RESUMO
BACKGROUND: The detection of antibodies against capripoxvirus has become easier with a commercially available ELISA validated for serum and plasma. In order to explore its suitability for immunological investigations on alternative samples, this study targeted milk as sample matrix available through non-invasive sampling. METHODS: Samples for this study were collected from dairy cows vaccinated against LSD in an area without reported LSD virus circulation. Paired serum and milk (individual and bulk) samples were tested by ELISA without and with modifications of the sample incubation time for the milk samples. For the evaluation of the test specificity, 352 milk samples from a milk repository in Germany were used as negative control. Receiver operating characteristic analysis was performed for determination of the Youden index and determination of the most suitable cut-off value for maximum specificity. RESULTS: From 154 analyzed serum samples from Serbia, 75 were detected as positive in the ELISA. Sensitivity and specificity of the ELISA test for milk samples reached values of 88 to 91% using Youden criteria. A cut-off of 10 was determined aiming for maximum specificity. This cut-off value was used for further analysis. Using the protocol for serum, out of 154 milk samples, 38 were detected as positive, number of positive detected milk samples increase up to 48 with modified protocol. Milk samples from Germany reacted negative, except two samples that had borderline results using modified protocol. Significant statistical difference (p < 0.05) was observed between two incubation protocols. The detection of LSD-specific antibodies from bulk milk samples (pools of 2-10 individuals) came along with a reduced sensitivity over the sample of individual animals. CONCLUSIONS: Results show that the detection of capripoxvirus specific antibodies in milk samples using the commercially available ELISA from IDvet is feasible and can represent a helpful tool for LSDV monitoring programs.
Assuntos
Anticorpos Antivirais/análise , Capripoxvirus/imunologia , Imunidade Humoral , Doença Nodular Cutânea/imunologia , Doença Nodular Cutânea/prevenção & controle , Leite/imunologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Ensaio de Imunoadsorção Enzimática , Feminino , Alemanha , Sensibilidade e Especificidade , VacinaçãoRESUMO
Goatpox virus (GTPV) is an important member of the Capripoxvirus genus of the Poxviridae Capripoxviruses have large and complex DNA genomes encoding many unknown proteins that may contribute to virulence. We identified that the 135 open reading frame of GTPV is an early gene that encodes an â¼18-kDa protein that is nonessential for viral replication in cells. This protein functioned as an inhibitor of NF-κB activation and apoptosis and is similar to the N1L protein of vaccinia virus. In the natural host, sheep, deletion of the 135 gene from the GTPV live vaccine strain AV41 resulted in less attenuation than that induced by deletion of the tk gene, a well-defined nonessential gene in the poxvirus genome. Using the 135 gene as the insertion site, a recombinant AV41 strain expressing hemagglutinin of peste des petits ruminants virus (PPRV) was generated and elicited stronger neutralization antibody responses than those obtained using the traditional tk gene as the insertion site. These results suggest that the 135 gene of GTPV encodes an immunomodulatory protein to suppress host innate immunity and may serve as an optimized insertion site to generate capripoxvirus-vectored live dual vaccines.IMPORTANCE Capripoxviruses are etiological agents of important diseases in sheep, goats, and cattle. There are rare reports about viral protein function related to capripoxviruses. In the present study, we found that the 135 protein of GTPV plays an important role in inhibition of innate immunity and apoptosis in host cells. Use of the 135 gene as the insertion site to generate a vectored vaccine resulted in stronger adaptive immune responses than those obtained using the tk locus as the insertion site. As capripoxviruses are promising virus-vectored vaccines against many important diseases in small ruminants and cattle, the 135 gene may serve as an improved insertion site to generate recombinant capripoxvirus-vectored live dual vaccines.
Assuntos
Apoptose/genética , Capripoxvirus/genética , NF-kappa B/antagonistas & inibidores , Proteínas Virais/genética , Vacinas Virais/genética , Animais , Capripoxvirus/imunologia , Capripoxvirus/patogenicidade , Vetores Genéticos , Células HEK293 , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Imunidade Inata , Fatores Imunológicos/imunologia , Mutagênese Insercional , NF-kappa B/genética , Fases de Leitura Aberta/genética , Vírus da Peste dos Pequenos Ruminantes/química , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/imunologia , Ovinos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Vacinas Virais/imunologiaRESUMO
BACKGROUND: Sheeppox (SPP) and goatpox (GTP) caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively of the genus Capripoxvirus in the family Poxviridae, are severely afflicting small ruminants' production systems in Africa and Asia. In endemic areas, SPP and GTP are controlled using vaccination with live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). Sometimes outbreaks occur following vaccination. In order to successfully control the spread of the virus, it is essential to identify whether the animals were infected by the field strain and the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or in rare occasions, re-gain virulence. Thus, diagnostic tools for differentiation of virulent strains from attenuated vaccine strains of the virus are needed. The aim of this study was to identify an appropriate diagnostic target region in the capripoxvirus genome by comparing the genomic sequences of SPPV field isolates with those of the most widely used SPP vaccine strains. RESULTS: A unique 84 base pair nucleotide deletion located between the DNA ligase gene and the VARV B22R homologue gene was found only in SPPV vaccines derived from the Romanian and Yugoslavian RM/65 strains and absent in SPPV field isolates originated from various geographical locations of Asia and Africa. In addition, we developed and evaluated a conventional PCR assay, exploiting the targeted intergenic region to differentiate SPPV vaccine virus from field isolates. The assay produced an amplicon size of 218 bp for the vaccine strains, while the SPPV field isolates resulted in a 302 bp PCR fragment. The assay showed good sensitivity and specificity, and the results were in full agreement with the sequencing data of the PCR amplicons. CONCLUSION: The developed assay is an improvement of currently existing diagnostic tools and, when combined with a capripox virus species-specific assay, will enhance SPP and GTP diagnosis and surveillance and facilitate epidemiological investigations in countries using live attenuated SPP vaccines. In addition, for laboratories with limited resources, the assay provides a simple and cost-effective alternative for sequencing.
Assuntos
Capripoxvirus/imunologia , Doenças das Cabras/prevenção & controle , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/prevenção & controle , Vacinas Virais/imunologia , Animais , Capripoxvirus/classificação , Capripoxvirus/genética , Linhagem Celular , Cabras , Reação em Cadeia da Polimerase , Ovinos , Especificidade da EspécieRESUMO
BACKGROUND: Sheeppox (SPP) is one of the priorities, high-impact animal diseases in many developing countries, where live attenuated vaccines are routinely used against sheeppox virus (SPPV). In an event of an SPP outbreak, historically disease-free countries would hesitate to use of live vaccines against SPPVdue to the safety and trade reasons. Currently no killed SPPV vaccines are commercially available. In this study, we developed an inactivated Romanian SPPVvaccine and assessed its efficacy and potency in comparison with a live attenuated Romanian SPPV vaccine. Four naïve sheep were vaccinated once with the Romanian SPPV live attenuated vaccine and16 sheep were vaccinated twice with the inactivated vaccine. All sheep in the live vaccine group were included in the challenge trial, which was conducted using a highly virulent Moroccan SPPV field strain. Eight sheep of the inactivated vaccine group were challenged and the remaining sheep were monitored for seroconversion. Experimental animals were closely monitored for the appearance of clinical signs, body temperature and inflammation at the injection site. Two naïve sheep were used as unvaccinated controls. RESULTS: The inactivated Romanian SPPV vaccine was found to be safe and confer a good protection, similar to the live vaccine. Specific antibodies appeared from seven days post vaccination and remained up to nine months. CONCLUSION: This study showed that the developed inactivated Romanian SPPV vaccine has a potential to replace attenuated vaccine to control and prevent sheep pox in disease-free or endemic countries.
Assuntos
Capripoxvirus/imunologia , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/prevenção & controle , Vacinas Virais/imunologia , Animais , Chlorocebus aethiops , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/prevenção & controle , Ovinos , Doenças dos Ovinos/imunologia , Potência de Vacina , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologia , Células VeroRESUMO
Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Capripoxvirus , Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Testes de Neutralização , Infecções por Poxviridae , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Vírus da Doença Nodular Cutânea/imunologia , Vírus da Doença Nodular Cutânea/genética , Capripoxvirus/imunologia , Capripoxvirus/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Ovinos , Doença Nodular Cutânea/prevenção & controle , Doença Nodular Cutânea/imunologia , Doença Nodular Cutânea/virologia , Infecções por Poxviridae/veterinária , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , Doenças dos Ovinos/virologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/prevenção & controle , CabrasRESUMO
In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD(50) was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.
Assuntos
Capripoxvirus/imunologia , Infecções por Poxviridae/imunologia , Doenças dos Ovinos/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Capripoxvirus/classificação , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Infecções por Poxviridae/prevenção & controle , Ovinos , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/virologia , Especificidade da Espécie , Fatores de Tempo , Resultado do Tratamento , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Vacinas Virais/administração & dosagemRESUMO
Viral interference is a common occurrence that has been reported in cell culture in many cases. In the present study, viral interference between two capripox viruses (sheeppox SPPV and lumpy skin disease virus LSDV in cattle) with Rift Valley fever virus (RVFV) was investigated in vitro and in their natural hosts, sheep and cattle. A combination of SPPV/RVFV and LSDV/RVFV was used to co-infect susceptible cells and animals to detect potential competition. In-vitro interference was evaluated by estimating viral infectivity and copies of viral RNA by a qPCR during three serial passages in cell cultures, whereas in-vivo interference was assessed through antibody responses to vaccination. When lamb testis primary cells were infected with the mixture of capripox and RVFV, the replication of both SPPV and LSDV was inhibited by RVFV. In animals, SPPV/RVFV or LSDV/RVFV combinations inhibited the replication SPPV and LSDV and the antibody response following vaccination. The combined SPPV/RVFV did not protect sheep after challenging with the virulent strain of SPPV and the LSDV/RVFV did not induce interferon Gamma to LSDV, while immunological response to RVFV remain unaffected. Our goal was to assess this interference response to RVFV/capripoxviruses' coinfection in order to develop effective combined live-attenuated vaccines as a control strategy for RVF and SPP/LSD diseases. Our findings indicated that this approach was not suitable for developing a combined SPPV/LSDV/RVFV vaccine candidate because of interference of replication and the immune response among these viruses.
Assuntos
Capripoxvirus/genética , Vírus da Febre do Vale do Rift/genética , Animais , Anticorpos Antivirais/biossíntese , Capripoxvirus/imunologia , Capripoxvirus/fisiologia , Bovinos , Células Cultivadas , Chlorocebus aethiops , Genes Virais , Técnicas In Vitro , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Vírus da Febre do Vale do Rift/imunologia , Vírus da Febre do Vale do Rift/fisiologia , Ovinos , Células Vero , Vacinas Virais/imunologia , Replicação ViralRESUMO
Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κß p65 (NF-κß), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κß, between IL-18 and IL-15, and between NF-κß and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 ß, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.
Assuntos
Capripoxvirus/imunologia , Imunidade Inata , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/prevenção & controle , Vacinas Virais/imunologia , Animais , Capripoxvirus/genética , Capripoxvirus/isolamento & purificação , Leucócitos Mononucleares/imunologia , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular , Ovinos , Vacinas Atenuadas/imunologiaRESUMO
In this comparative study, we examine the safety of the sheeppox (SPP) and goatpox (GTP) vaccines and the protective response of these vaccines in cattle against a virulent lumpy skin disease (LSD) field strain. The vaccine safety was tested in rabbits, mice and cattle using ten times recommended dose. In the safety trial, none of the vaccinated animals showed any deviation from physiological norms or fever, inappetence or local/ generalized skin reactions. In the challenge trial, both SPP and GTP vaccine groups developed virus-neutralizing antibodies with an average titre of 2.1 log2 at 21 days post-vaccination. No significant difference in seroconversion was found in cattle vaccinated with SPP and GTP vaccines (P ≥ 0.05). When challenged with a virulent LSD field strain, one animal vaccinated with the SPP Niskhi vaccine strain showed typical LSD skin lesions at the injection sites of different dilutions of the challenge virus. All animals vaccinated with GTP G20-LKV vaccine strain showed full protection. After infection with the challenge virus, unvaccinated fully susceptible control cattle showed characteristic clinical signs of LSD. The average protective index for SPP and GTP vaccine groups was 5.3 ± 1.42 and 5.9 ± 0.00, respectively.
Assuntos
Capripoxvirus/imunologia , Doenças dos Bovinos/prevenção & controle , Imunogenicidade da Vacina , Doença Nodular Cutânea/prevenção & controle , Vírus da Doença Nodular Cutânea/patogenicidade , Vacinas Virais/imunologia , Animais , Capripoxvirus/classificação , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Feminino , Vírus da Doença Nodular Cutânea/imunologia , Camundongos , Coelhos , Vacinação , Vacinas Virais/administração & dosagemRESUMO
Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV-28. Phylogenetic analysis of BTV-28 segment 2 (Seg-2) showed that it is related to Seg-2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%-66% identity in nucleotide sequences (nt) and 59%-62% in amino acid (aa) sequences of BTV VP2. BTV-28 Seg-6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg-5 was most closely related to a South African BTV-4 strain, and all other segments showed close similarity to BTV-26. Experimental infection by injection of 6-month-old ewes caused clinical signs in all injected animals, lasting from 2 to 3 days to several weeks post-infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV-injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact-infected animals. These findings demonstrate that BTV-28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts.
Assuntos
Vírus Bluetongue/imunologia , Bluetongue/virologia , Capripoxvirus/imunologia , Ceratopogonidae/virologia , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/virologia , Vacinas Virais , Animais , Bluetongue/transmissão , Vírus Bluetongue/isolamento & purificação , Feminino , Filogenia , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , RNA Viral/genética , Sorogrupo , Ovinos , Doenças dos Ovinos/transmissãoRESUMO
The genus Capripoxvirus in the subfamily Chordopoxvirinae, family Poxviridae, comprises sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which cause the eponymous diseases across parts of Africa, the Middle East and Asia. These diseases cause significant economic losses and can have a devastating impact on the livelihoods and food security of small farm holders. So far, only live classically attenuated SPPV, GTPV and LSDV vaccines are commercially available and the history, safety and efficacy of many have not been well established. Here, we report 13 new capripoxvirus genome sequences, including the hairpin telomeres, from both pathogenic field isolates and vaccine strains. We have also updated the genome annotations to incorporate recent advances in our understanding of poxvirus biology. These new genomes and genes grouped phenetically with other previously sequenced capripoxvirus strains, and these new alignments collectively identified several recurring alterations in genes thought to modulate virulence and host range. In particular, some of the many large capripoxvirus ankyrin and kelch-like proteins are commonly mutated in vaccine strains, while the variola virus B22R-like gene homolog has also been disrupted in many vaccine isolates. Among these vaccine isolates, frameshift mutations are especially common and clearly present a risk of reversion to wild type in vaccines bearing these mutations. A consistent pattern of gene inactivation from LSDV to GTPV and then SPPV is also observed, much like the pattern of gene loss in orthopoxviruses, but, rather surprisingly, the overall genome size of ~150 kbp remains relatively constant. These data provide new insights into the evolution of capripoxviruses and the determinants of pathogenicity and host range. They will find application in the development of new vaccines with better safety, efficacy and trade profiles.
Assuntos
Capripoxvirus/genética , Variação Genética , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/virologia , África , Animais , Ásia , Evolução Biológica , Capripoxvirus/imunologia , Capripoxvirus/patogenicidade , Capripoxvirus/fisiologia , Células Cultivadas , Especiação Genética , Índia , Masculino , Oriente Médio , Mutação , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , Ovinos , Doenças dos Ovinos/prevenção & controle , Testículo/virologia , Vacinas Virais/imunologia , VirulênciaRESUMO
The Capripoxvirus genus includes three agents: Sheeppox virus, Goatpox virus and Lumpy skin disease virus. Related diseases are of economic importance and present a major constraint to animals and animal products trade in addition to mortality and morbidity. Attenuated vaccines against these diseases are available, but afforded cross-protection is controversial in each specie. In this study, groups of sheep, goats and cattle were vaccinated with Romania SPPV vaccine and challenged with corresponding virulent strains. Sheep and cattle were also vaccinated with Neethling LSDV vaccine and challenged with both virulent SPPV and LSDV strains. Animals were monitored by clinical observation, rectal temperature as well as serological response. The study showed that sheep and goats vaccinated with Romania SPPV vaccine were fully protected against challenge with virulent SPPV and GTPV strains, respectively. However, small ruminants vaccinated with LSDV Neethling vaccine showed only partial protection against challenge with virulent SPPV strain. Cattle showed also only partial protection when vaccinated with Romania SPPV and were fully protected with Neethling LSDV vaccine. This study showed that SPPV and GTPV vaccines are closely related with cross-protection, while LSDV protects only cattle against the corresponding disease, which suggests that vaccination against LSDV should be carried out with homologous strain.
Assuntos
Capripoxvirus/fisiologia , Doenças dos Bovinos/prevenção & controle , Doenças das Cabras/prevenção & controle , Doenças dos Ovinos/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Animais , Anticorpos Antivirais/metabolismo , Capripoxvirus/classificação , Capripoxvirus/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Proteção Cruzada , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Romênia , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/virologia , Vacinação/veterinária , Vacinas Atenuadas/classificação , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/classificação , Vacinas Virais/imunologiaRESUMO
Semiconductor nanocrystals (or quantum dots, QDs) have the potential to overcome some of the limitations encountered by traditional fluorophores in fluorescence labeling applications. The unique spectroscopic properties of QDs make them hold immense promise as versatile labels for biological applications. In this work, we employ the layer-by-layer (LbL) method for the construction of bio-functional multicolor QD-encoded microspheres. Polystyrene microspheres with diameter of 3 microm were used as templates for the deposition of different sized CdTe QDs/polyelectrolyte multilayers. Two different antigens, Chicken newcastle disease (CND) antigen and goat pox virus (GPV) antigen, were conjugated to two kinds of biofunctional multicolor microspheres with different optical encoding. The multicolor microspheres can capture corresponding antibodies labeled with QDs, QDs-CND antibody and QDs-GPV antibody in the fluoroimmunoassays. The microspheres can be distinguished from each other based on their optical encoding.
Assuntos
Antígenos Virais/análise , Compostos de Cádmio/química , Capripoxvirus/isolamento & purificação , Fluorimunoensaio/métodos , Vírus da Doença de Newcastle/isolamento & purificação , Poliestirenos/química , Telúrio/química , Animais , Antígenos Virais/imunologia , Compostos de Cádmio/síntese química , Capripoxvirus/imunologia , Galinhas , Imunoconjugados/química , Imunoconjugados/imunologia , Microesferas , Vírus da Doença de Newcastle/imunologia , Poliestirenos/síntese química , Pontos Quânticos , Espectrometria de FluorescênciaRESUMO
Efficacy of live viral vaccine and vaccine-induced sero-conversion depends on the optimum number of live virus particles in a vaccine dose, which is one of the important aspects of quality control. In the present study, TaqMan probe quantitative polymerase chain reaction (QPCR) based on conserved DNA pol gene of capripoxvirus was developed for the quality control of attenuated monovalent goatpox and/or combined attenuated goatpox and peste des petits ruminants (PPR) vaccines. Cells infected with vaccine virus were harvested at critical time point and subjected to QPCR. A critical time point for harvest of Vero cells infected with various log10 dilutions of reference virus was determined to be 36 h (highest slope 3.062), by comparison of slopes of standard curves established with harvests at different time intervals. The assay method was evaluated using different batches of goatpox vaccine, and bivalent goatpox and PPR vaccine. The titers estimated by QPCR and TCID50 method were comparable to each other. The QPCR assay thus, could be used as an alternate method or supplementary tool for estimation of live GTPV particles in monovalent goatpox or bivalent goatpox and PPR vaccines.
Assuntos
Capripoxvirus/imunologia , Reação em Cadeia da Polimerase/métodos , Vacinas Virais , Animais , Sequência de Bases , Capripoxvirus/isolamento & purificação , Chlorocebus aethiops , Primers do DNA , Reação em Cadeia da Polimerase/normas , Controle de Qualidade , Células VeroRESUMO
Sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates.
Assuntos
Capripoxvirus/genética , Capripoxvirus/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/virologia , Vacinas Virais/imunologia , Animais , Capripoxvirus/classificação , Capripoxvirus/isolamento & purificação , DNA Viral , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Ovinos , Temperatura de TransiçãoRESUMO
The role of interferon (IFN)-induced protein kinase R (PKR) in capripoxvirus (CaPV)-infected cells remains unknown. In this study, we show that CaPV infection triggered PKR and eukaryotic translation initiation factor 2 alpha (eIF2α) protein phosphorylation in a dose-dependent manner, and that this leads to decreased CaPV replication. Overexpression of PKR compromised viral gene expression and inhibited sheeppox virus (SPPV) replication. Downregulation of PKR with siRNAs significantly decreased eIF2α phosphorylation and reduced the mRNA level of IFN-ß, which increased virus replication. In luciferase assays, species-different CaPVs K3L proteins inhibited sheep PKR (sPKR): goatpox virus K3L strongly inhibited sPKR and goat PKR (gPKR), but SPPV K3L only partially inhibited gPKR. These results are the first to show that SPPV infection induces phosphorylation of eIF2α through PKR activation, which then results in restriction of CaPV replication. Furthermore, our data show that CaPV K3L inhibits PKR in a species-specific manner. The results presented are consistent with the hypothesis that different levels of PKR inhibition by K3L orthologs from various viruses could potentially contribute to the host range function of K3L.
Assuntos
Capripoxvirus/crescimento & desenvolvimento , Infecções por Poxviridae/imunologia , Replicação Viral/fisiologia , eIF-2 Quinase/metabolismo , Animais , Capripoxvirus/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Cabras , Células HeLa , Humanos , Interferon gama/genética , Fosforilação , Infecções por Poxviridae/virologia , Interferência de RNA , RNA Interferente Pequeno/genética , Ovinos , Células Vero , Proteínas Virais/metabolismo , eIF-2 Quinase/genéticaRESUMO
Sheeppox virus from an outbreak of sheeppox that occurred in Srinagar (Jammu and Kashmir, India) in 2000 was isolated by inoculation of susceptible sheep and further re-isolated in cell culture. The field virus, adapted to grow in lamb testes culture, was evaluated for its potential use as challenge virus in potency testing of sheeppox vaccine currently in use. The virus (passage 6) produced severe disease in susceptible sheep when inoculated subcutaneously with a dose of 106.2 TCID50. The virus identity was confirmed by PCR, sequencing of P32 gene and species-specific signature residues identified in deduced aa sequence of the gene. The virus was successfully evaluated for its virulence using two batches of sheep pox vaccines. Use of this field virus enables consistent potency experiments of sheeppox vaccines avoiding use of animals for its propagation and titration.
Assuntos
Capripoxvirus/patogenicidade , Vacinas Virais/análise , Adaptação Fisiológica , Alternativas aos Testes com Animais , Animais , Capripoxvirus/genética , Capripoxvirus/imunologia , Capripoxvirus/fisiologia , Células Cultivadas , Efeito Citopatogênico Viral , Genes Virais , Masculino , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/veterinária , Infecções por Poxviridae/virologia , Ovinos , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/virologia , VirulênciaRESUMO
Sheep pox, a well-known endemic capripox infection, has significant impacts on small ruminant populations in Tunisia. It is responsible for high economic losses throughout North Africa due to its enzootic nature and to the active animal transhumance existing in some governorates in Tunisia. The aim of this review was to analyse data gathered on annual vaccination campaigns designed to control its spread by reducing the level of endemicity and to describe diagnostic and management tools adapted to the Tunisian situation. Seasonal, temporal and spatial distributions of sheep pox outbreaks, as well as related clinical features, were found. It was concluded from this review that establishing strong herd immunization through individual animal immunization, creating adequate infrastructure, increasing awareness among breeders, setting up a field-based surveillance network and improving routine diagnostic methods need to be the major components of a programme to eradicate the disease. It was also felt that cost-benefit analyses of the surveillance and control strategies used would help in controlling its persistence.
Assuntos
Capripoxvirus/imunologia , Imunização/veterinária , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/epidemiologia , Animais , Capripoxvirus/isolamento & purificação , Análise Custo-Benefício , Surtos de Doenças/veterinária , Infecções por Poxviridae/epidemiologia , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , Ovinos , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/virologia , Tunísia/epidemiologiaRESUMO
Sheep and goat pox (SGP) with peste des petits ruminants (PPR) are transboundary viral diseases of small ruminants that cause huge economic losses. Recombinant vaccines that can protect from both infections have been reported as a promising solution for the future. SGP was used as a vector to express two structural proteins hemagglutinin or the fusion protein of PPRV. We compared immunity conferred by recombinant capripoxvirus vaccines expressing H or F or both HF. Safety and efficacy were evaluated in goats and sheep. Two vaccine doses were tested in sheep, 104.5TCDI50 in 1ml dose was retained for the further experiment. Results showed that the recombinant HF confers an earlier and stronger immunity against both SGP and PPR. This recombinant vaccine protect also against the disease in exposed and unexposed sheep. The potential Differentiating Infected from Vaccinated Animals of recombinant vaccines is of great advantage in any eradication program.
Assuntos
Capripoxvirus/imunologia , Doenças das Cabras/prevenção & controle , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/prevenção & controle , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Capripoxvirus/genética , Capripoxvirus/isolamento & purificação , Capripoxvirus/fisiologia , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Hemaglutininas/administração & dosagem , Hemaglutininas/genética , Hemaglutininas/imunologia , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/isolamento & purificação , Vírus da Peste dos Pequenos Ruminantes/fisiologia , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/virologia , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Lumpy skin disease, sheeppox and goatpox are high-impact diseases of domestic ruminants with a devastating effect on cattle, sheep and goat farming industries in endemic regions. In this article, we review the current geographical distribution, economic impact of an outbreak, epidemiology, transmission and immunity of capripoxvirus. The special focus of the article is to scrutinize the use of currently available vaccines to investigate the resource needs and challenges that will have to be overcome to improve disease control and eradication, and progress on the development of safer and more effective vaccines. In addition, field evaluation of the efficacy of the vaccines and the genomic database available for poxviruses are discussed.