African swine fever (ASF) entered China in August 2018 and rapidly spread across the entire country, severely threatening the Chinese domestic pig population, which accounts for more than 50% of the pig population worldwide. In this study, an ASFV isolate, Pig/Heilongjiang/2018 (Pig/HLJ/18), was isolated in primary porcine alveolar macrophages (PAMs) from a pig sample from an ASF outbreak farm. The isolate was characterized by using the haemadsorption (HAD) test, Western blotting and immunofluorescence, and electronic microscopy. Phylogenetic analysis of the viral p72 gene revealed that Pig/HLJ/18 belongs to Genotype II. Infectious titres of virus propagated in primary PAMs and pig marrow macrophages were as high as 10^7.2 HAD₅₀/ml. Specific-pathogen-free pigs intramuscularly inoculated with different virus dosages at 10^3.5–10^6.5 HAD₅₀ showed acute disease with fever and haemorrhagic signs. The incubation periods were 3–5 days for virus-inoculated pigs and 9 days for contact pigs. All virus-inoculated pigs died between 6–9 days post-inoculation (p.i.), and the contact pigs died between 13–14 days post-contact (p.c.). Viremia started on day 2 p.i. in inoculated pigs and on day 9 p.c. in contact pigs. Viral genomic DNA started to be detected from oral and rectal swab samples on 2–5 days p.i. in virus-inoculated pigs, and 6–10 days p.c. in contact pigs. These results indicate that Pig/HLJ/18 is highly virulent and transmissible in domestic pigs. Our study demonstrates the threat of ASFV and emphasizes the need to control and eradicate ASF in China.

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.

African swine fever (ASF) is a severe hemorrhagic disease in swine characterized by massive lymphocyte depletion and cell death, with apoptosis and necrosis in infected lymphoid tissues. However, the molecular mechanism regarding ASFV-induced cell death remains largely unknown. In this study, 94 ASFV-encoded proteins were screened to determine the viral proteins involved in cell death in vitro, and pE199L showed the most significant effect. Ectopic expression of pE199L in porcine cells (CRL-2843) and human cells (HEK293T and HeLa cells) induced cell death remarkably, showing obvious shrinking, blistering, apoptotic bodies, and nuclear DNA fragments. Meanwhile, cell death was markedly alleviated when the expression of pE199L was knocked down during ASFV infection. Additionally, the expression of pE199L caused a loss of mitochondrial membrane potential, release of cytochrome C, and caspase-9 and -3/7 activation, indicating that the mitochondrial apoptotic pathway was involved in pE199L-induced apoptosis. Further investigations showed that pE199L interacted with several anti-apoptotic BCL-2 subfamily members (such as BCL-X_L, MCL-1, BCL-W, and BCL-2A1) and competed with BAK for BCL-X_L, which promoted BAK and BAX activation. Taken together, ASFV pE199L induces the mitochondrial-dependent apoptosis, which may provide clues for a comprehensive understanding of ASFV pathogenesis.

Macrophage polarization is a process by which macrophages acquire a distinct phenotypic and functional profile in response to microenvironmental signals. The classically and alternatively activated (M1 and M2, respectively) macrophage phenotypes are defined by the specific molecular characteristics induced in response to prototypic pro- and anti-inflammatory cues. In this study, we used LPS/IFN-γ and IL-4 to stimulate porcine alveolar macrophages (PAMs) in vitro and investigated the expression changes of several novel markers during macrophage polarization. Notably, we found that LPS/IFN-γ-stimulated PAMs express prototypical M1 molecules, whereas IL-4-stimulated PAMs express M2 molecules. We also demonstrated that replication of the highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) strain HuN4 was effectively suppressed in LPS/IFN-γ-stimulated M1 PAMs (M1 type), but not IL-4 stimulated M2 PAMs. However, this was not observed with the classic, less pathogenic CH-1a strain. Moreover, we found that M2 marker expression gradually increased after PAM infection with PRRSV, whereas no significant changes were found with M1 marker expression, suggesting that PRRSV infection may skew macrophage polarization towards an M2 phenotype. Finally, we found that anti-viral cytokine expression was significantly higher in M1 macrophages than in M2 macrophages or nonpolarized controls. In summary, our results show that PRRSV replication was significantly impaired in M1 PAMs, which may serve as a foundation for further understanding of the dynamic phenotypic changes during macrophage polarization and their effects on viral infection.

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