Abstract
We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.
Highlights
Alphaviruses are a group of 30 mosquito-borne arboviruses belonging to the genus Alphavirus in the family Togaviridae [1,2,3]
To generate defective replicons used for detecting alphaviruses, we first constructed the plasmids pVaXJ-EGFP and pVaXJ-GLuc containing the XJ-160 virus, a Sindbis virus (SINV) isolated from Xinjiang, China [21], and reporter genes that express enhanced green fluorescence protein (EGFP) or Gaussia luciferase (GLuc), respectively
Reporter gene expression assays showed that specific green fluorescence was observed after BHK-21 cells were transfected with pVaXJ-EGFP (Figure 2A), while no EGFP expression was observed following transfection with the defective replicon pVaXJEGFPDnsp4 (Figure 2B)
Summary
Alphaviruses are a group of 30 mosquito-borne arboviruses belonging to the genus Alphavirus in the family Togaviridae [1,2,3] Some alphaviruses, such as Sindbis virus (SINV) [4] and Chikungunya virus (CHIKV) [5,6], can cause fever, arthritis, rash, and other symptoms. Alphaviruses such as Western equine encephalitis virus (WEEV) [7], Eastern equine encephalitis virus (EEEV) [8], and Venezuelan equine encephalitis virus (VEEV) [9] can cause fever and viral encephalitis in humans. A rapid, specific detection technique for alphavirus pathogens plays an important role in monitoring infectious diseases and public security
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