Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (17): 3479-3486.doi: 10.3864/j.issn.0578-1752.2023.17.019

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Construction of Infectious cDNA Clone of GETV SC483 Strain

DU BingChen(), WANG Ming, LIU ChunGuo, WANG ShiDa, WEI XinYu, LU YaMan, SUN ZhenZhao, LIU ZaiSi, WEI LiLi, WANG JingFei(), YANG DeCheng()   

  1. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory of Animal Disease Control and Prevention, Harbin 150069
  • Received:2022-08-01 Accepted:2022-11-16 Online:2023-09-01 Published:2023-09-08
  • Contact: WANG JingFei, YANG DeCheng

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Abstract:

【Background】 Getah virus (GETV) is a mosquito-borne arbovirus, and belongs to the Alphavirus genus in the Togaviridae family. GETV has an extensive host range, which includes multiple mammals, such as pig, horse, cattle, fox and so on, although GETV is not known to cause disease in human. GETV infection often causes clinical symptoms, such as fever, urticarial rash, arthritis, reproductive disorders, and fetal death. GETV is widely prevalent in the world, and the prevalence in China is on the rise in recent years. In 2018, it suddenly broke out in many pig farms in southern China. The harm for livestock and poultry production and public health caused by GETV has got widespread attention. Currently, there is still no specific antiviral drug or vaccine against GETV. Due to the lack of research on GETV, its biological characteristics, pathogenicity, pathogenesis and epidemic properties are largely unknown. 【Objective】 The aim of this study was to develop a reverse genetics platform of GETV, which could provide a basis for investigating the structure and function of GETV genome, the genetic determinant of pathogenesis, and developing novel vaccine against the virus. 【Method】 The full-length genome of the GETV SC483 strain flanked by a hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequence was chemically synthesized and cloned into a low-copy plasmid pOK12-CMV to generate the recombinant plasmid pGETV-SC483, which was transfected into BHK-21 cells to rescue the virus. Then, compared with the parental virus, the rescued-virus was serially passaged, identified, and analyzed for biological characteristics. The genetic stability of the recombinant plasmid pGETV-SC483 in E. coli was also verified. 【Result】 The typical cell pathogenic effect (CPE) of GETV was observed in the cells after being transfected with the pGETV-SC483 for 48 h. The rescued GETV, namely rSC483, was successfully obtained, which had a G4332A mutation resulted in the deletion of a Not I restriction site to differentiate from the parental virus. The rescued virus was further verified by indirect immunofluorescence assay using GETV specific antibody as detection antibody and electron microscopic observation of virus particles. Furthermore, plaque phenotype and growth curve of the rescued virus were similar to that of its parental virus in BHK-21 cells. In addition, the infectious cloning plasmid pGETV-SC483 had good genetic stability in E. coli DH5α. 【Conclusion】 The successfully constructed full-length infectious cDNA clone of GETV SC483 strain provided a technical platform for the study of biological characteristics and pathogenesis of GETV as well as development of novel vaccine against GETV.

Key words: Getah virus, cDNA infectious clone, virus rescue, reverse genetics system

Fig. 1

The strategy for the construction of the full-length cDNA clone of GETV SC483 strain"

Fig. 2

CPE occurred on BHK-21 cells infected with the rescued GETV"

Fig. 3

Identification of the rescued GETV in BHK-21 cells by IFA"

Fig. 4

Morphology of the rescued virus observed with an electron microscope"

Fig. 5

Identification of site mutation in rescued virus as genetic marker A: Not I digestion analysis; B: Sequence analysis. M: 1.5 kb DNA ladder; 1: RT-PCR product of parental virus; 2: RT-PCR product of rescued virus; 3: RT-PCR product of parental virus digested with Not I; 4: RT-PCR product of rescued virus digested with Not I"

Fig. 6

Plaque phenotype of the rescued virus in BHK-21 cells"

Fig. 7

The one-step growth curve of the rescued virus in BHK-21 cells"

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