Construction and Characterization of an Infectious Clone of Chikungunya Virus Strain 37997

Chikungunya virus (CHIKV) is the pathogen that causes Chikungunya fever, and there is no specific antiviral drug treatment for CHIKV infection. A simple and efficient CHIKV infectious clone is an important tool for studying the mechanism of CHIKV infection and development of vaccine and drug. Based on the cDNA sequence of the virus published in GenBank, we obtained the whole sequence of the viral genome by cleavage and ligation of adjacent fragments, and inserted a mammalian intron sequence which does not affect the production of the virus to ensure the stable inheritance of the virus. We introduced an EcoR Ⅰ restriction endonuclease cleavage site and a T7 promoter sequence at the 5' end, and introduced a Not I restriction endonuclease cleavage site at the 3' end. Then the entire sequence was inserted into pVRC-L vector to obtain the infectious clone pCHIKV. The transfected supernatant was obtained by transcribing and transfecting into BHK-21 cells. The virus was identified by sequencing of the transfected supernatant, empty plaque, indirect immunofluorescence and mice infection test. The results showed that pCHIKV and the transfected supernatant were sequenced as expected, the viral titer was calculated as 5*10⁷ FFU/ml, the virus could infect Vero E6 cells, the virus-infected mice became ill on the 3rd day, and all of them died on the 4th day, and the virus were present in the liver tissues of the dead mice. These results indicated the successful construction of the CHIKV infected clone.