RESUMEN
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.
Asunto(s)
Antivirales/administración & dosificación , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Peste Porcina Clásica/patología , Peste Porcina Clásica/prevención & control , Interferón-alfa/administración & dosificación , Animales , Peste Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Humanos , Análisis de Supervivencia , Porcinos , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Vacunas Virales/inmunología , Animales , Línea Celular , Virus de la Fiebre Porcina Clásica/patogenicidad , Femenino , Porcinos , Vacunas Atenuadas/inmunología , Viremia , VirulenciaRESUMEN
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.
Asunto(s)
Virus de la Fiebre Porcina Clásica/metabolismo , Peste Porcina Clásica/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Proteínas de Neoplasias/metabolismo , Proteínas del Núcleo Viral/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Peste Porcina Clásica/genética , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/química , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/patogenicidad , Interacciones Huésped-Patógeno , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Unión Proteica , Porcinos , Técnicas del Sistema de Dos Híbridos , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , VirulenciaRESUMEN
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.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Sustitución de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Virus de la Fiebre Porcina Clásica/genética , Liposomas/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Genética Inversa , Porcinos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
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.
Asunto(s)
Virus de la Fiebre Aftosa/fisiología , Fiebre Aftosa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Línea Celular , Complejo Dinactina , Virus de la Fiebre Aftosa/patogenicidad , Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Alineación de Secuencia , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virulencia , Replicación ViralRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Epiteliales/virología , Virus de la Fiebre Aftosa/fisiología , Vimentina/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Proteínas Portadoras/genética , Línea Celular , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Técnicas del Sistema de Dos Híbridos , Vimentina/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Virus de la Fiebre Aftosa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Animales , Autofagia , Beclina-1 , Bovinos , Línea Celular , Línea Celular Tumoral , Cricetinae/metabolismo , Células Epiteliales/citología , Virus de la Fiebre Aftosa/genética , Biblioteca de Genes , Humanos , Glándulas Mamarias Humanas/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Cisteína/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Aminoácido , Porcinos , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155]. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues (207)ATI(209) (I), (210)VVE(212) (II), (213)GVK(215) (III), or (232)GLYHN(236) (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle.
Asunto(s)
Virus de la Fiebre Porcina Clásica/patogenicidad , Interacciones Huésped-Patógeno , Mapeo de Interacción de Proteínas , Proteínas del Núcleo Viral/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Células Cultivadas , Peste Porcina Clásica/patología , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Macrófagos/virología , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/genética , Mutagénesis Sitio-Dirigida , Unión Proteica , Homología de Secuencia de Aminoácido , Porcinos , Proteínas del Núcleo Viral/genética , VirulenciaRESUMEN
Here we have identified host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme that interact with classical swine fever virus (CSFV) Core protein. Five highly conserved lysine residues (K179, K180, K220, K221, and K246) within the CSFV Core were identified as putative SUMOylation sites. Analysis of these interactions showed that K179A, K180A, and K221A substitutions disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9 binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A, K221A) harboring those substitutions were attenuated in swine. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo.
Asunto(s)
Virus de la Fiebre Porcina Clásica/patogenicidad , Mapeo de Interacción de Proteínas , Proteína SUMO-1/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Peste Porcina Clásica/patología , Peste Porcina Clásica/virología , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Alineación de Secuencia , Porcinos , VirulenciaRESUMEN
NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Mutación , Proteínas no Estructurales Virales/fisiología , Virulencia/fisiología , Secuencia de Aminoácidos , Aminoquinolinas/farmacología , Animales , Secuencia de Bases , Cartilla de ADN , Imiquimod , Interleucina-6/genética , Macrófagos/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Porcinos , Receptores Toll-Like/fisiología , Transfección , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
E1, along with E(rns) and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previously we showed that glycosylation status of virulent CSFV strain Brescia E2 or E(rns) affects virus virulence. Here, the three putative glycosylation sites of E1 were serially removed by means of site directed mutagenesis of a CSFV Brescia infectious clone (BICv) and their effect on virulence assessed in swine. Removal of all three putative glycosylation sites in E1, at CSFV positions N500, N513 and N594, yielded nonviable progeny, while single or dual site mutants excluding N594 were viable. Individual N594A (E1.N3 virus) or combined N500A/N513A (E1.N1N2 virus) substitutions resulted in BICv attenuation. Furthermore infection with E1.N3 or E1.N1N2 viruses efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. As previously observed with E(rns) and E2 and here with E1 data suggest that modification of glycosylation patterns could be used for developing CSFV live-attenuated vaccines.
Asunto(s)
Virus de la Fiebre Porcina Clásica/patogenicidad , Proteínas Estructurales Virales/metabolismo , Factores de Virulencia/metabolismo , Sustitución de Aminoácidos/genética , Animales , Sangre/virología , Línea Celular , Glicosilación , Riñón/virología , Ganglios Linfáticos/virología , Viabilidad Microbiana , Mutagénesis Sitio-Dirigida , Mutación Missense , Cavidad Nasal/virología , Tonsila Palatina/virología , Bazo/virología , Porcinos , Vacunas Atenuadas/inmunología , Ensayo de Placa Viral , Proteínas Estructurales Virales/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
Until recently strategies for controlling Classical Swine Fever Virus (CSFV) involve either prophylactic vaccination or non-vaccination with elimination of infected herds depending on the epidemiological situation of the affected geographical area. Marker vaccines allowing distinction between naturally infected from vaccinated swine could complement "stamping out" measures. Here we developed a double antigenic marker live attenuated CSFV strain FlagT4v obtained by combining two genetic determinants of attenuation. FlagT4v harbors a positive antigenic marker, synthetic Flag(R) epitope, introduced via a 19mer insertion in E1 glycoprotein; and a negative marker resulting from mutations of the binding site of monoclonal antibody WH303 (mAbWH303) epitope in E2 glycoprotein. Intranasal or intramuscular administration of FlagT4v protected swine against virulent CSFV Brescia strain at early (2 or 3 days), and late (28 days) time post-inoculation. FlagT4v induced antibody response in pigs reacted strongly against the Flag(R) epitope but failed to inhibit binding of mAbWH303 to a synthetic peptide representing the WH303 epitope. These results constitute a proof-of-concept for rationally designing a CSFV antigenically marked live attenuated virus.
Asunto(s)
Antígenos Virales/análisis , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/inmunología , Vacunas Atenuadas , Vacunas Virales , Administración Intranasal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Marcadores Genéticos , Genoma Viral , Inyecciones Intramusculares , Datos de Secuencia Molecular , Mutación , Porcinos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/química , Vacunas Atenuadas/genéticaRESUMEN
We have previously reported [Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L. 2005a. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J. Virol. 79, 3787-3796] that chimeric virus 319.1v containing the E2 glycoprotein gene from Classical Swine Fever Virus (CSFV) vaccine strain CS with the genetic background of highly virulent CSFV strain Brescia (BICv) was markedly attenuated in pigs. To identify the amino acids mediating 319.1v attenuation a series of chimeric viruses containing CS E2 residues in the context of the Brescia strain were constructed. Chimera 357v, containing CS E2 residues 691 to 881 of CSFV polyprotein was virulent, while chimera 358v, containing CS E2 residues 882 to 1064, differing in thirteen amino acids from BICv, was attenuated in swine. Single or double substitutions of those amino acids in BICv E2 to CS E2 residues did not affect virulence. Groups of amino acids were then substituted in BICv E2 to CS E2 residues. Mutant 32v, with six substitutions between residues 975 and 1059, and mutant 33v, with six substitutions between 955 and 994, induced disease indistinguishable from BICv. Mutant 31v, with seven substitutions between residues 882 and 958, induced a delayed onset of lethal disease. Amino acids abrogating BICv virulence were then determined by progressively introducing six CS residues into 31v. Mutant 39v, containing nine residue substitutions, was virulent. Mutant 40v, containing ten residue substitutions, induced mild disease. Mutant 42v, containing twelve substitutions, and mutant 43v, with an amino acid composition identical to 358v, were attenuated in swine indicating that all substitutions were necessary for attenuation of the highly virulent strain Brescia. Importantly, 358v protected swine from challenge with virulent BICv at 3 and 28 days post-infection.
Asunto(s)
Virus de la Fiebre Porcina Clásica/genética , Proteínas del Envoltorio Viral/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Quimera/genética , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Virus de la Fiebre Porcina Clásica/patogenicidad , ADN Viral/genética , Genes Virales , Mutación , Fenotipo , Estructura Secundaria de Proteína , Porcinos , Proteínas del Envoltorio Viral/química , Vacunas Virales/genética , Vacunas Virales/farmacología , Virulencia/genéticaRESUMEN
E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.