Langat virus inhibits the gp130/JAK/STAT signaling by reducing the gp130 protein level.

The tick-borne encephalitis virus (TBEV) serocomplex includes several medically important flavivirus members endemic to Europe, Asia, and North America, which can induce severe neuroinvasive or viscerotropic diseases with unclear mechanisms of pathogenesis. Langat virus (LGTV) shares a high sequence identity with TBEV but exhibits lower pathogenic potential in humans and serves as a model for virus-host interactions. In this study, we demonstrated that LGTV infection inhibits the activation of gp130/JAK/STAT (Janus kinases (JAK) and signal transducer and activator of transcription (STAT)) signaling, which plays a pivotal role in numerous biological processes. Our data show that the LGTV-infected cells had significantly lower phosphorylated STAT3 (pSTAT3) protein upon oncostatin M (OSM) stimulation than the mock-infected control. LGTV infection blocked the nuclear translocation of STAT3 without a significant effect on total STAT3 protein level. LGTV inhibited JAK1 activation and reduced gp130 protein expression in infected cells, with the viral NS5 protein mediating this effect. TBEV infection also reduces gp130 level. On the other hand, pretreatment of Vero cells with OSM significantly reduces LGTV replication, and STAT1/STAT2 knockdown had little effect on OSM-mediated antiviral effect, which suggests it is independent of STAT1/STAT2 and, instead, it is potentially mediated by STAT3 signlaing. These findings shed light on the LGTV and TBEV-cell interactions, offering insights for the future development of antiviral therapeutics and improved vaccines.

Oxysterol binding protein (OSBP) contributes to hepatitis E virus replication.

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus and causes primarily acute self-limiting infections. The ORF1 of the HEV genome encodes a polyprotein around 190 kDa, which contains several putative domains, including helicase and RNA-dependent RNA polymerase. The HEV-encoded helicase is a member of the superfamily 1 helicase family and possesses multiple enzymatic functions, such as RNA 5'-triphosphatase, RNA unwinding, and NTPase, which are thought to contribute to viral RNA synthesis. However, the helicase interaction with cellular proteins remains less known. Oxysterol binding protein (OSBP) is a lipid regulator that shuffles between the Golgi apparatus and the endoplasmic reticulum for cholesterol and phosphatidylinositol-4-phosphate exchange and controls the efflux of cholesterol from cells. In this study, the RNAi-mediated silencing of OSBP significantly reduced HEV replication. Further studies indicate that the HEV helicase interacted with OSBP, shown by co-immunoprecipitation and co-localization in co-transfected cells. The presence of helicase blocked OSBP preferential translocation to the Golgi apparatus. These results demonstrate that OSBP contributes to HEV replication and enrich our understanding of the HEV-cell interactions.

Genetic characteristics and growth patterns of the hybrid grouper derived from the hybridization of Epinephelus fuscoguttatus (female) × Epinephelus polyphekadion (male).

Hybridization is one of the primary methods used to cultivate farmed grouper species. The hybrid grouper derived from crossing Epinephelus fuscoguttatus (♀) and E. polyphekadion (♂) exhibits growth superiority over its parents. The genetic characteristics and growth patterns of the hybrid grouper have not yet been defined. This study confirms the ploidy level of the hybrid grouper (2n = 48) using chromosome count analysis and flow cytometry. The 5S rDNA family was used to evaluate genetic diversity. Only one 5S class (~400 bp) was detected in the hybrid grouper, which could be used to distinguish between two different types based on nucleotide sequences, likely representing homologous unit classes from the female and male parental species. Growth patterns of 5-8-month-old hybrid groupers were also monitored. In this phase, a positive allometric growth pattern in body mass with total length was found. Body height and body mass were significantly correlated based on correlation and path coefficient, suggesting that body height could serve as an excellent index to increase body mass. These results aid our understanding of the genetic evolution of the hybrid grouper and inform the development of improved rearing techniques.

Effect of P-glycoprotein Inhibition on the Penetration of Ceftriaxone Across the Blood-Brain Barrier.

Recent studies indicate that inhibition of the efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) may represent a putative strategy to increase the BBB penetration of several antibiotics. Therefore, the present study aimed to investigate the effect of P-gp inhibition on the transport of ceftriaxone (CFX) across the BBB. Blood and brain microdialysis in rats was used to monitor blood and brain unbound CFX concentrations following intravenous administration (50 mg/kg), with or without pretreatment with one of the P-gp inhibitors, cyclosporin A (6.25, 12.5, 25 mg/kg) or verapamil (5, 10, 20 mg/kg). An inhibitory effect was demonstrated by an increase in the ratio of unbound brain to unbound blood concentration (Kp.uu.brain) of CFX. The concentrations of CFX in blood and brain from 0 to 180 min after intravenous administration (CFX, 50 mg/kg) ranged from 3 to 40 µg/ml and 1 to 10 µg/ml, respectively. The Kp.uu.brain of CFX was 24.74 ± 1.34%. Pretreatment with cyclosporin A increased the brain concentration and the Kp.uu.brain of CFX in a dose-dependent manner. However, pretreatment with verapamil increased the brain concentration of CFX but not the Kp.uu.brain. The present data shows that CFX might be a substrate of P-gp efflux transporter at the BBB and P-gp inhibition might enhance the brain concentration of CFX. Future studies involving more selective P-gp inhibitors or knockout mouse models should be conducted to specifically elucidate the impact of P-gp inhibition on penetration of CFX across the BBB.

Chloroperoxidase-catalyzed oxidative degradation of sulfur mustard.

As a natural heme protein catalyzing the oxidation of sulfides to sulfoxides without sulfone formation, chloroperoxidase (CPO) is well suited for the degradation of sulfur mustard (HD), a persistent chemical warfare agent that has been widely disposed since World War II and continuously leaks into aquatic environments. Herein, we report the first systematic investigation of CPO-catalyzed degradation of HD and the potential application of CPO in destroying chemical weapons under mild conditions. The related Michaelis-Menten parameters (Km=0.17 mM, Vmax=0.06 mM s-1 (R2 =0.935), and kcat= 2717 s-1) indicated nearly a prominent enzymatic efficiency. Under optimal conditions, 80% of HD was transformed to bis(2-chloroethyl) sulfoxide as identified by mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Other metabolites were also generated during the decontamination process. A plausible oxidation mechanism was proposed based on the degradation products, NMR titration experiments, and molecular dynamics simulations. CPO also promoted the degradation of other chemical weapon agents, namely, Lewisite (L) and venomous agent X (VX), thereby exhibiting a broad substrate scope. The high potential of the developed system for the decontamination of aquatic environments was demonstrated by the successful hatching of zebrafish embryos after HD degradation and the survival of zebrafish (Danio rerio, AB strain) larvae after the degradation of Agent Yellow (L+HD).

Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Induces STAT2 Degradation To Inhibit Interferon Signaling.

Interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce the expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen, causing huge losses to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced the STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5 h. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV nonstructural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and the coiled-coil domain. Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity.IMPORTANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling since it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 since a mutation of it led to loss of the ability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus.

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1ß but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1ß and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1ß depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1ß nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via nsp5, Which Induces STAT3 Degradation.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ∼3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. IMPORTANCE: The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

The middle half genome of interferon-inducing porcine reproductive and respiratory syndrome virus strain A2MC2 is essential for interferon induction.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to antagonize the innate immune response. An atypical PRRSV strain A2MC2 is capable of inducing synthesis of type I interferons (IFNs) in cultured cells. Here, we show that the middle half of the A2MC2 genome is needed for triggering the IFN synthesis. First, a cDNA infectious clone of this atypical strain was constructed as a DNA-launched version. Virus recovery was achieved from the infectious clone and the recovered virus, rA2MC2, was characterized. The rA2MC2 retained the feature of IFN induction in cultured cells. Infection of pigs with the rA2MC2 virus caused viremia similar to that of the wild-type virus. Chimeric infectious clones were constructed by swapping genomic fragments with a cDNA clone of a moderately virulent strain VR-2385 that antagonizes IFN induction. Analysis of the rescued chimeric viruses demonstrated that the middle two fragments, ranging from nt4545 to nt12709 of the A2MC2 genome, were needed for the IFN induction, whereas the chimeric viruses containing any one of the two A2MC2 fragments failed to do so. The results and the cDNA infectious clone of the IFN-inducing A2MC2 will facilitate further study of its biology, ultimately leading towards the development of an improved vaccine against PRRS.

Enhancement of interferon induction by ORF3 product of hepatitis E virus.

UNLABELLED: Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-ß mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-ß promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-ß expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion. IMPORTANCE: Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.

Hepatitis E virus inhibits type I interferon induction by ORF1 products.

Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-ß) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. Importance: Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host IFNs, though the viral genome was sequenced more than 2 decades ago. This is the first report of identification of the potential IFN antagonists encoded by HEV. By screening all the domains in the open reading frame 1 (ORF1) polyprotein, we identified two IFN antagonists and performed further research to determine how and at which step in the IFN induction pathway they antagonize host IFN induction. Our work provides valuable information about HEV-cell interaction and pathogenesis.

Porcine reproductive and respiratory syndrome virus Nsp1ß inhibits interferon-activated JAK/STAT signal transduction by inducing karyopherin-α1 degradation.

Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the interferon-mediated antiviral response. Type I interferons (IFNs) induce the expression of IFN-stimulated genes by activating phosphorylation of both signal transducer and activator of transcription 1 (STAT1) and STAT2, which form heterotrimers (interferon-stimulated gene factor 3 [ISGF3]) with interferon regulatory factor 9 (IRF9) and translocate to the nucleus. PRRSV Nsp1ß blocks the nuclear translocation of the ISGF3 complex by an unknown mechanism. In this study, we discovered that Nsp1ß induced the degradation of karyopherin-α1 (KPNA1, also called importin-α5), which is known to mediate the nuclear import of ISGF3. Overexpression of Nsp1ß resulted in a reduction of KPNA1 levels in a dose-dependent manner, and treatment of the cells with the proteasome inhibitor MG132 restored KPNA1 levels. Furthermore, the presence of Nsp1ß induced an elevation of KPNA1 ubiquitination and a shortening of its half-life. Our analysis of Nsp1ß deletion constructs showed that the N-terminal domain of Nsp1ß was involved in the ubiquitin-proteasomal degradation of KPNA1. A nucleotide substitution resulting in an amino acid change from valine to isoleucine at residue 19 of Nsp1ß diminished its ability to induce KPNA1 degradation and to inhibit IFN-mediated signaling. Interestingly, infection of MARC-145 cells by PRRSV strains VR-2332 and VR-2385 also resulted in KPNA1 reduction, whereas infection by an avirulent strain, Ingelvac PRRS modified live virus (MLV), did not. MLV Nsp1ß had no effect on KPNA1; however, a mutant with an amino acid change at residue 19 from isoleucine to valine induced KPNA1 degradation. These results indicate that Nsp1ß blocks ISGF3 nuclear translocation by inducing KPNA1 degradation and that valine-19 in Nsp1ß correlates with the inhibition.

Individualized prediction of conditional survival for colorectal signet-ring cell carcinoma patients.

Background: Conditional survival (CS) considers the time already survived after surgery and may provide additional survival information. The authors sought to construct and validate novel conditional survival nomograms for the prediction of conditional overall survival (OS) and cancer-specific survival (CSS) of colorectal signet-ring cell carcinoma (SRCC) patients. Methods: Patients diagnosed with stage I-III SRCC between 2010 and 2019 were identified from the Surveillance, Epidemiology, and End Results database. The formula calculating CS was: CS(x|y) = S(x+y)/S(x), where S(x) represents the survival at x years. CS nomograms were then constructed to predict the 5-year conditional OS and CSS, followed by internal validation. Results: A total of 944 colorectal SRCC patients were finally identified in this study. The 5-year OS and CSS improved gradually with additional survival time. Univariate and multivariate Cox regression analysis conducted in training set revealed that age, race, T stage, LNR, and perineural invasion were independent risk factors for both OS and CSS. Two nomograms with considerable predictive ability were successfully constructed [area under the curve (AUC) for OS: 0.788; AUC for CSS: 0.847] and validated (AUC for OS: 0.773; AUC for CSS: 0.799) for the prediction of 5-year OS and CSS, based on the duration of 1-4 years post-surgery survival. Conclusions: The probability of achieving 5-year OS and 5-year CSS in colorectal SRCC patients improved gradually with additional time. Conditional nomograms considering survival time will be more reliable and informative for risk stratification and postoperative follow-up.

SARS-CoV-2 ORF3a Protein as a Therapeutic Target against COVID-19 and Long-Term Post-Infection Effects.

The COVID-19 pandemic caused by SARS-CoV-2 has posed unparalleled challenges due to its rapid transmission, ability to mutate, high mortality and morbidity, and enduring health complications. Vaccines have exhibited effectiveness, but their efficacy diminishes over time while new variants continue to emerge. Antiviral medications offer a viable alternative, but their success has been inconsistent. Therefore, there remains an ongoing need to identify innovative antiviral drugs for treating COVID-19 and its post-infection complications. The ORF3a (open reading frame 3a) protein found in SARS-CoV-2, represents a promising target for antiviral treatment due to its multifaceted role in viral pathogenesis, cytokine storms, disease severity, and mortality. ORF3a contributes significantly to viral pathogenesis by facilitating viral assembly and release, essential processes in the viral life cycle, while also suppressing the body's antiviral responses, thus aiding viral replication. ORF3a also has been implicated in triggering excessive inflammation, characterized by NF-κB-mediated cytokine production, ultimately leading to apoptotic cell death and tissue damage in the lungs, kidneys, and the central nervous system. Additionally, ORF3a triggers the activation of the NLRP3 inflammasome, inciting a cytokine storm, which is a major contributor to the severity of the disease and subsequent mortality. As with the spike protein, ORF3a also undergoes mutations, and certain mutant variants correlate with heightened disease severity in COVID-19. These mutations may influence viral replication and host cellular inflammatory responses. While establishing a direct link between ORF3a and mortality is difficult, its involvement in promoting inflammation and exacerbating disease severity likely contributes to higher mortality rates in severe COVID-19 cases. This review offers a comprehensive and detailed exploration of ORF3a's potential as an innovative antiviral drug target. Additionally, we outline potential strategies for discovering and developing ORF3a inhibitor drugs to counteract its harmful effects, alleviate tissue damage, and reduce the severity of COVID-19 and its lingering complications.

The serological evidence in humans supports a negligible risk of zoonotic infection from porcine circovirus type 2.

There are two porcine circovirus (PCV) genotypes, PCV-1 and PCV-2. In pigs, PCV-1 infection is asymptomatic but PCV-2 infection can cause severe respiratory disease and other pathology. Although humans ingest PCV-contaminated foods and are exposed to PCV through other sources, the potential of PCV-2 as a zoonotic agent in humans and other species has not been fully explored. Here, four recombinant proteins derived from the PCV-2 capsid gene were examined as antigens using the Luciferase Immunoprecipitation System (LIPS) assay for serological analysis of PCV-2 infection. PCV-2-CAP-Δ1 was the optimum recombinant protein in the LIPS assay with a sensitivity of 93% and specificity of 100% using porcine samples. Testing of healthy human blood donors, equine and bovine serum samples failed to demonstrate the presence of anti-PCV-2 antibodies. Additionally, analysis of two high-risk human groups, cystic fibrosis patients taking porcine derived oral supplements and type I diabetes patients who had undergone porcine islet cell transplantation, showed no evidence of anti-PCV-2 antibodies. These results extend the extensively demonstrated use of LIPS as a robust approach for identifying humoral responses and provide evidence that PCV-2 is likely not infectious in humans.

Hepatitis E Virus: Isolation, Propagation, and Quantification.

Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver disease. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and propagation of hepatitis E virus in cultured cells from clinical HEV specimens Support Protocol 1: Quantification of HEV RNA by RT-qPCR Basic Protocol 2: Recovery of HEV from infectious cDNA clones and purification of the virus Support Protocol 2: Quantification of HEV live particles by infectivity assay.

Zika Virus Induces Degradation of the Numb Protein Required through Embryonic Neurogenesis.

Zika virus (ZIKV) is a mosquito-borne flavivirus and causes an infection associated with congenital Zika syndrome and Guillain-Barre syndrome. The mechanism of ZIKV-mediated neuropathogenesis is not well understood. In this study, we discovered that ZIKV induces degradation of the Numb protein, which plays a crucial role in neurogenesis by allowing asymmetric cell division during embryonic development. Our data show that ZIKV reduced the Numb protein level in a time- and dose-dependent manner. However, ZIKV infection appears to have minimal effect on the Numb transcript. Treatment of ZIKV-infected cells with a proteasome inhibitor restores the Numb protein level, which suggests the involvement of the ubiquitin-proteasome pathway. In addition, ZIKV infection shortens the half-life of the Numb protein. Among the ZIKV proteins, the capsid protein significantly reduces the Numb protein level. Immunoprecipitation of the Numb protein co-precipitates the capsid protein, indicating the interaction between these two proteins. These results provide insights into the ZIKV-cell interaction that might contribute to its impact on neurogenesis.

Acetylcholinesterase-capped mesoporous silica gated switches for selective detection of high-toxicity organophosphate compounds.

Organophosphate (OP) compounds are widely used in agriculture, industry, and even terrorism. It is important to distinguish high-toxicity OP compounds from low-toxicity OP compounds in dealing with chemical accidents. However, there are very few portable and simple detection methods. Mesoporous silica gated switches may provide an effective solution. In this study, a gated switch based on mesoporous silica as an inorganic scaffold loaded with sulforhodamine B and capped with acetylcholinesterase (AChE) was prepared for specific detection of OP compounds. Carbamate derivatives (G1-G6) were designed and synthetized as grafting compounds in consideration of the binding ability with AChE. Through further modification and optimization, grafting compound G6 with phenylpyridine as the substituent showed the best capping capacity, and it achieved excellent blocking of mesoporous silica gated switches for loaded sulforhodamine B. In the presence of high-toxicity OP compounds, low-toxicity OP compounds, AChE substrates and reversible AChE inhibitors respectively, only high-toxicity OP compounds could make the gated switch release loaded sulforhodamine B. The limit of detection for paraoxon-ethyl was 10.6 µΜ. Furthermore, the preparation process of the gated switch is fast and simple, and the prepared gated switch has good stability and rapid distinguishing ability. The results of this paper provide a new idea for rapidly distinguishing high-toxicity OP compounds from low-toxicity OP compounds and other related compounds on the spot.

Acyclovir Brain Disposition: Interactions with P-gp, Bcrp, Mrp2, and Oat3 at the Blood-Brain Barrier.

BACKGROUND AND OBJECTIVE: Acyclovir is effective in treating herpes simplex virus infections of the central nervous system. The purpose of this study was to investigate the interactions between acyclovir and the efflux pumps P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), multidrug resistance protein 2 (Mrp2), and organic anion transporter 3 (Oat3) at the blood-brain barrier (BBB). METHODS: Acyclovir concentrations in the blood and brain were evaluated by microdialysis and high-performance liquid chromatography. Acyclovir pharmacokinetic parameters, including the area under the unbound blood concentration-time curve (AUCu,blood), the area under the unbound brain concentration-time curve (AUCu,brain), and the ratio of AUCu,brain to AUCu,blood (Kp.uu.brain), were evaluated in the presence and absence of elacridar (P-gp/Bcrp inhibitor, 7.5 mg/kg), tariquidar (P-gp/Bcrp inhibitor, 7.5 mg/kg), MK571 (Mrp2 inhibitor, 7.5 mg/kg), cyclosporine (P-gp/Bcrp/Mrp2 inhibitor, 25 mg/kg), and probenecid (Oat3 inhibitor, 50 mg/kg). RESULTS: The average AUCu,blood, AUCu,brain, and Kp.uu.brain in rats who received acyclovir (25 mg/kg, intravenous) alone were 1377.7 min · µg/ml, 435.4 min · µg/ml, and 31.6%, respectively. Probenecid drastically increased the AUCu,blood of acyclovir 1.73-fold, whereas coadministration with elacridar, tariquidar, MK571, and cyclosporine did not alter the blood pharmacokinetic parameters of acyclovir. Elacridar, tariquidar, MK571, cyclosporine, and probenecid significantly increased the AUCu,brain of acyclovir 1.51-, 1.54-, 1.47-, 1.95-, and 2.34-fold, respectively. Additionally, the Kp.uu.brain of acyclovir markedly increased 1.48-, 1.63-, 1.39-, 1.90-, and 1.35-fold following elacridar, tariquidar, MK571, cyclosporine, and probenecid administration, respectively. CONCLUSION: The present study demonstrated that P-gp, Bcrp, Mrp2, and Oat3 inhibition increased the penetration of acyclovir across the BBB, supporting the hypothesis that these efflux pumps restrict the distribution of acyclovir in the brain.

A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport.

The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone's response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson's coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187-188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson's coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II ß-turn between the anti-parallel ß4 and ß5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.