African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boars, and has tremendous negative socioeconomic impact on the swine industry and food security worldwide. It is characterized as a notifiable disease by World Organisation for Animal Health (OIE). No effective vaccine or treatment against ASF has so far been available. Early detection and rapid diagnosis are of potential significance to control the spread of ASF. Recombinase-based isothermal amplification assay, recombinase polymerase amplification (RPA) developed by TwistDx (Cambridge, United Kingdom) or recombinase-aided amplification (RAA) by Qitian (Wuxi, China), is becoming a molecular tool for the rapid, specific, and cost-effective identification of multiple pathogens. In this study, we aim to investigate if RPA/RAA can be a potential candidate for on-site, rapid and primary detection of ASFV. A panel of 152 clinical samples previously well-characterized by OIE-recommended qPCR was enrolled in this study, including 20 weak positive (Ct value ≥ 30) samples. This panel was consisted of different types, such as EDTA-blood, spleen, lung, lymph node, kidney, tonsil, liver, brain. We evaluated two recombinase-based isothermal amplification assays, RPA or RAA, by targeting the ASFV B646L gene (p72), and validated the clinical performance in comparison with OIE real-time PCR. Our result showed that the analytical sensitivity of RPA and RAA was as 93.4 and 53.6 copies per reaction, respectively at 95% probability in 16 min, at 39°C. They were universally specific for all 24 genotypes of ASFV and no cross reaction to other pathogens including Classical swine fever virus (CSV), Foot-and-mouth disease virus (FMDV), Pseudorabies virus, Porcine circovirus 2 (PCV2), Porcine Reproductive and respiratory syndrome virus (PPRSV). The results on detection of various kinds of clinical samples indicated an excellent diagnostic agreement between RPA, RAA and OIE real-time PCR method, with the kappa value of 0.960 and 0.973, respectively. Compared to real-time PCR, the specificity of both RPA and RAA was 100% (94.40% ∼ 100%, 95% CI), while the sensitivity was 96.59% (90.36% ∼ 99.29%, 95% CI) and 97.73% (92.03% ∼ 99.72%, 95% CI), respectively. Our data demonstrate that the developed recombinase-based amplification assay (RPA/RAA), promisingly equipped with field-deployable instruments, offers a sensitive and specific platform for the rapid and reliable detection of ASFV, especially in the resource-limited settings for the purpose of screening and surveillance of ASF.

Japanese encephalitis virus (JEV) genotype dominance has shifted to genotype I (GI) from genotype III (GIII) in China as demonstrated by molecular epidemiological surveillance. In this study, we performed a serological survey in JEV-non-vaccinated pigs to confirm JEV genotype shift at the sero-epidemiological level. The average ratio of GI/GIII infection was 1.87, suggesting co-circulation of GI and GIII infections with GI infection being more prevalent in pigs in China. To gain an insight into the reasons for this JEV genotype shift, the replication kinetics of seven recently-isolated JEV isolates including three GI strains and four GIII strains were compared in mosquito C6/36 cells, chicken fibroblast cells (DF-1) and porcine iliac artery endothelial cells (PIEC). We observed that GI strains replicated more efficiently than GIII strains in DF-1 and PIEC cells, particularly in DF-1 cells with titers reaching 22.9–225.3 fold higher than GIII strains. This shows an enhanced replication efficiency of GI viruses in avian cells. To examine this enhanced replication efficiency in vivo, young domestic ducklings were used as the animal model and inoculated with GI and GIII strains at day 2 post-hatching. We observed that GI-inoculated ducklings developed higher viremia titers and displayed a comparatively longer viremic duration than GIII-inoculated ducklings. These results conform to the hypothesis of an enhanced replication efficiency for GI viruses in birds. There are 36 amino acid differences between GI and GIII viruses, some of which may be responsible for the enhanced replication efficiency of GI viruses in birds. Based on these findings, we speculated that the enhanced replication of GI viruses in birds would have resulted in higher exposure and therefore infection in mosquitoes, which could result in an increased transmission efficiency of GI viruses in the birds-mosquitoes-birds enzootic transmission cycle, thereby contributing to JEV genotype shift. Japanese encephalitis virus (JEV) causes encephalitis in humans and reproductive disorder in pigs. The enzootic transmission cycle of JEV is maintained in nature by several species of mosquitoes and vertebrates including birds and pigs. In recent years, JEV genotype I (GI) replaced genotype IIII (GIII) as the dominant genotype in Asian countries. Genotype shift has an impact on disease control, and understanding the reasons for this shift will offer valuable insight into avenues for future disease control. Therefore, we compared the replication efficiencies of GI and GIII viruses in vitro and in vivo. We observed that GI viruses show higher replication titers in avian cells and higher viremia levels in young domestic ducklings than GIII viruses, suggesting an enhanced replication efficiency of GI viruses in birds. Based on these findings, we speculated that the enhanced replication of GI viruses in birds could provide increased mosquito infection, leading to an increase in the birds-mosquitoes-birds transmission cycle, thereby contributing to JEV genotype shift.

The NF-κB pathway is an essential signalling cascade in the defence against viral infections, including African swine fever virus (ASFV) infection. ASFV encodes more than 151 proteins via its own transcription machinery and possesses a great capacity to evade or subvert antiviral innate immune responses. Although some of these viral proteins have been reported, many remain unknown. Here, we show that pD345L, an ASFV-encoded lambda-like exonuclease, acts as an inhibitor of cGAS/STING-mediated NF-κB signalling by blocking the IkappaB kinase (IKKα/β) activity. Specifically, we showed that overexpression of pD345L suppresses cGAS/STING-induced IFNβ and NF-κB activation, resulting in decreased transcription of IFNβ and several proinflammatory cytokines, including IL-1α, IL-6, IL-8, and TNFα. In addition, we showed that pD345L acts at or downstream of IKK and upstream of p65. Importantly, we found that pD345L associates with the KD and HLH domains of IKKα and the LZ domain of IKKβ and thus interrupts their kinase activity towards the downstream substrate IκBα. Finally, we showed that pD345L-mediated inhibition of NF-κB signalling was independent of its exonuclease activity. Considering these results collectively, we concluded that pD345L blocks IKKα/β kinase activity via protein–protein interactions and thus disrupts cGAS/STING-mediated NF-κB signalling.