Fei Gao

Integrins are cell surface adhesion molecules. They bridge the intracellular and extracellular environments, enabling bidirectional transmembrane signaling and regulating immune responses. However, it remains unclear whether integrin protein β1 (ITGβ1) is involved in innate immune responses. In our previous study, we demonstrated that porcine epidemic diarrhea virus (PEDV) can induce type I interferon (IFN-I) production. In this study, we observed that ITGβ1 expression is rapidly induced following PEDV infection and further established that PEDV infection primarily promoted ITGβ1 expression through upregulation of the transcription factor c-Myc. We hypothesized that ITGβ1 might be involved in PEDV-induced innate immune responses through IFN-I production. Our investigation revealed ITGβ1 overexpression promotes the phosphorylation and subsequent nuclear translocation of both interferon regulatory factor 3 (IRF3) and NF-κB, thereby enhancing SeV-induced IFN-β promoter activity. Furthermore, we showed that ITGβ1 functions as an activator in the melanoma differentiation-associated protein 5 (MDA5)-mediated IFN-I signaling pathway. More importantly, we demonstrated that ITGβ1 is critically involved in PEDV-induced IFN-I antiviral responses. Mechanistically, ITGβ1 facilitates MDA5 oligomerization by specifically interacting with its caspase activation and recruitment domain (CARD), thereby enhancing dsRNA-recruitment capacity. In summary, the findings of this study indicate that ITGβ1 acts as an activator of the MDA5-dependent IFN-I antiviral innate immune response and positively regulates the MDA5-mediated RIG-I-like receptor signaling pathway. IMPORTANCE: Porcine epidemic diarrhea virus (PEDV), an alpha coronavirus, severely impacts newborn piglets, leading to acute manifestations including vomiting, diarrhea, dehydration, and high mortality rates in suckling piglets. These consequences have devastating implications for the global swine industry. Within the host’s innate antiviral response, RIG-I-like receptors (RLRs) are critical for the activation of the interferon signaling pathway. Integrin proteins, known for their role in regulating bidirectional signal transduction across the cell membrane, are associated with numerous viral infections. In this study, utilizing PEDV as an infection model, we demonstrated that overexpression of ITGβ1 suppresses PEDV replication, while knockdown of ITGβ1 expression enhances it. Additionally, ITGβ1 significantly augments PEDV-induced type I interferon production in host cells. We further elucidated that ITGβ1 interacts with the 2CARD region of MDA5, promoting MDA5 oligomerization and the transmission of activation signals. These findings establish ITGβ1 as a positive regulatory factor in MDA5-mediated RLR signaling pathway. These findings not only identify ITGβ1 as a novel host antiviral protein against PEDV but also reveal, for the first time, a previously unrecognized function of ITGβ1 in the cellular innate antiviral immune response.

African swine fever (ASF), caused by African swine fever virus (ASFV), has inflicted severe economic losses on China’s pig industry. Existing ASFV nucleic acid detection methods struggle to identify infected pigs in the pre-viremic stage, especially for recently emerged recombinant ASFV strains that exhibit delayed clinical symptoms and prolonged virus shedding, posing great challenges to ASF prevention and control. To fit the problem, this study established a TaqMan duplex quantitative polymerase chain reaction (qPCR) assay targeting the ASFV p72 gene and porcine Hp gene for early diagnosis of ASFV infection. The qPCR reaction system (20 μL) and conditions were optimized and showed high sensitivity, with detection limits of 1.42 × 10¹ copies/μL for Hp and 2.23 × 10¹ copies/μL for ASFV, as well as excellent specificity and reproducibility. Serum cDNA samples from pigs infected with virulent or recombinant ASFV strains were tested, and the result showed that Hp was detectable as early as 1 day post-infection (DPI), however ASFV remained undetectable until 3DPI. Then cDNA samples from cohabitation infection were tested and 80% samples were Hp-positive, although ASFV test was negative.In conclusion, this duplex qPCR assay for simultaneous detection of Hp and ASFV enables pre-viremia diagnosis of ASF, providing a valuable tool for early screening of ASFV-infected pigs.

Background/Objectives: African Swine Fever (ASF), caused by the African Swine Fever Virus (ASFV), is a highly contagious and lethal disease in pigs, for which no recognized safe and effective vaccine is currently available. The ASFV EP153R gene, expressed during both early and late infection stages, exhibits strong protective potential. Utilizing advances in genetic engineering, recombinant PRRSV vector vaccines carrying ASFV exogenous genes were constructed. This study aims to prepare pEP153R-based polyclonal antibodies and an iELISA detection method using the constructed rPRRSV-EP153R as a specific target to verify the iELISA’s specificity and effectiveness. Methods: A prokaryotic plasmid, pCold-TF-EP153R, was constructed to express protein in BL21 (DE3). The purified soluble protein (2 mg/mL) was used to generate a murine polyclonal antibody and establish an indirect ELISA. The EP153R gene was inserted between ORF1b and ORF2a of PRRSV via reverse genetics, yielding recombinant rPRRSV-EP153R. Its biological properties were assessed in vitro and in vivo. Results: The pEP153R was specifically detected by both anti-His antibody and generated polyclonal antibodies. An established iELISA showed high specificity, sensitivity, and 98.18% accuracy. The antibodies specifically recognized pEP153R expressed in recombinant virus and eukaryotic systems. Additionally, the recombinant virus stably maintained EP153R without changes in virological characteristics relative to vHuN4-F112. In vaccinated piglets, the rPRRSV-EP153R induced a specific, consistent, and detectable immune response. Conclusions: The established iELISA, characterized by high specificity, sensitivity, and accuracy, furnishes reliable technical support for the serological diagnosis of ASFV. Meanwhile, the recombinant virus rPRRSV-EP153R demonstrates potential as a novel live vectored vaccine candidate, with the capability to induce specific immunity against both ASFV and PRRSV.

Classical swine fever virus (CSFV) incurs substantial economic losses in the global swine industry due to its persistent emergence and re-emergence across various countries. However, the precise mechanisms governing CSFV budding remain inadequately understood. Our study elucidates that the endosomal sorting complex required for transport (ESCRT)-associated protein ALIX, in conjunction with ESCRT-III, plays a pivotal role in orchestrating CSFV budding. Genomic sequence analysis identified a critical interaction between the YPXnL late domain on the E2 protein and ALIX. Through immunoprecipitation and structural domain deletion assays, we demonstrated that the ALIX Bro1 domain specifically recognized viral particles by binding to the YPXnL motif. Immunoelectron and transmission electron microscopy further confirmed that, upon infection, ALIX accumulated at the periphery of subcellular organelles, including COPII vesicles, endosomes, and the Golgi apparatus, thereby facilitating CSFV budding. Our findings also revealed that ESCRT-III subunits CHMP2B, CHMP4B, CHMP7, and VPS4A interacted with ALIX to expedite CSFV budding. Notably, Rab8 activated by Kif4A contributed to the release of CSFV particles by interacting with ALIX and directing ALIX-containing vesicles along microtubules toward the cytosol. Our study demonstrates that ALIX specifically recognizes E2 and orchestrates the recruitment of ESCRT-III and Rab8 to facilitate the vesicular budding of CSFV particles from the Golgi apparatus to the cytosol. Ultimately, virus-laden vesicles propelled by Kif4A are transported along microtubules to the plasma membrane for release. Our findings offer the first comprehensive elucidation of the CSFV budding process and contribute to the identification of antiviral targets, thereby advancing the development of antiviral therapeutics. IMPORTANCE: The endosomal sorting complex required for transport (ESCRT) machinery plays a pivotal role in the sorting of membrane proteins in eukaryotic cells and regulating various stages of infection for numerous viruses. Previous studies have underscored the indispensable role of ESCRT in the cellular entry and replication of classical swine fever virus (CSFV). However, the precise mechanisms by which ESCRT recognizes CSFV particles and initiates viral vesicle budding have remained elusive. This study reveals that the Bro1 domain of ALIX initiates viral budding proximal to the Golgi apparatus by specifically recognizing the YPXnL late domain on the CSFV E2 protein. Mechanistically, ALIX and ESCRT-III facilitate Rab8-regulated endosomal transport of CSFV particles from the Golgi apparatus to the plasma membrane. Subsequently, virions are propelled by the kinesin Kif4A along microtubules for egress into the extracellular space. In summary, these findings significantly advance our understanding of CSFV pathogenesis and offer valuable insights into the vesicular transport and budding mechanisms of CSFV particles.

Recombination is a significant factor driving the evolution of RNA viruses. The prevalence and variation of porcine reproductive and respiratory syndrome virus (PRRSV) in China have been increasing in complexity due to extensive interlineage recombination. When this recombination phenomenon occurs in live vaccine strains, it becomes increasingly difficult to prevent and control PRRSV. Reverse genetic manipulation to engineer a different transcriptional regulatory sequence (TRS) circuit introduces genetic traps into the viral genome that are lethal to recombinant RNA progeny viruses. In this study, major interlineage recombination patterns were identified between lineage 1 (L1) PRRSVs and lineage 8 (L8) PRRSVs in China, from 2019 to 2023. The recombinant mutant virus, vA-TRSall, was constructed and successfully rescued by rewiring the entire TRS circuit without changing the amino acid-coding sequence in the genome of the PRRSV live vaccine strain vHuN4-F112. The vA-TRSall, with a brand new TRS circuit, provided effective immune protection against the highly pathogenic L8 PRRSV (vHuN4) and epidemic NADC30-like L1 PRRSV (vZJqz21). Recombination analysis in vitro and in vivo showed that, compared with the vHuN4-F112 and vZJqz21 co-infection groups, the incidence rates of mutation breakpoints and template-switching recombination in the vA-TRSall and vZJqz21 co-infected groups were effectively reduced. The results have enriched our understanding of the critical role of TRS circuits in PRRSV recombination mechanisms and indicate a successful redesign that can endow PRRSV live vaccines with recombination-resistant capabilities. IMPORTANCE: Porcine reproductive and respiratory syndrome viruses (PRRSVs) are genetically diverse, and this is due in part to their extensive recombination. Live vaccines are widely used to prevent and control PRRS in China. However, owing to the wide variety of live vaccines, non-standard use, and the wild viruses prevalent on pig farms, new strains, generated via RNA recombination, are continuously emerging. Vaccine strains are also involved in PRRSV recombination, which leads to the emergence of new variants and alterations in virulence and pathogenesis. A recombination-resistant genome was engineered by rewiring the entire transcriptional regulatory sequence (TRS) circuit of the live PRRSV vaccine strain. Theoretically, after clinical application, once the virus recombines with the genome of the epidemic strain, the base pairing between the two sets of TRS circuits should be disrupted, resulting in a fatal genetic trap for the generation of an RNA recombinant progeny virus. Therefore, the remodeled PRRSV TRS mutant generated in this study can serve as a recombination-resistant platform for the rational design of safe PRRS vaccines in the future.