Genome-Wide mRNA and Long Non-Coding RNA Analysis of Porcine Trophoblast Cells Infected with Porcine Reproductive and Respiratory Syndrome Virus Associated with Reproductive Failure.

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.

Establishment of a Real-Time Quantitative PCR Assay for Porcine Circovirus-Like Virus and the First Evidence of Its Spread to Hainan and Jiangxi Provinces of China.

Porcine Circovirus-like (PCL) virus, a new emerging virus, has been widely detected in Guangdong, Guangxi, and Anhui provinces in China, which may be a novel agent causing severe diarrhea in newborn piglets and tending to spread widely. Evidence suggests that the virus is related to hemorrhagic enteritis and diarrhea, and many newborn piglets were emaciated to death after infection. Therefore, a sensitive, quick, and accurate detection system for virus detection and epidemiological investigation is necessary. In this study, we developed a real-time quantitative PCR assay based on SYBR green for the detection of PCL virus. The ORF4 conserved region of PCL virus was found by the alignment of the uploaded genome sequences to design specific primers, and the primers were tested and showed good specificity, sensitivity, and reproducibility. Approximately, 138 fecal samples were obtained from diarrheal pigs in South China from June to December 2021. Approximately, 22.46% (31/138) of the samples and 40% (8/20) of the pig farms were positive for PCL virus, respectively, by using this method. Moreover, it is worth noting that the virus was first detected in Hainan and Jiangxi Provinces of China, which means that the virus may spread widely in China. Through evolutionary tree analysis and partial sequence comparison, there are some differences of virus genes in each province, suggesting that there is a risk of variation, and the four PCL virus strains showed a sequence similarity of 86.7%-87.8% for the rep gene and 92.2%-92.9% for the Rep protein, respectively, with Bo-Circo-like virus that is detected in bovine, which further demonstrates a close relationship between the two viruses that originated from different animals. In conclusion, our study provides a useful diagnostic approach to PCL virus detection and epidemiological inquiry. Meanwhile, the epidemic data using this real-time qPCR assay provide evidence for the widespread variations and epidemic of the virus in South China, and warn the appropriate measures for prevention, and control of porcine circovirus-like virus infection should be under consideration in pig production.

A strain of highly pathogenic porcine reproductive and respiratory syndrome virus: genomic characterization, pathogenicity, and construction of an infectious full-length cDNA clone.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which inflicts major economic losses on the global pig farming industry. Based on its similarity to highly pathogenic strains, the GDzj strain isolated in this study was predicted to be highly pathogenic. We therefore analyzed the pathogenicity of this strain experimentally in piglets. All piglets challenged with this virus experienced fever or high fever, loss of appetite, decreased food intake, daily weight loss, shortness of breath, and listlessness, and the necropsy results showed that they had experienced severe interstitial pneumonia. We then used the BAC system to construct a full-length cDNA infectious clone of GDzj, and the rescued virus displayed in vitro proliferation characteristics similar to those of the parental PRRSV strain. In summary, we successfully isolated a highly pathogenic PRRSV strain and constructed a full-length infectious cDNA clone from it, thereby providing an effective reverse genetics platform for further study of viral pathogenesis.

Molecular Characteristics and Pathogenicity of Porcine Epidemic Diarrhea Virus Isolated in Some Areas of China in 2015-2018.

Since 2010, Porcine epidemic diarrhea virus (PEDV) has caused severe diarrhea disease in piglets in China, resulting in large economic losses. To understand the genetic characteristics of the PEDV strains that circulated in some provinces of China between 2015 and 2018, 375 samples of feces and small intestine were collected from pigs and tested. One hundred seventy-seven samples tested positive and the PEDV-positive rate was 47.20%. A phylogenetic tree analysis based on the entire S gene showed that these strains clustered into four subgroups, GI-a, GI-b, GII-a, and GII-b, and that the GII-b strains have become dominant in recent years. Compared with previous strains, these strains have multiple variations in the SP and S1-NTD domains and in the neutralizing epitopes of the S protein. We also successfully isolated and identified a new virulent GII-b strain, GDgh16, which is well-adapted to Vero cells and caused a high mortality rate in piglets in challenge experiments. Our study clarifies the genetic characteristics of the prevalent PEDV strains in parts of China, and suggests that the development of effective novel vaccines is both necessary and urgent.

Porcine Circovirus Type 3 Cap Inhibits Type I Interferon Induction Through Interaction With G3BP1.

Porcine circovirus 3 (PCV3) infections cause clinical diseases similar to those seen in porcine circovirus 2 (PCV2) infections. It is unclear whether PCV3 infections can also cause immunosuppression like that seen with PCV2. Here, we report that Cap inhibits DNA-induced IFN-ß mRNA transcription and IFN promoter activation. Cap was also found to inhibit cyclic GMP-AMP (cGAMP) synthase (cGAS) binding to interferon-stimulating DNA (ISD). Immunoprecipitation and mass spectrometry were used to identify cellular interaction partners of Cap. Cap interacted with G3BP1 and inhibited the interaction between GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) and cGAS. Furthermore, the destruction of endogenously expressed G3BP1 by siRNA significantly reduced IFN promoter activation, and phosphorylation of tank-binding kinase 1 (TBK1) was induced by ISD. Overexpression of G3BP1 attenuated the inhibition of ISD binding of cGAS by Cap and promoted phosphorylation of TBK1 and IRF3 induced by ISD. Collectively, our results show that the interaction between Cap and G3BP1 prevents cGAS from recognizing DNA, thereby inhibiting the IFN production.

Porcine circovirus 3 Cap inhibits type I interferon signaling through interaction with STAT2.

Porcine circovirus 3 (PCV3) is a novel circovirus that is associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multi-systemic inflammation. The type I Interferon (IFN) signaling pathway is an important innate immune signaling pathway for defense against viral infection. Many mammalian viruses inhibit host innate immune signaling through diverse strategies. Here, we found that the PCV3 capsid protein (Cap) significantly inhibited IFN-ß-stimulated response element (ISRE) promoter activity, suggesting that Cap suppresses IFN signaling. However, Cap did not affect expression and phosphorylation levels of STAT1 and STAT2 and did not interrupt the heterodimerization of pSTAT1 and pSTAT2. Although Cap interacted with KPAN1, it did not block the interaction between KPNA1 and pSTAT1 or the nuclear translocation of pSTAT1 and pSTAT2. Interestingly, we found that Cap inhibited the activation of ISRE promoter induced by IRF9-S2C. Mechanistically, Cap interacted with the transactivation domain of STAT2, a key protein in type I IFN signaling. In addition, we found that Cap bound to ISRE and prevented ISRE binding of IRF9-S2C. This work is the first to describe the mechanism of inhibition of IFN signaling by PCV3 Cap.