Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses

The FlagT4G Vaccine Confers a Strong and Regulated Immunity and Early Virological Protection against Classical Swine Fever

Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination, mostly using vaccines that do not allow for differentiation of vaccinated from infected animals (DIVA). FlagT4G vaccine is a novel candidate that confers robust immunity and shows DIVA capabilities. The present study assessed the immune response elicited by FlagT4G and its capacity to protect pigs for a short time after vaccination. Five days after a single dose of FlagT4G vaccine, animals were challenged with a highly virulent CSFV strain. A strong, but regulated, interferon-α response was found after vaccination. Vaccinated animals showed clinical and virological protection against the challenge, in the absence of antibody response at 5 days post-vaccination. Upon challenge, a rapid rise in the titers of CSFV neutralizing antibodies and an increase in the IFN-γ producing cells were noticed in all vaccinated-challenged pigs. Meanwhile, unvaccinated pigs showed severe clinical signs and high viral replication, being euthanized before the end of the trial. These animals were unable to generate neutralizing antibodies and IFN-γ responses after the CSFV challenge. The results from the present study assert the fast and efficient protection by FlagT4G, a highly promising tool for CSFV control worldwide.info:eu-repo/semantics/publishedVersio

The FlagT4G Vaccine Confers a Strong and Regulated Immunity and Early Virological Protection against Classical Swine Fever

Altres ajuts: Generalitat de Catalunya, programa Cerca RTI2018-100887-B-I00Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination, mostly using vaccines that do not allow for differentiation of vaccinated from infected animals (DIVA). FlagT4G vaccine is a novel candidate that confers robust immunity and shows DIVA capabilities. The present study assessed the immune response elicited by FlagT4G and its capacity to protect pigs for a short time after vaccination. Five days after a single dose of FlagT4G vaccine, animals were challenged with a highly virulent CSFV strain. A strong, but regulated, interferon-α response was found after vaccination. Vaccinated animals showed clinical and virological protection against the challenge, in the absence of antibody response at 5 days post-vaccination. Upon challenge, a rapid rise in the titers of CSFV neutralizing antibodies and an increase in the IFN-γ producing cells were noticed in all vaccinated-challenged pigs. Meanwhile, unvaccinated pigs showed severe clinical signs and high viral replication, being euthanized before the end of the trial. These animals were unable to generate neutralizing antibodies and IFN-γ responses after the CSFV challenge. The results from the present study assert the fast and efficient protection by FlagT4G, a highly promising tool for CSFV control worldwide

Protection induced by a glycoprotein E-deleted bovine herpesvirus type 1 marker strain used either as an inactivated or live attenuated vaccine in cattle

BACKGROUND: Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections; causing a high economic loss in all continents. Use of marker vaccines in IBR eradication programs is widely accepted since it allows for protection of the animals against the disease while adding the possibility of differentiating vaccinated from infected animals.The aim of the present study was the development and evaluation of safety and efficacy of a glycoprotein E-deleted (gE-) BoHV-1 marker vaccine strain (BoHV-1ΔgEβgal) generated by homologous recombination, replacing the viral gE gene with the β-galactosidase (βgal) gene. RESULTS: In vitro growth kinetics of the BoHV-1ΔgEβgal virus was similar to BoHV-1 LA. The immune response triggered by the new recombinant strain in cattle was characterized both as live attenuated vaccine (LAV) and as an inactivated vaccine. BoHV-1ΔgEβgal was highly immunogenic in both formulations, inducing specific humoral and cellular immune responses. Antibody titers found in animals vaccinated with the inactivated vaccine based on BoHV-1ΔgEβgal was similar to the titers found for the control vaccine (BoHV-1 LA). In the same way, titers of inactivated vaccine groups were significantly higher than any of the LAV immunized groups, independently of the inoculation route (p < 0.001). Levels of IFN-γ were significantly higher (p < 0.001) in those animals that received the LAV compared to those that received the inactivated vaccine. BoHV-1ΔgEβgal exhibited an evident attenuation when administered as a LAV; no virus was detected in nasal secretions of vaccinated or sentinel animals during the post-vaccination period. BoHV-1ΔgEβgal, when used in either formulation, elicited an efficient immune response that protected animals against challenge with virulent wild-type BoHV-1. Also, the deletion of the gE gene served as an immunological marker to differentiate vaccinated animals from infected animals. All animals vaccinated with the BoHV-1ΔgE βgal strain were protected against disease after challenge and shed significantly less virus than control calves, regardless of the route and formulation they were inoculated. CONCLUSIONS: Based on its attenuation, immunogenicity and protective effect after challenge, BoHV-1ΔgEβgal virus is an efficient and safe vaccine candidate when used either as inactivated or as live attenuated forms.Fil: Romera, Sonia. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronomicas; Argentina. Universidad del Salvador; ArgentinaFil: Puntel, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Quattrocchi, Valeria. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronomicas; ArgentinaFil: del Medico Zajac, Maria Paula. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronomicas; ArgentinaFil: Zamorano, Patricia Ines. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronomicas; Argentina. Universidad del Salvador; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco Viera, Javier. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronomicas; ArgentinaFil: Carrillo, Consuelo. USDA. Plum Island Animal Disease Center; Estados UnidosFil: Chowdhury, Shafiqul. Louisiana State University. Department of Pathobiological Sciences; Estados UnidosFil: Borca, Manuel V.. USDA. Plum Island Animal Disease Center; Estados UnidosFil: Sadir, Ana M.. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronomicas; Argentina. Universidad del Salvador; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post–vaccination monitoring

Background: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring. Results: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). Conclusions: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.info:eu-repo/semantics/publishedVersio

Classical Swine Fever Virus p7 Protein Interacts with Host Protein CAMLG and Regulates Calcium Permeability at the Endoplasmic Reticulum

We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CAMLG, efficiently mediates calcium permeability in the ER. Interestingly, viruses harboring some of those mutated forms of p7 have been previously shown to have a significantly decreased virulence in swine.ARS/USDA-University of Connecticut SCA# 58-1940-1-190 and ARS/USDA-University of the Basque Country NACA#8064-32000-056-18S

Deletion of E184L, a Putative DIVA Target from the Pandemic Strain of African Swine Fever Virus, Produces a Reduction in Virulence and Protection against Virulent Challenge

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-G-ΔE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-ΔE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-ΔE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker. IMPORTANCE No commercial vaccines are available to prevent African swine fever (ASF). The ASF pandemic caused by the ASF virus Georgia 2010 (ASFV-G) strain is seriously affecting pork production in a contiguous geographical area from Central Europe to East Asia. The only effective experimental vaccines are viruses attenuated by deleting ASFV genes associated with virus virulence. Therefore, identification of such genes is of critical importance for vaccine development. Here, we report the discovery of a novel determinant of ASFV virulence, the E184L gene. Deletion of the E184L gene from the ASFV-G genome (ASFV-G-ΔE184L) produced a reduction in virus virulence, and importantly, animals surviving infection with ASFV-G-ΔE184L were protected from developing ASF after challenge with the virulent parental virus ASFV-G. Importantly, the virus protein encoded by E184L is highly immunogenic, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that unlike what is observed in animals inoculated with the vaccine candidate ASFV-G-ΔMGF, ASFV-G-ΔE184L-inoculated animals do not mount a E184L-specific antibody response, indicating the feasibility of using the E184L deletion as the antigenic marker for the development of a DIVA vaccine in ASFV.info:eu-repo/semantics/acceptedVersio

Molecular Characterization of the Viroporin Function of Foot-and-Mouth Disease Virus Nonstructural Protein 2B

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains

Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model

Citation: Carlson, J.; O’Donnell, V.; Alfano, M.; Velazquez Salinas, L.; Holinka, L.G.; Krug, P.W.; Gladue, D.P.; Higgs, S.; Borca, M.V. Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model. Viruses 2016, 8, 291.African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus, ASF virus (ASFV). There is no vaccine to prevent the disease and current control measures are limited to culling and restricting animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pretoriuskop/96/4 (Pret4) virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 days post infection (dpi) showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response (ASFV-specific antibody and interferon (IFN)-γ responses, or specific cytokine profiles) and protection against challenge. With the exception of ASFV-specific antibodies in survivors challenged at 21 and 28 dpi, no association between the parameters assessed and protection could be established. These results, encompassing data from 65 immunized swine, underscore the complexity of the system under study, suggesting that protection relies on the concurrence of different host immune mechanisms