Transmissible gastroenteritis virus (TGEV) is a coronavirus associated with diarrhea and high mortality in piglets. To gain insight into the evolution and adaptation of TGEV, a comprehensive analysis of phylogeny and codon usage bias was performed. The phylogenetic analyses of maximum likelihood and Bayesian inference displayed two distinct genotypes: genotypes I and II, and genotype I was classified into subtypes Ia and Ib. The compositional properties revealed that the coding sequence contained a higher number of A/U nucleotides than G/C nucleotides, and that the synonymous codon third position was A/U-enriched. The principal component analysis based on the values of relative synonymous codon usage (RSCU) showed the genotype-specific codon usage patterns. The effective number of codons (ENC) indicated moderate codon usage bias in the TGEV genome. Dinucleotide analysis showed that CpA and UpG were over-represented and CpG was under-represented in the coding sequence of the TGEV genome. The analyses of Parity Rule 2 plot, ENC-plot, and neutrality plot displayed that natural selection was the dominant evolutionary driving force in shaping codon usage preference in genotypes Ia and II. In addition, natural selection played a major role, while mutation pressure had a minor role in driving the codon usage bias in genotype Ib. The codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses suggested that genotype I might be more adaptive to pigs than genotype II. Current findings contribute to understanding the evolution and adaptation of TGEV.

Porcine astrovirus (PAstV), associated with mild diarrhea and neurological disease, is transmitted in pig farms worldwide. The purpose of this study is to elucidate the main factors affecting codon usage to PAstVs. Phylogenetic analysis showed that the subtype PAstV-5 sat at the bottom of phylogenetic tree, followed by PAstV-3, PAstV-1, PAstV-2, and PAstV-4, indicating that the five existing subtypes (PAstV1-PAstV5) may be formed by multiple differentiations of PAstV ancestors. A codon usage bias was found in the PAstVs-2,3,4,5 from the analyses of effective number of codons (ENC) and relative synonymous codon usage (RSCU). Nucleotides A/U are more frequently used than nucleotides C/G in the genome CDSs of the PAstVs-3,4,5. Codon usage patterns of PAstV-5 are dominated by mutation pressure and natural selection, while natural selection is the main evolutionary force that affects the codon usage pattern of PAstVs-2,3,4. The analyses of codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) showed the codon usage similarities between the PAstV and animals might contribute to the broad host range and the cross-species transmission of astrovirus. Our results provide insight into understanding the PAstV evolution and codon usage patterns.

The innate immune DNA sensing cyclic GMP-AMP synthase (cGAS)–stimulator of IFN genes (STING) signaling pathway plays a key role in host antiviral function. Although the cGAS-STING pathway has been extensively studied, the cGAS-STING signaling in livestock and poultry is not well understood, and whether the species specificity exists is still unknown. In this study, we found that porcine and chicken STING, but not cGAS, exhibit species differences in regulation of IFN; that is, porcine (p)STING mediates good induction of IFN in mammalian cells and low IFN induction in chicken DF-1 cells; on the contrary, chicken (ch)STING mediates IFN induction only in chicken cells but not in mammalian cells. Furthermore, it was found that the motifs pLxIS of pSTING and pLxVS of chSTING are responsible for the species disparity, with the IFN activity of pSTING and chSTING exchanged by swapping the two pLxI/VS motifs. The pLxI/VS motifs mediated the interactions of various STING with downstream IFN regulatory factors (IRFs), reflecting the species-specific pIRF3 and chIRF7. Next, the STING, IRFs, and STING-IRFs were reconstituted in porcine and chicken macrophages that were genetically knocked out for STING and/or IRFs by the CRISPR-Cas9 approach. The results showed that pSTING plus pIRF3 or chIRF7 are able to induce IFN; however, chSTING plus chIRF7 but not pIRF3 are able to induce IFN, suggesting that pIRF3 is specific and stringent, which underlies the inability of chSTING to induce IFN in mammalian cells. In summary, our findings reveal the differential species specificity in the cGAS-STING pathway and the underlying mechanisms, thus providing valuable insights on the cGAS-STING-IRF signaling axis for comparative immunology.

Atypical porcine pestivirus (APPV) has been identified as the main causative agent for congenital tremor (CT) type A-II in piglets, which is threatening the health of the global swine herd. However, the evolution of APPV remains largely unknown. In this study, phylogenetic analysis showed that APPV could be divided into three phylogroups (I, II, and III). Phylogroups I and II included viral strains from China, while phylogroup III contained strains from Europe, North America, and Asia. Phylogroups I and II are tentatively thought to be of Chinese origin. Next, compositional properties analysis revealed that a high frequency of nucleotide A and A-end codons was used in the APPV genome. Intriguingly, the analysis of preferred codons revealed that the AGA[Arg] and AGG[Arg] were overrepresented. Dinucleotide CC was found to be overrepresented, and dinucleotide CG was underrepresented. Furthermore, it was found that the weak codon usage bias of APPV was mainly dominated by selection pressures versus mutational forces. The codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses showed that the codon usage patterns of phylogroup II and III were more similar to the one of pig than phylogroup I, suggesting that phylogroup II and III may be more adaptive to pigs. Overall, this study provides insights into APPV evolution through phylogeny and codon usage pattern analysis.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B–Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

Porcine deltacoronavirus (PDCoV) is an emerging virus that poses a significant threat to the global swine industry. Its membrane (M) protein is crucial for virion assembly and virus–host interactions. We selected the hydrophilic region of M protein for prokaryotic expression, purification, and recombinant protein production. Utilizing hybridoma technology, we prepared the monoclonal antibody (mAb) 24-A6 against M protein. The mAb 24-A6 was shown to be suitable for use in immunofluorescence assays, western blotting, and immunoprecipitation, with specificity for PDCoV and no cross-reactivity with other five porcine viruses. The M protein was observed to be expressed as early as 3 h after PDCoV infection, increasing its expression over the duration of infection. Notably, the antigenic epitope of the M protein identified as ¹⁰³SPESRL¹⁰⁸ recognized by mAb 24-A6 was found within a conserved structural domain (SWWSFNPETNNL) of the coronavirus M protein, indicating a crucial overlap between a functionally important viral assembly region and a region recognized by the immune system. Our findings provide valuable insights into mAb 24-A6 targeting the antigenic epitope of M protein and may contribute to the development of diagnostic tools for PDCoV infection and fundamental research into the function of PDCoV M protein.