Riding apoptotic bodies for cell-cell transmission by African swine fever virus.

African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell-cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the "eat-me" signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell-cell spread to evade host responses.

Viral evasion of PKR restriction by reprogramming cellular stress granules.

Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1ß. We show that nsp1ß is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1ß residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.

Modulation of Autophagy-Lysosome Axis by African Swine Fever Virus and Its Encoded Protein pEP153R.

The autophagy-lysosome axis is an evolutionarily conserved intracellular degradation pathway which constitutes an important component of host innate immunity against microbial infections. Here, we show that African swine fever virus (ASFV), one of most devastating pathogens to the worldwide swine industry, can reshape the autophagy-lysosome axis by recruiting the critical lysosome membrane proteins (LAMP1 and LAMP2) to viral factories while inhibiting autophagic induction in macrophages. The screening of viral membrane proteins led to the identification of several ASFV membrane proteins, exemplified by viral protein pEP153R, that could significantly alter the subcellular localization of LAMP1/2 when expressed alone in transfected cells. Further analysis showed that pEP153R was also a component of viral factories and could induce endoplasmic reticulum (ER) retention of LAMP1/2, leading to the inhibition of the fusion of autophagosomes with lysosomes. Interestingly, the ASFV mutant lacking EP153R could still actively recruit LAMP into viral factories (VFs) and inhibit autophagic flux, indicating the existence of a functional redundancy of other viral proteins in the absence of pEP153R and highlighting the complexity of ASFV replication biology. Taken together, our results reveal novel information about the interplay of ASFV with the autophagy-lysosome axis and a previously unrecognized function of ASFV protein pEP153R in regulating the cellular autophagic process.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

Characterization of the Protective Efficacy Against QX Strain of a Recombinant Infectious Bronchitis Virus With H120 Backbone and QX Spike Gene.

Infectious bronchitis virus (IBV) has been prevalent in chicken farms for many years, and its control relies on extensive vaccine administration. The continuous emergence of new variants and the low cross-protection efficiency prompt the development of new vaccines. In this study, we develop a reverse genetics technique based on the classical vaccine strain H120 genome, via in vitro ligation method. Using the H120 genome as the backbone, we constructed the recombinant virus rH120-QX(S) by replacing the H120 S gene with the QX S gene, a prevalent strain in China. Biological characteristics of the rH120-QX(S) virus, such as 50% egg lethal dose (ELD50), 50% egg infectious dose (EID50), dwarf embryo, growth curve, and genetic stability, are measured, which are comparable to the parental virus H120. There are no clinical symptoms and tissue lesions in the trachea and kidney in the rH120-QX(S)-infected specific-pathogen-free (SPF) chickens, demonstrating that this recombinant virus does not confer pathogenicity. Furthermore, protection studies show that there is 100% homologous protection of rH120-QX(S) to the virulent QX strain, as shown by the absence of clinical signs and no lethality. Taken together, our results demonstrate that swapping the S gene onto the H120 genetic backbone is a precise and effective way to produce genetically defined IBV vaccine candidates.