CRISPR-Cas9-mediated chicken prmt5 gene knockout and its critical role in interferon regulation.

Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-ß activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5-/- DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5-/- DF-1 cells had increased IFN-ß production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-ß production and interferon-stimulated gene expression.

Variety of femoral anteversion and its measurement in cementless total hip arthroplasty: Does robotic technology improve accuracy?

BACKGROUND: High-performance total hip arthroplasty (THA) depends on the accurate position of components. However, femoral anteversion is variable, and current studies only used traditional instruments to evaluate it, such as protractor and spirit level with limited cases. This study aimed to identify the variability in the measured femoral native anteversion and intraoperative stem anteversion under different measurement methods, including intraoperative robotic method. We hypothesized that robotic technology was more accurate than traditional instruments for femoral anteversion evaluation. METHODS: This study included 117 hips of patients who underwent robotic-assisted THA between November 2019 and March 2021. Preoperative native femoral anteversion was measured using a robotic system. Intraoperative femoral stem anteversion was evaluated visually, and then measured with a goniometer and a robotic system, respectively. Variability in the measured femoral native anteversion and intraoperative femoral stem anteversion was calculated and compared. Intraclass correlation coefficient (ICC) and Pearson correlation analysis were used to assess the consistency and correlation of anteversion of different measurements and postoperative CT-measured stem anteversion, respectively. RESULTS: The result of measurement for preoperative native femoral anteversion was more variable than the intraoperative robotic-measured stem anteversion. Intraoperative robotic-measured stem version showed the highest correlation with postoperative CT measurement of stem version (r = 0.806, P < 0.001), while intraoperative surgeon estimation had the lowest correlation coefficient (r = 0.281, P = 0.025). As for the consistency with postoperative CT measurement of femoral stem anteversion, the intraoperative robotic-measured femoral stem version also had the highest value (ICC = 0.892, P < 0.001). CONCLUSION: Native femoral anteversion was variable preoperatively. Using cementless stems, anteversion was also highly variable. Robotic assessment for stem anteversion during surgery was more consistent with the final position than the preoperative assessment and conventional intraoperative estimation.

Inhibition of PTEN-induced kinase 1 autophosphorylation may assist in preventing epileptogenesis induced by pentylenetetrazol.

PTEN-induced kinase 1 (PINK1) autophosphorylation-triggered mitophagy is the main mitophagic pathway in the nervous system. Moreover, multiple studies have confirmed that mitophagy is closely related to the occurrence and development of epilepsy. Therefore, we speculated that the PINK1 autophosphorylation may be involved in epileptogenesis by mediating mitophagic pathway. This study aimed to explore the contribution of activated PINK1 to epileptogenesis induced by pentylenetetrazol (PTZ) in Sprague‒Dawley rats. During PTZ-induced epileptogenesis, the levels of phosphorylated PINK1 were increased, accompanied by elevated mitophagy, mitochondria oxidative stress and neuronal damage. After microRNA intervention targeting translocase outer mitochondrial membrane 7 (TOM7) or overlapping with the m-AAA protease 1 homolog (OMA1), the levels of PINK1 phosphorylation, mitophagy, mitochondrial oxidative stress, neuronal injury were observed in the rats with induced epileptogenesis. Furthermore, inhibiting of the expression of TOM7, a positive regulator of PINK1 autophosphorylation, reversed the increase in PINK1 phosphorylation and alleviated mitophagy, neuronal injury, thereby preventing epileptogenesis. In contrast, reducing the levels of OMA1, a negative regulator of PINK1 autophosphorylation, led to increased phosphorylation of PINK1, accompanied by aggravated neuronal injury and ultimately, epileptogenesis. This study confirmed the contribution of activated PINK1 to PTZ-induced epileptogenesis and suggested that the inhibition of PINK1 autophosphorylation may assist in preventing epileptogenesis.

LncRNA H19 mediates BMP9-induced angiogenesis in mesenchymal stem cells by promoting the p53-Notch1 angiogenic signaling axis.

BMP9 mediated osteogenic differentiation mechanisms of MSCs were widely explored, however, mechanisms of BMP9-induced angiogenesis still need to be clarified. We previously characterized that Notch1 promoted BMP9-induced osteogenesis-angiogenesis coupling process in mesenchymal stem cells (MSCs). Here, we explored the underlying mechanisms of lncRNA H19 (H19) mediated regulation of BMP9-induced angiogenesis through activating Notch1 signaling. We demonstrated that basal expression level of H19 was high in MSCs, and silencing H19 attenuates BMP9-induced osteogenesis and angiogenesis of MSCs both in vitro and in vivo. Meanwhile, we identified that BMP9-induced production of CD31+ cells was indispensable for BMP9-induced bone formation, and silencing H19 dramatically blocked BMP9-induced production of CD31+ cells. In addition, we found that down-regulation of H19 inhibited BMP9 mediated blood vessel formation and followed subsequent bone formation in vivo. Mechanistically, we clarified that H19 promoted p53 phosphorylation by direct interacting and phosphorylating binding, and phosphorylated p53 potentiated Notch1 expression and activation of Notch1 targeting genes by binding on the promoter area of Notch1 gene. These findings suggested that H19 regulated BMP9-induced angiogenesis of MSCs by promoting the p53-Notch1 angiogenic signaling axis.

Metformin improves fibroblast metabolism and ameliorates arthrofibrosis in rats.

Background: Emerging studies have suggested an essential role of fibroblast metabolic reprogramming in the pathogenesis of arthrofibrosis. The metabolic modulator metformin appears to be a therapeutic candidate for fibrotic disorders. However, whether metformin could alleviate arthrofibrosis has not been defined. In this study we have determined if treatment with metformin has beneficial effect on arthrofibrosis and its underlying mechanism. Methods: Articular capsule samples were collected from patients with/without arthrofibrosis to perform gene and protein expression analysis. Arthrofibrosis animal model was established to examine the anti-fibrotic effect of metformin. Cell culture experiments were conducted to determine the mechanism by which metformin inhibits fibroblast activation. Results: We found that glycolysis was upregulated in human fibrotic articular capsules. In an arthrofibrosis animal model, intra-articular injection of metformin mitigated inflammatory reactions, downregulated expression of both fibrotic and glycolytic markers, improved range of motion (ROM) of the joint, and reduced capsular fibrosis and thickening. At the cellular level, metformin inhibited the activation of fibroblasts and mitigated the abundant influx of glucose into activated fibroblasts. Interestingly, metformin prompted a metabolic shift from oxidative phosphorylation to aerobic glycolysis in activated fibroblasts, resulting in the anti-fibrotic effect of metformin. Conclusion: Metformin decreased glycolysis, causing a metabolic shift toward aerobic glycolysis in activated fibroblasts and has beneficial effect on the treatment of arthrofibrosis.The translational potential of this article: The findings of this study demonstrated the therapeutic effect of metformin on arthrofibrosis and defined novel targets for the treatment of articular fibrotic disorders.

Single ethanol binge causes severe liver injury in mice fed Western diet.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) and NAFLD often coexist in Western societies that consume energy-rich and cholesterol-containing Western diets. Increased rates of ALD mortality in young people in these societies are likely attributable to binge drinking. It is largely unknown how alcohol binge causes liver damage in the setting of Western diets. APPROACH AND RESULTS: In this study, we showed that a single ethanol binge (5 g/kg body weight) induced severe liver injury as shown by marked increases in serum activities of the 2 aminotransferases AST and ALT in C57BL/6J mice that have been fed a Western diet for 3 weeks. The Western diet plus binge ethanol-fed mice also displayed severe lipid droplet deposition and high contents of triglycerides and cholesterol in the liver, which were associated with increased lipogenic and reduced fatty acid oxidative gene expression. These animals had the highest Cxcl1 mRNA expression and myeloperoxidase (MPO)-positive neutrophils in the liver. Their hepatic ROS and lipid peroxidation were the highest, but their hepatic levels of mitochondrial oxidative phosphorylation proteins remained largely unaltered. Hepatic levels of several ER stress markers, including mRNAs for CHOP, ERO1A, ERO1B, BIM, and BIP, as well as Xbp1 splicing and proteins for BIP/GRP78 and IRE-α were also the highest in these animals. Interestingly, Western diet feeding for 3 weeks or ethanol binge dramatically increased hepatic caspase 3 cleavage, and the combination of the 2 did not further increase it. Thus, we successfully established a murine model of acute liver injury by mimicking human diets and binge drinking. CONCLUSIONS: This simple Western diet plus single ethanol binge model recapitulates major hepatic phenotypes of ALD, including steatosis and steatohepatitis characterized by neutrophil infiltration, oxidative stress, and ER stress.

The Necessity of Implant Removal after Fixation of Thoracolumbar Burst Fractures-A Systematic Review.

BACKGROUND: Thoracolumbar burst fractures are a common traumatic vertebral fracture in the spine, and pedicle screw fixation has been widely performed as a safe and effective procedure. However, after the stabilization of the thoracolumbar burst fractures, whether or not to remove the pedicle screw implant remains controversial. This review aimed to assess the benefits and risks of pedicle screw instrument removal after fixation of thoracolumbar burst fractures. METHODS: Data sources, including PubMed, EMBASE, Cochrane Library, Web of Science, Google Scholar, and Clinical trials.gov, were comprehensively searched. All types of human studies that reported the benefits and risks of implant removal after thoracolumbar burst fractures, were selected for inclusion. Clinical outcomes after implant removal were collected for further evaluation. RESULTS: A total of 4051 papers were retrieved, of which 35 studies were eligible for inclusion in the review, including four case reports, four case series, and 27 observational studies. The possible risks of pedicle screw removal after fixation of thoracolumbar burst fractures include the progression of the kyphotic deformity and surgical complications (e.g., surgical site infection, neurovascular injury, worsening pain, revision surgery), while the potential benefits of pedicle screw removal mainly include improved segmental range of motion and alleviated pain and disability. Therefore, the potential benefits and possible risks should be weighed to support patient-specific clinical decision-making about the removal of pedicle screws after the successful fusion of thoracolumbar burst fractures. CONCLUSIONS: There was conflicting evidence regarding the benefits and harms of implant removal after successful fixation of thoracolumbar burst fractures, and the current literature does not support the general recommendation for removal of the pedicle screw instruments, which may expose the patients to unnecessary complications and costs. Both surgeons and patients should be aware of the indications and have appropriate expectations of the benefits and risks of implant removal. The decision to remove the implant or not should be made individually and cautiously by the surgeon in consultation with the patient. Further studies are warranted to clarify this issue. LEVEL OF EVIDENCE: level 1.

PRMT5 Facilitates Infectious Bursal Disease Virus Replication through Arginine Methylation of VP1.

The infectious bursal diseases virus (IBDV) polymerase, VP1 protein, is responsible for transcription, initial translation and viral genomic replication. Knowledge about the new kind of post-translational modification of VP1 supports identification of novel drugs against the virus. Because the arginine residue is known to be methylated by protein arginine methyltransferase (PRMT) enzyme, we investigated whether IBDV VP1 is a substrate for known PRMTs. In this study, we show that VP1 is specifically associated with and methylated by PRMT5 at the arginine 426 (R426) residue. IBDV infection causes the accumulation of PRMT5 in the cytoplasm, which colocalizes with VP1 as a punctate structure. In addition, ectopic expression of PRMT5 significantly enhances the viral replication. In the presence of PMRT5, enzyme inhibitor and knockout of PRMT5 remarkably decreased viral replication. The polymerase activity of VP1 was severely damaged when R426 mutated to alanine, resulting in impaired viral replication. Our study reports a novel form of post-translational modification of VP1, which supports its polymerase function to facilitate the viral replication. IMPORTANCE Post-translational modification of infectious bursal disease virus (IBDV) VP1 is important for the regulation of its polymerase activity. Investigation of the significance of specific modification of VP1 can lead to better understanding of viral replication and can probably also help in identifying novel targets for antiviral compounds. Our work demonstrates the molecular mechanism of VP1 methylation mediated by PRMT5, which is critical for viral polymerase activity, as well as viral replication. Our study expands a novel insight into the function of arginine methylation of VP1, which might be useful for limiting the replication of IBDV.

Identification of a novel porcine Teschovirus 2 strain as causative agent of encephalomyelitis in suckling piglets with high mortality in China.

BACKGROUND: Porcine Teschovirus (PTV), also named Teschovirus A, is prevalent in pig populations, mainly causing neurological symptoms, diarrhea, pneumonia, and reproductive failure, however the morbidity and mortality are usually low in pig farms. CASE PRESENTATION: In this study, we reported a PTV outbreak investigation in one large-scale pig farm in China with severe symptoms including diarrhea, lethargy, locomotor ataxia, nystagmus, paralysis of the hind limbs, and coma in piglets. More importantly, the mortality reached 38% in suckling pigs, which is remarkably high in PTV history. A novel PTV strain, named HeNZ1, was isolated from cerebral samples of one suckling pig and the genome sequence was obtained by NGS sequencing. Phylogenetic and evolutionary divergence analyses revealed that HeNZ1 belongs to PTV genotype 2. Surprisingly, the VP1 coding region of HeNZ1 shares the highest sequence similarity with European PTV-2 strains, instead of China domestic PTV-2 strains, implying it may not derive from China local PTV-2 strains. Multiple sequence alignment and B cell epitope prediction of PTV VP1 and VP2 protein revealed 10 B cell epitopes, 5 mutant clusters and 36 unique mutation sites, of which 19 unique mutation sites are located in B cell epitopes and exposed on the surface of VP1 or VP2, implying significant antigenic drift potential of HeNZ1. CONCLUSION: These results indicate that HeNZ1 is a highly virulent PTV-2 strain, which capable of causing severe neurological symptoms and high mortality in piglets. Bioinformatic analysis suggest that HeNZ1 is genetically and antigenically different from other Chinese PTV-2 strains. Overall, current case expanded our understanding of PTV-2 clinical spectrum and revealed the emergence of a highly virulent PTV-2 strain with substantial genetic diversity and antigenic drift potential in VP1 and VP2.

Chicken PRMT1 promotes infectious bursal disease virus replication via suppressing IFN-ß production.

The protein arginine methyltransferase (PRMT) family, such as PRMT1, regulates the arginine methylation of various substrates. Many studies have examined the role of PRMT1 in mammals, however, it is still unknown how PRMT1 works in chickens. To investigate the effect of chicken PRMT1 (chPRMT1) on regulating IFN-ß production and IBDV replication, chPRMT1 knock out DF-1 cells were constructed in this study. First, we found that chPRMT1 was widely expressed in a variety of chicken tissues and that it was distributed in the cytoplasm and nucleus of DF-1 cells. Additionally, IFN-ß activation was inhibited by chPRMT1 at the step of chMAVS. In addition, chPRMT1 knock out DF-1 cells were constructed using CRISPR-Cas9 technique. The morphology and viability of chPRMT1 knock out DF-1 cells were similar with the wild-type cells. In addition, the IFN-ß as well as interferon stimulate genes activation induced by chMAVS in PRMT1 knock out DF-1 cells were significantly higher than that in WT cells. Furthermore, ectopic expression of chPRMT1 significantly supports IBDV replication. We also found that the ability of IBDV replication in PRMT1 knock out DF-1 cells was remarkably lower than that of in WT cells, suggesting that PRMT1 negatively regulate IBDV replication via suppressing IFN-ß production. In conclusion, the PRMT1 knock out DF-1 cells were constructed, which was further used to demonstrate an inhibitory role of chPRMT1 in IFN-ß production, and a contributor of chPRMT1 in IBDV replication.

Robotic-assisted revision total joint arthroplasty: a state-of-the-art scoping review.

Background: During the past decades, robotic-assisted technology has experienced an incredible advancement in the field of total joint arthroplasty (TJA), which demonstrated promise in improving the accuracy and precision of implantation and alignment in both primary total hip arthroplasty (THA) and total knee arthroplasty (TKA). However, revision TJA remains a technically challenging procedure with issues of large-scale bone defects and damage to nearby anatomical structures. Thus, surgeons are trying to harness the abilities of robotic-assisted technology for revision TJA surgery. Methods: PubMed, Embase, Cochrane Library, and Google Scholar were comprehensively searched to identify relevant publications that reported the application of robotic-assisted technology in revision TJA. Results: Overall, ten studies reported the use of the robotic system in revision TJA, including active (ROBODOC) and semi-active (MAKO and NAVIO) systems. One clinical case reported conversion from hip fusion to THA, and three studies reported revision from primary THA to revision THA. Moreover, four studies reported that robotic-assisted technology is helpful in revising unicompartmental knee arthroplasty (UKA) to TKA, and two case reports converted primary TKA to revision TKA. In this study, we present the latest evolvements, applications, and technical obstacles of robotic-assisted technology in the revision of TJA and the current state-of-the-art. Conclusion: Current available evidence suggests that robotic-assisted technology may help surgeons to reproducibly perform preoperative plans and accurately achieve operative targets during revision TJA. However, concerns remain regarding preoperative metal artifacts, registration techniques, closed software platforms, further bone loss after implant removal, and whether robotic-assisted surgery will improve implant positioning and long-term survivorship.

Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.

Characterization and functional analysis of chicken dsRNA binding protein hnRNPU.

In mammals, heterogeneous ribonucleoprotein U (hnRNPU), also named as nuclear matrix protein-nuclear scaffold attachment factor (SAFA), was originally identified as a DNA/RNA interactor protein. It has been reported that human hnRNPU facilitates IFN-ß generation after vesicular stomatitis virus (VSV) infection. Nevertheless, the role of chicken hnRNPU (chhnRNPU) in IFN-ß regulation as well as in infectious bursal diseases virus (IBDV) replication is still unclear. Here, we found that chhnRNPU inhibits IFN-ß production via interacting with MDA5 and MAVS, and facilitates IBDV replication via associating with genomic dsRNA of IBDV. Firstly, chicken hnRNPU (chhnRNPU) was widely expressed in different tissues of chickens and was distributed in the nucleus of DF-1 cells. Overexpression of chhnRNPU significantly suppresses IFN-ß promoter activities induced by MDA5 and MAVS. Additionally, immunoprecipitated by dsRNA antibodies, which followed LC-MS analysis demonstrate that chhnRNPU is a partner of viral genomic dsRNA. chhnRNPU is translocated from nucleus to cytosol to co-localize with replication complex of IBDV after IBDV infection. Over-expression of chhnRNPU significantly promotes IBDV replication, which was determined by western blotting, qRT-PCR and TCID50 assay. Furthermore, knock down chhnRNPU by siRNA remarkably facilitates IFN-ß production, and inhibits IBDV proliferation. These data collectively reveal that chhnRNPU positively regulates IBDV replication via negatively regulating IFN-ß response.

Total Hip Arthroplasty Patients with Distinct Postoperative Fibrinolytic Phenotypes Require Different Antifibrinolytic Strategies.

Bleeding patients exhibit different fibrinolytic phenotypes after injury, and the universal use of tranexamic acid (TXA) is doubted. We aimed to evaluate the efficacy of postoperative antifibrinolytic treatment in total hip arthroplasty (THA) patients with different fibrinolytic phenotypes. A retrospective analysis was conducted in 238 patients who underwent THA. Patients were divided into two groups by different fibrinolytic phenotypes (non-fibrinolytic shutdown and fibrinolytic shutdown), determined by the LY30 level on postoperative day 1 (POD1). The two groups were further stratified into four sub-groups based on different postoperative TXA regimens (Group A received no TXA postoperatively, while Group B did). Hidden blood loss (HBL), decline of hemoglobin (ΔHb), D-dimer (D-D), fibrinogen/fibrin degradation product (FDP), prothrombin time (PT), activated partial thromboplastin time (APTT), and demographics were collected and compared. The clinical baseline data were comparable between the studied groups. In patients who presented non-fibrinolytic shutdown postoperatively, Group B suffered significantly lower HBL and ΔHb than Group A on POD3 and POD5. In patients who presented postoperative fibrinolytic shutdown, Group B failed to benefit from the postoperative administration of TXA when compared to Group A. No difference was found in postoperative levels of D-D, FDP, PT, and APTT. Postoperative antifibrinolytic therapy is beneficial for THA patients who presented non-fibrinolytic shutdown postoperatively, while the efficacy and necessity should be considered with caution in those with fibrinolytic shutdown. LY30 is a promising parameter to distinguish different fibrinolytic phenotypes and guide TXA administration. However, further prospective studies are needed to confirm these findings.

Frequency detection of porcine circovirus-like viruses in pigs with porcine respiratory disease.

The emergence of the circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses in different hosts has been associated with serious diseases, such as porcine diarrhea. The prevalence and pathogenicity of porcine circovirus-like virus (Po-Circo-like virus (PCLV)), a member of CRESS DNA virus, has not been fully illustrated. In order to understand the frequency of PCLV in pigs with respiratory disease, 519 healthy tissues (268 lungs, 201 lymph nodes and 50 hearts) and 380 tissues (212 lungs, 124 lymph nodes and 44 hearts) diagnosed with respiratory disease were collected for analyzing the prevalence of the PCLV infection using the Tag-Man qPCR assay. In addition, the complete genome of 43 PCLV strains were then sequenced, which were subsequently to analyze their characteristics. We found that 31.7 % (285/899) samples were tested positive for PCL virus. It is interesting to note that just 9.6 % (50/519) of the healthy samples were tested positive for PCLV, 61.8 % (235/380) of the diseased samples were PCLV positive. Analysis of the full genome of 43 PCLV strains showed that the genome of 42 PCLV strains included two distinct stem-loop structures, but the genome of PCLV FJ5-2020 strain contained no stem-loop structures. A phylogenetic tree analysis based on the Rep protein, PCLV could be classified into four genotypes: PCLVa, PCLVb, PCLVc, and PCLVd. In conclusion, this is the first report that the high frequency of PCLV in association in respiratory diseased pigs. PCLV strains were divided into four new genotypes of PCLV.

An Improved, Dual-Direction, Promoter-Driven, Reverse Genetics System for the Infectious Bursal Disease Virus (IBDV).

The infectious bursal disease virus (IBDV), one member of the Birnaviridae family, causes immunosuppression in young chickens by damaging the mature B cells of the bursa of Fabricius (BF), the central immune system of young chickens. The genome of IBDV is a bisegmented, double-strand RNA (dsRNA). Reverse genetics systems for IBDV allow the generation of genetically manipulated infectious virus via transfected plasmid DNA, encoding the two genomic viral RNA segments as well as major viral proteins. For this purpose, the minus-sense of both segment A and segment B are inserted into vectors between the polymerase I promoter and the corresponding terminator I. These plasmids facilitate the transcription of the viral minus-sense genome but copy the plus-sense genome as well viral protein translation depends on the activity of VP1 and VP3, when transfected into 293T cells. To further improve rescue efficiency, dual-direction promoters were generated based on the polymerase II promoter in the reverse direction in the backbone of the pCDNA3.0 vector. Therefore, the polymerase I promoter transcribes the viral minus-sense genome in the forward direction and the polymerase II promoter transcribes viral mRNA, translated into viral proteins that produce infectious IBDV. We also found that the rescue efficiency of transfecting two plasmids is significantly higher than that of transfecting four plasmids. In addition, this dual-direction promoter rescue system was used to generate R186A mutant IBDV since Arg186 is the arginine monomer-methylation site identified by LC-MS. Our data furtherly showed that the Arg186 monomer methylation mutant was due to a reduction in VP1 polymerase activity as well as virus replication, suggesting that the Arg186 methylation site is essential for IBDV replication.

Development and Primary Application of an Indirect ELISA Based on Rep Protein to Analyze Antibodies against Porcine Cocirvirus-like Virus (PCLV).

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome replication. The development of serological detection methods for PCLV is of great necessity for clinical diagnosis, as well as epidemiological investigations. Therefore, this study attempted to build an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCLV based on the His-tagged recombinant Rep protein. Full-length PCLV Rep protein was induced and expressed in E. coli and was purified as an antigen to establish an ELISA detection kit. The purified Rep protein was used to inject into mice to produce specific antibodies. There was no cross-reaction of Rep-based ELISA with antisera against other porcine viruses. The intra-assay and inter-assay coefficient variations (CVs) were 0.644-8.211% and 0.859-7.246%, respectively, indicating good repeatability. The non-cross-reaction with TGEV, PRRSV and PCV2 testing showed high sensitivity and high specificity for this ELISA assay. A total of 1593 serum samples collected from different pig farms in Jiangxi Province were tested for anti-PCLV Rep antibodies, and 284 (17.83%) of the 1593 samples were Rep antibody positive. Altogether, the indirect ELISA detection tool developed in this study could be applied to examine serum of PCLV antibodies with good repeatability, high sensitivity and high specificity. In addition, field sample detection results suggested that the PCLV antibody has a low prevalence in pig populations in Jiangxi Province of China.

Chicken TAX1BP1 suppresses type I interferon production via degrading chicken MAVS and facilitates infectious bursal diseases virus replication.

Mammalian TAX1BP1 (TAX1 binding protein 1), originally identified as a partner of the HTLV-1 viral oncoprotein, functions in regulation of cellular cytokine production. TAX1BP1 plays an important signal transduction regulator, specifically modulating innate immune signaling pathways including NF-B and IRF3. The function of TAX1BP1, which regulates the innate immune response in mammals, has been well studied in previous reports, but the role of chicken TAX1BP1 (chTAX1) in IFN regulation and infectious bursal disease virus (IBDV) replication is still unclear. In this report, chTAX1 was successfully cloned and sub-inserted into a eukaryotic expression vector. The critical regions of chTAX1, such as LC3 binding motif, ubiquitin binding motif, are highly conserved compared to other organisms. We also found that chTAX1 inhibits IFN expression by promoting degradation of chicken MAVS (chMAVS). In addition, the distribution of chTAX1 altered and translocated to co-localize with both VP1 and VP3 after IBDV infection. Overexpression of chTAX1 promotes IBDV replication and knockdown of chTAX1 by RNA interference suppresses IBDV replication. In summary, our data initially indicate that chTAX1 is a suppressor of IFN expression as well as a promoter of IBDV replication.