rey FA. Structure and interactions at the viral surface of the envelope protein E1 of semliki forest virus. Structure. 2006;14:75–86. doi: 10.1016/j.str.2005.09.014. [DOI] [PubMed] [Google Scholar]
  14. Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, Lavenir R, Pardigon N, Reynes J-M, Pettinelli F, Biscornet L, Diancourt L, Michel S, Duquerroy S, Guigon G, Frenkiel MP, Bréhin AC, Cubito N, Desprès P, Kunst F, Rey FA, Zeller H, Brisse S. Genome microevolution of chikungunya viruses causing the indian ocean outbreak. PLoS Medicine. 2006;3:e263. doi: 10.1371/journal.pmed.0030263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Taubitz W, Cramer JP, Kapaun A, Pfeffer M, Drosten C, Dobler G, Burchard GD, Löscher T. Chikungunya fever in travelers: Clinical presentation and course. Clin Infect Dis. 2007;45:e1–e4. doi: 10.1086/518701. [DOI] [PubMed] [Google Scholar]
  16. Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathogens. 2007;3:e201. doi: 10.1371/journal.ppat.0030201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tsetsarkin KA, Chen R, Sherman MB, Weaver SC. Chikungunya virus: Evolution and genetic determinants of emergence. Curr Opin Virol. 2011;1:310–317. doi: 10.1016/j.coviro.2011.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tsetsarkin KA, Chen R, Leal G, Forrester N, Higgs S, Huang J, Weaver SC. Chikungunya virus emergence is constrained in Asia by lineage-specific adaptive landscapes. Proc Natl Acad Sci U S A. 2011;108:7872–7877. doi: 10.1073/pnas.1018344108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vashishtha M, Phalen T, Marquardt MT, Ryu JS, Ng AC, Kielian M. A single point mutation controls the cholesterol dependence of semliki forest virus entry and exit. The J Cell Biol. 1998;140:91–99. doi: 10.1083/jcb.140.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Volk SM, Chen R, Tsetsarkin KA, Adams AP, Garcia TI, Sall AA, Nasar F, Schuh AJ, Holmes EC, Higgs S, Maharaj PD, Brault AC, Weaver SC. Genome-scale phylogenetic analyses of chikungunya virus reveal independent emergences of recent epidemics and various evolutionary rates. J Virol. 2010;84:6497–6504. doi: 10.1128/JVI.01603-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

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