MDA5 belongs to the RIG-I-like receptor family, which is involved in innate immunity. During viral infection, MDA5 generates an antiviral response by recognizing the ligand to activate interferon. However, the role and mechanism of MDA5 in canine influenza virus (CIV) infection are unclear. To understand the mechanism of canine MDA5-mediated innate immunity during CIV infection, we detected the distribution of MDA5 in beagles, and the structural prediction showed that MDA5 was mainly composed of a CARD domain, RD domain, and DExD/H helix structure. Moreover, we found that MDA5 inhibits CIV replication. Furthermore, in the dual luciferase assay, we revealed that the CARD region of MDA5 strongly activated the IFN-β promoter and mainly transmitted signals through the CARD region. Overexpression of the CARD region of MDA5 revealed that the MDA5-mediated signaling pathway could transmit signals by activating the IRF3/NF-κB and IRF3 promoters, promoting the expression of antiviral proteins and cytokine release, thereby inhibiting CIV replication. Upon silencing of MDA5, cytokine production decreased, while the replication ability of CIV was increased. Thus, this study revealed a novel mechanism by which MDA5 mediated CIV infection and provided new avenues for the development of antiviral strategies.

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I–mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

Lactate dehydrogenase A (LDHA) plays a crucial role in regulating lactate synthesis in various biological processes. Lactate, a byproduct of glycometabolism, has been recognized as a unique molecule with implications in both metabolism and immunity. Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is a highly contagious and severe infectious disease that primarily affects pigs. Prior research has shown that CSFV infection disrupts the normal glycolytic process, leading to an accumulation of lactate within the host. Nevertheless, it remains unclear whether there is mutual regulation between the CSFV and LDHA-lactate axis. Here, we have found that CSFV infection increases LDHA expression in vivo and in vitro , which may be attributed to attenuated ISGylation of LDHA. Furthermore, CSFV infection induces L-lactate production via LDHA dependence in vitro . The cellular biology research on LDHA has revealed that LDHA not only localizes to the mitochondria but also inhibits PINK1-Parkin-mediated mitophagy. Through various experimental techniques such as western blot to detect mitophagy marker proteins, laser confocal microscopy to observe the flow of mitophagy, and transmission electron microscopy to assess changes in the number of mitochondria enclosed within autophagosome-like vesicles, it has been discovered that the addition of exogenous lactate can inhibit PINK1-Parkin-mediated mitophagy. Importantly, we have observed that lactate activates the JAK1-STAT1-ISG15 network and suppresses CSFV replication by antagonizing CCCP-induced mitophagy. These results represent the first report on the mechanisms through which the LDHA-lactate axis regulates mitophagy, the JAK-STAT pathway, and CSFV replication. This study provides novel insights into the roles of the LDHA-lactate axis in glycometabolism and viral replication. IMPORTANCE: This research unveils how CSFV interacts with cellular metabolism through LDHA. By revealing LDHA’s dual role and how lactate influences cellular processes during CSFV infection, this study uncovers new pathways for viral replication. These findings not only deepen our understanding of viral-host interactions but also open doors for innovative antiviral strategies centered around manipulating cellular metabolism.

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host’s immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-β promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2′-5′oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1β. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-β, and IL-1β. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies.

The canine influenza virus (CIV) outbreaks have raised concerns as they pose a threat to the health of dogs. The successful construction of a canine influenza (CI) infection model is essential to study the CIV. Here we investigated the pathogenicity of different infectious doses of H3N2 CIV in Beagle dogs. Thirty-seven healthy Beagle dogs were used in the experiment and were infected with 10³, 10⁴, 10⁵, and 10⁶ 50% egg-infectious doses (EID₅₀). Compared to the dogs in the other three groups, those in the 10⁶ EID₅₀ group presented with obvious clinical symptoms, high virus titer, and typical pathological changes. Considering the ensemble of clinical scores, body temperature, virus shedding, lung lesions, pathological section scores, and visceral virus titers, we determined that 10⁶ EID₅₀ is the minimum infectious dose for the Beagle infection model. The other three infectious doses had almost no clinical symptoms. These results indicate that 10⁶ EID₅₀ is the minimum infectious dose of H3N2 CIV that can cause obvious clinical manifestations and pathological changes associated with CI in Beagle dogs. The theoretical framework developed in this research will guide the establishment of an infection model of CIV for future research.