A Recombinant Genotype I Japanese Encephalitis Virus Expressing a Gaussia Luciferase Gene for Antiviral Drug Screening Assay and Neutralizing Antibodies Detection.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in humans throughout Asia. In the past twenty years, the emergence of the genotype I (GI) JEV as the dominant genotype in Asian countries has raised a significant threat to public health security. However, no clinically approved drug is available for the specific treatment of JEV infection, and the commercial vaccines derived from the genotype III JEV strains merely provided partial protection against the GI JEV. Thus, an easy-to-perform platform in high-throughput is urgently needed for the antiviral drug screening and assessment of neutralizing antibodies specific against the GI JEV. In this study, we established a reverse genetics system for the GI JEV strain (YZ-1) using a homologous recombination strategy. Using this reverse genetic system, a gaussia luciferase (Gluc) expression cassette was inserted into the JEV genome to generate a reporter virus (rGI-Gluc). The reporter virus exhibited similar growth kinetics to the parental virus and remained genetically stable for at least ten passages in vitro. Of note, the bioluminescence signal strength of Gluc in the culture supernatants was well correlated with the viral progenies determined by viral titration. Taking advantage of this reporter virus, we established Gluc readout-based assays for antiviral drug screening and neutralizing antibody detection against the GI JEV. These Gluc readout-based assays exhibited comparable performance to the assays using an actual virus and are less time consuming and are applicable for a high-throughput format. Taken together, we generated a GI JEV reporter virus expressing a Gluc gene that could be a valuable tool for an antiviral drug screening assay and neutralization assay.

Evaluation of the genotype I Japanese encephalitis virus as a stable viral vector for foreign gene expression.

Manipulation of the flavivirus genome to accommodate and express a heterologous gene of interest has become an attractive approach for gene delivery and the development of viral-vectored vaccines. However, due to the inherent genetic instability of the flavivirus genomes, the construction of recombinant viruses carrying a foreign gene could be problematic and heavily resistant. In this study, the possibility of the Japanese encephalitis virus (JEV) as a stable flavivirus vector for the expression of a foreign gene was assessed using reverse genetics. The full-length cDNA genome of genotype I (GI) JEV inherently possessed excellent stability and manipulability in a bacterial host, while mutations and deletions accumulated in the cDNA genomes of genotype Ⅲ (GⅢ) JEV strains. Using the GI JEV as backbones, we generate a panel of recombinant viruses expressing various foreign genes. All recombinant viruses exhibited excellent genetic stability and efficiently express foreign genes for at least ten serial passages in vitro. In application, a convenient, rapid and reliable image-based assay for neutralizing antibody testing and antiviral drug discovery was established with a mCherry-reporter recombinant virus (rBJ-mCherry). Meanwhile, the recombinant viruses expressing the antigens of the African swine fever virus (ASFV) or Classical swine fever virus (CSFV) could effectively induce antibody responses to the JEV vector and foreign antigens in a mouse vaccination model. Therefore, GI JEV strains could serve as viral vectors accommodating the expression of large foreign genes.

Development of a highly sensitive Gaussia luciferase immunoprecipitation assay for the detection of antibodies against African swine fever virus.

In recent years, African swine fever (ASF) has caused a devastating blow to the swine industry globally. Since no effective vaccine is available, strict biosafety measures and rapid diagnosis are the most effective strategies for ASF control. ASFV p30 is one of the most antigenic viral proteins that have been widely used in the field for serological diagnosis of ASF infection. In this study, we developed a luciferase immunoprecipitation system (LIPS) assay for the detection of ASFV antibodies in pig serum using Gaussia luciferase (GLuc)-tagged p30 as a diagnostic antigen. The optimal GLuc-p30 input of 107 luminance units (LU) and optimal serum dilution factor of 1/100 were set to achieve the highest P/N ratio. Based on 87 ASFV-positive and negative pig sera, the cutoff value of the S/N ratio could be set between 2.298 and 30.59 to achieve 100% sensitivity and 100% specificity. Moreover, the diagnostic sensitivity of this LIPS is comparable to that of a commercial enzyme-linked immunosorbent assay (ELISA) and the specificity of LIPS is even superior to the tested ELISA. In conclusion, we have established a LIPS assay for ASFV antibody detection, which could be a potential method for ASFV diagnosis in laboratories and farms.

Porcine TRIM35 positively regulate TRAF3-mediated IFN-ß production and inhibit Japanese encephalitis virus replication.

Tripartite motif 35 (TRIM35) protein is a ubiquitin E3 ligase that mediates interferon-beta (IFN-ß) production via regulating ubiquitination of multiple adaptor proteins in innate immune signaling pathways. Here, we cloned the porcine TRIM35 (porTRIM35) gene and analyzed its involvement in IFN-ß expression as well as the antiviral response against Japanese encephalitis virus (JEV). The full-length porTRIM35 gene encoded a 493-amino acid protein and exhibited 79.6%-89.5% sequence similarity with its orthologues in humans, mice, monkeys and rabbits. porTRIM35 possessed typical structural features of TRIMs, including a RING domain, a B-box domain, a coiled-coil domain and a PRY/SPYR domain. Exogenous overexpression of porTRIM35 significantly up-regulated the mRNA expression level of IFN-ß in swine testicular (ST) cell in response to poly(I:C) stimulation, whereas knockdown endogenous expression of porTRIM35 lead to a decrease in the expression level of IFN-ß. Mechanically, porTRIM35 directly interacted with porcine TNF-receptor associated factor 3 (TRAF3) and catalyzed its Lys63-linked polyubiquitination, thereby leading to the up-regulation of IFN-ß production. Meanwhile, we demonstrated that the RING and PRY/SPRY domains were essential for the E3 ligase activity of porTRIM35. In response to JEV infection, the endogenous expression of porTRIM35 was markedly inhibited at the mRNA level, while exogenous expression of porTRIM35 significantly elevated the expression of IFN-ß induced by JEV infection and reduced viral titers in ST cells, suggesting that porTRIM35 is a negative regulator for JEV replication. These data demonstrate the importance of porTRIM35 in IFN-ß expression as well as the antiviral response against JEV replication.