Eukaryotic circular Rep encoding single-stranded DNA (CRESS DNA) viruses exhibit high genomic diversity. Four species of circoviruses within the genus Circovirus have been identified in pigs, including the non-pathogenic porcine circovirus type 1 (PCV1), and the pathogenic PCV2, PCV3, and PCV4. In this study, a novel single circular DNA virus was identified in pigs suffering from respiratory, diarrheal, and reproductive failure diseases. The viral Rep and Cap showed low amino acid sequence identities to the four PCV species, with the highest identity of 26.2% to PCV2 Rep and 20.8% to PCV4 Cap. The classification of CRESS DNA viruses primarily relies on Rep, and biochemical analyses of the viral Rep confirm its classification as a CRESS DNA virus. Thus, it was tentatively named porcine circovirus type 5 (PCV5). This tentative designation reflects its host and genomic organization; however, phylogenetic analysis indicates that it clusters outside the family Circoviridae and is most closely related to the fur seal faces-associated circular DNA virus (FSfaCV). The positive detection rate for PCV5 in tested samples was 54.4% (294 out of 540), with 24.3% (17 out of 70) of pig farms in China testing positive for the virus. A close correlation between the copy number of PCV5 and the severity of infectious diseases was observed. Moreover, a PCV5 virus-like particles (VLPs)-based enzyme-linked immunosorbent assay, which was developed, also elucidated that PCV5 has been circulating. We subsequently established an in vitro culture system for PCV5, successfully purifying the virus. Morphological identification revealed that the purified PCV5 has a diameter of approximately 20 nm, and the recombinant PCV5 Cap could assemble into VLPs similar to purified PCV5. These results indicate that PCV5, associated with porcine circovirus-associated disease, commonly circulates within Chinese swine. IMPORTANCE: The single-stranded circular DNA virus, tentatively named porcine circovirus type 5 (PCV5), belongs to the circular Rep encoding single-stranded DNA (CRESS DNA) viruses. Notably, PCV5 is not classified within the same family as the previously reported PCV1–PCV4. PCV5, which was associated with porcine circovirus associated-disease, is widely prevalent in Southern China. PCV5 is characterized by a larger genome (2,904 nt), along with longer Cap (1,182 nt) and Rep (1,056 nt) regions, as well as an extended stem-loop structure (stem: 9 nt and loop: 17 nt). Importantly, we subsequently established an in vitro culture system for PCV5, successfully purifying the virus. Morphological identification revealed that the purified PCV5 has a diameter of approximately 20 nm. Additionally, a variant of the PCV5 Cap, lacking the N-terminal 63 residues, was expressed in Escherichia coli , and purified Cap could assemble into virus-like particles similar to purified PCV5. These results hold significant implications for the study of CRESS DNA viruses. The emergence of PCV5 warrants further study.

Porcine reproductive and respiratory syndrome (PRRS) is a vertically transmitted reproductive disorder that is typically characterized by miscarriage, premature birth, and stillbirth in pregnant sows after infection. Such characteristics indicate that PRRSV can infect and penetrate the porcine placental barrier to infect fetus piglets. The porcine trophoblast is an important component of the placental barrier, and secretes various hormones, including estrogen and progesterone, to maintain normal pregnancy and embryonic development during pregnancy. It is conceivable that the pathogenic effects of PRRSV infection on porcine trophoblast cells may lead to reproductive failure; however, the underlying detailed mechanism of the interaction between porcine trophoblast (PTR2) cells and PRRSV is unknown. Therefore, we conducted genome-wide mRNA and long non-coding RNA (lncRNA) analysis profiling in PRRSV-infected PTR2. The results showed that 672 mRNAs and 476 lncRNAs were significantly different from the control group after viral infection. Target genes of the co-expression and co-location of differential mRNAs and lncRNAs were enriched by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, revealing that most of the pathways were involved in cell nutrient metabolism, cell proliferation, and differentiation. Specifically, the estrogen signaling pathway, the PI3K (PhosphoInositide-3 Kinase)-Akt (serine/threonine kinase) signaling pathway, and the insulin secretion related to embryonic development were selected for analysis. Further research found that PRRSV inhibits the expression of G-protein-coupled estrogen receptor 1 (GPER1), thereby reducing estrogen-induced phosphorylation of AKT and the mammalian target of rapamycin (mTOR). The reduction in the phosphorylation of AKT and mTOR blocks the activation of the GPER1- PI3K-AKT-mTOR signaling pathway, consequently restraining insulin secretion, impacting PTR2 cell proliferation, differentiation, and nutrient metabolism. We also found that PRRSV triggered trophoblast cell apoptosis, interrupting the integrity of the placental villus barrier. Furthermore, the interaction network diagram of lncRNA, regulating GPER1 and apoptosis-related genes, was constructed, providing a reference for enriching the functions of these lncRNA in the future. In summary, this article elucidated the differential expression of mRNA and lncRNA in trophoblast cells infected with PRRSV. This infection could inhibit the PI3K-AKT-mTOR pathway and trigger apoptosis, providing insight into the mechanism of the vertical transmission of PRRSV and the manifestation of reproductive failure.

Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic losses to the global swine industry. Previously, researchers have believed that porcine aminopeptidase-N (pAPN) was the primary receptor for PEDV, but it has been found that PEDV can infect pAPN knockout pigs. Currently, the functional receptor for PEDV remains unspecified. In the present study, we performed virus overlay protein binding assay (VOPBA), found that ATP1A1 was the highest scoring protein in the mass spectrometry results, and confirmed that the CT structural domain of ATP1A1 interacts with PEDV S1. First, we investigated the effect of ATP1A1 on PEDV replication. Inhibition of hosts ATP1A1 protein expression using small interfering RNA (siRNAs) significantly reduced the cells susceptibility to PEDV. The ATP1A1-specific inhibitors Ouabain (a cardiac steroid) and PST2238 (a digitalis toxin derivative), which specifically bind ATP1A1, could block the ATP1A1 protein internalization and degradation, and consequently reduce the infection rate of host cells by PEDV significantly. Additionally, as expected, overexpression of ATP1A1 notably enhanced PEDV infection. Next, we observed that PEDV infection of target cells resulted in upregulation of ATP1A1 at the mRNA and protein levels. Furthermore, we found that the host protein ATP1A1 was involved in PEDV attachment and co-localized with PEDV S1 protein in the early stage of infection. In addition, pretreatment of IPEC-J2 and Vero-E6 cells with ATP1A1 mAb significantly reduced PEDV attachment. Our observations provided a perspective on identifying key factors in PEDV infection, and may provide valuable targets for PEDV infection, PEDV functional receptor, related pathogenesis, and the development of new antiviral drugs.

The continuous emergence of circular Rep‐encoding single‐stranded (CRESS) DNA viruses across diverse hosts has been closely associated with the occurrence of severe diseases. Four circoviruses within the genus Circovirus have been identified in pigs, including porcine circovirus Type 1 (PCV1), PCV2, PCV3, PCV4, and PCV5. In late 2021, a large pig farm experienced an outbreak of reproductive disorders that were undiagnosed by standard tests. Subsequent viral metagenomic analysis of stillborn piglets identified a novel single‐stranded circular DNA virus, designated porcine megalocircovirus (PMCV). PMCV has a large genome of 9426 nt and encodes nine open reading frames. Biochemical analyses of Rep confirm PMCV as a CRESS DNA virus. However, PMCV Rep showed low amino acid sequence identities to the four PCV species and several human CRESS DNA viruses, with the highest identity of 23.6% to PCV4 Rep. The genetic evolutionary tree indicates that PMCV belongs to an unknown family of the CRESS DNA viruses. The positive detection rate for PMCV in tested samples was 24% (30/125), while the positive rate regarding pig farms was 41.18% (14/34) in China. The emergence of PMCV warrants further investigation.

Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus, is primarily recognized as an enteric pathogen causing severe gastrointestinal disease in newborn piglets. PEDV has been endemic in most parts of the world, leading to high mortality in neonatal piglets and causing massive economic losses to the pig industry. However, reproductive disorders have been repeatedly observed in sows following PEDV outbreaks, and the underlying mechanisms remain poorly understood. Here, we provide evidence that PEDV can infect the porcine uterus in vivo and exhibits a pronounced cellular tropism toward uterine stromal cells. Using single-cell RNA sequencing, immunofluorescence analyses, and primary cell infection models, we show that PEDV-derived viral transcripts and proteins are preferentially detected in stromal cells, whereas epithelial and other uterine cell populations display minimal viral signals. PEDV efficiently replicates in primary porcine uterine endometrial cells in a trypsin-independent manner, indicating tissue-specific differences in viral entry requirements. Transcriptomic profiling reveals that PEDV infection is associated with extensive inflammatory activation, disruption of extracellular matrix organization, and altered intercellular communication. Notably, stromal cells emerge as a central signaling hub within the infected primary uterine endometrial cell culture system and likely within the uterine microenvironment as well. Correlation analyses further suggest that PEDV infection is linked to stromal cell-restricted expression patterns of candidate host factors rather than canonical enteric coronavirus receptors. Together, these findings redefine the cellular landscape of PEDV infection beyond the intestine and provide a mechanistic framework linking PEDV infection of uterine stromal cells to PEDV-associated reproductive disorders. IMPORTANCE: This study identifies uterine stromal cells as a novel target of porcine epidemic diarrhea virus (PEDV), breaking the long-held notion that PEDV is strictly enteric and expanding its known tissue tropism beyond the intestine. Leveraging single-cell RNA sequencing and multi-layered functional validation, it uncovers that PEDV replicates efficiently in uterine stromal cells via a trypsin-independent mechanism—distinct from its intestinal infection mode. This infection triggers inflammatory activation, extracellular matrix disruption, and altered intercellular communication, which are potential key contributors of PEDV-associated reproductive disorders plaguing the pig industry. These findings redefine alphacoronavirus pathogenesis and provide a critical mechanistic framework for developing targeted interventions. Additionally, this work highlights the value of single-cell approaches in unraveling extraintestinal viral infection, advancing both coronavirus biology research and veterinary disease control.