Schematic diagram of <i>Alphavirus</i> detection using defective XJ-160 replicons.

<p>Due to the deletion in the non-structural protein coding region, the defective XJ-160 replicons (pVaXJ-EGFPΔ nsp4 and pVaXJ-GLucΔ nsp4) failed to express the reporter genes efficiently. When cells were infected with alphavirus, the alphavirus synthesized the viral non-structural proteins, and these proteins acted on the defective XJ-160 replicons in <i>trans</i>, resulting in high-level expression of the reporter genes, which could be easily measured.</p

GLuc expression in replicon-defective transfected cells infected with varying PFUs of alphaviruses.

<p>BHK-21 cells were transfected with pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent, 6 h before mock-infection or infection with varying PFU levels of alphaviruses. GLuc activity was measured 44 h post-infection. Each data point represents the mean ±SEM of three independent experiments. RLU, relative light units.</p

EGFP and GLuc expression in replicon-defective transfected cells infected with SINV (XJ-160).

<p>BHK-21 cells were transfected with pVaXJ-EGFPΔnsp4 or pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent, 6 h before mock-infection or infection with 1 MOI SINV (XJ-160). (A, B) EGFP expression was examined 44 h post-infection. Green color indicates EGFP, and blue color indicates nucleus. (C) GLuc activity was measured at different time points (from 6–68 h) post-infection. *<i>p</i><0.05, (pVaXJ-GLucΔnsp4+SINXJ160) vs. pVaXJ-GLucΔnsp4.</p

Diagram of the generation of defective XJ-160 replicons.

<p>(A) The pVa-XJ replicon was constructed by inserting the XJ-160 virus genome into the eukaryotic expression vector pVAX1 and replacing the structural gene with the multiple cloning site (MCS) sequence. (B) The constructs containing the reporter gene (pVaXJ-EGFP or pVaXJ-GLuc) were generated by digesting the <i>enhanced green fluorescent protein</i> (<i>EGFP</i>) gene or <i>Gaussia luciferase</i> (<i>GLuc</i>) gene with the restriction enzymes <i>Fse</i>I and <i>Asc</i>I, and ligating them into the MCS of pVa-XJ. (C) The defective XJ-160 replicons (pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4) were produced by introducing an 1139-nt deletion mutation in the non-structural protein coding regions of pVaXJ-EGFP and pVaXJ-GLuc using <i>Acl</i>I digestion. The deleted regions of the XJ-160 genome in defective replicons are denoted by dotted lines and the non-structural protein gene deletion is designated by a “Δ”.</p

EGFP and GLuc expression in replicon-transfected cells.

<p>BHK-21 cells were transfected with pVaXJ-EGFP, pVaXJ-EGFPΔnsp4, pVaXJ-GLuc, or pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent. (A), (B) EGFP expression was examined 48 h post-transfection using an Olympus IX51 fluorescence microscope. Green color indicates EGFP, and blue color indicates nucleus. (C) GLuc activity was measured at different time points (from 18–170 h) post-transfection using the BioLux™ Gaussia Luciferase Assay Kit and a luminometer. Each data point represents the mean ±SEM of three independent experiments. RLU, relative light units.</p

EGFP and GLuc expression in replicon-defective transfected cells infected with various viruses.

<p>BHK-21 cells were transfected with pVaXJ-EGFPΔnsp4 or pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent, 6 h before mock-infection or infection with 1 MOI of various viruses, including alphaviruses [SINV (XJ-160, YN87448, and MX10), CHIKV (SD08Pan), and GETAV (HB0234)], and non-alphaviruses [JEV (P3) and TAHV (XJ0625)]. (A–H) EGFP expression was examined 44 h post-infection. Green color indicates EGFP, and blue color indicates nucleus. (I) GLuc activity was measured 44 h post-infection. Each data point represents the mean ±SEM of three independent experiments. RLU, relative light units.</p