A Cell Culture-Adapted Vaccine Virus against the Current African Swine Fever Virus Pandemic Strain.

African swine fever virus (ASFV) causes a virulent, deadly infection in wild and domestic swine and is currently causing a pandemic covering a contiguous geographical area from Central and Eastern Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in devastating economic losses to the swine industry. The most advanced vaccine candidates are live attenuated strains developed using a genetically modified virulent parental virus. Recently, we developed a vaccine candidate, ASFV-G-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV pandemic strain Georgia (ASFV-G). ASFV-G-ΔI177L is safe and highly efficacious in challenge studies using parental ASFV-G. Large-scale production of ASFV-G-ΔI177L has been limited because it can replicate efficiently only in primary swine macrophages. Here, we present the development of an ASFV-G-ΔI177L derivative strain, ASFV-G-ΔI177L/ΔLVR, that replicates efficiently in a stable porcine cell line. In challenge studies, ASFV-G-ΔI177L/ΔLVR maintained the same level of attenuation, immunogenic characteristics, and protective efficacy as ASFV-G-ΔI177L. ASFV-G-ΔI177L/ΔLVR is the first rationally designed ASF vaccine candidate that can be used for large-scale commercial vaccine manufacture. IMPORTANCE African swine fever is currently causing a pandemic resulting in devastating losses to the swine industry. Experimental ASF vaccines rely on the production of vaccine in primary swine macrophages, which are difficult to use for the production of a vaccine on a commercial level. Here, we report a vaccine for ASFV with a deletion in the left variable region (LVR). This deletion allows for growth in stable cell cultures while maintaining the potency and efficacy of the parental vaccine strain. This discovery will allow for the production of an ASF vaccine on a commercial scale.

Structural Glycoprotein E2 of Classical Swine Fever Virus Critically Interacts with Host Protein Torsin-1A during the Virus Infectious Cycle.

The classical swine fever virus (CSFV) glycoprotein E2 is the major structural component of the virus particle. E2 is involved in several functions, such as virus adsorption to the cell, the elicitation of protective immune responses, and virus virulence in swine. Using a yeast two-hybrid system, we previously identified the swine host protein Torsin-1A, an ATPase protein residing in the endoplasmic reticulum and inner nucleus membrane of the cell, as a specific binding partner for E2. The interaction between Torsin-1A and E2 proteins was confirmed to occur in CSFV-infected swine cells using three independent methods: coimmunoprecipitation, confocal microscopy, and proximity ligation assay (PLA). Furthermore, the E2 residue critical to mediate the protein-protein interaction with Torsin-1A was identified by a reverse yeast two-hybrid assay using a randomly mutated E2 library. A recombinant CSFV E2 mutant protein with a Q316L substitution failed to bind swine Torsin-1A in the yeast two-hybrid model. In addition, a CSFV infectious clone harboring the E2 Q316L substitution, although expressing substantial levels of E2 protein, repetitively failed to produce virus progeny when the corresponding RNA was transfected into susceptible SK6 cells. Importantly, PLA analysis of the transfected cells demonstrated an abolishment of the interaction between E2 Q316L and Torsin-1A, indicating a critical role for that interaction during CSFV replication.IMPORTANCE Structural glycoprotein E2 is an important structural component of the CSFV particle. E2 is involved in several virus functions, particularly virus-host interactions. Here, we characterized the interaction between CSFV E2 and swine protein Torsin-1A during virus infection. The critical amino acid residue in E2 mediating the interaction with Torsin-1A was identified and the effect of disrupting the E2-Torsin-1A protein-protein interaction was studied using reverse genetics. It is shown that the amino acid substitution abrogating E2-Torsin-1A interaction constitutes a lethal mutation, demonstrating that this virus-host protein-protein interaction is a critical factor during CSFV replication. This highlights the potential importance of the E2-Torsin-1A protein-protein interaction during CSFV replication and provides a potential pathway toward blocking virus replication, an important step toward the potential development of novel virus countermeasures.

Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle.

The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence.IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

Identification of structural glycoprotein E2 domain critical to mediate replication of Classical Swine Fever Virus in SK6 cells.

Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical virus functions. To analyze the role of E2 in virus replication, a series of recombinant CSFVs harboring chimeric forms of E2 CSFV and Bovine viral diarrhea virus (BVDV) were created and tested for their ability to infect swine or bovine cell lines. Substitution of native CSFV E2 by BVDV E2 abrogates virus replication in both cell lines. Substitution of individual domains in CSFV Brescia E2 by the homologous from BVDV produces chimeras that efficiently replicate in SK6 cells with the exception of a chimera harboring BVDV E2 residues 93-168. Further mapping revealed a critical area in E2 required for CSFV replication in SK6 cells between protein residues 136-156. This is the first report categorically defining a discrete portion of E2 as essential to pestivirus infection in susceptible cells.

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.IMPORTANCE FMDV nonstructural protein 2B is able to insert itself into cellular membranes to form a pore. This pore allows the passage of ions and small molecules through the membrane. In this study, we were able to show that both current and small molecules are able to pass though the pore made by 2B. We also discovered for the first time a virus with a pore-forming protein that contains two independent functional pores. By making mutations in our infectious clone of FMDV, we determined that mutations in either pore resulted in nonviable virus. This suggests that both pore-forming functions are independently required during FMDV infection.

Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine.

E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, as well as two aromatic residues (871W and 875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2. IMPORTANCE: This report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain.

Systemic Foot-and-Mouth Disease Vaccination in Cattle Promotes Specific Antibody-Secreting Cells at the Respiratory Tract and Triggers Local Anamnestic Responses upon Aerosol Infection.

UNLABELLED: Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting biungulate species. Commercial vaccines, formulated with inactivated FMD virus (FMDV), are regularly used worldwide to control the disease. Here, we studied the generation of antibody responses in local lymphoid tissues along the respiratory system in vaccinated and further aerosol-infected cattle. Animals immunized with a high-payload monovalent FMD vaccine developed high titers of neutralizing antibodies at 7 days postvaccination (dpv), reaching a plateau at 29 dpv. FMDV-specific antibody-secreting cells (ASC), predominantly IgM, were evident at 7 dpv in the prescapular lymph node (LN) draining the vaccination site and in distal LN draining the respiratory mucosa, although in lower numbers. At 29 dpv, a significant switch to IgG1 was clear in prescapular LN, while FMDV-specific ASC were detected in all lymphoid tissues draining the respiratory tract, mostly as IgM-secreting cells. None of the animals (n = 10) exhibited FMD symptoms after oronasal challenge at 30 dpv. Three days postinfection, a large increase in ASC numbers and rapid isotype switches to IgG1 were observed, particularly in LN-draining virus replication sites already described. These results indicate for the first time that systemic FMD vaccination in cattle effectively promotes the presence of anti-FMDV ASC in lymphoid tissues associated with the respiratory system. Oronasal infection triggered an immune reaction compatible with a local anamnestic response upon contact with the replicating FMDV, suggesting that FMD vaccination induces the circulation of virus-specific B lymphocytes, including memory B cells that differentiate into ASC soon after contact with the infective virus. IMPORTANCE: Over recent decades, world animal health organizations as well as national sanitary authorities have supported the use of vaccination as an essential component of the official FMD control programs in both endemic and disease-free settings. Very few works studied the local immunity induced by FMD vaccines at the respiratory mucosa, and local responses induced in vaccinated animals after aerosol infection have not been described yet. In this work, we demonstrate for the first time that systemic FMD vaccination (i) induced the early presence of active antigen-specific ASC along the respiratory tract and (ii) prompted a rapid local antibody response in the respiratory mucosa, triggered upon oronasal challenge and congruent with a memory B-cell response. This information may help to understand novel aspects of protective responses induced by current FMD vaccines as well as to provide alternative parameters to establish protection efficiency for new vaccine developments.

Treatment with interferon-alpha delays disease in swine infected with a highly virulent CSFV strain.

Interferon-alpha (IFNα) can effectively inhibit or abort a viral infection within the host. It has been reported that IFN induction and production is hindered during classical swine fever virus (CSFV) infection. Most of those studies have been performed in vitro, making it difficult to elucidate the actual role of IFNs during CSFV infection in swine. Here, we report the effect of IFNα treatment (delivered by a replication defective recombinant human adenovirus type 5, Ad5) in swine experimentally infected with highly virulent CSFV strain Brescia. Treatment with two different subtypes of IFNα delayed the appearance of CSF-related clinical signs and virus replication although it did not prevent lethal disease. This is the first report describing the effect of IFNα treatment during CSFV infection in swine.

Heterogeneity in the Antibody Response to Foot-and-Mouth Disease Primo-vaccinated Calves.

Foot-and-mouth disease (FMD) vaccines are routinely used as effective control tools in large regions worldwide and to limit outbreaks during epidemics. Vaccine-induced protection in cattle has been largely correlated with the FMD virus (FMDV)-specific antibodies. Genetic control of cattle immune adaptive responses has been demonstrated only for peptide antigens derived from FMDV structural proteins. Here, we quantify the heterogeneity in the antibody response of cattle primo-vaccinated against FMD and study its association with the genetic background in Holstein and Jersey sires. A total of 377 FMDV-seronegative calves (122 and 255 calves from 16 and 15 Holstein and Jersey sires, respectively) were included in the study. Samples were taken the day prior to primo-vaccination and 45 days post-vaccination (dpv). Animals received commercial tetravalent FMD single emulsion oil vaccines formulated with inactivated FMDV. Total FMDV-specific antibody responses were studied against three viral strains included in the vaccine, and antibody titres were determined by liquid-phase blocking ELISA. Three linear hierarchical mixed regression models, one for each strain, were formulated to assess the heterogeneity in the immune responses to vaccination. The dependent variables were the antibody titres induced against each FMDV strain at 45 dpv, whereas sire's 'breed' was included as a fixed effect, 'sire' was included as a random effect, and 'farm' was considered as a hierarchical factor to account for lack of independence of within herd measurements. A significant association was found between anti-FMDV antibody responses and sire's breed, with lower immune responses found in the Jersey sires' offspring compared with those from Holstein sires. No significant intrabreed variation was detected. In addition, farm management practices were similar in this study, and results of the serological assays were shown to be repeatable. It therefore seems plausible that differences in the immune response may be expected in the event of a mass vaccination campaigns.

Development of an improved live attenuated antigenic marker CSF vaccine strain candidate with an increased genetic stability.

Controlling classical swine fever (CSF) involves vaccination in endemic regions and preemptive slaughter of infected swine herds during epidemics. Live attenuated marker vaccines that confer effective protection against the disease and allow differentiation between infected and vaccinated animals (DIVA) could impact CSF control policies. Previously, we reported the development of FlagT4 virus (FlagT4v), a rationally designed live attenuated marker vaccine. During its vaccine assessment, FlagT4v reverted to a virulent virus during successive passages in piglets. Sequence analysis revealed deletions and substitutions almost exclusively in the areas of E1 and E2. To improve genetic stability of FlagT4v, we introduced changes in the codon usage in those areas. The newly developed virus, FlagT4Gv, was shown to retain the attenuated phenotype after successive passages in piglets. As observed with FlagT4v, the newly developed FlagT4Gv conferred effective protection against challenge with virulent CSFV at early (7 days) and at late (28 days) times post-vaccination.

Virus-host interactions in persistently FMDV-infected cells derived from bovine pharynx.

Foot-and-mouth disease virus (FMDV) produces a disease in cattle characterized by vesicular lesions and a persistent infection with asymptomatic low-level production of virus in pharyngeal tissues. Here we describe the establishment of a persistently infected primary cell culture derived from bovine pharynx tissue (PBPT) infected with FMDV serotype O1 Manisa, where surviving cells were serially passed until a persistently infected culture was generated. Characterization of the persistent virus demonstrated changes in its plaque size, ability to grow in different cell lines, and change in the use of integrins as receptors, when compared with the parental virus. These results demonstrate the establishment of persistently infected PBPT cell cultures where co-adaptation has taken place between the virus and host cells. This in vitro model for FMDV persistence may help further understanding of the molecular mechanisms of the cattle carrier state.

Interaction of structural core protein of classical swine fever virus with endoplasmic reticulum-associated degradation pathway protein OS9.

Classical swine fever virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, the osteosarcoma amplified 9 protein (OS9) was further studied. Using alanine scanning mutagenesis, the OS9 binding site in the CSFV Core protein was identified, between Core residues (90)IAIM(93), near a putative cleavage site. Truncated versions of Core were used to show that OS9 binds a polypeptide representing the 12 C-terminal Core residues. Cells transfected with a double-fluorescent labeled Core construct demonstrated that co-localization of OS9 and Core occurred only on unprocessed forms of Core protein. A recombinant CSFV containing Core protein where residues (90)IAIM(93) were substituted by alanines showed no altered virulence in swine, but a significant decreased ability to replicate in cell cultures.

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication.

E2, along with E(rns) and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, (818)CPIGWTGVIEC(828), containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a ß-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP (818)CPIGWTGVIEC(828) indicates a membrane fusion activity and a critical role in virus replication.

Interaction of foot-and-mouth disease virus nonstructural protein 3A with host protein DCTN3 is important for viral virulence in cattle.

UNLABELLED: Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular protein DCTN3 as a specific host binding partner for 3A. DCTN3 is a subunit of the dynactin complex, a cofactor for dynein, a motor protein. The dynactin-dynein duplex has been implicated in several subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction identified by the yeast two-hybrid approach was further confirmed in mammalian cells. Overexpression of DCTN3 or proteins known to disrupt dynein, p150/Glued and 50/dynamitin, resulted in decreased FMDV replication in infected cells. We mapped the critical amino acid residues in the 3A protein that mediate the protein interaction with DCTN3 by mutational analysis and, based on that information, we developed a mutant harboring the same mutations in O1 Campos FMDV (O1C3A-PLDGv). Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in LFBK-αvß6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a delayed disease in cattle compared to the virulent parental O1Campus (O1Cv). Virus isolated from lesions of animals inoculated with O1C3A-PLDGv virus contained amino acid substitutions in the area of 3A mediating binding to DCTN3. Importantly, 3A protein harboring similar amino acid substitutions regained interaction with DCTN3, supporting the hypothesis that DCTN3 interaction likely contributes to virulence in cattle. IMPORTANCE: The objective of this study was to understand the possible role of a FMD virus protein 3A, in causing disease in cattle. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. It was shown that manipulation of DCTN3 has a profound effect in virus replication. We developed a FMDV mutant virus that could not bind DCTN3. This mutant virus exhibited a delayed disease in cattle compared to the parental strain highlighting the role of the 3A-DCTN3 interaction in virulence in cattle. Interestingly, virus isolated from lesions of animals inoculated with mutant virus contained mutations in the area of 3A that allowed binding to DCTN3. This highlights the importance of the 3A-DCTN3 interaction in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

Foot-and-mouth disease virus modulates cellular vimentin for virus survival.

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Aphthovirus within the Picornaviridae family. During infection with FMDV, several host cell membrane rearrangements occur to form sites of viral replication. FMDV protein 2C is part of the replication complex and thought to have multiple roles during virus replication. To better understand the role of 2C in the process of virus replication, we have been using a yeast two-hybrid approach to identify host proteins that interact with 2C. We recently reported that cellular Beclin1 is a natural ligand of 2C and that it is involved in the autophagy pathway, which was shown to be important for FMDV replication. Here, we report that cellular vimentin is also a specific host binding partner for 2C. The 2C-vimentin interaction was further confirmed by coimmunoprecipitation and immunofluorescence staining to occur in FMDV-infected cells. It was shown that upon infection a vimentin structure forms around 2C and that this structure is later resolved or disappears. Interestingly, overexpression of vimentin had no effect on virus replication; however, overexpression of a truncated dominant-negative form of vimentin resulted in a significant decrease in viral yield. Acrylamide, which causes disruption of vimentin filaments, also inhibited viral yield. Alanine scanning mutagenesis was used to map the specific amino acid residues in 2C critical for vimentin binding. Using reverse genetics, we identified 2C residues that are necessary for virus growth, suggesting that the interaction between FMDV 2C and cellular vimentin is essential for virus replication.

Early adaptive immune responses in the respiratory tract of foot-and-mouth disease virus-infected cattle.

Foot-and-mouth disease (FMD) is a highly contagious viral disease which affects both domestic and wild biungulate species. This acute disease, caused by the FMD virus (FMDV), usually includes an active replication phase in the respiratory tract for up to 72 h postinfection, followed by hematogenous dissemination and vesicular lesions at oral and foot epithelia. The role of the early local adaptive immunity of the host in the outcome of the infection is not well understood. Here we report the kinetics of appearance of FMDV-specific antibody-secreting cells (ASC) in lymphoid organs along the respiratory tract and the spleen in cattle infected by aerosol exposure. While no responses were observed for up to 3 days postinfection (dpi), all animals developed FMDV-ASC in all the lymphoid organs studied at 4 dpi. Tracheobronchial lymph nodes were the most reactive organs at this time, and IgM was the predominant isotype, followed by IgG1. Numbers of FMDV-ASC were further augmented at 5 and 6 dpi, with an increasing prevalence in upper respiratory organs. Systemic antibody responses were slightly delayed compared with the local reaction. Also, IgM was the dominant isotype in serum at 5 dpi, coinciding with a sharp decrease of viral RNA detection in peripheral blood. These results indicate that following aerogenous administration, cattle develop a rapid and vigorous genuine local antibody response throughout the respiratory tract. Time course and isotype profiles indicate the presence of an efficient T cell-independent antibody response which drives the IgM-mediated virus clearance in cattle infected by FMDV aerosol exposure.

Foot-and-mouth disease virus nonstructural protein 2C interacts with Beclin1, modulating virus replication.

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.

Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine.

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.