PCBP1 Deficient Pigs Hold the Potential to Inhibit CSFV Infection

Classical swine fever virus (CSFV), pathogen of classic swine fever, has caused severe economic losses worldwide. Poly (rC)-binding protein 1 (PCBP1), interacting with Npro of CSFV, plays a vital role in CSFV growth. Here, our research is the first report to generate PCBP1 knockout pigs via gene editing technology. The PCBP1 knockout pigs exhibited normal birth weight, reproductive-performance traits, and developed normally. Viral challenge results indicated that primary cells isolated from F0 and F1 generation pigs could significantly reduce CSFV infection. Additional mechanism exploration further confirmed that PCBP1 KO mediated antiviral effect is related with the activation of type I interferon. Beyond showing that gene editing strategy can be used to generate PCBP1 KO pigs, our study introduces a valuable animal model for further investigating infection mechanisms of CSFV that help to develop better antiviral solution.

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TNF-Mediated Inhibition of Classical Swine Fever Virus Replication Is IRF1-, NF-κB- and JAK/STAT Signaling-Dependent

Viruses ◽

10.3390/v13102017 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2017

Author(s):

Matthias Liniger ◽

Markus Gerber ◽

Sandra Renzullo ◽

Obdulio García-Nicolás ◽

Nicolas Ruggli

Keyword(s):

Signaling Pathways ◽

Classical Swine Fever Virus ◽

Proinflammatory Cytokine ◽

Classical Swine Fever ◽

Antiviral Effect ◽

Fever Virus ◽

Type I ◽

Type I Ifn ◽

Interferon Regulatory Factor 1 ◽

Necrosis Factor

The sera from pigs infected with virulent classical swine fever virus (CSFV) contain substantial amounts of tumor necrosis factor (TNF), a prototype proinflammatory cytokine with pleiotropic activities. TNF limits the replication of CSFV in cell culture. In order to investigate the signaling involved in the antiviral activity of TNF, we employed small-molecule inhibitors to interfere specifically with JAK/STAT and NF-κB signaling pathways in near-to-primary endothelial PEDSV.15 cells. In addition, we knocked out selected factors of the interferon (IFN) induction and signaling pathways using CRISPR/Cas9. We found that the anti-CSFV effect of TNF was sensitive to JAK/STAT inhibitors, suggesting that TNF induces IFN signaling. Accordingly, we observed that the antiviral effect of TNF was dependent on intact type I IFN signaling as PEDSV.15 cells with the disrupted type I IFN receptor lost their capacity to limit the replication of CSFV after TNF treatment. Consequently, we examined whether TNF activates the type I IFN induction pathway. With genetically modified PEDSV.15 cells deficient in functional interferon regulatory factor 1 or 3 (IRF1 or IRF3), we observed that the anti-CSFV activity exhibited by TNF was dependent on IRF1, whereas IRF3 was dispensable. This was distinct from the lipopolysaccharide (LPS)-driven antiviral effect that relied on both IRF1 and IRF3. In agreement with the requirement of IRF1 to induce TNF- and LPS-mediated antiviral effects, intact IRF1 was also essential for TNF- and LPS-mediated induction of IFN-β mRNA, while the activation of NF-κB was not dependent on IRF1. Nevertheless, NF-κB activation was essential for the TNF-mediated antiviral effect. Finally, we observed that CSFV failed to counteract the TNF-mediated induction of the IFN-β mRNA in PEDSV.15 cells, suggesting that CSFV does not interfere with IRF1-dependent signaling. In summary, we report that the proinflammatory cytokine TNF limits the replication of CSFV in PEDSV.15 cells by specific induction of an IRF1-dependent antiviral type I IFN response.

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Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

Journal of Virology ◽

10.1128/jvi.02718-15 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4412-4426 ◽

Cited By ~ 30

Author(s):

Lian-Feng Li ◽

Jiahui Yu ◽

Yongfeng Li ◽

Jinghan Wang ◽

Su Li ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Binding Protein ◽

Classical Swine Fever ◽

Antiviral Effect ◽

Viral Disease ◽

Fever Virus ◽

Type I ◽

Gtpase Activity ◽

Interferon Stimulated Genes ◽

Guanylate Binding Protein

ABSTRACTMany viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathioneS-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.IMPORTANCEClassical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.

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Immunological Aspects Related to Viral Infections in Severe Asthma and the Role of Omalizumab

Biomedicines ◽

10.3390/biomedicines9040348 ◽

2021 ◽

Vol 9 (4) ◽

pp. 348

Author(s):

Francesco Menzella ◽

Giulia Ghidoni ◽

Carla Galeone ◽

Silvia Capobelli ◽

Chiara Scelfo ◽

...

Keyword(s):

Innate Immunity ◽

Severe Asthma ◽

Viral Infections ◽

Respiratory Infections ◽

Antiviral Effect ◽

Point Of View ◽

Type I ◽

Effector Cells ◽

Viral Spread ◽

Increased Risk

Viral respiratory infections are recognized risk factors for the loss of control of allergic asthma and the induction of exacerbations, both in adults and children. Severe asthma is more susceptible to virus-induced asthma exacerbations, especially in the presence of high IgE levels. In the course of immune responses to viruses, an initial activation of innate immunity typically occurs and the production of type I and III interferons is essential in the control of viral spread. However, the Th2 inflammatory environment still appears to be protective against viral infections in general and in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections as well. As for now, literature data, although extremely limited and preliminary, show that severe asthma patients treated with biologics don’t have an increased risk of SARS-CoV-2 infection or progression to severe forms compared to the non-asthmatic population. Omalizumab, an anti-IgE monoclonal antibody, exerts a profound cellular effect, which can stabilize the effector cells, and is becoming much more efficient from the point of view of innate immunity in contrasting respiratory viral infections. In addition to the antiviral effect, clinical efficacy and safety of this biological allow a great improvement in the management of asthma.

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Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells

Virology ◽

10.1016/j.virol.2005.02.004 ◽

2005 ◽

Vol 335 (1) ◽

pp. 131-144 ◽

Cited By ~ 18

Author(s):

Elizabeth K. Wansley ◽

Patrick J. Dillon ◽

Maria D. Gainey ◽

James Tam ◽

Scott D. Cramer ◽

...

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Type I Interferon ◽

Simian Virus ◽

Type I ◽

Tumor Cell Lines ◽

Primary Cells ◽

Simian Virus 5

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Simultaneous detection of astrovirus, rotavirus, reovirus and adenovirus type I in broiler chicken flocks

Polish Journal of Veterinary Sciences ◽

10.2478/v10181-012-0052-0 ◽

2012 ◽

Vol 15 (2) ◽

pp. 337-344 ◽

Cited By ~ 11

Author(s):

D. Roussan ◽

I. Shaheen ◽

G. Khawaldeh ◽

W. Totanji ◽

R. Al-Rifai

Keyword(s):

Polymerase Chain Reaction ◽

Broiler Chicken ◽

Simultaneous Detection ◽

Adenovirus Type ◽

Economic Losses ◽

Type I ◽

Chain Reaction ◽

Group I ◽

Avian Nephritis Virus ◽

Polymerase Chain

Simultaneous detection of astrovirus, rotavirus, reovirus and adenovirus type I in broiler chicken flocksEnteric diseases cause substantial economic losses to the poultry industry. Astroviruses, rotaviruses, reoviruses, and adenovirus type 1 have been reported as a significant cause of intestinal symptoms in poultry. In the present study, intestinal samples from 70 commercial broiler chicken flocks were examined for the presence of astroviruses, rotavirus, and reovirus by reverse transcription-polymerase chain reaction, and for the presence of group I adenovirus by polymerase chain reaction. Astroviruses were identified in 38.6% of samples tested. Both avian nephritis virus and chicken astrovirus were identified in the astrovirus positive flocks, where 74.1% of these flocks were positive for only one type of astrovirus, whereas, 25.9% of these flocks were positive for both types of astrovirus. Reoviruses, rotaviruses, and adenoviruses were identified in 21.4, 18.6, and 14.3% of these flocks, respectively. Concomitant infection with two or more viruses in the same flock were also prominent, where 5.7, 5.7, 2.9, 2.9, 1.4, and 1.4% of these flocks were positive with both astrovirus and rotavirus; astrovirus and adenovirus; astrovirus and reovirus; rotavirus and adenovirus; rotavirus and reovirus; and reovirus and adenovirus respectively. Moreover, 4.3 and 2.7% of these flocks were positive for astrovirus, reovirus, and adenovirus; and astrovirus, reovirus, and rotavirus, respectively. Further studies will focus on identifying specific viral factors or subtypes/subgroups associated with disease through pathogenesis studies, economic losses caused by infections and co-infections of these pathogens, and the costs and benefits of countermeasures.

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Efficient homology-directed gene editing by CRISPR/Cas9 in human stem and primary cells using tube electroporation

Scientific Reports ◽

10.1038/s41598-018-30227-w ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 21

Author(s):

Xiaoyun Xu ◽

Dongbing Gao ◽

Ping Wang ◽

Jian Chen ◽

Jinxue Ruan ◽

...

Keyword(s):

Gene Editing ◽

Primary Cells

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The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

Journal of Virology ◽

10.1128/jvi.00655-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8780-8794 ◽

Cited By ~ 46

Author(s):

Subhajit Poddar ◽

Jennifer L. Hyde ◽

Matthew J. Gorman ◽

Michael Farzan ◽

Michael S. Diamond

Keyword(s):

Viral Infections ◽

Transmembrane Protein ◽

Flow Cytometric Analysis ◽

Antiviral Effect ◽

Host Cells ◽

Type I ◽

Targeted Gene Deletion ◽

Multiple Organs ◽

Viral Burden ◽

Alphavirus Infection

ABSTRACTHost cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon-stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses, including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased inIfitm3−/−andIfitmlocus deletion (Ifitm-del) fibroblasts and, reciprocally, reduced in fibroblasts transcomplemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis,Ifitm3−/−mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 postinfection, and this correlated with higher levels of proinflammatory cytokines and viral burden. Flow cytometric analysis suggested thatIfitm3−/−macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments withIfitm3−/−bone marrow-derived macrophages.Ifitm3−/−mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses.IMPORTANCEThe interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture. Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion ofIfitm3as well as deficient cells transcomplemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses.

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Metagenomic discovery of CRISPR-associated transposons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2112279118 ◽

2021 ◽

Vol 118 (49) ◽

pp. e2112279118

Author(s):

James R. Rybarski ◽

Kuang Hu ◽

Alexis M. Hill ◽

Claus O. Wilke ◽

Ilya J. Finkelstein

Keyword(s):

Gene Editing ◽

Type I ◽

Genomic Databases ◽

Type Iv ◽

Shed Light

CRISPR-associated Tn7 transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover architectures for all known CASTs, including arrangements of the Cascade effectors, target homing modalities, and minimal V-K systems. We also describe families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These systems shed light on how CRISPR systems have coevolved with transposases and expand the programmable gene-editing toolkit.

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Identification of E2 with improved secretion and immunogenicity against CSFV in piglets

10.21203/rs.2.18234/v2 ◽

2020 ◽

Author(s):

Huiling Xu ◽

Yanli Wang ◽

Guangwei Han ◽

Weihuan Fang ◽

fang he

Keyword(s):

Single Dose ◽

Signal Peptide ◽

Classical Swine Fever Virus ◽

Subunit Vaccine ◽

Classical Swine Fever ◽

Cost Effective ◽

Fever Virus ◽

Economic Losses ◽

Swine Industry ◽

Virus Challenge

Abstract Background: Outbreaks of Classical swine fever virus (CSFV) cause significant economic losses in the swine industry. Vaccination is the major method to prevent and control the disease. As live attenuated vaccines fail to elicit differentiable immunity between infected and vaccinated animals, subunit vaccine was considered as an alternative candidate to prevent and eradicate CSFV. Subunit vaccines present advantages in DIVA immunogenicity and safety. The technology was limited due to the low yield and the high cost with multiple and large doses. The native E2 signal peptide has not been well defined before. Here, the aim of this study is to develop a cost-effective and efficacious E2 vaccine candidate against CSFV with signal peptide and E2 sequence selection. Results: A novel CSFV E2 sequence (E2ZJ) was identified from an epidemic strain of Zhejiang for outstanding secretion in baculovirus and enhanced immunogenicity. E2 secretion induced with the selected signal peptide, SPZJ (SP23), increase at least 50% as compared to any other signal peptides tested. Besides, unique antigenic features were identified in E2ZJ. E2ZJ elicited CSFV antibodies at the earlier stage than other E2 types tested in mice. Moreover, higher level of neutralization antibodies against both genotypes 1 and 2 CSFV with E2ZJ was detected than other E2s with the same dosage. Further, in piglets, E2ZJ successfully elicited neutralizing immunity. A single dose of 5 μg of E2ZJ was sufficient to induce protective antibodies against CSFV in piglets and provided 100% protection against lethal virus challenge. Conclusions: Our studies provide evidence that E2ZJ guided by a novel E2 signal peptide (SPZJ) was efficiently secreted and presented significantly improved immunogenicity than conventional E2 vaccines. Moreover, a single dose of 5 μg E2ZJ is efficacious against CSFV in piglets. Keywords: Classical swine fever virus; novel signal peptide; SPZJ-E2ZJ; subunit vaccine; protective immunity

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Mycobacterial infection of precision cut lung slices reveals that the type 1 interferon pathway is locally induced by Mycobacterium bovis but not M. tuberculosis in different cattle breeds

10.1101/2021.04.16.440039 ◽

2021 ◽

Author(s):

Aude Remot ◽

Florence Carreras ◽

Anthony Coupe ◽

Emilie Doz-Deblauwe ◽

Maria-Laura Boschiroli ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Animal Health ◽

Mycobacterial Infection ◽

Developed Countries ◽

Host Cells ◽

Economic Losses ◽

Reference Laboratory ◽

Type I ◽

Cattle Breeds ◽

Interferon Pathway

Tuberculosis exacts a terrible toll on human and animal health. While Mycobacterium tuberculosis (Mtb) is restricted to humans, Mycobacterium bovis (Mb) is present in a large range of mammalian hosts. In cattle, bovine TB (bTB) is a notifiable disease responsible for important economic losses in developed countries and underestimated zoonosis in the developing world. Early interactions that take place between mycobacteria and the lung tissue early after aerosol infection govern the outcome of the disease. In cattle, these early steps remain poorly characterized. The precision-cut lung slice (PCLS) model preserves the structure and cell diversity of the lung. We developed this model in cattle in order to study the early lung response to mycobacterial infection. In situ imaging of PCLS infected with fluorescent Mb revealed bacilli in the alveolar compartment, adjacent or inside alveolar macrophages (AMPs) and in close contact with pneumocytes. We analyzed the global transcriptional lung inflammation signature following infection of PCLS with Mb and Mtb in two French beef breeds: Blonde d'Aquitaine and Charolaise. Whereas lungs from the Blonde d'Aquitaine produced high levels of mediators of neutrophil and monocyte recruitment in response to infection, such signatures were not observed in the Charolaise in our study. In the Blonde d'Aquitaine lung, whereas the inflammatory response was highly induced by two Mb strains, AF2122 isolated from cattle in the UK and Mb3601 circulating in France, the response against two Mtb strains, H37Rv the reference laboratory strain and BTB1558 isolated from zebu in Ethiopia, was very low. Strikingly, the type I interferon pathway was only induced by Mb but not Mtb strains indicating that this pathway may be involved in mycobacterial virulence and host tropism. Hence, the PCLS model in cattle is a valuable tool to deepen our understanding of early interactions between lung host cells and mycobacteria. It revealed striking differences between cattle breeds and mycobacterial strains. This model could help deciphering biomarkers of resistance versus susceptibility to bTB in cattle as such information is still critically needed for bovine genetic selection programs and would greatly help the global effort to eradicate bTB.

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