RESUMO
Reticuloendotheliosis virus (REV) is a retroviral pathogen capable of infecting several avian hosts and is associated with immunosuppression, anemia, proventriculitis, neoplasia, and runting-stunting syndrome. Its genome contains the three major genes, gag, pol, and env, and two flanking long terminal repeat (LTR) regions. Complete genome sequences of REV are limited in terms of geographical origin. The aim of this study was to characterize the complete genome of REV detected in Brazilian chickens with multiple viral coinfections and analyze the polymorphisms in the deduced amino acids sequences corresponding to its encoded proteins. We tested the presence and completeness of REV as well as other viral pathogens in samples from Brazilian poultry farms by qPCR. The complete genomes of two REV strains were sequenced by overlapping fragments through the dideoxy method. Phylogenetic analysis, pairwise identity matrix, polymorphism identification and protein modeling were performed along the entire genome. We detected REV in 65% (26/40) of the tested samples. Concomitant viral infections were detected in 82.5% (33/40) of the samples and in 90% (9/10) of the farms. Multiple infections included up to seven viruses. Phylogenetic analysis classified both Brazilian strains into REV subtype 3, and the pairwise comparison indicated that strains from the USA and fowlpox virus (FWPV)-related strains were the most identical. The subdomain p18 in gag, the reverse transcriptase/ribonuclease H in pol, and the surface (SU) in the env protein were the most polymorphic in genomic comparisons. The relevant motifs for each protein were highly conserved, with fewer polymorphisms in the fusion peptide, immunosuppression domain, and disulfide bonds on the surface (SU) and transmembrane (TM) of env. This is the first study to include complete genomes of REV in Brazil and South America detected in farms with multiple viral coinfections. Our findings suggest an involvement of REV as an immunosuppressor and active agent in the emergence and progression of multiple infectious diseases. We also found a possible etiological relationship between Brazilian strains and the USA and FWPV recombinant strains. This information highlights the need for epidemiological vigilance regarding REV in association with another pathogens.
Assuntos
Coinfecção , Vírus da Varíola das Aves Domésticas , Doenças das Aves Domésticas , Vírus da Reticuloendoteliose , Animais , Brasil/epidemiologia , Galinhas/genética , Coinfecção/genética , Coinfecção/veterinária , Vírus da Varíola das Aves Domésticas/genética , Genoma Viral , Filogenia , Vírus da Reticuloendoteliose/genéticaRESUMO
Fowlpox is one of the oldest diseases reported in birds. The causative genus Avipoxvirus affects ~232 domestic and wild species. We present herein the history, clinical findings, and macroscopic and histologic lesions caused by a clade C poxvirus in an exotic psittacine breeding colony in southern Brazil. Clinical signs included yellow nodular lesions at the commissure of the beak and on the periocular skin, loss of appetite, and death. Fifty birds were autopsied, and fragments of periocular skin, tongue, and trachea were examined histologically, which revealed hyperkeratosis associated with eosinophilic intracytoplasmic inclusion bodies. Tracheal fragments and periocular skin were subjected to nested PCR and phylogenetic analyses. The sequenced strain showed 99.58% identity with the nucleotide sequences of Avipoxvirus strains AY53011, KC018069, AM050383, and AM05382 isolated from birds in Germany, United States, and United Kingdom. The strain was grouped under clade C, which represents isolates exclusively from the Psittacidae family. The infection caused by clade C Avipoxvirus in the exotic psittacines examined ( Platycercus sp. and Psephotus haematonotus) demonstrates the circulation of this clade in this breeding colony.
Assuntos
Doenças das Aves/epidemiologia , Surtos de Doenças/veterinária , Vírus da Varíola das Aves Domésticas/isolamento & purificação , Infecções por Poxviridae/veterinária , Animais , Doenças das Aves/virologia , Brasil/epidemiologia , Vírus da Varíola das Aves Domésticas/genética , Papagaios , Filogenia , Reação em Cadeia da Polimerase/veterinária , Infecções por Poxviridae/epidemiologiaRESUMO
Two 1-mo-old local breed chickens, with gross lesions in the skin of the head region suspected to be fowl poxvirus infection, were submitted to the Diagnostic Laboratory of the School of Veterinary Medicine, Grenada, West Indies. Cutaneous lesions were collected from these birds for virus isolation, histopathologic diagnosis, and molecular analysis. Fowl poxvirus infection was confirmed by virus isolation in chicken embryo and by histopathology. Molecular characterization of the fowl poxvirus was conducted by PCR amplification of selected genomic fragments and by nucleotide sequencing. Integration of reticuloendotheliosis virus fragments into the fowl poxvirus genome was confirmed by PCR and DNA sequencing. This is the first report from the Caribbean region on the preliminary molecular characterization of a fowl poxvirus isolate.
Assuntos
Galinhas , Vírus da Varíola das Aves Domésticas/genética , Varíola Aviária/virologia , Animais , Sequência de Bases , DNA Viral/genética , Varíola Aviária/epidemiologia , Granada/epidemiologia , Filogenia , Reação em Cadeia da PolimeraseRESUMO
HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core-E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-gamma secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.
Assuntos
Chlorocebus aethiops/imunologia , Vírus da Varíola das Aves Domésticas/genética , Hepatite C/imunologia , Imunização , Polimorfismo de Nucleotídeo Único/imunologia , Vaccinia virus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Chlorocebus aethiops/virologia , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Vírus da Varíola das Aves Domésticas/imunologia , Hepatite C/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genéticaRESUMO
The presence of avian pox in endemic birds in the Galápagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction-based method for identification and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction-amplified loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), finches (Geospiza spp.), and Galápagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in Galápagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% different from the canarypox/Galápagos group viruses in the regions sequenced. These results indicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on Galápagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in combination with mutation led to the canarypox-like viruses that are now prevalent in the Galápagos.
Assuntos
Avipoxvirus/isolamento & purificação , Doenças das Aves/virologia , Galinhas/virologia , Passeriformes/virologia , Doenças das Aves Domésticas/virologia , Infecções por Poxviridae/veterinária , Sequência de Aminoácidos , Animais , Animais Domésticos , Animais Selvagens , Avipoxvirus/classificação , Avipoxvirus/genética , Doenças das Aves/epidemiologia , Doenças das Aves/transmissão , Aves , Vírus da Varíola dos Canários/classificação , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/isolamento & purificação , DNA Viral/análise , Equador/epidemiologia , Varíola Aviária/epidemiologia , Varíola Aviária/transmissão , Varíola Aviária/virologia , Vírus da Varíola das Aves Domésticas/classificação , Vírus da Varíola das Aves Domésticas/genética , Vírus da Varíola das Aves Domésticas/isolamento & purificação , Dados de Sequência Molecular , Mutação , Filogenia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/transmissão , Infecções por Poxviridae/epidemiologia , Infecções por Poxviridae/transmissão , Infecções por Poxviridae/virologia , Alinhamento de Sequência/veterináriaRESUMO
The use of DNA vectors based on the SFV (Semliki Forest virus) replicon have not been reported in the modality of DNA prime virus boost. In the present study, SFV DNA vectors (DNA vectors based on the SFV replicon) bearing the HIV-1 TAB9 multiepitopic polypeptide minigene were evaluated as priming DNA immunogens followed by a recombinant fowlpox expressing the TAB9 mutiepitope (FPTAB9LZ) boost. The results indicated that mice primed with pSFV(k)tab9 and boosted with FPTAB9LZ significantly decreased the HIV-1 recombinant (VVTAB13, a recombinant vaccinia virus expressing the TAB13 multiepitope) vaccinia virus replication, compared with groups given pSFV(k)tab9 vector and FPTAB9LZ virus alone. Additionally, the viral titre in ovary correlated with the number of specific gamma-interferon-secreting T-cells in spleen. These results support the possible use of SFV DNA vectors in prime-boost approaches implemented in therapeutic/prophylactic treatments for infectious diseases such as HIV-1.
Assuntos
DNA Viral/administração & dosagem , Vírus da Varíola das Aves Domésticas/genética , Terapia Genética/métodos , Infecções por HIV/prevenção & controle , Imunização Secundária/métodos , Vírus da Floresta de Semliki/genética , Vacinas de DNA/administração & dosagem , Animais , Terapia Combinada , Feminino , Vírus da Varíola das Aves Domésticas/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Floresta de Semliki/imunologia , Resultado do Tratamento , Vacinação/métodos , Vacinas de DNA/imunologiaRESUMO
A prime-boost strategy combining FWPV (fowlpox virus) and the MVA (modified vaccinia virus Ankara), both expressing HIV-1 multi-V3 epitope polypeptides, was compared with a DNA-based Semliki Forest virus replicon/poxvirus approach for the induction of a CD8(+) T-cell response. Priming mice with recombinant MVA and boosting with recombinant FWPV, and not in the reverse order, increased the number of specific interferon-gamma-secreting cells in relation to the homologous combinations. Moreover, the improvement of the CD8(+) T-cell response with this combination was remarkably higher than that obtained by priming with a DNA vector containing a Semliki Forest virus replicon expressing the multi-epitope polypeptide and boosting either with recombinant MVA or FWPV. These results open a new and attractive alternative for vaccine preparation against HIV-1 using different immunogens.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Vírus da Varíola das Aves Domésticas/imunologia , HIV-1/imunologia , Imunização Secundária , Vacinas de DNA , Vaccinia virus/imunologia , Animais , Linhagem Celular Tumoral , Epitopos/genética , Feminino , Vírus da Varíola das Aves Domésticas/genética , HIV-1/genética , Interferon gama/metabolismo , Mastocitoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Recombinação Genética , Baço/citologia , Vacinação , Vaccinia virus/genéticaRESUMO
HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carbidopa/imunologia , Epitopos de Linfócito T/imunologia , Epitopos/imunologia , Vírus da Varíola das Aves Domésticas/imunologia , Levodopa/imunologia , Animais , Linhagem Celular , Galinhas , Combinação de Medicamentos , Epitopos/genética , Epitopos de Linfócito T/genética , Feminino , Vírus da Varíola das Aves Domésticas/genética , Regulação Viral da Expressão Gênica , Antígenos HIV/genética , Antígenos HIV/imunologia , Imunidade Celular/imunologia , Levodopa/genética , Camundongos , Peptídeos/genética , Peptídeos/imunologia , Regiões Promotoras Genéticas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Recombinação Genética , Linfócitos T Citotóxicos/imunologia , Vaccinia virus/imunologiaRESUMO
Recombinant avipoxvirus vectors are attractive candidates for use in vaccination strategies for infections such as human immunodeficiency virus type 1 (HIV-1), where induction of a CD8+ T cell response is thought to be an important component of protective immunity. Here, we report the expression of a multiepitope polypeptide (TAB9) composed of the central 15 amino acids of the V3 loop from six different isolates of HIV-1 in a fowlpox virus (FWPV) vector, and the use of this vector (FPTAB9LZ) to induce strong HIV-specific CD8+ T cell responses in mice. In animals immunized twice intravenously with FPTAB9LZ, almost 2% of the CD8+ T cells in the spleen were shown to produce IFN-gamma in response to stimulation with HIV-1 peptides 1 week after the second immunization. The most dominant response was to the HIV-1 IIIB peptide. A strong HIV-specific response was also induced by intraperitoneal immunization of mice with FPTAB9LZ, whilst subcutaneous immunization elicited a weaker response. Intraperitoneal immunization with FPTAB9LZ was also shown to provide protection against challenge with a recombinant vaccinia virus expressing antigens, including those in TAB9. These results confirm the potential of FWPV vectors for use in HIV vaccination strategies.