Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2=-O-methyltransferase (2=-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE 4A , with quadruple alanine substitutions in the catalytic tetrad of the 2=-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE 4A -SYA, by abolishing the endocytosis signal of the spike protein of KDKE 4A . Compared with icPC22A, the KDKE 4A and KDKE 4A -SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE 4A -SYA and KDKE 4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE 4A -, and KDKE 4A -SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE 4A -SYA-and KDKE 4A -inoculated pigs were protected from the challenge, because no KDKE 4A -SYA-and one KDKE 4A -inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE 4A -SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE 4A -SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2=-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2=-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE 4A -SYA mutant. KDKE 4A -SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs. Downloaded fromthe endocytosis signal of the S protein, we showed that the KDKE 4A -SYA mutant was more attenuated and induced better protection than KDKE 4A against the virulent-PEDV challenge. We also confirmed that the introduced mutations in the KDKE 4A -SYA genome were stable after passaging the mutant virus in Gn pigs three times. Currently, both G1 and G2 PEDV strains are circulating among pigs in different countries....

Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1␤) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1␤, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1␤. The nsp1␤ L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1␤-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-␣/␤), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFNinduced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1␤, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1␤ protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1␤ binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1␤ binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.KEYWORDS Nuclear pore complex, nucleoporin, Nup62, arterivirus, nsp1, nucleocytoplasmic trafficking, porcine reproductive and respiratory syndrome virus P orcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that belongs to the family Arteriviridae in the order Nidovirales (https://talk.ictvonline .org/taxonomy). The PRRSV genome is a single-stranded positive-sense RNA approximately 15 kb in length with a 5= cap and a 3= polyadenylated tail and thus is translated FIG 8 Nup62 binding by nsp1 subunits of different arteriviruses and nuclear rim disintegration. (A) HeLa cells were cotransfected with the HA-Nup62 gene together with the indicated plasmids...

Porcine reproductive and respiratory syndrome virus (PRRSV) activates NF-κB during infection. We examined the ability of all 22 PRRSV genes for NF-κB regulation and determined the nucleocapsid (N) protein as the NF-κB activator. Protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1) (PIAS1) was identified as a cellular protein binding to N. PIAS1 is known to bind to p65 (RelA) in the nucleus and blocks its DNA binding, thus functions as a repressor of NF-κB. Binding of N to PIAS1 released p65 for NF-κB activation. The N-terminal half of PIAS1 was mapped as the N-binding domain, and this region overlapped its p65-binding domain. For N, the region between 37 and 72 aa was identified as the binding domain to PIAS1, and this domain alone was able to activate NF-κB. A nuclear localization signal (NLS) knock-out mutant N did not activate NF-κB, and this is mostly likely due to the lack of its interaction with PIAS1 in the nucleus, demonstrating the positive correlation between the binding of N to PIAS1 and the NF-κB activation. Our study reveals a role of N in the nucleus for NF-κB activation and proinflammatory cytokine production during infection.

BackgroundWhether or not a single-dose Ad26.COV2.S prime and boost vaccination induces sufficient immunity is unclear. Concerns about the increased risk of breakthrough infections in the Ad26.COV2.S-primed population have also been raised.MethodsA prospective cohort study was conducted. Participants included healthy adults who were Ad26.COV2.S primed and scheduled to receive a booster vaccination with BNT162b2, mRNA-1273, or Ad26.COV2.S. The IgG anti-receptor binding domain (RBD) antibody titers, neutralizing antibody (NAb) titers (against wild type [WT] and Omicron [BA.1 and BA.5]), and Spike-specific interferon-γ responses of the participants were estimated at baseline, 3–4 weeks, 3 months, and 6 months after booster vaccination.ResultsA total of 89 participants were recruited (26 boosted with BNT162b2, 57 with mRNA-1273, and 7 with Ad26.COV2.S). The IgG anti-RBD antibody titers of all participants were significantly higher at 6 months post-vaccination than at baseline. The NAb titers against WT at 3 months post-vaccination were 359, 258, and 166 in the participants from the BNT162b2-, mRNA-1273-, and Ad26.COV2.S-boosted groups, respectively. Compared with those against WT, the NAb titers against BA.1/BA.5 were lower by 23.9/10.9-, 16.6/7.4-, and 13.8/7.2-fold in the participants from the BNT162b2-, mRNA-1273-, and Ad26.COV2.S-boosted groups, respectively, at 3 months post-vaccination. Notably, the NAb titers against BA.1 were not boosted after Ad26.COV2.S vaccination. Breakthrough infections occurred in 53.8%, 62.5%, and 42.9% of the participants from the BNT162b2-, mRNA-1273-, and Ad26.COV2.S-boosted groups, respectively. No significant difference in humoral and cellular immunity was found between individuals with and without SARS-CoV-2 breakthrough infections.ConclusionBooster vaccination elicited acceptable humoral and cellular immune responses in Ad26.COV2.S-primed individuals. However, the neutralizing activities against Omicron subvariants were negligible, and breakthrough infection rates were remarkably high at 3 months post-booster vaccination, irrespective of the vaccine type. A booster dose of a vaccine containing the Omicron variant antigen would be required.

Background Several genome-wide association studies (GWAS) have been performed to identify variants related to chronic diseases. Somatic variants in cancer tissues are associated with cancer development and prognosis. Expression quantitative trait loci (eQTL) and methylation QTL (mQTL) analyses were performed on chronic disease-related variants in TCGA dataset. Methods MuTect2 calling variants for 33 carcinomas from TCGA and 296 GWAS variants provided by LocusZoom were used. At least one mutation was found in TCGA 22 carcinomas and LocusZoom 23 studies. Differentially expressed genes (DEGs) and differentially methylated regions (DMRs) from the three carcinomas (TCGA-COAD, TCGA-STAD, and TCGA-UCEC). Variants were mapped to the world map using population locations of the 1000 Genomes Project (1GP) populations. Decision tree analysis was performed on the discovered features and survival analysis was performed according to the cluster. Results Based on the DEGs and DMRs with clinical data, the decision tree model classified seven and three nodes in TCGA-COAD and TCGA-STAD, respectively. A total of 11 variants were commonly detected from TCGA and LocusZoom, and eight variants were selected from the 1GP variants, and the distribution patterns were visualized on the world map. Conclusions Variants related to tumors and chronic diseases were selected, and their geological regional 1GP-based proportions are presented. The variant distribution patterns could provide clues for regional clinical trial designs and personalized medicine.