Supplemental L-arginine promotes hepatocyte proliferation and alters liver fatty acid metabolism in the late embryonic phase: an RNA-seq analysis.

The in ovo feeding (IOF) of L-arginine (L-Arg) to chick embryos is a viable method for improving early intestinal development, subsequently leading to an acceleration in growth rate during the posthatch stage. However, the liver, being the pivotal organ for energy metabolism in poultry, the precise effects and mechanisms of L-Arg on the liver development and metabolism remain unclear. To elucidate these, the present study injected 2 doses of L-Arg (10 mg/egg and 15 mg/egg) into the embryos of Hongyao chickens at 17.5 d of incubation, subsequently incubating them until d 19 for further analysis. IOF of 15 mg L-Arg/egg significantly increased the organ indices of liver and small intestine, as well as the duodenal villus height/crypt depth. RNA-Seq analysis of liver tissues showed that the metabolism of xenobiotics, amino acid metabolism, and the fatty acid metabolism were significantly enriched in L-Arg injection group. The core differentially expressed genes (DEGs) were primarily involved in cell proliferation and fatty acid metabolism. The CCK8 assays revealed that supplemental L-Arg significantly enhanced the proliferation of primary embryo hepatocytes and leghorn male hepatoma (LMH) cells. Upregulation of core DEGs, including HBEGF, HES4, NEK3, EGR1, and USP2, significantly promoted the proliferation of liver cells. Additionally, analysis of triglyceride and total cholesterol content, as well as oil red O staining, indicated that supplemental L-Arg effectively reduced lipid accumulation. Overall, L-Arg supplementation in late chick embryos may promote early liver and small intestine development by reducing liver lipid deposition and enhancing energy efficiency, necessitating further experimental validation. This study provides profound insights into the molecular regulatory network of L-Arg in promoting the development of chicken embryos. The identified DEGs that promote cell proliferation and lipid metabolism can serve as novel targets for further developing methods to enhance early development of chicken embryos.

Phylogenetic and Genetic Variation Analysis of Porcine Epidemic Diarrhea Virus in East Central China during 2020-2023.

Porcine epidemic diarrhea virus (PEDV) is a major causative pathogen of a highly contagious, acute enteric viral disease. This study evaluated the emergence of nine variants in Jiangsu and Anhui provinces of China from 2020 to 2023. S gene-based phylogenetic analysis indicated that three variants belong to the G1c subgroup, while the other six strains are clustered within the G2c subgroup. Recombination analyses supported that three variants of the G1c subgroup were likely derived from recombination of parental variants FR0012014 and a donor variant AJ1102. In addition, there are novel mutations on amino acid 141-148 and these likely resulted in changes in antigenicity in the three variants. These results illustrated that the study provides novel insights into the epidemiology, evolution, and transmission of PEDV in China.

Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus by Disrupting Virus Entry.

The porcine epidemic diarrhea virus (PEDV), belonging to the α-coronavirus, is the causative agent of porcine epidemic diarrhea (PED). Presently, protection from the existing PEDV vaccine is not effective. Therefore, anti-PEDV compounds should be studied. Berbamine (BBM), Fangchinoline (FAN), and (+)-Fangchinoline (+FAN), are types of bis-benzylisoquinoline alkaloids that are extracted from natural medicinal plants. These bis-benzylisoquinoline alkaloids have various biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, we found that BBM, FAN, and +FAN suppressed PEDV activity with a 50% inhibitory concentration of 9.00 µM, 3.54 µM, and 4.68 µM, respectively. Furthermore, these alkaloids can decrease the PEDV-N protein levels and virus titers in vitro. The time-of-addition assay results showed that these alkaloids mainly inhibit PEDV entry. We also found that the inhibitory effects of BBM, FAN, and +FAN on PEDV rely on decreasing the activity of Cathepsin L (CTSL) and Cathepsin B (CTSB) by suppressing lysosome acidification. Taken together, these results indicated that BBM, FAN, and +FAN were effective anti-PEDV natural products that prevented PEDV entry and may be considered novel antiviral drugs.

The economic impact of porcine reproductive and respiratory syndrome outbreak in four Chinese farms: Based on cost and revenue analysis.

The economic impact after the outbreak of porcine reproductive and respiratory syndrome (PRRS) has been proven to be tremendous for pig production worldwide. However, the economic impact of the disease is not well understood in China. In our previous study, we acquired and analyzed the main production data (the number of weaned piglets, health costs, delayed marketing age, etc.) from the management system before and after the PRRS outbreaks occurring in November 2014, March 2015, December 2016, and February 2017. This study aimed to analyze and quantify the economic losses of the four PRRS outbreaks in Chinese herds. A straightforward approach was used to calculate additional costs and decreased revenues based on the PRRS-induced production deficiencies by average cost-of-production indices calculated from annual estimates of costs between 2014 and 2017. The results showed that economic losses varied between ¥668.14 and ¥1004.43 per sow in breeding herds from the outbreaks to regain the basic performance, with an average of ¥822.75 per sow, and the mean costs in the fattening herds (including nursery pigs) were ¥601.62 per sow, ranging from ¥318.64 to ¥937.14. Overall, the economic impact of PRRS on the whole herd was ¥1424.37 per sow. The majority of the losses were due to the reduction in the number of weaned piglets for breeding herds, and the increased feed cost (occupying 44.88%) was the primary source of loss for fattening herds. Our study fills the gap in knowledge of PRRS economics in China, enriches the data for veterinary economics, and re-stresses the necessity for producers and veterinarians to control PRRS effectively.

Porcine Enteric Coronavirus PEDV Induces the ROS-ATM and Caspase7-CAD-γH2AX Signaling Pathways to Foster Its Replication.

DNA damage response (DDR) is an evolutionarily conserved mechanism by which eukaryotic cells sense DNA lesions caused by intrinsic and extrinsic stimuli, including virus infection. Although interactions between DNA viruses and DDR have been extensively studied, how RNA viruses, especially coronaviruses, regulate DDR remains unknown. A previous study showed that the porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the Coronaviridae family, induces DDR in infected cells. However, the underlying mechanism was unclear. This study showed that PEDV activates the ATM-Chk2 signaling, while inhibition of ATM or Chk2 dampens the early stage of PEDV infection. Additionally, we found that PEDV-activated ATM signaling correlates with intracellular ROS production. Interestingly, we showed that, unlike the typical γH2AX foci, PEDV infection leads to a unique γH2AX staining pattern, including phase I (nuclear ring staining), II (pan-nuclear staining), and III (co-staining with apoptotic bodies), which highly resembles the apoptosis process. Furthermore, we demonstrated that PEDV-induced H2AX phosphorylation depends on the activation of caspase-7 and caspase-activated DNAse (CAD), but not ATM-Chk2. Finally, we showed that the knockdown of H2AX attenuates PEDV replication. Taken together, we conclude that PEDV induces DDR through the ROS-ATM and caspase7-CAD-γH2AX signaling pathways to foster its early replication.

Production Performance of Four Pig Herds Infected With Porcine Reproductive and Respiratory Syndrome Using the "Load-Close-Exposure" Approach in China.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases causing enormous losses to pig producers all over the world. The intervention measure of "load-close-exposure" [interrupting the introduction of replacement pigs combined with whole-herd exposure to live PRRS virus (PRRSV)] has been widely used in North America and has shown wonderful outcomes in controlling PRRS in the field. In the present study, we performed analyses of the production performance of four herds acutely infected with PRRSV by adopting this measure for the first time in China. Our results showed that the development rate of gilts decreased by a mean of 8.56%, the farrowing rate of breeding sows decreased from 86.18 to 77.61%, the number of piglets born alive per sow decreased by a mean of 0.73 pigs, and the pre-weaning and post-weaning mortality of piglets increased by a mean of 2.74-4.97% compared to the parameters of 6 months before an outbreak. The time to PRRSV stability (TTS), defined as the time in weeks it took to produce PRRSV-negative pigs at weaning, is an important indicator of successful control of PRRSV. The median TTS among herds A, C, and D was 21.8 weeks (21.6 22.1 weeks). In herd B, TTS was 42.3 weeks, which could be explained by the double introduction of gilts. Our study suggests that the "load-close-exposure" strategy may be a good alternative for Chinese producers and veterinaries to control PRRS in the field.

Pseudorabies virus kinase UL13 phosphorylates H2AX to foster viral replication.

The DNA damage response (DDR) pathway is critical for maintaining genomic integrity and sustaining organismal development. Viruses can either utilize or circumvent the DDR to facilitate their replication. Pseudorabies virus (PRV) infection was shown to induce apoptosis via stimulating DDR. However, the underlying mechanisms have not been fully explored to date. This study showed that PRV infection robustly activates the ATM and DNA-PK signaling pathways shortly after infection. However, inhibition of ATM, but not DNA-PK, could dampen PRV replication in cells. Importantly, we found that PRV-encoded serine/threonine kinase UL13 interacts with and subsequently phosphorylates H2AX. Furthermore, we found that UL13 deletion largely attenuates PRV neuroinvasiveness and virulence in vivo. In addtion, we showed that UL13 contributes to H2AX phosphorylation upon PRV infection both in vitro and in vivo, but does not affect ATM phosphorylation. Finally, we showed that knockdown of H2AX reduces PRV replication, while this reduction can be further enhanced by deletion of UL13. Taken together, we conclude that PRV-encoded kinase UL13 regulates DNA damage marker γH2AX and UL13-mediated H2AX phosphorylation plays a pivotal role in efficient PRV replication and progeny production.

Porcine Epidemic Diarrhea Virus Infection Induces Caspase-8-Mediated G3BP1 Cleavage and Subverts Stress Granules To Promote Viral Replication.

Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus causing severe diarrhea and high mortality rates in suckling piglets and posing significant economic impact. PEDV replication is completed and results in a large amount of RNA in the cytoplasm. Stress granules (SGs) are dynamic cytosolic RNA granules formed under various stress conditions, including viral infections. Several previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. However, the underlying mechanisms are poorly understood. This study aimed to delineate the molecular mechanisms regulating the SG response to PEDV infection. SG formation is induced early during PEDV infection, but as infection proceeds, this ability is lost and SGs disappear at late stages of infection (>18 h postinfection). PEDV infection resulted in the cleavage of Ras-GTPase-activating protein-binding protein 1 (G3BP1) mediated by caspase-8. Using mutational analysis, the PEDV-induced cleavage site within G3BP1 was identified, which differed from the 3C protease cleavage site previously identified. Furthermore, G3BP1 cleavage by caspase-8 at D168 and D169 was confirmed in vitro as well as in vivo The overexpression of cleavage-resistant G3BP1 conferred persistent SG formation and suppression of viral replication. Additionally, the knockdown of endogenous G3BP1 abolished SG formation and potentiated viral replication. Taken together, these data provide new insights into novel strategies in which PEDV limits the host stress response and antiviral responses and indicate that caspase-8-mediated G3BP1 cleavage is important in the failure of host defense against PEDV infection.IMPORTANCE Coronaviruses (CoVs) are drawing extensive attention again since the outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. CoVs are prone to variation and own the transmission capability by crossing the species barrier resulting in reemergence. How CoVs manipulate the antiviral responses of their hosts needs to be explored. Overall, the study provides new insight into how porcine epidemic diarrhea virus (PEDV) impaired SG assembly by targeting G3BP1 via the host proteinase caspase-8. These findings enhanced the understanding of PEDV infection and might help identify new antiviral targets that could inhibit viral replication and limit the pathogenesis of PEDV.

PRV UL13 inhibits cGAS-STING-mediated IFN-ß production by phosphorylating IRF3.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky's disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-ß production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-ß secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS-STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS-STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-ß gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS-STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-ß gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS-STING-mediated IFN-ß production by phosphorylating IRF3.