Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

African Swine Fever Virus E184L Protein Interacts with Innate Immune Adaptor STING to Block IFN Production for Viral Replication and Pathogenesis.

African swine fever is one of the most serious viral diseases that affects domestic and wild pigs. The causative agent, African swine fever virus (ASFV), has evolved sophisticated immune evasion mechanisms that target both innate and adaptive immune responses. However, the underlying molecular mechanisms have not been fully understood. Here, we report that ASFV E184L protein inhibits host innate immune response via targeting the stimulator of IFN genes (STING)-mediated signaling pathway in both human embryonic kidney HEK-293T cells and porcine pulmonary alveolar macrophages. E184L interacts with STING, impairing dimerization and oligomerization of STING but not affecting its puncta formation at the perinuclear region. Furthermore, E184L disrupts STING-TBK1-IRF3 complex formation, leading to inhibition of STING phosphorylation, and IRF3 dimerization and nuclear translocation. The 1-20 aa region in E184L is essential for E184L-STING interaction and blocking IL-1ß and type I IFN production. Deletion of E184L in ASFV considerably impairs antagonistic function of the virus in suppression of the STING-mediated antiviral response, an effect that is reversible by introduction of E184L. Importantly, the virulence of mutant ASFV lacking E184L is reduced in pigs compared with its parental virus due to induction of higher IFN production in vivo. Our findings indicate that ASFV E184L is an important antagonist of IFN signaling to evade host innate immune antiviral responses, which improves our understanding of immune evasion mechanisms of ASFV.

Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge.

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.

Metabolomic analysis of pig spleen reveals African swine fever virus infection increased acylcarnitine levels to facilitate viral replication.

African swine fever (ASF) is a devastating disease caused by the African swine fever virus (ASFV) that adversely affects the pig industry. The spleen is the main target organ of ASFV; however, the function of metabolites in the spleen during ASFV infection is yet to be investigated. To define the metabolic changes in the spleen after ASFV infection, untargeted and targeted metabolomics analyses of spleens from ASFV-infected pigs were conducted. Untargeted metabolomics analysis revealed 540 metabolites with significant differential levels. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that these metabolites were mainly enriched in metabolic pathways, including nucleotide metabolism, purine metabolism, arginine biosynthesis, and neuroactive ligand-receptor interaction. Moreover, 134 of 540 metabolites quantified by targeted metabolomics analysis had differential levels and were enriched in metabolic pathways such as the biosynthesis of cofactors, ABC transporters, and biosynthesis of amino acids. Furthermore, coalition analysis of untargeted and targeted metabolomics data revealed that the levels of acylcarnitines, which are intermediates of fatty acid ß-oxidation, were significantly increased in ASFV-infected spleens compared with those in the uninfected spleens. Moreover, inhibiting fatty acid ß-oxidation significantly reduced ASFV replication, indicating that fatty acid ß-oxidation is essential for this process. To our knowledge, this is the first report presenting the metabolite profiles of ASFV-infected pigs. This study revealed a new mechanism of ASFV-mediated regulation of host metabolism. These findings provide new insights into the pathogenic mechanisms of ASFV, which will benefit the development of target drugs for ASFV replication. IMPORTANCE African swine fever virus, the only member of the Asfarviridae family, relies on hijacking host metabolism to meet the demand for self-replication. However, the change in host metabolism after African swine fever virus (ASFV) infection remains unknown. Here, we analyzed the metabolic changes in the pig spleen after ASFV infection for the first time. ASFV infection increased the levels of acylcarnitines. Inhibition of the production and metabolism of acylcarnitines inhibited ASFV replication. Acylcarnitines are the vital intermediates of fatty acid ß-oxidation. This study highlights the critical role of fatty acid ß-oxidation in ASFV infection, which may help identify target drugs to control African swine fever disease.

Deletion of DP148R, DP71L, and DP96R Attenuates African Swine Fever Virus, and the Mutant Strain Confers Complete Protection against Homologous Challenges in Pigs.

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.

African swine fever virus I267L acts as an important virulence factor by inhibiting RNA polymerase III-RIG-I-mediated innate immunity.

ASFV is a large DNA virus that is highly pathogenic in domestic pigs. How this virus is sensed by the innate immune system as well as why it is so virulent remains enigmatic. In this study, we show that the ASFV genome contains AT-rich regions that are recognized by the DNA-directed RNA polymerase III (Pol-III), leading to viral RNA sensor RIG-I-mediated innate immune responses. We further show that ASFV protein I267L inhibits RNA Pol-III-RIG-I-mediated innate antiviral responses. I267L interacts with the E3 ubiquitin ligase Riplet, disrupts Riplet-RIG-I interaction and impairs Riplet-mediated K63-polyubiquitination and activation of RIG-I. I267L-deficient ASFV induces higher levels of interferon-ß, and displays compromised replication both in primary macrophages and pigs compared with wild-type ASFV. Furthermore, I267L-deficiency attenuates the virulence and pathogenesis of ASFV in pigs. These findings suggest that ASFV I267L is an important virulence factor by impairing innate immune responses mediated by the RNA Pol-III-RIG-I axis.

A ferritin-based nanoparticle displaying a neutralizing epitope for foot-and-mouth disease virus (FMDV) confers partial protection in guinea pigs.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140-158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines. RESULTS: This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs. CONCLUSIONS: The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

A QP509L/QP383R-Deleted African Swine Fever Virus Is Highly Attenuated in Swine but Does Not Confer Protection against Parental Virus Challenge.

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.

Combinational Deletions of MGF360-9L and MGF505-7R Attenuated Highly Virulent African Swine Fever Virus and Conferred Protection against Homologous Challenge.

Multigene family (MGF) gene products are increasingly reported to be implicated in African swine fever virus (ASFV) virulence and attenuation of host defenses, among which the MGF360-9L and MGF505-7R gene products are characterized by convergent but distinct mechanisms of immune evasion. Herein, a recombinant ASFV mutant, ASFV-Δ9L/Δ7R, bearing combinational deletions of MGF360-9L and MGF505-7R, was constructed from the highly virulent ASFV strain CN/GS/2018 of genotype II that is currently circulating in China. Pigs inoculated intramuscularly with 104 50% hemadsorption doses (HAD50) of the mutant remained clinically healthy without any serious side effects. Importantly, in a virulence challenge, all four within-pen contact pigs demonstrated clinical signs and pathological findings consistent with ASF. In contrast, vaccinated pigs (5/6) were protected and clinical indicators tended to be normal, accompanied by extensive tissue repairs. Similar to most viral infections, innate immunity and both humoral and cellular immune responses appeared to be vital for protection. Notably, transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR) analysis revealed a regulatory function of the mutant in dramatic and sustained expression of type I/III interferons and inflammatory and innate immune genes in vitro. Furthermore, infection with the mutant elicited an early and robust p30-specific IgG response, which coincided and was strongly correlated with the protective efficacy. Analysis of the cellular response revealed a strong ASFV-specific interferon gamma (IFN-γ) response and immunostaining of CD4+ T cells coupled with a high level of CD163+ macrophage infiltration in spleens of vaccinated pigs. Our study identifies a new mechanism of immunological regulation by ASFV MGFs that rationalizes the design of live attenuated vaccine for implementation of improved control strategies to eradicate ASFV. IMPORTANCE Currently, the deficiency in commercially available vaccines or therapeutic options against African swine fever constitutes a matter of major concern in the swine industry globally. Here, we report the design and construction of a recombinant ASFV mutant harboring combinational deletions of interferon inhibitors MGF360-9L and MGF505-7R based on a genotype II ASFV CN/GS/2018 strain currently circulating in China. The mutant was completely attenuated when inoculated at a high dose of 104 HAD50. In the virulence challenge with homologous virus, sterile immunity was achieved, demonstrating the mutant's potential as a promising vaccine candidate. This sufficiency of effectiveness supports the claim that this live attenuated virus may be a viable vaccine option with which to fight ASF.

Sequential Deletions of Interferon Inhibitors MGF110-9L and MGF505-7R Result in Sterile Immunity against the Eurasia Strain of Africa Swine Fever.

African swine fever virus (ASFV) causes significant morbidity and mortality in pigs worldwide. The lack of vaccines or therapeutic options warrants urgent further investigation. To this aim, we developed a rationally designed live attenuated ASFV-Δ110-9L/505-7R mutant based on the highly pathogenic Genotype II ASFV CN/GS/2018 backbone by deleting 2 well-characterized interferon inhibitors MGF110-9L and MGF505-7R. The mutant was slightly attenuated in vitro compared to parental ASFV but highly tolerant to genetic modifications even after 30 successive passages in vitro. Groups of 5 pigs were intramuscularly inoculated with increasing doses of the mutant, ranging from 103 to 106 hemadsorption units (HAD50). Thirty-five days later, all groups were challenged with 102 HAD50 of virulent parental ASFV. All the animals were clinically normal and devoid of clinical signs consistent with ASFV at the period of inoculation. In the virulent challenge, 2 animals from 103 HAD50-inoculated group and 1 animal from 104 HAD50-inoculated group were unprotected with severe postmortem and histological lesions. The rest of animals survived and manifested with relatively normal clinical appearance accompanied by tangible histological improvements in the extent of tissue damage. Meanwhile, antibody response, as represented by p30-specific antibody titers was positively correlated to protective efficacy, potentializing its usage as an indicator of protection. Moreover, compared to 1 dose, 2 doses provided additional protection, proving that 2 doses were better than 1 dose. The sufficiency in effectiveness supports the claim that our attenuated mutant may be a viable vaccine option with which to fight ASF. IMPORTANCE African swine fever virus (ASFV) is a causative agent of acute viral hemorrhagic disease of domestic swine which is associated with significant economic losses in the pig industry. The lack of vaccines or treatment options requires urgent further investigation. ASFV MGF110-9L and MGF505-7R, 2 well-characterized interferon inhibitors, were associated with viral virulence, host range, and immune modulation. In this study, a recombinant two-gene deletion ASFV mutant with deletion of MGF110-9L and MGF505-7R was constructed. The result showed that the mutant was safe, and also highly resistant to genetic modification even after 30 successive passages. High doses of our mutant (105 and 106 HAD50) provided sterile immunity and complete protection in a virulent challenge. Two doses were superior to 1 dose and provided additional protection. This study develops a new ASFV-specific live attenuated vaccine and may be a viable vaccine option against ASF.

Comparison of multifidus degeneration between scoliosis and lumbar disc herniation.

OBJECTIVE: To assess and compare the pathological and radiological outcomes of multifidus degeneration in scoliosis and lumbar disc herniation patients. METHODS: We performed a retrospective review on 24 patients with scoliosis and 26 patients with lumbar disc herniation (LDH) in the Third Hospital of Hebei Medical University from January 2017 to March2021. The patients were divided into scoliosis group and LDH group according to the treatment. The MRI fatty infiltration rate (FIR) of multifidus and strength of back muscle were calculated to evaluate muscle condition. Multifidus biopsy samples were obtained during surgery in the affected side at L4 or L5 segment in LDH group and on the concavity side of apical vertebrae in scoliosis group. The biopsy fatty infiltration degree (FID) and FIR in two groups, the FIR of affected and unaffected side in LDH group, and the FIR of concavity and convexity side in scoliosis group were compared. The correlation between concavity-convexity FIR difference and cobb angle in scoliosis group, back muscle strength and FIR in LDH group, FID and FIR in both groups was calculated respectively. RESULTS: The FIR was higher in scoliosis group than in LDH group, higher in concavity side than convexity side in scoliosis group (both P < 0.05). The FID was higher in scoliosis group than in LDH group (P < 0.05). No significant difference was found between affected and unaffected side in LDH group (P > 0.05). There was a positive correlation between concavity-convexity FIR difference and cobb angle, FIR and FID (both P < 0.01). There was a negative correlation between back muscle strength and FIR (P < 0.01). The biopsy staining results showed that both two groups were found the existence of rimmed vacuoles, nuclear aggregation, and abnormal enzyme activity, indicating that the scoliosis and LDH may be associated with myogenic diseases. CONCLUSION: The scoliosis patients showed more serious fatty infiltration than LDH patients and rare pathological findings were found in both diseases.

Genetic Determinants of Altered Virulence of Type O Foot-and-Mouth Disease Virus.

Under different circumstances, the alteration of several viral genes may give an evolutionary advantage to the virus to maintain its prevalence in nature. In this study, a 70-nucleotide deletion in the small fragment (S fragment) of the viral 5'-untranslated region (5'-UTR) together with one amino acid insertion in the leader protein (Lpro) that naturally occurred in several serotype O foot-and-mouth disease virus (FMDV) strains in China was identified. The properties of two field serotype O FMDV strains, with or without the 70-nucleotide deletion in the S fragment and the amino acid insertion in Lpro, were compared in vitro and in vivo Clinical manifestations of FMD were clearly observed in cattle and pigs infected by the virus without the mutations. However, the virus with the mentioned mutations caused FMD outcomes only in pigs, not in cattle. To determine the role of the 70-nucleotide deletion in the S fragment and the single amino acid insertion in Lpro in the pathogenicity and host range of FMDV, four recombinant viruses, with complete genomes and a 70-nucleotide deletion in the S fragment, a single amino acid insertion in Lpro, or both mutations, were constructed and rescued. It showed that deletion of 70 nucleotides in the S fragment or insertion of one amino acid (leucine) at position 10 of Lpro partly decreased the viral pathogenicity of Mya-98 lineage virus in cattle and pigs. However, the virus with dual mutations caused clinical disease only in pigs, not in cattle. This suggested that the S fragment and Lpro are significantly associated with the virulence and host specificity of FMDV. The naturally occurring dual mutation in the S fragment and Lpro is a novel determinant of viral pathogenicity and host range for serotype O FMDV.IMPORTANCE FMD is probably the most important livestock disease in the world due to the severe economic consequences caused. The alteration of several viral genes may give the virus selective advantage to maintain its prevalence in nature. Here, we identified that a 70-nucleotide deletion in the S fragment combined with a single leucine insertion in the leader protein (Lpro) is a novel determinant of restricted growth on bovine cells, which significantly contributes to the altered virulence of serotype O FMDV in cattle. A synergistic and additive effect of the 70-nucleotide deletion in the S fragment and the single leucine insertion in Lpro on the virulence and host specificity of the virus was determined. These results will benefit efforts to understand the vial pathogenicity mechanism and molecular characteristics of FMDV.

Amber codon is genetically unstable in generation of premature termination codon (PTC)-harbouring Foot-and-mouth disease virus (FMDV) via genetic code expansion.

The foot-and-mouth disease virus (FMDV) is the causative agent of FMD, a highly infectious and devastating viral disease of domestic and wild cloven-hoofed animals. FMD affects livestock and animal products' national and international trade, causing severe economic losses and social consequences. Currently, inactivated vaccines play a vital role in FMD control, but they have several limitations. The genetic code expansion technology provides powerful strategies for generating premature termination codon (PTC)-harbouring virus as a live but replication-incompetent viral vaccine. However, this technology has not been explored for the design and development of new FMD vaccines. In this study, we first expanded the genetic code of the FMDV genome via a transgenic cell line containing an orthogonal translation machinery. We demonstrated that the transgenic cells stably integrated the orthogonal pyltRNA/pylRS pair into the genome and enabled efficient, homogeneous incorporation of unnatural amino acids into target proteins in mammalian cells. Next, we constructed 129 single-PTC FMDV mutants and four dual-PTC FMDV mutants after considering the tolerance, location, and potential functions of those mutated sites. Amber stop codons individually substituted the selected amino acid codons in four viral proteins (3D, L, VP1, and VP4) of FMDV. We successfully rescued PTC-FMDV mutants, but the amber codon unexpectedly showed a highly degree of mutation rate during PTC-FMDV packaging and replication. Our findings highlight that the genetic code expansion technology for the generation of PTC-FMD vaccines needs to be further improved and that the genetic stability of amber codons during the packaging and replication of FMDV is a concern.

The Pseudoknot Region of the 5' Untranslated Region Is a Determinant of Viral Tropism and Virulence of Foot-and-Mouth Disease Virus.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is characterized by genetic instability and different antigenic properties. The nonstructural protein 3A is a primary determinant of the tropism and virulence of Cathay topotype FMDVs. However, several other determinants are also speculated to be involved in viral tropism and virulence. Deletion of 43 nucleotides (nt) in the pseudoknot (PK) region of the 5' untranslated region (UTR) has been found to coexist with the identified 3A deletion in Cathay topotype FMDV genomes. In this study, we isolated an O/ME-SA/PanAsia lineage FMDV strain, O/GD/CHA/2015, that includes an 86-nt deletion in the PK region and shows a porcinophilic phenotype. To investigate the potential role of the PK region in viral pathogenicity, we generated a recombinant FMDV strain with an incomplete PK region and compared its virulence and pathogenesis to the intact FMDV strain in swine and bovines. Deletion of the 86 nt in the PKs had no major effects on the pathogenicity of the virus in swine but significantly attenuated its ability to infect bovine cells and cattle, indicating that the PK region is a newly discovered determinant of viral tropism and virulence. The role of the 43-nt deletion existing in the Cathay topotype FMDV was also investigated by evaluating the infection properties of genetically engineered viruses. Consistently, the 43-nt deletion in the PK region significantly decreased the pathogenicity of the virus in bovines. Overall, our findings suggest that the PK region deletion occurred naturally in the FMDV genome and that the PK region is highly associated with viral host range and functions as a novel determinant for FMDV pathogenesis.IMPORTANCE This study demonstrates that the deletion in the PK region occurred naturally in the FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in the PK region showed a pig-adapted characteristic that could cause clinical signs in swine but not bovines. Compared to the wild-type FMDV strain, which possesses full infection capacity in both swine and bovines, the recombinant virus with the 86-nt deletion in the PK region is deficient in causing disease in bovines. Deletion of the previously reported 43 nt in the PK region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PK region is a novel determinant of the tropism and virulence of FMDV.

The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-ß signaling pathway.

Foot-and-mouth disease is a frequently occurring disease of cloven-hoofed animals that is caused by infection with the foot-and-mouth virus (FMDV). FMDV circumvents the type-I IFN response by expressing proteins that antagonize cellular innate immunity, such as leader protease and 3C protease. We identified the FMDV structural protein VP3 as a negative regulator of the virus-triggered IFN-ß signaling pathway. Expression of FMDV VP3 inhibited the Sendai virus-triggered activation of IFN regulatory factor-3 and the expression of retinoic acid-inducible gene-I/melanoma differentiation-associated protein-5. Transient transfection and coimmunoprecipitation confirmed that the structural protein VP3 interacts with virus-induced signaling adapter (VISA), which is dependent on the C-terminal aa 111-220 of VP3. In addition, we found that FMDV VP3 inhibits the expression of VISA by disrupting its mRNA. Taken together, our findings reveal a novel strategy used by the structural VP3 protein of FMDV to evade host innate immunity.-Li, D., Yang, W., Yang, F., Liu, H., Zhu, Z., Lian, K., Lei, C., Li, S., Liu, X., Zheng, H., Shu, H. The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-ß signaling pathway.

Highly lipophilic 3-epi-betulinic acid derivatives as potent and selective TGR5 agonists with improved cellular efficacy.

AIM: TGR5 is a G protein-coupled receptor that is expressed in intestinal L-cells and stimulates glucagon-like peptide 1 (GLP-1) secretion. TGR5 may represent a novel target for the treatment of metabolic disorder. Here, we sought to design and synthesize a series of TGR5 agonists derived from the natural product betulinic acid. METHODS: A series of betulinic acid derivatives were designed and synthesized. A cAMP assay was established using a HEK293 cell line expressing human TGR5. Luciferase reporter assay was established using HEK293 cells transfected with plasmids encoding human FXR and luciferase reporter. A human intestinal L-cell line NCI-H716 was used to evaluate the effects of the betulinic acid derivatives on GLP-1 secretion in vitro. RESULTS: Biological data revealed that the 3-α-OH triterpenoids consistently show increased potency for TGR5 compared to their 3-ß-OH epimers. 3-OH esterification increased the lipophilicity and TGR5 activity of 3-α betulinic derivatives and enhanced the activity differences between 3-α and 3-ß derivatives. The 3-α-acyloxy betulinic acids also exhibited a significant dose-dependent GLP-1 secretion effect. CONCLUSION: This study demonstrates that highly lipophilic 3-epi-betulinic acid derivatives can be potent and selective TGR5 agonists with improved cellular efficacy, and our research here provides a new strategy for the design and development of potent TGR5 agonists.

Application of New Polymer Soil Amendment in Ecological Restoration of High-Steep Rocky Slope in Seasonally Frozen Soil Areas.

In seasonally frozen soil areas, high-steep rocky slopes resulting from open-pit mining and slope cutting during road construction undergo slow natural restoration, making ecological restoration generally challenging. In order to improve the problems of external soil attachment and long-term vegetation growth in the ecological restoration of high-steep rocky slopes in seasonally frozen areas, this study conducted a series of experiments through the combined application of polyacrylamide (PAM) and carboxymethyl cellulose (CMC) to assess the effects of soil amendments on soil shear strength, water stability, freeze-thaw resistance, erosion resistance, and vegetation growth. This study showed that the addition of PAM-CMC significantly increased the shear resistance and cohesion of the soil, as well as improving the water stability, freeze-thaw resistance, and erosion resistance, but the internal friction angle of the soil was not significantly increased after reaching a certain content. Moderate amounts of PAM-CMC can extend the survival of vegetation, but overuse may cause soil hardening and inhibit vegetation growth by limiting air permeability. It was observed by a scanning electron microscope (SEM) that the gel membrane formed by PAM-CMC helped to "bridge" and bind the soil particles. After discussion and analysis, the optimum application rate of PAM-CMC was 3%, which not only improved the soil structure but also ensured the growth of vegetation in the later stage under the optimum application rate. Field application studies have shown that 3% PAM-CMC-amended soil stably attaches to high-steep rocky slopes, with stable vegetation growth, and continues to grow after five months of freeze-thaw action, with no need for manual maintenance after one year.

ASFV infection induces macrophage necroptosis and releases proinflammatory cytokine by ZBP1-RIPK3-MLKL necrosome activation.

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, which is highly pathogenic and causes severe mortality in domestic pigs. It is caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and the effect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV infection activates the necroptosis signaling in vivo and macrophage necroptosis in vitro. Further evidence showed that ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. The Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis provoked the extracellular release of proinflammatory cytokines, including TNF-α and IL-1ß induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

Total Thoracoscopic Surgery for Late Mitral Paravalvular Leakage Repair in A Beating Heart.

Paravalvular leakage (PVL) after mitral valve replacement is a troublesome complication that may lead to severe symptoms and reoperation. Previous case reports on total thoracoscopic cardiac surgery without aortic cross-clamping for repairing late PVL are rare. We describe a 64-year-old man who had undergone aortic and mitral valve replacement via median sternotomy eight years earlier, and who recently developed cardiac failure due to severe tricuspid regurgitation (TR) and PVL in the posterior mitral annulus. During total thoracoscopic surgery with using the beating heart technique, direct closure of the PVL was achieved via pledgeted mattress sutures, and tricuspid valvuloplasty was routinely performed to treat TR. This case indicated that total thoracoscopic surgery on a beating heart may be an excellent option for treating PVL concomitant with TR.