RESUMO
Canine parvovirus 2 (CPV-2), a highly contagious virus, affects dogs worldwide. Infected animals present severe and acute gastroenteritis which may culminate in death. CPV-2 VP2 protein is responsible for important biological functions related to virus-host interactions. Herein we obtained VP2 full-length gene sequences from Brazilian dogs with bloody diarrhea (n=15) and vaccine strains (n=7) produced by seven different laboratories and marketed in Brazil. All wild sequences and one vaccine strain were classified as CPV-2b and six vaccines were the classic CVP-2. Mutations in VP2 protein from vaccine and wild strains obtained in Brazil and worldwide were analyzed (n=906). Amino acid sequences from vaccine strains remarkably diverge from each other, even that classic CPV-2. Phylogenetic analysis based on VP2 gene and conducted with sequences displaying mutations in epitope regions previously described shows that vaccine strains are distantly related from the wide range of wild CPV-2. The impact of amino acid mutations over VP2 protein structure shows that vaccine and wild strains obtained in this study diverge in loop 3, an epitope region that plays a role in the CPV-2 host range. This is the first analysis of CPV-2 VP2 from commercial vaccine strains in Brazil and wild ones from Minas Gerais State, Brazil, and the first detailed attempt to vaccinal VP2 molecular and structural analyses.
Assuntos
Doenças do Cão , Infecções por Parvoviridae , Parvovirus Canino , Vacinas , Animais , Brasil , Cães , Infecções por Parvoviridae/veterinária , Parvovirus Canino/genética , FilogeniaRESUMO
Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Varíola dos Canários/genética , Portadores de Fármacos , Vetores Genéticos , Doenças das Aves Domésticas/prevenção & controle , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Galinhas , Doenças das Aves Domésticas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.(AU)
Assuntos
Animais , Galinhas/virologia , Vírus da Varíola dos Canários , Proteínas Virais , Vírus da Doença Infecciosa da BursaRESUMO
Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.
Assuntos
Animais , Galinhas/virologia , Proteínas Virais , Vírus da Doença Infecciosa da Bursa , Vírus da Varíola dos CanáriosRESUMO
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
RESUMO
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
RESUMO
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection. Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The isolate was defined as CPV-2a (JN403045) subtype by sequencing. The VP2 ORF was inserted into pET-32a by T4 ligase and introduced into the E. coli Bal21. VP2 protein was produced by the induction of the E. coli Bal21 containing pET-32a-VP2 with isopropyl-ß-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant VP2 protein (rVP2) fused with His-tag was analyzed by SDS-PAGE and detected by western blotting using anti-His monoclonal antibody. The rVP2 was purified by Ni+-affinity purification chromatography under denature condition and dialyzed against PBS. The concentration of the rVP2 was determined by Bradford method. After immunizing the chickens with rVP2, anti-VP2 IgY was isolated by PEG 6000 precipitation and analyzed by SDS-PAGE. The activity and specificity of the IgY antibody were analyzed by indirect ELISA and western blotting. SDS-PAGE analysis showed that the rVP2 fused with His-tag had a molecular of 85 kDa, accompanied with the low molecular fractions around 50 kDa and 40 kDa. The rVP2 could be recognized be the anti-His monoclonal antibody. The IgY antibody isolated by PEG 6000 method and analyzed by SDS-PAGE showed the IgY mainly contained two parts, 23 kDa and 67 kDa, which corresponded to light and heavy chain, respectively. In additional, some lower bands around 40 kDa were presented on the gel. The anti-VP2 IgY reached to 1:40960 after the fourth immunization. The anti-VP2-IgY could recognize the VP2 specially in Western blotting, while no reaction was seen with the low molecular. Discussion: The emergence of the low molecular proteins in 50 kDa and 40 kDa in the pellets of the bacterial lysates may be due to the degradation of the rVP2 by endogenous proteases in E. coli. The fact that IgY could recognize the entire VP2 fraction suggests the lost of the antigenic sites of the low molecular proteins.