SQSTM1 downregulates avian metapneumovirus subgroup C replication via mediating selective autophagic degradation of viral M2-2 protein.

Avian metapneumovirus subgroup C (aMPV/C), an important pathogen causing acute respiratory infection in chickens and turkeys, contributes to substantial economic losses in the poultry industry worldwide. aMPV/C has been reported to induce autophagy, which is beneficial to virus replication. Sequestosome 1 (SQSTM1/P62), a selective autophagic receptor, plays a crucial role in viral replication by clearing ubiquitinated proteins. However, the relationship between SQSTM1-mediated selective autophagy and aMPV/C replication is unclear. In this study, we found that the expression of SQSTM1 negatively regulates aMPV/C replication by reducing viral protein expression and viral titers. Further studies revealed that the interaction between SQSTM1 and aMPV/C M2-2 protein is mediated via the Phox and Bem1 (PB1) domain of the former, which recognizes a ubiquitinated lysine at position 67 of the M2-2 protein, and finally degrades M2-2 via SQSTM1-mediated selective autophagy. Collectively, our results reveal that SQSTM1 degrades M2-2 via a process of selective autophagy to suppress aMPV/C replication, thereby providing novel insights for the prevention and control of aMPV/C infection.IMPORTANCEThe selective autophagy plays an important role in virus replication. As an emerging pathogen of avian respiratory virus, clarification of the effect of SQSTM1, a selective autophagic receptor, on aMPV/C replication in host cells enables us to better understand the viral pathogenesis. Previous study showed that aMPV/C infection reduced the SQSTM1 expression accompanied by virus proliferation, but the specific regulatory mechanism between them was still unclear. In this study, we demonstrated for the first time that SQSTM1 recognizes the 67th amino acid of M2-2 protein by the interaction between them, followed by M2-2 degradation via the SQSTM1-mediated selective autophagy, and finally inhibits aMPV/C replication. This information supplies the mechanism by which SQSTM1 negatively regulates viral replication, and provides new insights for preventing and controlling aMPV/C infection.

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

Ubiquitination-dependent degradation of nucleolin mediated by porcine circovirus type 3 capsid protein.

IMPORTANCE: Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon ß. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

RSAD2 suppresses viral replication by interacting with the Senecavirus A 2 C protein.

Senecavirus A (SVA), an emerging virus that causes blisters on the nose and hooves, reduces the production performance of pigs. RSAD2 is a radical S-adenosylmethionine (SAM) enzyme, and its expression can suppress various viruses due to its broad antiviral activity. However, the regulatory relationship between SVA and RSAD2 and the mechanism of action remain unclear. Here, we demonstrated that SVA infection increased RSAD2 mRNA levels, whereas RSAD2 expression negatively regulated viral replication, as evidenced by decreased viral VP1 protein expression, viral titres, and infected cell numbers. Viral proteins that interact with RSAD2 were screened, and the interaction between the 2 C protein and RSAD2 was found to be stronger than that between other proteins. Additionally, amino acids (aa) 43-70 of RSAD2 were crucial for interacting with the 2 C protein and played an important role in its anti-SVA activity. RSAD2 was induced by type I interferon (IFN-I) via Janus kinase signal transducer and activator of transcription (JAK-STAT), and had antiviral activity. Ruxolitinib, a JAK-STAT pathway inhibitor, and the knockdown of JAK1 expression substantially reduced RSAD2 expression levels and antiviral activity. Taken together, these results revealed that RSAD2 blocked SVA infection by interacting with the viral 2 C protein and provide a strategy for preventing and controlling SVA infection.

A Receptor Integrin ß1 Promotes Infection of Avian Metapneumovirus Subgroup C by Recognizing a Viral Fusion Protein RSD Motif.

Avian metapneumovirus subgroup C (aMPV/C) causes respiratory diseases and egg dropping in chickens and turkeys, resulting in severe economic losses to the poultry industry worldwide. Integrin ß1 (ITGB1), a transmembrane cell adhesion molecule, is present in various cells and mediates numerous viral infections. Herein, we demonstrate that ITGB1 is essential for aMPV/C infection in cultured DF-1 cells, as evidenced by the inhibition of viral binding by EDTA blockade, Arg-Ser-Asp (RSD) peptide, monoclonal antibody against ITGB1, and ITGB1 short interfering (si) RNA knockdown in cultured DF-1 cells. Simulation of the binding process between the aMPV/C fusion (F) protein and avian-derived ITGB1 using molecular dynamics showed that ITGB1 may be a host factor benefiting aMPV/C attachment or internalization. The transient expression of avian ITGB1-rendered porcine and feline non-permissive cells (DQ cells and CRFK cells, respectively) is susceptible to aMPV/C infection. Kinetic replication of aMPV/C in siRNA-knockdown cells revealed that ITGB1 plays an important role in aMPV/C infection at the early stage (attachment and internalization). aMPV/C was also able to efficiently infect human non-small cell lung cancer (A549) cells. This may be a consequence of the similar structures of both metapneumovirus F protein-specific motifs (RSD for aMPV/C and RGD for human metapneumovirus) recognized by ITGB1. Overexpression of avian-derived ITGB1 and human-derived ITGB1 in A549 cells enhanced aMPV/C infectivity. Taken together, this study demonstrated that ITGB1 acts as an essential receptor for aMPV/C attachment and internalization into host cells, facilitating aMPV/C infection.

Seneca Valley Virus Enters PK-15 Cells via Caveolae-Mediated Endocytosis and Macropinocytosis Dependent on Low-pH, Dynamin, Rab5, and Rab7.

Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. IMPORTANCE Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family Picornaviridae enter host cells, and provides new insights for preventing and controlling SVV infection.

DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication.

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases that pose a serious threat to the swine industry. PCV2 capsid (Cap) protein has been shown to interact with DEAD-box RNA helicase 21 (DDX21), an important protein that regulates RNA virus replication. However, whether the interaction between DDX21 and the PCV2 Cap regulates PCV2 replication remains unclear. Herein, by using western blotting, interaction assays, and knockdown analysis, we found that PCV2 infection induced the cytoplasmic relocation of DDX21 from the nucleolus in cultured PK-15 cells. Moreover, the nuclear localization signal (NLS) of PCV2 Cap interacted directly with DDX21. The NLS of PCV2 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were essential for the dual interaction. Upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV2 replication via a lentivirus-delivered system, as evidenced by decreased levels of viral protein expression and virus production. In contrast, the replication of PCV2 increased in transiently DDX21-overexpressing cells. Our results indicate that DDX21 interacts with PCV2 Cap and plays a crucial role in virus replication. These results provide a reference for developing novel potential targets for prevention and control of PCV2 infection.

Heme Oxygenase-1 and Its Metabolites Carbon Monoxide and Biliverdin, but Not Iron, Exert Antiviral Activity against Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) is a newly discovered pathogen that causes porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, multisystemic inflammation, and reproductive failure. Heme oxygenase-1 (HO-1), a stress-inducible enzyme, exerts protective functions by converting heme into carbon monoxide (CO), biliverdin (BV), and iron. However, the effects of HO-1 and its metabolites on PCV3 replication remain unknown. In this study, experiments involving specific inhibitors, lentivirus transduction, and small interfering RNA (siRNA) transfection revealed that active PCV3 infection reduced HO-1 expression and that the expression of HO-1 negatively regulated virus replication in cultured cells, depending on its enzymatic activity. Subsequently, the effects of the HO-1 metabolites (CO, BV, and iron) on PCV3 infection were investigated. The CO inducers (cobalt protoporphyrin IX [CoPP] or tricarbonyl dichloro ruthenium [II] dimer [CORM-2]) mediate PCV3 inhibition by generating CO, and this inhibition is reversed by hemoglobin (Hb; a CO scavenger). The inhibition of PCV3 replication by BV depended on BV-mediated reactive oxygen species (ROS) reduction, as N-acetyl-l-cysteine affected PCV3 replication while reducing ROS production. The reduction product of BV, bilirubin (BR), specifically promoted nitric oxide (NO) generation and further activated the cyclic GMP/protein kinase G (cGMP/PKG) pathway to attenuate PCV3 infection. Both the iron provided by FeCl3 and the iron chelated by deferoxamine (DFO) with CoPP treatment failed to affect PCV3 replication. Our data demonstrate that the HO-1-CO-cGMP/PKG, HO-1-BV-ROS, and HO-1-BV-BR-NO-cGMP/PKG pathways contribute crucially to the inhibition of PCV3 replication. These results provide important insights regarding preventing and controlling PCV3 infection. IMPORTANCE The regulation of host protein expression by virus infection is the key to facilitating self-replication. As an important emerging pathogen of swine, clarification of the interaction between PCV3 infection and the host enables us to understand the viral life cycle and pathogenesis better. Heme oxygenase-1 (HO-1) and its metabolites carbon monoxide (CO), biliverdin (BV), and iron have been demonstrated to involve a wealth of viral replications. Here, we, for the first time, demonstrated that HO-1 expression decreases in PCV3-infected cells and negatively regulates PCV3 replication and that the HO-1 metabolic products CO and BV inhibit PCV3 replication by the CO- or BV/BR/NO-dependent cGMP/PKG pathway or BV-mediated ROS reduction, but the iron (the third metabolic product) does not. Specifically, PCV3 infection maintains normal proliferation by downregulating HO-1 expression. These findings clarify the mechanism by which HO-1 modulates PCV3 replication in cells and provide important targets for preventing and controlling PCV3 infection.

Evolutionary Origin, Genetic Recombination, and Phylogeography of Porcine Kobuvirus.

The newly identified porcine Kobuvirus (PKV) has raised concerns owing to its association with diarrheal symptom in pigs worldwide. The process involving the emergence and global spread of PKV remains largely unknown. Here, the origin, genetic diversity, and geographic distribution of PKV were determined based on the available PKV sequence information. PKV might be derived from the rabbit Kobuvirus and sheep were an important intermediate host. The most recent ancestor of PKV could be traced back to 1975. Two major clades are identified, PKVa and PKVb, and recombination events increase PKV genetic diversity. Cross-species transmission of PKV might be linked to interspecies conserved amino acids at 13-17 and 25-40 residue motifs of Kobuvirus VP1 proteins. Phylogeographic analysis showed that Spain was the most likely location of PKV origin, which then spread to pig-rearing countries in Asia, Africa, and Europe. Within China, the Hubei province was identified as a primary hub of PKV, transmitting to the east, southwest, and northeast regions of the country. Taken together, our findings have important implications for understanding the evolutionary origin, genetic recombination, and geographic distribution of PKV thereby facilitating the design of preventive and containment measures to combat PKV infection.

Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation.

Avian metapneumovirus subgroup C (aMPV/C) is an important pathogen that causes upper respiratory symptoms and egg production decline in turkeys and chickens. aMPV/C infection leads to inhibition of the host antiviral immune response. However, our understanding of the molecular mechanisms underlying host immune response antagonized by aMPV/C infection is limited. In this study, we demonstrated that the aMPV/C phosphoprotein (P) inhibits the IFN antiviral signaling pathway triggered by melanoma differentiation gene 5 (MDA5) and reduces interferon ß (IFN-ß) production and IFN-stimulated genes (ISGs) by targeting IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) in DF-1 cells. Moreover, we found that aMPV/C P protein only blocks the nuclear translocation of IRF3 by interacting with IRF3 in HEK-293T cells, instead of affecting IRF3 phosphorylation and inducing IRF3 degradation, which suppresses IRF3 signaling activation and results in a decrease in IFN-ß production. Collectively, these results reveal a novel mechanism by which aMPV/C infection disrupts IFN-ß production in the host. IMPORTANCE The innate immune response is the first defense line of host cells and organisms against viral infections. When RNA viruses infect cells, viral RNA induces activation of retinoic acid-induced gene I and melanoma differentiation gene 5, which initiates downstream molecules and finally produces type I interferon (IFN-I) to regulate antiviral immune responses. The mechanism for avian metapneumovirus (aMPV) modulating IFN-I production to benefit its replication remains unknown. Here, we demonstrate that phosphoprotein of aMPV subgroup C (aMPV/C) selectively inhibits the nuclear translocation of interferon regulatory 3 (IRF3), instead of affecting the expression and phosphorylation of IRF3, which finally downregulates IFN-I production. This study showed a novel mechanism for aMPV/C infection antagonizing the host IFN response.

Porcine Circovirus Type 2 Vaccines: Commercial Application and Research Advances.

Porcine circovirus type 2 (PCV2) infection can lead to porcine circovirus-associated disease (PCVAD), causing great economic losses to the global swine industry. Conventional vaccination programs are a major measure in the prevention and control of this disease. Currently, there are 5 commercially available PCV2 vaccines in the international market and 10 kinds commercially available PCV2 vaccines in the Chinese market that confer good efficacy against this virus by alleviating clinicopathological manifestations and enhancing growth performance in pigs. In addition, diverse experimental PCV2 vaccines with protective efficiency have been developed, including attenuated chimeric, nucleic acid, subunit, multivalent, and viral-vectored vaccines. These experimental vaccines have been shown to be relatively effective in improving the efficiency of pig production and simplifying prevention procedures. Adjuvants can be used to promote vaccines with higher protective immunity. Herein, we review the application of multiple commercial vaccines over the years and research advances in experimental vaccines, which provide the possibility for the development of superior vaccines to successfully prevent and control PCV2 infection in the future.

Interaction of Nucleolin with the Fusion Protein of Avian Metapneumovirus Subgroup C Contributes to Viral Replication.

Avian metapneumovirus subgroup C (aMPV/C) is highly pathogenic to various avian species with acute respiratory tract clinicopathology and/or drops in egg production. Nucleolin (NCL), an important nucleolar protein, has been shown to regulate multiple viral replication and serve as a functional receptor for viral entry and internalization. Whether NCL is involved in aMPV/C pathogenesis is not known. In this study, we found that aMPV/C infection altered the subcellular localization of NCL in cultured cells. siRNA-targeted NCL resulted in a remarkable decline in aMPV/C replication in Vero cells. DF-1 cells showed a similar response after CRISPR/Cas9-mediated knock out of NCL during aMPV/C infection. Conversely, NCL overexpression significantly increased aMPV/C replication. Pretreatment with AS1411-a aptamer, a guanine (G)-rich oligonucleotide that forms four-stranded structures and competitively binding to NCL, decreased aMPV/C replication and viral titers in cultured cells. Additionally, we found that the aMPV/C fusion (F) protein specifically interacts with NCL through its central domain and that AS1411 disrupts this interaction, thus inhibiting viral replication. Taken together, these results reveal that the aMPV/C F protein interacts with NCL, which is employed by aMPV/C for efficient replication, thereby highlighting the strategic potential for control and therapy of aMPV/C infection.

Reconstruction of the Evolutionary Origin, Phylodynamics, and Phylogeography of the Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) is a newly identified virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and multisystemic inflammatory responses in pigs. Recent studies suggests that PCV3 originated from bat circoviruses; however, the origin time, mode of spread, and geographic distribution of PCV3 remain unclear. In this study, the evolutionary origin, phylodynamics, and phylogeography of PCV3 were reconstructed based on the available complete genome sequences. PCV3 showed a closer relationship with bird circovirus than with bat circovirus, but their common ancestor was bat circovirus, indicating that birds may be intermediate hosts for the spread of circoviruses in pigs. Using the BEAST and phylogenetic analyses, three different clades of PCV3 (PCV3a, PCV3b, and PCV3c) were identified, with PCV3a being the most prevalent PCV3 clade. Further studies indicated that the earliest origin of PCV3 can be traced back to 1907.53-1923.44, with a substitution rate of 3.104 × 10-4 to 6.8524 × 10-4 substitution/site/year. A phylogeographic analysis highlighted Malaysia as the earliest location of the original PCV3, which migrated to Asia, America, and Europe. Overall, this study provides novel insights into the evolutionary origin, spread mode, and geographic distribution of PCV3, which will facilitate the prevention and control of PCV3 epidemics in the future.

The Evolution, Genomic Epidemiology, and Transmission Dynamics of Tembusu Virus.

Tembusu virus (TMUV) can induce severe egg drop syndrome in ducks, causing significant economic losses. In this study, the possible origin, genomic epidemiology, and transmission dynamics of TMUV were determined. The time to the most recent common ancestor of TMUV was found to be 1924, earlier than that previously reported. The effective population size of TMUV increased rapidly from 2010 to 2013 and was associated with the diversification of different TMUV clusters. TMUV was classified into three clusters (clusters 1, 2, and 3) based on the envelope (E) protein. Subcluster 2.2, within cluster 2, is the most prevalent, and the occurrence of these mutations is accompanied by changes in the virulence and infectivity of the virus. Two positive selections on codons located in the NS3 and NS5 genes (591 of NS3 and 883 of NS5) were identified, which might have caused changes in the ability of the virus to replicate. Based on phylogeographic analysis, Malaysia was the most likely country of origin for TMUV, while Shandong Province was the earliest province of origin in China. This study has important implications for understanding TMUV and provides suggestions for its prevention and control.

The Network of Interactions between the Porcine Epidemic Diarrhea Virus Nucleocapsid and Host Cellular Proteins.

Host-virus protein interactions are critical for intracellular viral propagation. Understanding the interactions between cellular and viral proteins may help us develop new antiviral strategies. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe damage to the global swine industry. Here, we employed co-immunoprecipitation and liquid chromatography-mass spectrometry to characterize 426 unique PEDV nucleocapsid (N) protein-binding proteins in infected Vero cells. A protein-protein interaction network (PPI) was created, and gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses revealed that the PEDV N-bound proteins belong to different cellular pathways, such as nucleic acid binding, ribonucleoprotein complex binding, RNA methyltransferase, and polymerase activities. Interactions of the PEDV N protein with 11 putative proteins: tripartite motif containing 21, DEAD-box RNA helicase 24, G3BP stress granule assembly factor 1, heat shock protein family A member 8, heat shock protein 90 alpha family class B member 1, YTH domain containing 1, nucleolin, Y-box binding protein 1, vimentin, heterogeneous nuclear ribonucleoprotein A2/B1, and karyopherin subunit alpha 1, were further confirmed by in vitro co-immunoprecipitation assay. In summary, studying an interaction network can facilitate the identification of antiviral therapeutic strategies and novel targets for PEDV infection.

Characterization and pathogenicity of a naturally reassortant and recombinant infectious bursal disease virus in China.

Infectious bursal disease virus (IBDV), an Avibirnavirus, is the pathogen of infectious bursal disease, which is a severely immunosuppressive disease in 3-15-week-old chickens. Different phenotypes of IBDV, including classical, variant, very virulent (vv) and attenuated IBDV, have been reported in many chicken-rearing countries worldwide. Here, we isolated and identified a naturally reassortant and recombinant IBDV (designated GXB02) from 20-day-old chickens with clinicopathological changes of infectious bursal disease (IBD) in Guangxi Province, China. Whole genomic sequencing showed that the strain GXB02 simultaneously has both reassortant and recombinant characteristics with segments A and B being derived from recombinant intermediate vaccine strain and classic strains of IBDV. Segment A of strain GXB02 was incorporated into the skeleton of an intermediate IBDV vaccine strain (W2512), where the breakpoints of two recombinant events located at nucleotide positions 1468 and 1648 were replaced by reassortant vvIBDV (PK2) and vvIBDV (D6948) of segment A, respectively. We used this GXB02 strain to inoculate 21-day-old specific-pathogen-free chickens to evaluate its pathogenicity. Strain GXB02 has clinicopathologic characteristics of IBD with severe bursal lesions, as evidenced by necrosis, depletion of lymphocytes, and follicle atrophy, indicating that reassortment with classical strains in segment B or/and recombination with very virulent strains increased pathogenicity of the strain GXB02 in chickens. These findings provide important insights into the genetic exchange between classic and attenuated strains of IBDV with two recombinant events occurring at the intermediate derivative segment A with vvIBDV strains, thereby increasing the difficulty of prevention and control of IBD due to novel reassortant-recombinant strains.

Avian Metapneumovirus Subgroup C Induces Mitochondrial Antiviral Signaling Protein Degradation through the Ubiquitin-Proteasome Pathway.

The mitochondrial antiviral signaling (MAVS) protein, a critical adapter, links the upstream recognition of viral RNA to downstream antiviral signal transduction. However, the interaction mechanism between avian metapneumovirus subgroup C (aMPV/C) infection and MAVS remains unclear. Here, we confirmed that aMPV/C infection induced a reduction in MAVS expression in Vero cells in a dose-dependent manner, and active aMPV/C replication was required for MAVS decrease. We also found that the reduction in MAVS occurred at the post-translational level rather than at the transcriptional level. Different inhibitors were used to examine the effect of proteasome or autophagy on the regulation of MAVS. Treatment with a proteasome inhibitor MG132 effectively blocked MAVS degradation. Moreover, we demonstrated that MAVS mainly underwent K48-linked ubiquitination in the presence of MG132 in aMPV/C-infected cells, with amino acids 363, 462, and 501 of MAVS being pivotal sites in the formation of polyubiquitin chains. Finally, E3 ubiquitin ligases for MAVS degradation were screened and identified and RNF5 targeting MAVS at Lysine 363 and 462 was shown to involve in MAVS degradation in aMPV/C-infected Vero cells. Overall, these results reveal the molecular mechanism underlying aMPV/C infection-induced MAVS degradation by the ubiquitin-proteasome pathway.