Primary Porcine Alveolar Macrophages Research Articles

Porcine reproductive and respiratory syndrome virus infection activates ADAM17 to induce inflammatory responses

Porcine reproductive and respiratory syndrome (PRRS), which has posed substantial threats to the swine industry worldwide, is primarily characterized by interstitial pneumonia. A disintegrin and metalloproteinase 17 (ADAM17) is a multifunctional sheddase involved in various inflammatory diseases. Herein, our study showed that PRRS virus (PRRSV) infection elevated ADAM17 activity, as demonstrated in primary porcine alveolar macrophages (PAMs), an immortalized PAM cell line (IPAM cells), and the lung tissues of PRRSV-infected piglets. We found that PRRSV infection promoted ADAM17 translocation from the endoplasmic reticulum to the Golgi by enhancing its interaction with inactive rhomboid protein 2 (iRhom2), a newly identified ADAM17 regulator, which in turn elevated ADAM17 activity. By screening for PRRSV-encoded structural proteins, viral envelope (E) and nucleocapsid (N) proteins were identified as the predominant ADAM17 activators. E and N proteins bind with both ADAM17 and iRhom2 to form ternary protein complexes, ultimately strengthening their interactions. Additionally, we demonstrated, using an ADAM17-knockout cell line, that ADAM17 augmented the shedding of soluble TNF-α, a pivotal inflammatory mediator. We also discovered that ADAM17-mediated cleavage of porcine TNF-α occurred between Arg-78 and Ser-79. By constructing a precision mutant cell line with Arg-78-Glu/Ser-79-Glu substitution mutations in TNF-α, we further revealed that the ADAM17-mediated production of soluble TNF-α contributed to the induction of inflammatory responses by PRRSV and its E and N proteins. Taken together, our results elucidate the mechanism by which PRRSV infection activates the iRhom2/ADAM17/TNF-α axis to enhance inflammatory responses, providing valuable insights into the elucidation of PRRSV pathogenesis.

Characterization of in vitro viral neutralization targets of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in alveolar macrophage and evaluation of protection potential against HP-PRRSV challenged based on combination of HP-PRRSV-structure proteins in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global pork industry, resulting in substantial economic losses. Current control measures rely on modified live virus (MLV) vaccines with safety concerns. However, the lack of consensus on protective PRRSV antigens is impeding the development of effective and safety subunit vaccines. In this study, we conducted in vitro virus neutralization (VN) assays in MARC-145 and CRL-2843CD163/CD169 cell lines and primary porcine alveolar macrophages (PAMs) to systemically identify PRRSV structural proteins (SPs) recognized by virus-neutralizing antibodies in hyperimmune serum collected from piglets infected with highly pathogenic PRRSV (HP-PRRSV). Additionally, piglets immunized with different combinations of recombinant PRRSV-SPs were challenged with HP-PRRSV to evaluate their in vivo protection potential. Intriguingly, different in vitro VN activities of serum antibodies elicited by each PRRSV SP were observed depending on the cell type used in the VN assay. Notably, antibodies specific for GP3, GP4, and M exhibited highest in vitro VN activities in PAMs, correlating with complete protection (100% survival) against HP-PRRSV challenge in vivo after immunization of piglets with combination of GP3, GP4, M and N (GP3/GP4/M/N). Further analysis of lung pathology, weight gain, and viremia post-challenge revealed that the combination of GP3/GP4/M/N provided superior protective efficacy against severe infection. These findings underscore the potential of this SP combination to serve as an effective PRRSV subunit vaccine, marking a significant advancement in pork industry disease management.

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.

CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

Vidofludimus inhibits porcine reproductive and respiratory syndrome virus infection by targeting dihydroorotate dehydrogenase

Porcine reproductive and respiratory syndrome virus (PRRSV) infection has caused huge economic losses in global swine industry over the last 37 years. PRRSV commercial vaccines are not effective against all epidemic PRRSV strains. In this study we performed a high-throughput screening (HTS) of an FDA-approved drug library, which contained 2339 compounds, and found vidofludimus (Vi) could significantly inhibits PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages (PAMs). Compounds target prediction, molecular docking analysis, and target protein interference assay showed that Vi interacts with dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in the de novo pyrimidine synthesis pathway. Furthermore, PRRSV infection was restored in the presence of excess uridine and cytidine which promote pyrimidine salvage, or excess orotate which is the product of DHODH in the de novo pyrimidine biosynthesis pathway, thus confirming that the antiviral effect of Vi against PRRSV relies on the inhibition of DHODH. In addition, Vi also has antiviral activity against Seneca virus A (SVA), encephalomyocarditis virus (EMCV), porcine epidemic diarrhea virus (PEDV), and pseudorabies virus (PRV) in vitro. These findings should be helpful for developing a novel prophylactic and therapeutic strategy against PRRSV and other swine viral infections.

Probiotic Bacillus spp. enhance TLR3-mediated TNF signalling in macrophages.

Probiotics have been reported to have immunomodulatory properties in the context of infectious disease and inflammation, although the underlying mechanisms are not fully understood. Here, we aimed to determine how different probiotic bacterial strains modulated macrophage function during TLR3 stimulation mimicking viral infection. We screened 14 different strains for their ability to modulate TNF-α, IL-6 IL-10, IFN-α, IFN-β and IFN-γ secretion in RAW 264.7 macrophages with or without poly(I:C) stimulation. Seven strains were selected for further analysis using primary porcine alveolar macrophages. In-depth transcriptomic analysis on alveolar macrophages was conducted for two strains. Most strains induced a synergistic effect when co-incubated with poly(I:C) resulting in increased levels of IL-6 and TNF-α secretion from RAW 264.7 cells. This synergistic effect was found to be TLR2 independent. Only strains of Bacillus spp. could induce this effect in alveolar macrophages. Transcriptomic analysis indicated that the increased TNF-α secretion in alveolar macrophages after co-incubation with poly(I:C) correlated with significant upregulation of TNF and IL23A-related pathways. Collectively, our data show that probiotic bacteria possess strain-dependent immunomodulatory properties that may be harnessed to enhance innate immune responses to pathogens.

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.

The D129L protein of African swine fever virus interferes with the binding of transcriptional coactivator p300 and IRF3 to prevent beta interferon induction.

African swine fever (ASF) is an acute, hemorrhagic, and severe porcine infectious disease caused by African swine fever virus (ASFV). ASF outbreaks severely threaten the global pig industries and result in serious economic losses. No safe and efficacious commercial vaccine is currently available except in Vietnam. To date, large gaps in the knowledge concerning viral biological characteristics and immunoevasion strategies have hindered the ASF vaccine design. In this study, we demonstrate that pD129L negatively regulates the type I interferon (IFN) signaling pathway by interfering with the interaction of the transcriptional coactivator p300 and IRF3, thereby inhibiting the induction of type I IFNs. This study reveals a novel immunoevasion strategy employed by ASFV, shedding new light on the intricate mechanisms for ASFV to evade the host immune responses.

The African swine fever virus I10L protein inhibits the NF-κB signaling pathway by targeting IKKβ.

Proinflammatory factors play important roles in the pathogenesis of African swine fever virus (ASFV), which is the causative agent of African swine fever (ASF), a highly contagious and severe hemorrhagic disease. Efforts in the prevention and treatment of ASF have been severely hindered by knowledge gaps in viral proteins responsible for modulating host antiviral responses. In this study, we identified the I10L protein (pI10L) of ASFV as a potential inhibitor of the TNF-α- and IL-1β-triggered NF-κB signaling pathway, the most canonical and important part of host inflammatory responses. The ectopically expressed pI10L remarkably suppressed the activation of NF-κB signaling in HEK293T and PK-15 cells. The ASFV mutant lacking the I10L gene (ASFVΔI10L) induced higher levels of proinflammatory cytokines production in primary porcine alveolar macrophages (PAMs) compared with its parental ASFV HLJ/2018 strain (ASFVWT). Mechanistic studies suggest that pI10L inhibits IKKβ phosphorylation by reducing the K63-linked ubiquitination of NEMO, which is necessary for the activation of IKKβ. Morever, pI10L interacts with the kinase domain of IKKβ through its N-terminus, and consequently blocks the association of IKKβ with its substrates IκBα and p65, leading to reduced phosphorylation. In addition, the nuclear translocation efficiency of p65 was also altered by pI10L. Further biochemical evidence supported that the amino acids 1-102 on pI10L were essential for the pI10L-mediated suppression of the NF-κB signaling pathway. The present study clarifies the immunosuppressive activity of pI10L, and provides novel insights into the understanding of ASFV pathobiology and the development of vaccines against ASF. IMPORTANCE African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. To date, few safe and effective vaccines or antiviral strategies have been marketed due to large gaps in knowledge regarding ASFV pathobiology and immune evasion mechanisms. In this study, we deciphered the important role of the ASFV-encoded I10L protein in the TNF-α-/IL-1β-triggered NF-κB signaling pathway. This study provides novel insights into the pathogenesis of ASFV and thus contributes to the development of vaccines against ASF.

The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKα and IKKβ through the recruitment of TOLLIP.

The multigene family genes (MGFs) in the left variable region (LVR) of the African swine fever virus (ASFV) genome have been reported to be involved in viral replication in primary porcine alveolar macrophages (PAMs) and virulence in pigs. However, the exact functions of key MGFs in the LVR that regulate the replication and virulence of ASFV remain unclear. In this study, we identified the MGF300-2R gene to be critical for viral replication in PAMs by deleting different sets of MGFs in the LVR from the highly virulent strain ASFV HLJ/18 (ASFV-WT). The ASFV mutant lacking the MGF300-2R gene (Del2R) showed a 1-log reduction in viral titer, and induced higher IL-1β and TNF-α production in PAMs than did ASFV-WT. Mechanistically, the MGF300-2R protein was found to interact with and degrade IKKα and IKKβ via the selective autophagy pathway. Furthermore, we showed that MGF300-2R promoted the K27-linked polyubiquitination of IKKα and IKKβ, which subsequently served as a recognition signal for the cargo receptor TOLLIP-mediated selective autophagic degradation. Importantly, Del2R exhibited a significant reduction in both replication and virulence compared with ASFV-WT in pigs, likely due to the increased IL-1β and TNF-α, indicating that MGF300-2R is a virulence determinant. These findings reveal that MGF300-2R suppresses host innate immune responses by mediating the degradation of IKKα and IKKβ, which provides clues to paving the way for the rational design of live attenuated vaccines to control ASF.

Theaflavin inhibits African swine fever virus replication by disrupting lipid metabolism through activation of the AMPK signaling pathway in virto

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), which is one of the most harmful swine diseases in the pig industry because of its nearly 100% mortality rate in domestic pigs and results in incalculable economic loss. Ever since ASF was initially reported, scientists have worked to develop anti-ASF vaccines; however, currently no clinically effective vaccine for ASF is available. Therefore, the development of novel measures to prevent ASFV infection and transmission is essential. In this study, we aimed to investigate the anti-ASF activity of theaflavin (TF), a natural compound mainly isolated from black tea. We found that TF potently inhibited ASFV replication at non-cytotoxic concentrations ex vivo in primary porcine alveolar macrophages (PAMs). Mechanistically, we found that TF inhibited ASFV replication by acting on cells rather than interacting directly with ASFV to inhibit viral replication. Further, we found that TF upregulated the AMPK (5′-AMP-activated protein kinase) signaling pathway in ASFV-infected and uninfected cells, and treatment with the AMPK agonist MK8722 upregulated the AMPK signaling pathway and inhibited ASFV proliferation in a dose-dependent manner. Notably, the effects of TF on AMPK activation and ASFV inhibition were partially reversed by the AMPK inhibitor dorsomorphin. In addition, we found that TF down-regulated the expression of genes related to lipid synthesis and decreased the intracellular accumulation of total cholesterol and total triglycerides in ASFV-infected cells, suggesting that TF may inhibit ASFV replication by disrupting lipid metabolism. In summary, our results demonstrated that TF is an ASFV infection inhibitor and revealed the mechanism by which ASFV replication is inhibited, providing a novel mechanism and potential lead compound for the development of anti-ASFV drugs.

CD1d facilitates African swine fever virus entry into the host cells via clathrin-mediated endocytosis

ABSTRACT African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with high morbidity and mortality in domestic pigs and wild boars. The disease has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. However, the molecular mechanism underlying ASF virus (ASFV) entry of the host cells is not fully understood, which restricts the development of vaccines and antiviral-drugs of ASFV. In this study, we found that the host protein CD1d acts as a host factor, which mediates ASFV entry into the host cells. As the main capsid protein on the surface of ASFV virions, p72 can mediate viral entry. Using IP-MS assay, CD1d was identified as a binding partner of p72 on surface of ASFV virions. Knockdown of CD1d expression and blocking the cells with anti-pCD1d antibody, or incubating ASFV virions with soluble CD1d protein could significantly inhibit ASFV infection. CD1d is located on the membrane surface of primary porcine alveolar macrophages (PAMs) and mediates the virus entry via binding to p72. CD1d knockout or CD1d knockdown assay showed that CD1d could facilitate ASFV virions internalization via clathrin-mediated endocytosis (CME). Furthermore, CD1d interacts with EPS15 to mediate ASFV entry via clathrin-mediated endocytosis. Overall, our findings revealed that CD1d is a novel host-entry factor involved in ASFV internalization via the EPS15-clathrin endocytosis axis and a potential target for antiviral intervention.

Brincidofovir is a robust replication inhibitor against African swine fever virus in vivo and in vitro

ABSTRACT African swine fever virus (ASFV) infection is a major public and socioeconomic concern that has a serious impact on the global swine industry. Unfortunately, there are currently no commercially available vaccines or antiviral agents that are both safe and effective against ASFV. In the study, we use primary porcine alveolar macrophages to screen a kinase inhibitor library for anti-ASFV compounds. Six candidate compounds that inhibited ASFV infection with inhibition of > 90% were identified, among which brincidofovir exhibited optimal inhibitory effects on ASFV. Brincidofovir reduces ASFV replication in a dose-dependent manner (IC50 = 2.76 nM) without cytotoxicity (CC50 = 58 μM). It possesses the ability to reduce viral titres and inhibit viral structural protein expression. Time-of-addition assays suggest that the compound interferes with the post-invasion stage of the viral infection cycle. In pig challenge experiments, brincidofovir was indicated to protect pigs against ASFV-induced lethality by decreasing the viral load in organs and peripheral blood, while it alleviated the histopathological changes associated with ASFV infection. Furthermore, brincidofovir also decreased viral shedding in pigs with ASFV infection. Our data together demonstrate that brincidofovir may serve as a potentially effective agent for the prevention and control of ASFV infection, whereas further investigations are still required.

First Identification and Genomic Characterization of NADC34-Like PRRSV Strains Isolated from MLV-Vaccinated Pigs in Korea

NADC34-like porcine reproductive and respiratory syndrome viruses (PRRSVs) are emerging globally. Although lineage 1 PRRSVs are emerging in Korea, NADC34-like PRRSV strains have not been detected to date. This study reports the isolation of the first NADC34-like PRRSV strains from samples of MLV-vaccinated animals on a Korean farm with a severe PRRS outbreak causing high mortality and abortion rates in July 2022. Three PRRSV strains with a whole genome length of 15,088 bp were isolated from the outbreaks, and their phylogenetic, recombination properties, and in vitro growth kinetics were evaluated comprehensively. These Korean strains clustered with those of American and Chinese sublineage 1.5 (NADC34-like strains), according to phylogenetic analyses based on the ORF5 gene and the whole genome. All three complete genome sequences originated from recombination between the RespPRRS MLV and NADC34-like strains, according to recombination analysis. Full-length Nsp2 sequence alignment indicated that all three isolates had a continuous deletion in 100 aa, which was similar to that of the IA/2014/NADC34. Based on phylogenetic analysis and the Nsp2 deletion pattern, the isolates are identified as NADC34-like PRRSV. Growth kinetic analysis based on primary porcine alveolar macrophages (PAMs) and MARC-145 cells has revealed that Korean NADC34-like PRRSV strains had better replication ability in primary PAMs than other Korean lineage 1 viruses or the reference strain VR2332, implying higher pathogenicity in pigs. Considering the fact that NADC34-like PRRSV strains were detected in MLV-vaccinated animals on a farm in the area of the highest pig population density in Korea, additional vaccine failure cases and the spread of the virus could be detected in the near future. Collectively, these results aid in comprehending the current epidemic state of PRRSVs in Korea and highlight the need for continuous surveillance.

African Swine Fever Virus L83L Negatively Regulates the cGAS-STING-Mediated IFN-I Pathway by Recruiting Tollip To Promote STING Autophagic Degradation.

African swine fever (ASF) is a devastating infectious disease of pigs caused by the African swine fever virus (ASFV), which poses a great danger to the global pig industry. Many viral proteins can suppress with interferon signaling to evade the host's innate immune responses. Therefore, the development of an effective vaccine against ASFV has been dampened. Recent studies have suggested that the L83L gene may be integrated into the host genome, weakening the host immune system, but the underlying mechanism is unknown. Our study found that L83L negatively regulates the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. Overexpression of L83L inhibited IFN-β promoter and ISRE activity, and knockdown of L83L induced higher transcriptional levels of interferon-stimulated genes (ISGs) and phosphorylation levels of IRF3 in primary porcine alveolar macrophages. Mechanistically, L83L interacted with cGAS and STING to promote autophagy-lysosomal degradation of STING by recruiting Tollip, thereby blocking the phosphorylation of the downstream signaling molecules TBK1, IRF3, and IκBα and reducing IFN-I production. Altogether, our study reveals a negative regulatory mechanism involving the L83L-cGAS-STING-IFN-I axis and provides insights into an evasion strategy involving autophagy and innate signaling pathways employed by ASFV. IMPORTANCE African swine fever virus (ASFV) is a large double-stranded DNA virus that primarily infects porcine macrophages. The ASFV genome encodes a large number of immunosuppressive proteins. Current options for the prevention and control of this pathogen remain pretty limited. Our study showed that overexpression of L83L inhibited the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. In contrast, the knockdown of L83L during ASFV infection enhanced IFN-I production in porcine alveolar macrophages. Additional analysis revealed that L83L protein downregulated IFN-I signaling by recruiting Tollip to promote STING autophagic degradation. Although L83L deletion has been reported to have little effect on viral replication, its immune evade mechanism has not been elucidated. The present study extends our understanding of the functions of ASFV-encoded pL83L and its immune evasion strategy, which may provide a new basis for developing a live attenuated vaccine for ASF.

Interaction between Porcine Alveolar Macrophage-Tang Cells and Streptococcus suis Strains of Different Virulence: Phagocytosis and Apoptosis

Streptococcus suis is an important swine bacterial pathogen that activates macrophages to secrete inflammatory cytokines. Primary porcine alveolar macrophages (PAMs) are inconvenient to obtain, but it is unknown whether immortalized PAM-Tang cells can replace them as a better cell model for the study of the interaction between S. suis and macrophages. In this study, the phagocytic integrity, polarization, and pro-inflammatory cytokine secretion of PAM-Tang cells were confirmed by live-cell imaging, electron microscopy, confocal microscopy, and ELISA. Interestingly, the S. suis serotype 9 avirulent strain W7119 induced higher levels of adhesion and pro-inflammatory cytokines in PAM-Tang cells than the S. suis serotype 2 virulent strain 700794. Prolonged incubation with S. suis caused more cytotoxic cell damage, and the virulent strain induced higher levels of cytotoxicity to PAM-Tang cells. The virulent strain also induced higher levels of apoptosis in PAM-Tang cells, as shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. In addition, it is the first report of virulent and avirulent S. suis inducing PAM-Tang polarization towards pro-inflammatory M1 macrophages and p53- and caspase-dependent apoptosis in PAMs. Taken together, this study contributes to a better understand of interactions between macrophages and S. suis isolates of different virulence, and confirms that PAM-Tang cells provide a long-term, renewable resource for investigating macrophage infections with bacteria.

Inactivation rates of African swine fever virus by compound disinfectants

African swine fever is an important viral disease of domestic pigs and wild boars. Inactivation of the African swine fever virus (ASFV) contaminating equipment and premises is an effective biosecurity risk management measure to prevent the introduction of ASFV through contaminated fomites and the environment. Even though some chemical agents' virucidal activity against ASFV was previously demonstrated, compound disinfectants are still limited. For the fact that the ASFV titer in some excretions from pigs was far >4 log. Even after the application of disinfectants as instructed by the manufacturers, the excessive ASFV load in the excretions from pigs could contaminate fomites or the environment. To solve this dilemma, the concept of contact time of disinfectant for 1–log reduction was introduced, as the decimal reduction time or D value. Therefore, the objective of this study was to assess the inactivation rate of four compound disinfectants against ASFV as the D value. Four compound disinfectants (iodine and phenol, quaternary ammonium and alcohol, and two quaternary ammonium and glutaraldehyde compounds) currently used in the veterinary field were evaluated in a suspension test at 25 °C in the absence of interfering substances. Primary porcine alveolar macrophages were used to determine the titer of ASFV genotype II (strain VNUA/HY-ASF1/Vietnam/2019). The virucidal activity of four compound disinfectants achieved 4–log ASFV reduction. The mean D0.5%, D0.25%, and D0.125% values of all disinfectants inactivating ASFV were 1.62–3.60, 2.60–3.8, and 2.89–3.90 min, respectively. The D value of disinfectants allows users to manipulate the appropriate concentration and/or contact time of the disinfectant, depending on the magnitude of ASFV contamination, to completely inactivate the ASFV titer on the fomites to minimise a potential biosecurity risk.

The 24.5-kb Left Variable Region Is Not a Determinant for African Swine Fever Virus to Replicate in Primary Porcine Alveolar Macrophages.

African swine fever (ASF) is a widespread hemorrhagic and highly contagious infectious disease caused by African swine fever virus (ASFV), currently threatening the pig industry worldwide. Here, we demonstrated that the cell-adapted strain ASFV-P121 with a 24.5-kb deletion in the left variable region (LVR) lost the ability to replicate in primary porcine alveolar macrophages (PAMs). To explore whether this deletion determines the inability of ASFV-P121 replication in PAMs, a mutant virus (ASFV-ΔLVR) with the same LVR deletion as ASFV-P121 was constructed based on the wild-type ASFV HLJ/18 (ASFV-WT). However, the growth titer of ASFV-ΔLVR only reduced 10-fold compared with ASFV-WT in PAMs. Furthermore, we found that the large deletion of the LVR does not affect the formation of virus factories and virion morphogenesis. These findings reveal important implications for analyzing the molecular mechanism of ASFV cell tropism change.

Interaction network of African swine fever virus structural protein p30 with host proteins.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a lethal hemorrhagic disease that is currently threatening the global pig industry. ASFV structural protein p30 is a membrane phosphoprotein that suggests it may play a regulatory role, possibly in signal transduction. Despite its significance in internalization into host cells, the interaction between p30 and host proteins is relatively unknown. In this study, we describe the application of a DUALmembrane yeast two-hybrid assay to screen a primary porcine alveolar macrophages cDNA library and analyze the interactome of p30 protein. Our data identify seven host cellular proteins (DAB2, RPSA, OAS1, PARP9, CAPG, ARPC5, and VBP1) that putatively interact with the p30. We further verified the interaction between p30 and host proteins by laser confocal microscopy, co-immunoprecipitation, and GST-pulldown assay. To further understand the relationship between host proteins and p30, we drew the interaction network diagram and analyzed the functional enrichment of each host protein. Enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes indicated that host proteins were mainly related to endocytosis, actin cytoskeleton regulation, and innate immunity. Collectively, we identified the interaction between p30 and host cell protein using a membrane protein yeast two-hybrid system, which increases our knowledge of the interaction between ASFV and the host and informs future research on antiviral strategies.

Toosendanin activates caspase-1 and induces maturation of IL-1β to inhibit type 2 porcine reproductive and respiratory syndrome virus replication via an IFI16-dependent pathway

Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent and endemic swine pathogen which causes significant economic losses in the global swine industry. Multiple vaccines have been developed to prevent PRRSV infection. However, they provide limited protection. Moreover, no effective therapeutic drugs are yet available. Therefore, there is an urgent need to develop novel antiviral strategies to prevent PRRSV infection and transmission. Here we report that Toosendanin (TSN), a tetracyclic triterpene found in the bark or fruits of Melia toosendan Sieb. et Zucc., strongly suppressed type 2 PRRSV replication in vitro in Marc-145 cells and ex vivo in primary porcine alveolar macrophages (PAMs) at sub-micromolar concentrations. The results of transcriptomics revealed that TSN up-regulated the expression of IFI16 in Marc-145 cells. Furthermore, we found that IFI16 silencing enhanced the replication of PRRSV in Marc-145 cells and that the anti-PRRSV activity of TSN was dampened by IFI16 silencing, suggesting that the inhibition of TSN against PRRSV replication is IFI16-dependent. In addition, we showed that TSN activated caspase-1 and induced maturation of IL-1β in an IFI16-dependent pathway. To verify the role of IL-1β in PRRSV infection, we analyzed the effect of exogenous rmIL-1β on PRRSV replication, and the results showed that exogenous IL-1β significantly inhibited PRRSV replication in Marc-145 cells and PAMs in a dose-dependent manner. Altogether, our findings indicate that TSN significantly inhibits PRRSV replication at very low concentrations (EC50: 0.16–0.20 μM) and may provide opportunities for developing novel anti-PRRSV agents.