Type III interferons (IFNs) play a vital role in maintaining the antiviral state of the mucosal epithelial surface in the gut, and in turn, enteric viruses may have evolved to evade the type III IFN responses during infection. To study the possible immune evasion of the type III IFN response by porcine epidemic diarrhea virus (PEDV), a line of porcine intestinal epithelial cells was developed as a cell model for PEDV replication. IFN-λ1 and IFN-λ3 inhibited PEDV replication, indicating the anti-PEDV activity of type III IFNs. Of the 21 PEDV proteins, nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, open reading frame 3 (ORF3), E, M, and N were found to suppress type III IFN activities, and IRF1 (interferon regulatory factor 1) signaling mediated the suppression. PEDV specifically inhibited IRF1 nuclear translocation. The peroxisome is the innate antiviral signaling platform for the activation of IRF1-mediated IFN-λ production, and the numbers of peroxisomes were found to be decreased in PEDV-infected cells. PEDV nsp1 blocked the nuclear translocation of IRF1 and reduced the number of peroxisomes to suppress IRF1-mediated type III IFNs. Mutational studies showed that the conserved residues of nsp1 were crucial for IRF1-mediated IFN-λ suppression. Our study for the first time provides evidence that the porcine enteric virus PEDV downregulates and evades IRF1-mediated type III IFN responses by reducing the number of peroxisomes. Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that emerged in swine in the United States and has caused severe economic losses. PEDV targets intestinal epithelial cells in the gut, and intestinal epithelial cells selectively induce and respond to the production of type III interferons (IFNs). However, little is known about the modulation of the type III IFN response by PEDV in intestinal epithelial cells. In this study, we established a porcine intestinal epithelial cell model for PEDV replication. We found that PEDV inhibited IRF1-mediated type III IFN production by decreasing the number of peroxisomes in porcine intestinal epithelial cells. We also demonstrated that the conserved residues in the PEDV nsp1 protein were crucial for IFN suppression. This study for the first time shows PEDV evasion of the type III IFN response in intestinal epithelial cells, and it provides valuable information on host cell-virus interactions not only for PEDV but also for other enteric viral infections in swine.

Type I interferons (IFN-α/β) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11. Since nsp11 is an endoribonuclease (EndoU), the structure function relationship was examined using a series of nsp11 EndoU mutant plasmids. The mutants that impaired the EndoU activity failed to suppress IFN and led to the normal expression of MAVS. Seven single amino acid substitutions (4 in subdomain A and 3 in subdomain B) plus one insertion (frame-shift in nsp11) were then introduced into PRRSV infectious cDNA clones to generate nsp11 mutant viruses. Unfortunately, all EndoU knock-out nsp11 mutant viruses appeared replication-defective and no progenies were produced. Three mutations in EndoU subdomain A expressed the N and nsp2/3 proteins but their infectivity diminished after 2 passages. Taken together, our data show that PRRSV nsp11 endoribonuclease activity is critical for both viral replication and IFN antagonism. More importantly, the endoribonuclease activity of nsp11 demonstrates the substrate specificity towards MAVS and RIG-I (transcripts and proteins) over p65 and IRF3 in the context of gene transfection and overexpression. This is likely a mechanism of nsp11 suppression of type I IFN production.

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....

The contribution of heritable factors to antibody function and diversity is not fully understood, but has profound implications for delineating variation in the antibody response observed at the population-level. We performed matched long-read-based characterization of the immunoglobulin heavy chain (IGH) locus and expressed antibody repertoire profiling at population-scale to examine, for the first time, the impact of IGH genomic variation on the antibody repertoire. We characterized extensive IGH polymorphism, including novel structural variants (SVs), small insertion/deletions (indels), single nucleotide variants (SNVs), and IG genes and alleles. Countering models that antibody repertoire diversity is driven largely by stochastic processes, we demonstrate that IGH genetic factors make significant contributions to gene usage in both the naive and antigen-experienced repertoire. Specifically, the usage of 73% of IGH genes was associated with common polymorphisms, including those capable of explaining >70% of variance in gene usage. These variants were enriched in transcription factor binding sites and other functional elements associated with V(D)J recombination, and overlapped polymorphisms from genome-wide association studies. Furthermore, we found evidence for the coordinated regulation of IGH genes across the repertoire, demonstrating complex interactions between IGH variants and gene usage. These results refine our understanding of variation observed in the antibody repertoire, and will advance the study of antibody function in disease.