RESUMO
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
Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.
Assuntos
Capripoxvirus , Vírus da Doença Nodular Cutânea , Infecções por Poxviridae , Doenças dos Ovinos , Vacinas Virais , Ovinos , Bovinos , Animais , Capripoxvirus/genética , Mutação , Genoma Viral , Vírus da Doença Nodular Cutânea/genética , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/veterinária , Vacinas Virais/genética , Doenças dos Ovinos/epidemiologia , CabrasRESUMO
Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015-2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0-88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4-62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.
Assuntos
Antílopes , Peste dos Pequenos Ruminantes , Vírus da Peste dos Pequenos Ruminantes , Animais , Ovinos , Soroconversão , Peste dos Pequenos Ruminantes/diagnóstico , Anticorpos , Animais Selvagens , Búfalos , Camelus , CabrasRESUMO
Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.
RESUMO
Goatpox virus (GTPV) belongs to the genus Capripoxvirus, together with sheeppox virus (SPPV) and lumpy skin disease virus (LSDV). GTPV primarily affects sheep, goats and some wild ruminants. Although GTPV is only present in Africa and Asia, the recent spread of LSDV in Europe and Asia shows capripoxviruses could escape their traditional geographical regions to cause severe outbreaks in new areas. Therefore, it is crucial to develop effective source tracing of capripoxvirus infections. Earlier, conventional phylogenetic methods, based on limited samples, identified three different nucleotide sequence profiles in the G-protein-coupled chemokine receptor (GPCR) gene of GTPVs. However, this method did not differentiate GTPV strains by their geographical origins. We have sequenced the GPCR gene of additional GTPVs and analyzed them with publicly available sequences, using conventional alignment-based methods and an alignment-free approach exploiting k-mer frequencies. Using the alignment-free method, we can now classify GTPVs based on their geographical origin: African GTPVs and Asian GTPVs, which further split into Western and Central Asian (WCA) GTPVs and Eastern and Southern Asian (ESA) GTPVs. This approach will help determine the source of introduction in GTPV emergence in disease-free regions and detect the importation of additional strains in disease-endemic areas.
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The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen's kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/veterinária , Vison/imunologia , SARS-CoV-2/imunologia , Animais , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/imunologia , Teste Sorológico para COVID-19 , Quirópteros/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Monitoramento Epidemiológico , Furões/imunologia , Imunoprecipitação , Mesocricetus/imunologia , Fosfoproteínas/imunologia , Coelhos/imunologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
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
Lumpy skin disease (LSD), an economically significant disease in cattle caused by lumpy skin disease virus (LSDV), is endemic to nearly all of Africa. Since 2012, LSDV has emerged as a significant epizootic pathogen given its rapid spread into new geographical locations outside Africa, including the Middle East, Eastern Europe, and Asia. To assess the genetic diversity of LSDVs in East Africa, we sequenced and analyzed the RPO30 and GPCR genes of LSDV in twenty-two archive samples collected in Ethiopia, Kenya, and Sudan before the appearance of LSD in the Middle East and its incursion into Europe. We compared them to publicly available sequences of LSDVs from the same region and those collected elsewhere. The results showed that the East African field isolates in this study were remarkably similar to each other and to previously sequenced field isolates of LSDV for the RPO30 and GPCR genes. The only exception was LSDV Embu/B338/2011, a field virus collected in Kenya, which displayed mixed features between the LSDV Neethling vaccine and field isolates. LSDV Embu/B338/2011 had the same 12-nucleotide insertion found in LSDV Neethling and KS-1 vaccines. Further analysis of the partial EEV glycoprotein, B22R, RNA helicase, virion core protein, NTPase, and N1R/p28-like protein genes showed that LSDV Embu/B338/2011 differs from previously described LSDV variants carrying the 12-nucleotide insertion in the GPCR gene. These findings highlight the importance of the constant monitoring of genetic variation among LSDV isolates.
RESUMO
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
Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR.
Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Cabras/imunologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Vírus da Peste Bovina/imunologia , Ovinos/imunologia , Animais , Anticorpos Antivirais/imunologia , Cabras/sangue , Cabras/virologia , Imunoprecipitação , Luciferases , Peste dos Pequenos Ruminantes/diagnóstico , Peste dos Pequenos Ruminantes/imunologia , Peste dos Pequenos Ruminantes/virologia , Sensibilidade e Especificidade , Ovinos/sangue , Ovinos/virologiaRESUMO
The acute myelogenous leukemia-1 (AML1)-ETO fusion protein is generated by the t(8;21), which is found in 40% of AMLs of the French-American-British M2 subtype. AML1-ETO interferes with the function of the AML1 (RUNX1, CBFA2) transcription factor in a dominant-negative fashion and represses transcription by binding its consensus DNA-binding site and via protein-protein interactions with other transcription factors. AML1 activity is critical for the development of definitive hematopoiesis, and haploinsufficiency of AML1 has been linked to a propensity to develop AML. Murine experiments suggest that AML1-ETO expression may not be sufficient for leukemogenesis; however, like the BCR-ABL isoforms, the cellular background in which these fusion proteins are expressed may be critical to the phenotype observed. Retroviral gene transfer was used to examine the effect of AML1-ETO on the in vitro behavior of human hematopoietic stem and progenitor cells. Following transduction of CD34(+) cells, stem and progenitor cells were quantified in clonogenic assays, cytokine-driven expansion cultures, and long-term stromal cocultures. Expression of AML1-ETO inhibited colony formation by committed progenitors, but enhanced the growth of stem cells (cobblestone area-forming cells), resulting in a profound survival advantage of transduced over nontransduced cells. AML1-ETO-expressing cells retained progenitor activity and continued to express CD34 throughout the 5-week long-term culture. Thus, AML1-ETO enhances the self-renewal of pluripotent stem cells, the physiological target of many acute myeloid leukemias.
Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas de Fusão Oncogênica/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antígenos CD34/análise , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Subunidade alfa 2 de Fator de Ligação ao Core , Citocinas/farmacologia , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Luminescentes/genética , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1 , Retroviridae/genética , Células Estromais/fisiologia , Fatores de Transcrição/genética , TransfecçãoRESUMO
The CCAAT/enhancer binding protein alpha (C/EBPalpha) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPalpha(-/-) mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPalpha in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPalpha gene and down-regulation of C/EBPalpha by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPalpha in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34(+) cells with a retroviral construct that expresses the C/EBPalpha cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPalpha function in primary human CD34(+) cells, by the addition of beta-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPalpha target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor of differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras , Sítios de Ligação , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/deficiência , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Eritrócitos/citologia , Células Precursoras Eritroides/química , Estradiol/farmacologia , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Proteína 1 Inibidora de Diferenciação , Neutrófilos/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA/análise , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , TransfecçãoRESUMO
Hematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo.