RESUMEN
African swine fever, caused by a large icosahedral DNA virus (African swine fever virus, ASFV), is a highly contagious disease in domestic and feral swine, thus posing a significant economic threat to the global swine industry. Currently, there are no effective vaccines or the available methods to control ASFV infection. Attenuated live viruses with deleted virulence factors are considered to be the most promising vaccine candidates; however, the mechanism by which these attenuated viruses confer protection is unclear. Here, we used the Chinese ASFV CN/GS/2018 as a backbone and used homologous recombination to generate a virus in which MGF110-9L and MGF360-9L, two genes antagonize host innate antiviral immune response, were deleted (ASFV-ΔMGF110/360-9L). This genetically modified virus was highly attenuated in pigs and provided effective protection of pigs against parental ASFV challenge. Importantly, we found ASFV-ΔMGF110/360-9L infection induced higher expression of Toll-like receptor 2 (TLR2) mRNA compared with parental ASFV as determined by RNA-Seq and RT-PCR analysis. Further immunoblotting results showed that parental ASFV and ASFV-ΔMGF110/360-9L infection inhibited Pam3CSK4-triggered activating phosphorylation of proinflammatory transcription factor NF-κB subunit p65 and phosphorylation of NF-κB inhibitor IκBα levels, although NF-κB activation was higher in ASFV-ΔMGF110/360-9L-infected cells compared with parental ASFV-infected cells. Additionally, we show overexpression of TLR2 inhibited ASFV replication and the expression of ASFV p72 protein, whereas knockdown of TLR2 had the opposite effect. Our findings suggest that the attenuated virulence of ASFV-ΔMGF110/360-9L might be mediated by increased NF-κB and TLR2 signaling.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Proteínas Virales , Animales , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/patogenicidad , Formación de Anticuerpos/inmunología , Eliminación de Gen , FN-kappa B/genética , Porcinos , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Transcriptoma , Proteínas Virales/genética , Proteínas Virales/inmunología , Replicación Viral/inmunologíaRESUMEN
The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Eliminación de Gen , Genes Virales , Vacunas Virales , Factores de Virulencia , Animales , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/patogenicidad , Células Cultivadas , Genes Virales/genética , Histonas/genética , Porcinos , Vacunas Atenuadas/inmunología , Vacunas Virales/inmunología , Factores de Virulencia/genéticaRESUMEN
African swine fever (ASF), a devastating infectious disease in swine, severely threatens the global pig farming industry. Disease control has been hampered by the unavailability of vaccines. Here, we report that deletion of the QP509L and QP383R genes (ASFV-ΔQP509L/QP383R) from the highly virulent ASF virus (ASFV) CN/GS/2018 strain results in complete viral attenuation in swine. Animals inoculated with ASFV-ΔQP509L/QP383R at a 104 50% hemadsorbing dose (HAD50) remained clinically normal during the 17-day observational period. All ASFV-ΔQP509L/QP383R-infected animals had low viremia titers and developed a low-level p30-specific antibody response. However, ASFV-ΔQP509L/QP383R did not induce protection against challenge with the virulent parental ASFV CN/GS/2018 isolate. RNA-sequencing analysis revealed that innate immune-related genes (Ifnb, Traf2, Cxcl10, Isg15, Rantes, and Mx1) were significantly lower in ASFV-ΔQP509L/QP383R-infected than in ASFV-infected porcine alveolar macrophages. In addition, ASFV-ΔQP509L/QP383R-infected pigs had low levels of interferon-ß (IFN-ß) based on enzyme-linked immunosorbent assay (ELISA). These data suggest that deletion of ASFV QP509L/383R reduces virulence but does not induce protection against lethal ASFV challenge. IMPORTANCE African swine fever (ASF) is endemic to several parts of the word, with outbreaks of the disease devastating the swine farming industry; currently, no commercially available vaccine exists. Here, we report that deletion of the previously uncharacterized QP509L and QP383R viral genes completely attenuates virulence in the ASF virus (ASFV) CN/GS/2018 isolate. However, ASFV-ΔQP509L/QP383R-infected animals were not protected from developing an ASF infection after challenge with the virulent parental virus. ASFV-ΔQP509L/QP383R induced lower levels of innate immune-related genes and IFN-ß than the parental virus. Our results increase our knowledge of developing an effective and live ASF attenuated vaccine.
Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/virología , Interacciones Huésped-Patógeno , Eliminación de Secuencia , Proteínas Virales/genética , Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/inmunología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Células Cultivadas , Resistencia a la Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunización , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Mutagénesis , Porcinos , Transcriptoma , Virulencia/genética , Factores de Virulencia/genética , Replicación ViralRESUMEN
African swine fever is a devastating disease of swine caused by African swine fever virus (ASFV). The pathogenesis of the disease remains largely unknown, leaving the spread of the disease uncontrolled in many countries and regions. Here, we identified E120R, a structural protein of ASFV, as a key virulence factor and late-phase-expressed protein of the virus. E120R revealed an activity to suppress the host antiviral response through blocking beta interferon (IFN-ß) production, and the amino acids (aa) at sites 72 and 73 (amino acids 72-73) in the C-terminal domain were essential for this function. E120R interacted with interferon regulatory factor 3 (IRF3) and interfered with the recruitment of IRF3 to TANK-binding kinase 1 (TBK1), which in turn suppressed IRF3 phosphorylation, decreasing interferon production. A recombinant mutant ASFV was further constructed to confirm the claimed mechanism. The ASFV lacking the complete E120R region could not be rescued, whereas the virus could tolerate the deletion of the 72nd and 73rd residues in E120R (ASFV E120R-Δ72-73aa). ASFV E120R with the two-amino-acid deletion failed to interact with IRF3 during ASFV E120R-Δ72-73aa infection, and the viral infection activated IRF3 phosphorylation highly and induced more robust type I interferon production than its parental ASFV. An unbiased transcriptome-wide analysis of gene expression also confirmed that considerably more IFN-stimulated genes (ISGs) were detected in ASFV E120R-Δ72-73aa-infected porcine alveolar macrophages (PAMs) than in wild-type ASFV-infected PAMs. Together, our findings have identified a novel mechanism evolved by ASFV to inhibit the host antiviral response, and they provide a new target for guiding the development of ASFV live-attenuated vaccine. IMPORTANCE African swine fever is a highly contagious animal disease affecting the pig industry worldwide, which has brought enormous economic losses. Infection by the causative agent, African swine fever virus (ASFV), causes severe immunosuppression during viral infection, contributing to serious clinical manifestations. Therefore, identification of the viral proteins involved in immunosuppression is critical for ASFV vaccine design and development. Here, for the first time, we demonstrated that E120R protein, a structural protein of ASFV, played an important role in suppression of interferon regulatory factor 3 (IRF3) phosphorylation and type I interferon production by binding to IRF3 and blocking the recruitment of IRF3 to TANK-binding kinase 1 (TBK1). Deletion of the crucial binding sites in E120R critically increased the interferon response during ASFV infection. This study explored a novel antagonistic mechanism of ASFV, which is critical for guiding the development of ASFV live-attenuated vaccines.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Fiebre Porcina Africana/virología , Interacciones Huésped-Patógeno , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Mutación , Proteínas Virales/metabolismo , Fiebre Porcina Africana/genética , Fiebre Porcina Africana/metabolismo , Animales , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Fosforilación , Transducción de Señal , Porcinos , Proteínas Virales/genética , VirulenciaRESUMEN
African swine fever (ASF) is an acute, severe, and highly contagious disease caused by the African swine fever virus (ASFV), which infects domestic pigs and wild boars. The incidence and mortality rates of swine infected with virulent strains of ASFV can reach up to 100%. The large genome, its complex structure, multiple genotypes, and a lack of understanding regarding ASFV gene function are serious obstacles to the development of safe and effective vaccines. Here, ASFV I329L was identified as a relatively conserved gene that is expressed during the late stage of infection. A recombinant virus with I329L gene deletion (ASFV CN/GS/2018-ΔI329L) was produced by replacing I329L with an enhanced green fluorescent protein (EGFP) cassette. In order to explore the function of the ASFV I329L gene, transcriptome sequencing (RNA-seq) was performed on porcine alveolar macrophages (PAMs) infected with ASFV CN/GS/2018 and ASFV CN/GS/2018-ΔI329L. GO functional and KEGG pathway analyses were performed to analyze differentially expressed genes, and different alternative splicing (AS) events were also analyzed. We compared the sequencing data for each sample with the ASFV CN/GS/2018 reference sequence. Interestingly, we found 3 and 1 up-regulated genes and 12 and 19 down-regulated genes at 12 and 24 h post-infection, respectively. In addition, we verified the expression of 5 up-regulated and 5 down-regulated genes by RT-qPCR, and the results were consistent with those obtained based on RNA-seq. In summary, the results obtained in this study provide new insights for further elucidation of ASFV proteins and ASFV-host interactions. These findings will contribute to implementing a comprehensive strategy for controlling the spread of ASF.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Enfermedades de los Porcinos , Porcinos , Animales , Sus scrofa , Genotipo , Perfilación de la Expresión Génica/veterinariaRESUMEN
African swine fever virus (ASFV) causes acute hemorrhagic infectious disease in pigs. The ASFV genome encodes various proteins that enable the virus to escape innate immunity; however, the underlying mechanisms are poorly understood. The present study found that ASFV MGF-360-10L significantly inhibits interferon (IFN)-ß-triggered STAT1/2 promoter activation and the production of downstream IFN-stimulated genes (ISGs). ASFV MGF-360-10L deletion (ASFV-Δ10L) replication was impaired compared with the parental ASFV CN/GS/2018 strain, and more ISGs were induced by the ASFV-Δ10L in porcine alveolar macrophages in vitro. We found that MGF-360-10L mainly targets JAK1 and mediates its degradation in a dose-dependent manner. Meanwhile, MGF-360-10L also mediates the K48-linked ubiquitination of JAK1 at lysine residues 245 and 269 by recruiting the E3 ubiquitin ligase HERC5 (HECT and RLD domain-containing E3 ubiquitin protein ligase 5). The virulence of ASFV-Δ10L was significantly lower than that of the parental strain in vivo, which indicates that MGF-360-10L is a novel virulence factor of ASFV. Our findings elaborate the novel mechanism of MGF-360-10L on the STAT1/2 signaling pathway, expanding our understanding of the inhibition of host innate immunity by ASFV-encoded proteins and providing novel insights that could contribute to the development of African swine fever vaccines. IMPORTANCE African swine fever outbreaks remain a concern in some areas. There is no effective drug or commercial vaccine to prevent African swine fever virus (ASFV) infection. In the present study, we found that overexpression of MGF-360-10L strongly inhibited the interferon (IFN)-ß-induced STAT1/2 signaling pathway and the production of IFN-stimulated genes (ISGs). Furthermore, we demonstrated that MGF-360-10L mediates the degradation and K48-linked ubiquitination of JAK1 by recruiting the E3 ubiquitin ligase HERC5. The virulence of ASFV with MGF-360-10L deletion was significantly less than parental ASFV CN/GS/2018. Our study identified a new virulence factor and revealed a novel mechanism by which MGF-360-10L inhibits the immune response, thus providing new insights into the vaccination strategies against ASFV.