Epitope mapping and establishment of a blocking ELISA for mAb targeting the p72 protein of African swine fever virus.

The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20-39 aa, 8A9 and 5E10 are recognized at 263-282 aa, which is consistent with the reported 265-280 aa epitopes. Conserved analysis revealed 20-39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. KEY POINTS: • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.

Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection eliminates production of type I interferons (IFNs) in host cells, which triggers an antiviral immune response through the induction of downstream IFN-stimulated genes (ISGs), thus escaping the fate of host-mediated clearance. The IFN-induced transmembrane 3 (IFITM3) has recently been identified as an ISG and plays a pivotal role against enveloped RNA viruses by restricting cell entry. However, the role of IFITM3 in PRRSV replication is unknown. The present study demonstrated that overexpression of IFITM3 suppresses PRRSV replication, while silencing of endogenous IFITM3 prominently promoted PRRSV replication. Additionally, it was shown that IFITM3 undergoes S-palmitoylation and ubiquitination modification, and both posttranslational modifications contribute to the anti-PRRSV activity of IFITM3. Further study showed that PRRSV particles are transported into endosomes and then into lysosomes during the early stages of infection, and confocal microscopy results revealed that PRRSV particles are transported to IFITM3-positive cellular vesicles. By using a single virus particle fluorescent labeling technique, we confirmed that IFITM3 can restrict PRRSV membrane fusion by inducing accumulation of cholesterol in cellular vesicles. Additionally, we found that both endogenous and exogenous IFITM3 are incorporated into newly producing PRRS virions and diminish viral intrinsic infectivity. By using cell coculture systems, we found that IFITM3 effectively restricted PRRSV intercellular transmission, which may have been caused by disrupted membrane fusion and reduced viral infectivity. In conclusion, our results demonstrate, for the first time, that swine IFITM3 interferes with the life cycle of PRRSV, and possibly other enveloped arteritis viruses, at multiple steps.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an interferon (IFN)-stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions, and this decreased their intrinsic infectivity.

WITHDRAWN: Development of a double-antigen sandwich ELISA for African swine fever virus antibody detection based on K205R protein.

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