Nanobody peptide conjugate: a novel CD163 based broad neutralizing strategy against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

Porcine Circovirus 2 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Porcine Circovirus 2 Manipulates the PERK-ERO1α Axis of the Endoplasmic Reticulum To Favor Its Replication by Derepressing Viral DNA from HMGB1 Sequestration within Nuclei.

Porcine circovirus type 2 (PCV2) causes several disease syndromes in grower pigs. PCV2 infection triggers endoplasmic reticulum (ER) stress, autophagy, and oxidative stress, all of which support PCV2 replication. We have recently reported that nuclear HMGB1 is an anti-PCV2 factor by binding to viral genomic DNA. However, how PCV2 manipulates host cell responses to favor its replication has not been explored. Here, we demonstrate that PCV2 infection increased expression of ERO1α, generation of reactive oxygen species (ROS), and nucleocytoplasmic migration of HMGB1 via protein kinase R-like endoplasmic reticulum kinase (PERK) activation in PK-15 cells. Inhibition of PERK or ERO1α repressed ROS production in PCV2-infected cells and increased HMGB1 retention within nuclei. These findings indicate that PCV2-induced activation of the PERK-ERO1α axis would lead to enhanced generation of ROS sufficient to decrease HMGB1 retention in the nuclei, thus derepressing viral DNA from HMGB1 sequestration. The viral Rep and Cap proteins were able to induce PERK-ERO1α-mediated ROS accumulation. Cysteine residues 107 and 305 of Rep or 108 of Cap played important roles in PCV2-induced PERK activation and distribution of HMGB1. Of the mutant viruses, only the mutant PCV2 with substitution of all three cysteine residues failed to activate PERK with reduced ROS generation and decreased nucleocytoplasmic migration of HMGB1. Collectively, this study offers novel insight into the mechanism of enhanced viral replication in which PCV2 manipulates ER to perturb its redox homeostasis via the PERK-ERO1α axis, and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei-hence the release of HMGB1-bound viral DNA for replication. IMPORTANCE Considering the fact that clinical porcine circovirus-associated diseases (PCVAD) mostly results from activation of latent PCV2 infection by confounding factors such as coinfection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals, where PCV2 in infected cells might utilize the elevated reactive oxygen species (ROS) to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication, and severity of PCVAD. This will help decide the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD. Further investigation could examine if similar strategies are employed by DNA viruses, such as PCV3 and BFDV and if there is cross talk among endoplasmic reticulum (ER) stress, autophagy/mitophagy, and mitochondrial-sourced ROS in favor of PCV2 replication.

A modular and self-adjuvanted multivalent vaccine platform based on porcine circovirus virus-like nanoparticles.

BACKGROUND: Virus-like particles (VLPs) are supramolecular structures composed of multiple protein subunits and resemble natural virus particles in structure and size, making them highly immunogenic materials for the development of next-generation subunit vaccines. The orderly and repetitive display of antigenic epitopes on particle surface allows efficient recognition and cross-link by B cell receptors (BCRs), thereby inducing higher levels of neutralizing antibodies and cellular immune responses than regular subunit vaccines. Here, we present a novel multiple antigen delivery system using SpyCatcher/Spytag strategy and self-assembled VLPs formed by porcine circovirus type 2 (PCV2) Cap, a widely used swine vaccine in solo. RESULTS: Cap-SC, recombinant Cap with a truncated SpyCatcher polypeptide at its C-terminal, self-assembled into 26-nm VLPs. Based on isopeptide bonds formed between SpyCatcher and SpyTag, classical swine fever virus (CSFV) E2, the antigen of interest, was linked to SpyTag and readily surface-displayed on SpyCatcher decorated Cap-SC via in vitro covalent conjugation. E2-conjugated Cap VLPs (Cap-E2 NPs) could be preferentially captured by antigen presenting cells (APCs) and effectively stimulate APC maturation and cytokine production. In vivo studies confirmed that Cap-E2 NPs elicited an enhanced E2 specific IgG response, which was significantly higher than soluble E2, or the admixture of Cap VLPs and E2. Moreover, E2 displayed on the surface did not mask the immunodominant epitopes of Cap-SC VLPs, and Cap-E2 NPs induced Cap-specific antibody levels and neutralizing antibody levels comparable to native Cap VLPs. CONCLUSION: These results demonstrate that this modularly assembled Cap-E2 NPs retains the immune potential of Cap VLP backbone, while the surface-displayed antigen significantly elevated E2-induced immune potency. This immune strategy provides distinctly improved efficacy than conventional vaccine combination. It can be further applied to the development of dual or multiple nanoparticle vaccines to prevent co-infection of PCV2 and other swine pathogens.

Controlling Nutritional Status (CONUT) Score is Associated with Overall Survival in Patients with Hepatocellular Carcinoma Treated with Conventional Transcatheter Arterial Chemoembolization: A Propensity Score Matched Analysis.

PURPOSE: This study aims to evaluate the prognostic value of controlling nutritional status (CONUT) score in determining the prognosis of patients with hepatocellular carcinoma (HCC) treated with conventional transcatheter arterial chemoembolization (cTACE). METHODS: This study retrospectively analyzed 936 patients who underwent cTACE for HCC between January 2012 and December 2018, and divided them into two groups based on their CONUT score. To balance the bias in baseline characteristics, propensity score matched (PSM) analysis was conducted. The Kaplan-Meier method was used to establish a cumulative survival curve, and the log-rank test was employed to determine differences in overall survival (OS) and progression-free survival (PFS) among the CONUT score groups. Furthermore, the Cox proportional hazard model was employed to assess the correlation between CONUT score and OS and PFS, whereby hazard ratios (HRs) and 95% confidence intervals (95% CIs) were computed. RESULTS: Before PSM, the median OS for the low (≤ 3) and high (≥ 4) CONUT group (558 vs. 378 patients) was 21.7 and 15.6 months, respectively, and the median PFS was 5.7 and 5 months. Following PSM, both the low and high CONUT score groups comprised 142 patients. The low CONUT score group exhibited a significantly longer OS compared to the high CONUT score group, as determined by the log-rank test (median OS 22.2 vs. 17.0 months, P = 0.014). No significant association was observed between CONUT group and PFS (median PFS 6.4 vs. 4.7 months, log-rank test, P = 0.121). Cox proportional hazard regression analysis revealed that a CONUT score of ≥ 4 was an independent risk factor for OS in patients with HCC who underwent cTACE (HR = 1.361; 95% CI: 1.047-1.771; P = 0.022). These findings were consistent across most subgroup analyses. CONCLUSION: A high CONUT score has been found to be a prognostic factor for poorer OS in patients with HCC who underwent cTACE. LEVEL OF EVIDENCE: Level 3, Non-randomized controlled cohort.

Predictors of liver failure after transarterial chemoembolization in patients with spontaneously ruptured hepatocellular carcinoma: A retrospective study.

Background: Spontaneously ruptured hepatocellular carcinoma (rHCC) is a life-threatening condition. Transarterial chemoembolization (TACE) is a widely accepted treatment; however, it can lead to serious complications, especially liver failure. We sought to identify preoperative predictors of liver failure in patients with rHCC undergoing TACE. Methods: Patients with rHCC who received TACE as the initial therapy were retrospectively studied at our institution between January 2016 and December 2021. Based on the occurrence of liver failure after TACE, the patients were divided into liver failure and no-liver failure groups. Predictors of liver failure after TACE were analyzed using univariate and multivariate regression analyses. The predictive performance was assessed using the area under the curve (AUC). Delong's test was used to compare predictive efficiency. Results: Sixty patients (19 and 41 in the liver failure and non-liver failure groups, respectively) were included. Multivariate analysis showed that preoperative prothrombin activity (PTA) level (odds ratio [OR], 0.956; 95% confidence interval [CI], 0.920-0.994; P â€‹= â€‹0.024) and Child-Pugh grade B (OR, 6.419; 95% CI, 1.123-36.677; P â€‹= â€‹0.037) were independent predictors of liver failure after TACE in patients with rHCC. The AUCs of the preoperative PTA levels and Child-Pugh grade B for predicting liver failure after TACE in patients with rHCC were 0.783 and 0.764, respectively. Conclusion: Preoperative PTA level and Child-Pugh grade B were significant independent risk factors for liver failure after TACE in patients with rHCC. These can be used to predict liver failure after TACE in patients with rHCC for individual decision-making regarding treatment planning.

PNI-Based Nomograms to Predict Tumor Progression and Survival for Patients with Unresectable Hepatocellular Carcinoma Undergoing Transcatheter Arterial Chemoembolization.

BACKGROUND: The relationship between the prognostic nutritional index (PNI) and the prognosis of malignancy has been increasingly mentioned in recent research. This study aimed to construct nomograms based on the PNI to predict tumor progression and survival in patients with unresectable hepatocellular carcinoma (HCC) undergoing transcatheter arterial chemoembolization (TACE). MATERIALS AND METHODS: The development set included 785 patients who underwent their first TACE between 2012 and 2016, and the validation set included 336 patients who underwent their first TACE between 2017 and 2018. The clinical outcomes included the time to progression (TTP) and overall survival (OS). Cox regression was applied to screen for independent risk factors of TTP and OS in the development set, and PNI-based nomograms were constructed for TTP and OS. The predictive performance of nomograms was conducted through the C-index, calibration curves, and decision analysis curves in the development set and validation set. RESULTS: After multivariate analysis, the prognostic predictors of both TTP and OS included portal vessel invasion, extrahepatic metastasis, tumor number, alpha-fetoprotein (AFP) level, longest tumor diameter, and PNI. Furthermore, the Child-Pugh classification and platelets (PLTs) were independent risk factors for OS only. Nomograms for predicting TTP and OS were constructed using TTP and OS prognostic factors. In the development set and the validation set, the C-index of the TTP nomograms was 0.699 (95% confidence interval (CI): 0.680-0.718) and 0.670 (95%CI: 0.638-0.702), and the C-index of the OS nomograms was 0.730 (95%CI: 0.712-0.748) and 0.700 (95%CI: 0.665-0.723), respectively. CONCLUSION: Nomograms based on the PNI can effectively predict tumor progression and survival in patients with unresectable HCC undergoing TACE.

A novel nomogram based on the hematological prognosis risk scoring system can predict the overall survival of patients with hepatocellular carcinoma.

BACKGROUND: This study aimed to establish and validate a nomogram based on a hematological prognostic risk scoring system to predict the overall survival in patients with unresectable hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Patients diagnosed with unresectable HCC undergoing transcatheter arterial chemoembolization (TACE) in 2012-2016 and 2017-2018 were included in the development set and validation set, respectively. The clinical outcome was overall survival (OS). The LASSO regression analysis was used to construct a hematological prognostic risk scoring system (HPR) by using the 18 hematological markers of patients in the development set. Combining the features of oncology on the basis of HPR to construct a nomogram for OS. In the development set and validation sets, the C-index, calibration curve, and decision curve analysis (DCA) were used to evaluate the prediction performance of the nomogram. RESULTS: Multiple markers of immunity, coagulation, liver function, and nutrition, including red blood cell distribution width-coefficient of variation (RDW-CV), platelet (PLT), aspartate transferase (AST), alkaline phosphatase (ALP), prognostic nutritional index (PNI), and fibrinogen (Fib), construct the HPR. HPR was an independent risk factor for OS in patients with HCC. The C-index of the nomogram was 0.731 (95% confidence interval (CI) 0.712-0.749) and 0.696 (95% CI 0.668-0.725) in the development set and the validation set, respectively. CONCLUSIONS: HPR was a complement to the clinical features of patients with unresectable HCC. The nomogram based on HPR proved to be a practical and effective method for prognosticating HCC patients who undergo TACE.

Nanobodies against porcine CD163 as PRRSV broad inhibitor.

PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is a major swine pathogen causing economic losses. To the date, effective broad PRRSV inhibitory strategies have not been available in practice yet. Targeting the key viral receptor CD163 to block PRRSV entry has emerged as an alternative approach beside vaccines for PRRSV inhibition. As an effective therapeutic tool, nanoantibodies (Nbs) have been widely used in antiviral research. In this study, a phage display VHH library was constructed for the selection of Nbs against porcine CD163 scavenger receptor cysteine-rich 5-9 domain (SRCR5-9). After five rounds of bio-panning and indirect ELISA, seven CD163-specific Nbs (Nb1-Nb7) were identified. All obtained Nbs displayed strong affinity to CD163 receptor and excellent antiviral activity. In particular, Nb2 exhibited significant broad inhibitory effects on variable PRRSV lineages and downregulated virus-related NF-κB signaling. Further studies suggested that Nbs mainly exerted antiviral functions by interfering with virus attachment stage, and also decreased the transcription of CD163. The conformational epitopes recognized by Nbs were localized in the SRCR5 domain of CD163, a crucial region in PRRSV infection. Overall, our findings provide a novel insight into the biofunction of CD163 in antiviral infection and the development of broad-spectrum strategies against PRRSV.

Physical and mental health impairments experienced by operating surgeons and camera-holder assistants during laparoscopic surgery: a cross-sectional survey.

Introduction: Surgeons may experience physical and mental health problems because of their jobs, which may lead to chronic muscle damage, burnout, or even withdrawal. However, these are often ignored in camera-holder assistants during laparoscopic surgery. We aimed to analyze the differences between operating surgeons and camera-holder assistants. Methods: From January 1, 2022, to December 31, 2022, a cross-sectional survey was conducted to evaluate the muscle pain, fatigue, verbal scolding, and task load for operating surgeons and camera-holder assistants. The Nordic Musculoskeletal Questionnaire, the Space Administration Task Load Index, and the Surgical Task Load Index (SURG-TLX) were combined in the questionnaire. Results: 2,184 operations were performed by a total of 94 operating surgeons and 220 camera assistants. 81% of operating surgeons and 78% of camera-holder assistants reported muscle pain/discomfort during the procedure. The most affected anatomic region was the shoulders for operating surgeons, and the lower back for camera-holder assistants. Intraoperative fatigue was reported by 41.7% of operating surgeons and 51.7% of camera-holder assistants. 55.2% of camera-holder assistants reported verbal scolding from the operating surgeons, primarily attributed to lapses in laparoscope movement coordination. The SURG-TLX results showed that the distributions of mental, physical, and situational stress for operating surgeons and camera-holder assistants were comparable. Conclusion: Like operating surgeons, camera-holder assistants also face similar physical and mental health impairments while performing laparoscopic surgery. Improvements to the working conditions of the camera-holder assistant should not be overlooked.

The truncated form of flagellin (tFlic) provides the 2dCap subunit vaccine with better immunogenicity and protective effects in mice.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated diseases, and it causes substantial economic losses in the swine industry each year. It is crucial to develop an effective vaccine against the circulating strain PCV2d, which is prone to substantial degrees of mutation. In this study, a truncated form of flagellin (tFlic: 85-111 aa) was inserted into the C-terminal sequence of 2dCap, and Western blotting results showed that recombinant Cap-tFlic VLPs were successfully expressed. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) data indicated that purified recombinant Cap-tFlic fusion proteins existed in the form of polymers and that tFlic could not affect the formation and internalization of VLPs. Integrated Cap-tFlic VLPs induced the expression of antigen presentation-related factors (MHC-II and CD86) by bone marrow-derived dendritic cells (BM-DCs), and the expression of TLR5-related factors (TNF-α) was dramatically elevated. Mice intramuscularly immunized with Cap-tFlic VLPs exhibited significantly higher levels of Cap-specific antibodies and neutralizing antibodies than mice immunized with wild-type Cap VLPs. The data obtained in the current study indicate that Cap-tFlic may be a candidate for a subunit vaccine against PCV2 in the future.

Porcine Reproductive and Respiratory Syndrome Virus Adapts Antiviral Innate Immunity via Manipulating MALT1.

To fulfill virus replication and persistent infection in hosts, viruses have to find ways to compromise innate immunity, including timely impedance on antiviral RNases and inflammatory responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen causing immune suppression. MALT1 is a central immune regulator in both innate and adaptive immunity. In this study, MALT1 was confirmed to be induced rapidly upon PRRSV infection and mediate the degradation of two anti-PRRSV RNases, MCPIP1 and N4BP1, relying on its proteolytic activity, consequently facilitating PRRSV replication. Multiple PRRSV nsps, including nsp11, nsp7ß, and nsp4, contributed to MALT1 elicitation. Interestingly, the elevated expression of MALT1 began to decrease once intracellular viral expression reached a high enough level. Higher infection dose brought earlier MALT1 inflection. Further, PRRSV nsp6 mediated significant MALT1 degradation via ubiquitination-proteasome pathway. Downregulation of MALT1 suppressed NF-κB signals, leading to the decrease in proinflammatory cytokine expression. In conclusion, MALT1 expression was manipulated by PRRSV in an elaborate manner to antagonize precisely the antiviral effects of host RNases without excessive and continuous activation of inflammatory responses. These findings throw light on the machinery of PRRSV to build homeostasis in infected immune system for viral settlement. IMPORTANCE PRRSV is a major swine pathogen, suppresses innate immunity, and causes persistent infection and coinfection with other pathogens. As a central immune mediator, MALT1 plays essential roles in regulating immunity and inflammation. Here, PRRSV was confirmed to manipulate MALT1 expression in an accurate way to moderate the antiviral immunity. Briefly, multiple PRRSV nsps induced MALT1 protease to antagonize anti-PRRSV RNases N4BP1 and MCPIP1 upon infection, thereby facilitating viral replication. In contrast, PRRSV nsp6 downregulated MALT1 expression via ubiquitination-proteasome pathway to suppress the inflammatory responses upon infection aggravation, contributing to immune defense alleviation and virus survival. These findings revealed the precise expression control on MALT1 by PRRSV for antagonizing antiviral RNases, along with recovering immune homeostasis. For the first time, this study enlightens a new mechanism of PRRSV adapting antiviral innate immunity by modulating MALT1 expression.