TRIM32 inhibits Venezuelan Equine Encephalitis Virus Infection by targeting a late step in viral entry.

Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.

Enteric coronavirus nsp2 is a virulence determinant that recruits NBR1 for autophagic targeting of TBK1 to diminish the innate immune response.

Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete nsp2 deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication in vitro. Intriguingly, rPEDV-Δnsp2 was entirely avirulent in vivo, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs.Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2'-5'-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus.

Cross-species transmission and animal infection model of hepatitis E virus.

Zoonotic hepatitis E virus (HEV) infection is an emerging global public health concern, and understanding the dynamics of HEV transmission between animals and humans is crucial for public health. Animal models are critical to advancing the understanding of HEV pathogenesis, drug screening, vaccine development, and other related areas. Here, we provide an overview of recent studies investigating the cross-species transmission of HEV, and also delve into the current research and application of animal HEV infection models including non-human primates, rodents, pigs, and chickens, offering a comprehensive assessment of the advantages and disadvantages of each model. This review highlights the findings related to viral replication, shedding patterns, and immune response in these animal models, and discusses the implications for our understanding of HEV transmission to humans. These advancements in the field enhance our understanding of the biological traits and pathogenic mechanisms of HEV, offering robust support for the development of highly effective and targeted prevention and treatment strategies.

Fucoidan from Ascophyllum nodosum and Undaria pinnatifida attenuate SARS-CoV-2 infection in vitro and in vivo by suppressing ACE2 and alleviating inflammation.

The global healthcare challenge posed by COVID-19 necessitates the continuous exploration for novel antiviral agents. Fucoidans have demonstrated antiviral activity. However, the underlying structure-activity mechanism responsible for the inhibitory activity of fucoidans from Ascophyllum nodosum (FUCA) and Undaria pinnatifida (FUCU) against SARS-CoV-2 remains unclear. FUCA was characterized as a homopolymer with a backbone structure of repeating (1 â†’ 3) and (1 â†’ 4) linked α-l-fucopyranose residues, whereas FUCU was a heteropolysaccharide composed of Fuc1-3Gal1-6 repeats. Furthermore, FUCA demonstrated significantly higher anti-SARS-CoV-2 activity than FUCU (EC50: 48.66 vs 69.52 µg/mL), suggesting the degree of branching rather than sulfate content affected the antiviral activity. Additionally, FUCA exhibited a dose-dependent inhibitory effect on ACE2, surpassing the inhibitory activity of FUCU. In vitro, both FUCA and FUCU treatments downregulated the expression of pro-inflammatory cytokines (IL-6, IFN-α, IFN-γ, and TNF-α) and anti-inflammatory cytokines (IL-10 and IFN-ß) induced by viral infection. In hamsters, FUCA demonstrated greater effectiveness in attenuating lung and gastrointestinal injury and reducing ACE2 expression, compared to FUCU. Analysis of the 16S rRNA gene sequencing revealed that only FUCU partially alleviated the gut microbiota dysbiosis caused by SARS-CoV-2. Consequently, our study provides a scientific basis for considering fucoidans as poteintial prophylactic food components against SARS-CoV-2.

Immunological pathways in viral hepatitis-induced hepato-cellular carcinoma. / è‚ç‚Žç— æ¯’æ„ŸæŸ“å¯¼è‡´è‚ç»†èƒžç™Œå‘ç”Ÿçš„å ç–«æœºåˆ¶ç ”ç©¶è¿›å±•.

Hepatocellular carcinoma (HCC) is a serious neoplastic disease with increasing incidence and mortality, accounting for 90% of all liver cancers. Hepatitis viruses are the major causative agents in the development of HCC. Hepatitis A virus (HAV) primarily causes acute infections, which is associated with HCC to a certain extent, as shown by clinicopathological studies. Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections lead to persistent liver inflammation and cirrhosis, disrupt multiple pathways associated with cellular apoptosis and proliferation, and are the most common viral precursors of HCC. Mutations in the HBV X protein (HBx) gene are closely associated with the incidence of HCC, while the expression of HCV core proteins contributes to hepatocellular lipid accumulation, thereby promoting tumorigenesis. In the clinical setting, hepatitis D virus (HDV) frequently co-infects with HBV, increasing the risk of chronic hepatitis. Hepatitis E virus (HEV) usually causes acute infections. However, chronic infections of HEV have been increasing recently, particularly in immuno-compromised patients and organ transplant recipients, which may increase the risk of progression to cirrhosis and the occurrence of HCC. Early detection, effective intervention and vaccination against these viruses may significantly reduce the incidence of liver cancer, while mechanistic insights into the interplay between hepatitis viruses and HCC may facilitate the development of more effective intervention strategies. This article provides a comprehensive overview of hepatitis viruses and reviews recent advances in research on aberrant hepatic immune responses and the pathogenesis of HCC due to viral infection.

T cell proliferation requires ribosomal maturation in nucleolar condensates dependent on DCAF13.

T cells require rapid proliferation to initiate adaptive immunity to prevent pathogen attacks. The nucleolus, a distinct subnuclear membrane-less compartment for ribosomal biogenesis, is indispensable for cell proliferation. However, specific nucleolar proteins involved in rapid T cell proliferation and their underlying molecular regulatory mechanism remain elusive. Here, we identified an essential nucleolar protein, DCAF13, in T cells and revealed its significant regulation of rapid T cell proliferation. Its depletion drastically impairs T cell proliferation due to severe 18S rRNA maturation failure, consequent abnormal ribosome assembly in nucleoli, and insufficient production of nascent proteins. Mechanistically, we propose that DCAF13 promotes NPM1 phase separation to accelerate pre-RNA enrichment and its endonuclease UTP23 for 18S rRNA maturation during T cell proliferation. Our findings reveal the modulatory effect of nucleolar NPM1/DCAF13 phase separation on ribosomal maturation to ensure rapid T cell proliferation and further pathogen clearance for the first time.

Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.

Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90ß on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90ß in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

Role of heat shock protein 90 as an antiviral target for swine enteric coronaviruses.

A variety of swine enteric coronaviruses (SECoVs) have emerged and are prevalent in pig populations, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome (SADS)-CoV, a newly identified bat-origin CoV with zoonotic potential. Unfortunately, available traditional, inactivated and attenuated SECoV vaccines are of limited efficacy against the variants currently circulating in most pig populations. In this study, we evaluated the role of host factor heat shock protein 90 (Hsp90) as an antiviral target against SECoVs, exemplified by SADS-CoV. Pharmacological inhibition of Hsp90 diminished SADS-CoV replication significantly in porcine and human cell lines, and also decreased replication of SADS-CoV in a porcine intestinal enteroid model. Further mechanistic experiments revealed that both porcine and human isoforms of Hsp90 interact with the SADS-CoV nucleocapsid (N) protein, and inhibition of Hsp90 resulted in autophagic degradation of N protein. Moreover, we linked Hsp90 to virus-induced cellular pyroptosis, as SADS-CoV was found to trigger caspase-1/gasdermin-d-mediated pyroptotic cell death, which was mitigated by inhibition of Hsp90. Finally, we demonstrated that Hsp90 also associated with N proteins and was involved in propagation of PEDV, PDCoV and TGEV. This study thus extends our understanding of immune responses to SADS-CoV infection and offers a new potential therapeutic option against four SECoVs.

Targeting proteostasis of the HEV replicase to combat infection in preclinical models.

BACKGROUND & AIMS: Appropriate treatment options are lacking for hepatitis E virus (HEV)-infected pregnant women and immunocompromised individuals. Thus, we aimed to identify efficient anti-HEV drugs through high-throughput screening, validate them in vitro and in vivo (in a preclinical animal study), and elucidate their underlying antiviral mechanism of action. METHODS: Using appropriate cellular and rodent HEV infection models, we studied a critical pathway for host-HEV interactions and performed a preclinical study of the corresponding antivirals, which target proteostasis of the HEV replicase. RESULTS: We found 17 inhibitors that target HEV-HSP90 interactions by unbiased compound library screening on human hepatocytes harboring an HEV replicon. Inhibitors of HSP90 (iHSP90) markedly suppressed HEV replication with efficacy exceeding that of conventional antivirals (IFNα and ribavirin) in vitro. Mechanistically, iHSP90 treatment released the viral replicase ORF1 protein from the ORF1-HSP90 complex and triggered rapid ubiquitin/proteasome-mediated degradation of ORF1, resulting in abrogated HEV replication. Furthermore, a preclinical trial in a Mongolian gerbil HEV infection model showed this novel anti-HEV strategy to be safe, efficient, and able to prevent HEV-induced liver damage. CONCLUSIONS: In this study, we uncover a proteostatic pathway that is critical for host-HEV interactions and we provide a foundation from which to translate this new understanding of the HEV life cycle into clinically promising antivirals. IMPACT AND IMPLICATIONS: Appropriate treatment options for hepatitis E virus (HEV)-infected pregnant women and immunocompromised patients are lacking; hence, there is an urgent need for safe and effective HEV-specific therapies. This study identified new antivirals (inhibitors of HSP90) that significantly limit HEV infection by targeting the viral replicase for degradation. Moreover, these anti-HEV drugs were validated in an HEV rodent model and were found to be safe and efficient for prevention of HEV-induced liver injury in preclinical experiments. Our findings substantially promote the understanding of HEV pathobiology and pave the way for antiviral development.

Surface Display of Peptides Corresponding to the Heptad Repeat 2 Domain of the Feline Enteric Coronavirus Spike Protein on Bacillus subtilis Spores Elicits Protective Immune Responses Against Homologous Infection in a Feline Aminopeptidase-N-Transduced Mouse Model.

Although feline coronavirus (FCoV) infection is extremely common in cats, there are currently few effective treatments. A peptide derived from the heptad repeat 2 (HR2) domain of the coronavirus (CoV) spike protein has shown effective for inhibition of various human and animal CoVs in vitro, but further use of FCoV-HR2 in vivo has been limited by lack of practical delivery vectors and small animal infection model. To overcome these technical challenges, we first constructed a recombinant Bacillus subtilis (rBSCotB-HR2P) expressing spore coat protein B (CotB) fused to an HR2-derived peptide (HR2P) from a serotype II feline enteric CoV (FECV). Immunogenic capacity was evaluated in mice after intragastric or intranasal administration, showing that recombinant spores could trigger strong specific cellular and humoral immune responses. Furthermore, we developed a novel mouse model for FECV infection by transduction with its primary receptor (feline aminopeptidase N) using an E1/E3-deleted adenovirus type 5 vector. This model can be used to study the antiviral immune response and evaluate vaccines or drugs, and is an applicable choice to replace cats for the study of FECV. Oral administration of rBSCotB-HR2P in this mouse model effectively protected against FECV challenge and significantly reduced pathology in the digestive tract. Owing to its safety, low cost, and probiotic features, rBSCotB-HR2P is a promising oral vaccine candidate for use against FECV/FCoV infection in cats.

A Novel Nomogram Based on a Competing Risk Model Predicting Cardiovascular Death Risk in Patients With Chronic Kidney Disease.

Objective: Chronic kidney disease (CKD) patients are more likely to die from cardiovascular disease (CVD) than develop renal failure. This study aimed to develop a new nomogram for predicting the risk of cardiovascular death in CKD patients. Methods: This study enrolled 1656 CKD patients from NHANES 2003 to 2006 survey. Data sets from 2005 to 2006 survey population were used to build a nomogram for predicting the risk of cardiovascular death, and the nomogram was validated using data from 2003 to 2004 survey population. To identify the main determinants of cardiovascular death, we performed univariate analysis and backward-stepwise regression to select the key factors. The probability of cardiovascular death for each patient in 5, 7, and 9 years was calculated using a nomogram based on the predictors. To assess the nomogram's performance, the area under receiver operating characteristic curve (AUC) and the calibration curve with 1,000 bootstraps resamples were utilized. The prediction model's discrimination was examined using cumulative incidence function (CIF). Results: Age, homocysteine, potassium levels, CKD stage, and anemia were included in the nomogram after screening risk factors using univariate analysis and backward-stepwise regression. Internal validation revealed that this nomogram possesses high discrimination and calibration (AUC values of 5-, 7-, and 9-years were 0.79, 0.81, and 0.81, respectively). External validation confirmed the same findings (AUC values of 5-, 7- and 9-years were 0.76, 0.73, and 0.73, respectively). According to CIF, the established nomogram effectively differentiates patients at a high risk of cardiovascular death from those at low risk. Conclusion: This work develops a novel nomogram that integrates age, homocysteine, potassium levels, CKD stage, and anemia and can be used to more easily predict cardiovascular death in CKD patients, highlighting its potential value in clinical application.

Revisiting the Mongolian Gerbil Model for Hepatitis E Virus by Reverse Genetics.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. A convenient small mammalian model for basic research and antiviral testing is still greatly needed. Although a small rodent, the Mongolian gerbil, was reported to be susceptible to swine genotype-4 HEV infection, whether the previous results were reliable and consistent needs to be validated by using biologically pure HEV stocks or infectious RNA. In this study, we revisited this gerbil infection model for human HEV of genotype 1, 3, or 4 (G1, G3, or G4) by HEV reverse genetics. Gerbils inoculated intrahepatically with capped G3 HEV RNA transcripts or intraperitoneally with infectious G3 cloned HEV produced robust infection, as evidenced by presence of HEV in livers, spleens, and feces for up to 7 weeks post inoculation, seroconversion, and pathological liver lesions. Furthermore, the value of the gerbil model in antiviral testing and type I IFN in host defense was assessed. We demonstrated the effectiveness of peg-IFNα-2a and ribavirin in inhibiting HEV replication in gerbils. By treatment with two molecule inhibitors of TBK1, we also revealed a role of RIG-I like receptor-interferon regulatory factor 3 in host anti-HEV innate immune sensing in this in vivo model. Finally, susceptibility of G4 HEV was demonstrated in intrahepatically inoculated gerbils with infectious HEV RNA transcripts, whereas no evidence for G1 HEV susceptibility was found. The availability of the convenient gerbil model will greatly facilitate HEV-specific antiviral development and assess the mechanism of host immune response during HEV infection. IMPORTANCE HEV infects >20 million people annually, causing acute viral hepatitis as well as chronic hepatitis, neurological diseases, and pregnancy-associated high mortality, which require therapeutic intervention. The HEV antiviral research is largely limited by the lack of a convenient small animal model. Here we revisit the Mongolian gerbil model for three genotypes of human HEV by infectious HEV clones and recognized standards of experimental procedures. Fecal virus shedding, seroconversion, and pathological liver lesions could be detected in HEV-inoculated gerbils. We demonstrate the effectiveness and usefulness of this model in testing antiviral drugs, and in assessing the mechanism of host innate immune response upon HEV infection. This conventional rodent model will aid in future antiviral development and delineating mechanism of host immune response.

Expression of the human or porcine C-type lectins DC-SIGN/L-SIGN confers susceptibility to porcine epidemic diarrhea virus entry and infection in otherwise refractory cell lines.

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that causes great economic losses in the porcine industry. Although the functional receptor for the virus has not been identified, multiple isolates are able to infect different cell lines. Recently, it has been shown that the human C-type lectin DC-SIGN/L-SIGN (hDC-SIGN/L-SIGN) can promote entry of several coronaviruses. Here we examined whether hDC-SIGN/L-SIGN and its porcine homolog (pDC-SIGN) are entry determinants for PEDV. Expression of hDC-SIGN/L-SIGN or pDC-SIGN in refractory cells dramatically increased infection by a recombinant PEDV expressing green fluorescent protein. In both cases, lectin-mediated infection was inhibited by mannan or anti-hDC-SIGN/L-SIGN or pDC-SIGN antibodies; however, d-galactose had no effect on the virus-infected cells. Our results demonstrate that hDC-SIGN/L-SIGN or pDC-SIGN can mediate the cellular entry and propagation of PEDV, which provides a new theoretical basis for further understanding the infection mechanism of PEDV, and will be helpful for the development of novel therapeutic agents.

Efficacy and Safety of Chinese Herbal Formula Granules in Treating Chronic Kidney Disease Stage 3: A Multicenter, Randomized, Placebo-Controlled, Double-Blind Clinical Trial.

BACKGROUND: It is generally considered that traditional Chinese medicine (TCM) therapy postpones the progression of some chronic kidney diseases (CKDs). Chinese medicine herbs are widely applied in TCM therapy. We aimed to evaluate clinical efficacy and safety of Chinese herbal formula granules in patients with CKD stage 3 through a prospective randomized controlled study. METHODS: A total of 343 participants with CKD stage 3 were recruited from 9 hospitals in Jiangsu Province between April 2014 and October 2016. Participants were randomly assigned to a treatment or control group. Patients in the treatment group orally took Chinese herbal formula granules twice a day, while controls received placebo granules. The duration of intervention was 24 weeks. Primary outcomes were 24-hour proteinuria, serum creatinine, and eGFR, which were measured every 4 weeks. RESULTS: There was no statistical difference in 24-hour proteinuria between the two groups (0.97 ± 1.14 g/d vs. 0.97 ± 1.25 g/d). Patients in the treatment group had significantly lower serum creatinine level (130.78 ± 32.55 µmol/L versus 149.12 ± 41.27 µmol/L) and significantly higher eGFR level (55.74 ± 50.82 ml/min/1.73·m2 versus 44.46 ± 12.60 ml/min/1.73·m2) than those in the control group (P < 0.05). There was no significant difference between two groups in the incidence of adverse events. CONCLUSION: The treatment adopting Chinese herbal formula granules for 24 weeks improved kidney function of patients with CKD stage 3.

Stable Expression of a Hepatitis E Virus (HEV) RNA Replicon in Two Mammalian Cell Lines to Assess Mechanism of Innate Immunity and Antiviral Response.

Hepatitis E virus (HEV) is one of the major etiological agents responsible for acute hepatitis. Hepatitis E virus does not replicate efficiently in mammalian cell cultures, thus a useful model that mimics persistent HEV replication is needed to dissect the molecular mechanism of pathogenesis. Here we report a genotype-3 HEV RNA replicon expressing an EGFP-Zeocin (EZ) resistant gene (p6-EZ) that persistently self-replicated in cell lines of human (Huh-7-S10-3) or hamster (BHK-21) origin after transfection with in vitro RNA transcripts and subsequent drug screening. Two cell lines, S10-3-EZ and BHK-21-EZ, stably expressed EGFP in the presence of Zeocin during continuous passages. Both genomic and subgenomic HEV RNAs and viral replicase proteins were stably expressed in persistent HEV replicon cells. The values of the cell models in antiviral testing, innate immune RNA sensing and type I IFN in host defense were further demonstrated. We revealed a role of RIG-I like receptor-interferon regulatory factor 3 in host antiviral innate immune sensing during HEV replication. We further demonstrated that treatment with interferon (IFN-α) or ribavirin significantly reduced expression of replicon RNA in a dose-dependent manner. The availability of the models will greatly facilitate HEV-specific antiviral development, and delineate mechanisms of HEV replication.

Porcine Torovirus (PToV)-A Brief Review of Etiology, Diagnostic Assays and Current Epidemiology.

Porcine torovirus (PToV) is a potential enteric swine pathogen, found at especially high rates in piglets with diarrhea. It was first reported in the Netherlands in 1998 and has emerged in many countries around the world. Infections are generally asymptomatic and have not directly caused large economic losses, though co-infections with other swine pathogens and intertype recombination may lead to unpredictable outcomes. This review introduces progress in PToV research regarding its discovery, relationship with other Toroviruses, virion morphological characteristics, genetic structure and variation, recent epidemiology, diagnostic methods, and possibilities for future research.

Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry.

Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to an understanding of pathogenesis and to development of intervention strategies. The fourth CoV genus, Deltacoronavirus, evolutionarily related to the Gammacoronavirus, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine deltacoronovirus (PDCoV) and alphacoronovirus (AlphaCoV) (transmissible gastroenteritis virus [TGEV]) based upon three lines of evidence. First, the soluble S1 protein of PDCoV bound to the surface of target porcine cell lines known to express pAPN as efficiently as TGEV-S1, which could be blocked by soluble pAPN pretreatment. Second, both PDCoV-S1 and TGEV-S1 physically recognized and interacted with pAPN by coimmunoprecipitation in pAPN cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and to PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells than TGEV-S1, suggesting that there may be differences between these two viruses in the virus-binding regions in pAPN. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection.IMPORTANCE The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by a species-specific receptor of the host. Among the four genera of the Coronavirinae, a couple of functional receptors for the representative members in the genera Alphacoronavirus and Betacoronavirus have been identified, whereas receptors for Gammacoronavirus and Deltacoronavirus, which are believed to originate from birds, are still unknown. Porcine coronaviruses, including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets, have posed a serious threat to the pork industry in Asia and North America. Here, we report that PDCoV employs the alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of the PDCoV receptor provides another example of the expanded host range of CoV and paves the way for further investigation of PDCoV-host interaction and pathogenesis.

Effects of Astragaloside IV Against the TGF-ß1-Induced Epithelial-to-Mesenchymal Transition in Peritoneal Mesothelial Cells by Promoting Smad 7 Expression.

BACKGROUND/AIMS: To investigate the effect of Astragaloside IV (AS-IV) on the regulation of the TGF-ß1/Smad signaling pathway in peritoneal mesothelial cells with an epithelial-to-mesenchymal transition (EMT). METHODS: EMT of human peritoneal mesothelial cells (HMrSV5) was induced using 2 ng/ml TGF-ß1. Cells were randomly divided into a vehicle group, a vehicle group with AS-IV, a TGF-ß1 treated group, and a TGF-ß1 treated group receiving varied doses of AS-IV or NAC. Real-time quantitative PCR and western blot were used to detect the expression of genes and proteins associated with the TGF-ß1/Smad signaling pathway and EMT. DCFH-DA was used to detect the generation of ROS in HMrSV5 cells, and a transwell migration assay was used to verify the capacity of AS-IV to inhibit EMT in HMrSV5 cells. Lentiviruses were used as carriers for the overexpression or knockdown of the Smad7 gene. RESULTS: Expression levels of E-cadherin (epithelial marker) was decreased and vimentin, α-SMA (EMT markers) and collagen I (extracellular matrix protein) phospho-Smad2/3, Snail1 and Snail2 was increased significantly in the TGF-ß1-treated HMrSV5 cells. AS-IV was associated with downregulated expression of vimentin and phospho-Smad2/3 in a dose-dependent manner, while the expression of Smad7 increased. Silenced or forced expression of Smad7 verified its role in the inhibitory effect of AS-IV on TGF-ß1-induced EMT in HMrSV5 cells. CONCLUSION: AS-IV effectively promotes the upregulation of Smad7 in the TGF-ß1/Smad signaling pathway during the EMT of HMrSV5 cells, indicating its potential therapeutic effect for the control of PF.