Generation of Full-Length Infectious cDNA Clones of Middle East Respiratory Syndrome Coronavirus

J Microbiol Biotechnol. 2019 Jun 28;29(6):999-1007. doi: 10.4014/jmb.0905.05061.

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virusspecific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patientisolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.

Keywords: MERS-CoV; full-length cDNA; reverse genetics.

MeSH terms

  • Coronavirus Infections / virology
  • DNA, Complementary / genetics*
  • Genes, Viral / genetics
  • Genetic Engineering
  • Genome, Viral / genetics
  • Humans
  • Middle East Respiratory Syndrome Coronavirus / genetics*
  • Middle East Respiratory Syndrome Coronavirus / isolation & purification
  • RNA, Viral / genetics
  • Reverse Genetics*
  • Transcription, Genetic

Substances

  • DNA, Complementary
  • RNA, Viral