Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1ß to promote viral replication by regulating K48 ubiquitination.

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1ß (Nsp1ß) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1ß protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (Nsp1ß) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1ß, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1ß, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Fully Attenuated meq and pp38 Double Gene Deletion Mutant Virus Confers Superior Immunological Protection against Highly Virulent Marek's Disease Virus Infection.

Marek's disease virus (MDV) induces immunosuppression and neoplastic disease in chickens. The virus is controllable via an attenuated meq deletion mutant virus, which has the disadvantage of retaining the ability to induce lymphoid organ atrophy. To overcome this deficiency and produce more vaccine candidates, a recombinant MDV was generated from the highly virulent Md5BAC strain, in which both meq and a cytolytic replication-related gene, pp38, were deleted. Replication of the double deletion virus, Md5BAC ΔmeqΔpp38, was comparable with that of the parental virus in vitro. The double deletion virus was shown to be fully attenuated and to reduce lymphoid organ atrophy in vivo. Crucially, Md5BAC ΔmeqΔpp38 confers superior protection against highly virulent virus compared with a commercial vaccine strain, CVI988/Rispens. Transcriptomic profiling indicated that Md5BAC ΔmeqΔpp38 induced a different host immune response from CVI988/Rispens. In summary, a novel, effective, and safe vaccine candidate for prevention and control of MD caused by highly virulent MDV is reported. IMPORTANCE MDV is a highly contagious immunosuppressive and neoplastic pathogen. The virus can be controlled through vaccination via an attenuated meq deletion mutant virus that retains the ability to induce lymphoid organ atrophy. In this study, we overcame the deficiency by generating meq and pp38 double deletion mutant virus. Indeed, the successfully generated meq and pp38 double deletion mutant virus had significantly reduced replication capacity in vivo but not in vitro. It was fully attenuated and conferred superior protection efficacy against very virulent MDV challenge. In addition, the possible immunological protective mechanism of the double deletion mutant virus was shown to be different from that of the gold standard MDV vaccine, CVI988/Rispens. Overall, we successfully generated an attenuated meq deletion mutant virus and widened the range of potential vaccine candidates. Importantly, this study provides for the first time the theoretical basis of vaccination induced by fully attenuated gene-deletion mutant virus.

Pseudorabies Virus Tegument Protein UL13 Suppresses RLR-Mediated Antiviral Innate Immunity through Regulating Receptor Transcription.

Pseudorabies virus (PRV) has evolved various strategies to escape host antiviral immune responses. However, it remains unclear whether and how PRV-encoded proteins modulate the RIG-I-like receptor (RLR)-mediated signals for immune evasion. Here, we show that the PRV tegument protein UL13 functions as an antagonist of RLR-mediated antiviral responses via suppression of the transcription of RIG-I and MDA5, but not LGP2. UL13 overexpression significantly inhibits both the mRNA and protein levels of RIG-I and MDA5, along with RIG-I- or MDA5-mediated antiviral immune responses, whereas overexpression of RIG-I or MDA5 counteracts such UL13-induced suppression. Mechanistically, UL13 suppresses the expression of RIG-I and MDA5 by inhibiting activation of the transcription factor NF-κB. Consequently, overexpression of p65 promotes the activation of RIG-I and MDA5 promoters. Moreover, deletion of the p65-binding sites in the promoters of RIG-I or MDA5 abolishes the suppression role of UL13. As a result, mutant PRV lacking UL13 elicits stronger host antiviral immune responses than PRV-WT. Hence, our results provide a novel functional role of UL13-induced suppression of host antiviral immunity through modulating receptors' transcription.

Pseudorabies virus tegument protein UL13 recruits RNF5 to inhibit STING-mediated antiviral immunity.

Pseudorabies virus (PRV) has evolved various immune evasion mechanisms that target host antiviral immune responses. However, it is unclear whether and how PRV encoded proteins modulate the cGAS-STING axis for immune evasion. Here, we show that PRV tegument protein UL13 inhibits STING-mediated antiviral signaling via regulation of STING stability. Mechanistically, UL13 interacts with the CDN domain of STING and recruits the E3 ligase RING-finger protein 5 (RNF5) to promote K27-/K29-linked ubiquitination and degradation of STING. Consequently, deficiency of RNF5 enhances host antiviral immune responses triggered by PRV infection. In addition, mutant PRV lacking UL13 impaired in antagonism of STING-mediated production of type I IFNs and shows attenuated pathogenicity in mice. Our findings suggest that PRV UL13 functions as an antagonist of IFN signaling via a novel mechanism by targeting STING to persistently evade host antiviral responses.

Natural infection of a variant pseudorabies virus leads to bovine death in China.

Pseudorabies virus (PRV) infects numerous species of domestic and wild animals leading to severe diseases especially in swine and cattle. Since 2011, the variant PRVs were identified in pigs, which were genetically different from classic strains. Although variant PRV infection is widely observed in pigs, there is still no report of variant PRV infection in cattle. Here, we reported a natural infection of variant PRV leading to acute bovine death in Eastern China. Our study suggests that the new variant PRV strains could be a potential threat to cattle industry and possibly to the public health of human.

Newcastle Disease Virus Infection Interferes With the Formation of Intestinal Microflora in Newly Hatched Specific-Pathogen-Free Chicks.

Newcastle disease virus (NDV) infection leads to disproportion of intestinal tract microbiol population in chickens. Whether vertical infection of NDV affects the formation of a healthy and diverse intestinal community in newly hatched chicks, which might further perturb the establishment of a normal intestinal mucosal immunity, is unclear. This study examined the effects of NDV infection of chick embryos on the formation of the intestinal microbiome of chicks at hatch using 16S rRNA genes pyrosequencing. Eleven-day-old specific-pathogen-free chicken eggs were inoculated via intra-allantoic way with Class I NDV strain. At hatch, chicks were randomly selected and their duodenal and cecal contents were extracted and examined for the composition of gut microflora by Illumina sequencing of the V3+V4 region of the 16S rRNA genes. The results showed that the duodenal flora possesses a greater sample richness and higher microbial diversity as compared with the ceca flora in newly hatched chicks. In addition, there is a clear association with loss of important bacterial population in concert with an enrichment of potentially pathogenic population and NDV infections, both in the duodenum and ceca. It is also increasingly observed that the NDV infection may be associated with the dysbiosis of gut flora. This study presented a profile of the early intestinal microbiota in specific-pathogen-free chicks at hatch and strongly indicates that NDV infection interferes with the formation of intestinal microbiome in newly hatched chicks.

Characterization of duck (Anas platyrhynchos) MHC class I gene in two duck lines.

To enrich gene polymorphism ofDuMHCI and provide data for further studies on disease resistance, 14DuMHCI genes from Weishan Ma duck and Cherry Valley duck were cloned, and their characterization were investigated. The overall conservation of the 14 alleles could be observed within the sequences, and relative conservation were also displayed in the peptide-binding domain and CD8 interaction sites. Based on full-length amino acid homology, MHC class I from different duck lines could be divided into 13 gene groups and three novel gene groups existed.Moreover, 14 key variable residues corresponding to gene groups division were exhibited on the homology modelling constructed based on the resolved protein structure of DuMHC I. This study explicit the characteristics of DuMHC I in the two duck lines and could contribute to design effective diagnostics and vaccines for the species against various infections.

Exploration of the BF2*15 major histocompatibility complex class I binding motif and identification of cytotoxic T lymphocyte epitopes from the H5N1 influenza virus nucleoprotein in chickens.

The binding motif of BF2*15 major histocompatibility complex (MHC) class I was explored by analyzing the interaction between an infectious bronchitis virus octapeptide and BF2*15, and the cytotoxic T lymphocyte (CTL) epitope from the nucleoprotein (NP) of H5N1 virus was identified using experimental methods. Computational methods, including homology modeling, molecular dynamics simulation, and molecular docking analysis, were used. The recombinant plasmid pCAGGS-NP was constructed, and NP expression was confirmed by indirect immunofluorescence and Western blot in transfected 293T cells. Antibodies against NP in pCAGGS-NP-inoculated specific-pathogen-free chickens were detected by enzyme-linked immunosorbent assay (ELISA). Interferon γ (IFN-γ) mRNA was quantified, and IFN-γ production was evaluated using quantitative reverse transcription PCR and capture ELISA, respectively. CD8(+) T-lymphocyte proliferation was detected using flow cytometric analysis. The BF2*15 MHC class I binding motif "x-Arg/Lys-x-x-x-Arg/Lys" was explored. Quantification of chicken IFN-γ mRNA, evaluation of IFN-γ production, and measurement of CD8(+) T-lymphocyte proliferation confirmed that the peptide NP67-74 of H5N1 was the BF2*15 MHC-class-I-restricted CTL epitope.

Interactive mechanism between avian infectious bronchitis S1 protein T cell peptide and avian MHC I molecule.

This study aims to construct a 3D structure of the avian major histocompatibility complex (MHC)-ß2M complex through homology modelling technology, perform molecular docking of the predicted infectious bronchitis virus (IBV) S1 protein potential epitope peptide Sp6 (NQFYIKLT) and the avian MHC-ß2M complex, and demonstrate the interactive mechanism between Sp6 and MHC using molecular dynamical simulations. The peptide Sp6 and the non-related peptide NP89-97 (PKKTGGPIY) were used to stimulate in vitro recombinant plasmid (pCAGGS-S1) avian splenic lymphocytes. Flow cytometric results show that CD8(+) T lymphocytes reproduce stimulated by the Sp6 and the nonrelated peptide proliferate by 34.8% and 2.6%, respectively. Meanwhile, fluorescent quantitative PCR results show that the secretion of IFN-γ in avian splenic lymphocytes increases after Sp6 stimulation. These data suggest that Sp6 can induce the activated avian lymphocytes in vitro to produce CTL, which is the CTL epitope in IBV S1.