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1.
J Virol ; 96(6): e0202721, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35107377

RESUMO

Marek's disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek's disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. IMPORTANCE Marek's disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.


Assuntos
Herpesvirus Galináceo 3 , Linfoma , Doença de Marek , Proteínas do Envelope Viral , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Embrião de Galinha , Galinhas , Herpesvirus Galináceo 3/genética , Linfoma/veterinária , Linfoma/virologia , Proteínas do Envelope Viral/genética
2.
Avian Dis ; 65(2): 241-249, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34412454

RESUMO

Marek's disease virus (MDV) is an important poultry pathogen that is controlled through widespread vaccination with avirulent and attenuated strains. However, continued evolution of field viruses to higher virulence has required ongoing improvement of available vaccine strains, and these vaccine strains offer an attractive platform for designing recombinant vector vaccines with cross-protection against MDV and additional pathogens. Recent reports of failures in vaccine licensing trials of positive controls to reach appropriately high levels of Marek's disease incidence prompted us to evaluate possible combinations of outbred specific-pathogen-free layer lines and alternative virulent challenge strains that could provide more consistent models for serotype 3 vectored vaccine development. Choice of layer line and virulent MDV challenge strain each contributed to the ability of a challenge model to reach 80% virulence in unvaccinated positive control groups in the majority of trials, without overwhelming serotype 3 vectored vaccine protection in vaccinated groups. Conversely, reducing challenge virus dose by a factor of four, or vaccine dose by half, had no consistent effect across these models. Although MDV strain 617A had the most potential as an alternative to strains that are currently approved for licensing trials, no combination of layer line and challenge virus consistently met the goals for a successful challenge model in all study replicates, indicating that high variability is an inherent difficulty in MDV challenge studies, at least when outbred birds are used.


Artículo regular­Comparación de las cepas de desafío del virus de la enfermedad de Marek y los tipos de aves para la obtención de licencias de vacunas. El virus de la enfermedad de Marek (MDV) es un patógeno importante en la avicultura que se controla mediante la vacunación generalizada con cepas avirulentas y atenuadas. Sin embargo, la evolución continua de los virus de campo hacia una mayor virulencia ha requerido una mejora continua de las cepas vacunales disponibles y estas cepas vacunales ofrecen una plataforma atractiva para diseñar vacunas con vectores recombinantes que induzcan protección cruzada contra el virus de la enfermedad de Marek y patógenos adicionales. Los reportes recientes de fallas en los controles positivos para alcanzar niveles apropiadamente altos de incidencia de la enfermedad de Marek en los ensayos para obtener la licencia de vacunas llevaron a evaluar posibles combinaciones de líneas de postura híbridas libres de patógenos específicos y cepas de desafío virulentas alternativas que podrían proporcionar modelos más consistentes para el desarrollo de vacunas con vectores de serotipo 3. Tanto la elección de la línea de postura como de la cepa de desafío virulenta de Marek contribuyeron a obtener un modelo de desafío con capacidad para alcanzar el 80% de virulencia en grupos controles positivo no vacunados en la mayoría de los ensayos, sin una protección abrumadora de la vacuna con vector de serotipo 3 en los grupos vacunados. Por el contrario, la reducción de la dosis del virus de desafío en un factor de cuatro, o la dosis de vacuna a la mitad, no tuvieron un efecto constante en estos modelos. Aunque la cepa 617A de Marek mostró el mayor potencial como alternativa a las cepas que actualmente están aprobadas para ensayos de licenciar vacunas, ninguna combinación de línea de postura y virus de desafío cumplió consistentemente los objetivos de un modelo de desafío exitoso en todas las réplicas del estudio, lo que indica que la alta variabilidad es una dificultad inherente en los estudios de desafío para la enfermedad de Marek, al menos cuando se utilizan aves híbridas.


Assuntos
Galinhas/classificação , Herpesvirus Galináceo 3/classificação , Herpesvirus Galináceo 3/imunologia , Vacinas Virais/classificação , Animais , Galinhas/imunologia , Herpesvirus Galináceo 3/patogenicidade , Complexo Principal de Histocompatibilidade/genética , Organismos Livres de Patógenos Específicos , Vacinas Virais/normas , Virulência
3.
Avian Dis ; 64(2): 174-182, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32550618

RESUMO

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus type 2. Gallid alphaherpesvirus type 3 (GaHV-3) strain 301B/1 was previously shown to be an effective MD vaccine with synergistic efficacy when used as a bivalent vaccine with turkey herpesvirus. Since the nucleotide sequences of only two GaHV-3 strains have been determined, we sought to sequence the 301B/1 genome using Illumina MiSeq technology. Phylogenomic analysis indicated that 301B/1 is more closely related to other GaHV-3 strains (SB-1 and HPRS24) than to virulent or attenuated strains of GaHV-2. One hundred and twenty-six open reading frames (ORFs) have been identified within the 301B/1 genome with 108 ORFs showing a high degree of similarity to homologs found in the genomes of SB-1 and HPRS24; 14 ORFs are highly homologous (> 90% identity) with the corresponding ORFs within the SB-1 genome. The R-LORF8 and R-LORF9 genes are the most dissimilar to the collinear genes found in the SB-1 genome but are highly homologous (99%-100% identity) with those within the HPRS24 genome. Overall the 301B/1 genome is most similar to the SB-1 virus genome (99.1%) and to a lesser degree with the HPRS24 virus genome (97.7%). However, six 301B/1 ORFs (UL47, UL48, UL52, pp38, ICP4, and US10) have been identified that contain nonsynonymous substitutions relative to homologs found in the SB-1 genome. Notably, unlike the avian retrovirus long terminal repeat sequences found within the SB-1 genome, none were identified within the 301B/1 genome.


Caracterización molecular comparativa de cepas de Alfaherpesvirus del pollo tipo 3 cepas 301B/1, HPRS24 y SB-1. La enfermedad de Marek (MD) es una enfermedad linfoproliferativa altamente contagiosa de los pollos causada por el Alfaherpesvirus del pollo tipo 2. Se demostró previamente que la cepa 301B/1 del Alfaherpesvirus del pollo tipo 3 (GaHV-3) es una vacuna eficaz contra la enfermedad de Marek con eficacia sinérgica cuando se usa como una vacuna bivalente con el herpesvirus del pavo. Dado que se han determinado las secuencias de nucleótidos de solo dos cepas de GaHV-3, se buscó secuenciar el genoma de la cepa 301B/1 utilizando la tecnología Illumina MiSeq. El análisis filogenómico indicó que la cepa 301B/1 está más estrechamente relacionado con otras cepas de GaHV-3 (SB-1 y HPRS24) en comparación con cepas virulentas o atenuadas de GaHV-2. Se han identificado 126 marcos de lectura continuos (ORF) dentro del genoma de la cepa 301B/1 con 108 marcos de lectura continuos que muestran un alto grado de similitud con los secuencias homólogas encontrados en los genomas de las cepas SB-1 y HPRS24; 14 marcos de lectura continuo son altamente similares (> 90% de identidad) con los correspondientes dentro del genoma de SB-1. Los genes R-LORF8 y R-LORF9 fueron los más diferentes a los genes colineales encontrados en el genoma de SB-1, pero son altamente similares (99% -100% de identidad) con aquellos dentro del genoma HPRS24. En general, el genoma de la cepa 301B/1 es más similar al genoma del virus SB-1 (99.1%) y en menor grado con el genoma del virus HPRS24 (97.7%). Sin embargo, se han identificado seis marcos de lectura continuos en 301B/1 (UL47, UL48, UL52, pp38, ICP4 y US10) que contienen sustituciones no sinónimas en relación con las secuencias homólogas encontradas en el genoma SB-1. Notablemente, a diferencia de las secuencias repetidas terminales largas del retrovirus aviar encontradas dentro del genoma de SB-1, ninguna se identificó dentro del genoma 301B/1.


Assuntos
Antígenos Virais/genética , Herpesvirus Galináceo 3/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Transativadores/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/metabolismo , Embrião de Galinha , Galinhas , Doença de Marek/virologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Doenças das Aves Domésticas/virologia , Alinhamento de Sequência , Transativadores/química , Transativadores/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
4.
Avian Dis ; 63(4): 670-680, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31865682

RESUMO

Marek's disease (MD) is a complex pathology of chickens caused by MD virus (MDV) 1 and is observed as paralysis, immune suppression, neurologic signs, and the rapid formation of T-cell lymphomas. The incidence of MD in commercial broilers is largely controlled via vaccination, either in ovo or at hatch with live attenuated vaccines, i.e., turkey herpesvirus (HVT) or a bivalent combination of HVT with the MDV 2 strain (SB1). To further extend the protection conferred by bivalent HVT/SB-1, recombinant HVTs encoding transgenes of other avian viruses have similarly been used for in ovo administration. Despite decades of use, the specific mechanisms associated with vaccine-induced protection remain obscure. Additionally, the mechanistic basis for vaccine synergism conferred by bivalent HVT/SB-1, compared with HVT or SB-1 administered alone, is largely unknown. In the present study, we report on temporal changes in innate and acquired immune-patterning gene expression by using ex vivo splenocyte infection and in ovo vaccination models. We report that in the ex vivo splenocyte infection model, by 72 hr postinfection, vaccines induced IFN and IFN-stimulated gene expression, with lesser proinflammatory cytokine induction. For several genes (TLR3, IFN-γ, OASL, Mx1, NOS2A, and IL-1ß), the effects on gene expression were additive for HVT, SB1, and HVT/SB1 infection. We observed similar patterns of induction in in ovo-vaccinated commercial broiler embryos and chicks with HVT/SB-1 or recombinant HVT-based bivalent combination (HVT-LT/SB-1). Furthermore, HVT/SB-1 or HVT-LT/SB-1 in ovo vaccination appeared to hasten immune maturation, with expression patterns suggesting accelerated migration of T and natural killer cells into the spleen. Finally, HVT/SB-1 vaccination resulted in a coordinated induction of IL-12p40 and downregulation of suppressors of cytokine signaling 1 and 3, indicative of classical macrophage 1 and T-helper 1 patterning.


Análisis transcripcionales de patrones inmunes innatos y adquiridos inducidos por cepas vacunales del virus de la enfermedad de Marek: virus herpes del pavo (HVT), virus de Marek 2 (cepa SB1) y vacunas bivalentes (HVT/SB1 y HVT-LT/SB1). La enfermedad de Marek (MD) es una patología compleja de los pollos causada por el virus de Marek (MDV) 1 y se observa como parálisis, depresión inmune, signos neurológicos y la formación rápida de linfomas de células T. La incidencia de la enfermedad de Marek en pollos de engorde comerciales se controla en gran medida a través de la vacunación, ya sea in ovo o al momento de la eclosión con vacunas vivas atenuadas, por ejemplo, herpesvirus de pavo (HVT) o una combinación bivalente de HVT con la cepa SB1. Para ampliar aún más la protección conferida por la vacuna bivalente HVT/SB-1, los HVT recombinantes que codifican transgenes de otros virus aviares se han utilizado de forma similar para la administración in ovo. A pesar de décadas de uso, los mecanismos específicos asociados con la protección inducida por la vacuna siguen sin ser esclarecidos completamente. Además, el mecanismo para la sinergia de la vacuna conferida por la vacuna bivalente HVT/SB-1, en comparación con la administración de la cepa HVT o de la cepa SB-1 por sí solas, es en gran medida desconocida. En el presente estudio, se informa sobre los cambios temporales en la expresión genética de patrones inmunes innatos y adquiridos mediante la infección de esplenocitos ex vivo y en modelos de vacunación in ovo. Se reporta que en el modelo de infección de esplenocitos ex vivo, por 72 horas después de la infección, las vacunas indujeron IFN y la expresión de genes estimulada por IFN, con menor inducción de citocinas proinflamatorias. Para varios genes (TLR3, IFNc, OASL, Mx1, NOS2A e IL-1ß), los efectos sobre la expresión de genes fueron aditivos para la infección por HVT, SB1 y HVT/SB1. Se Observaron patrones de inducción similares en embriones de pollo y pollos de engorde comerciales vacunados in ovo con HVT/SB-1 o con la combinación bivalente recombinante basada en HVT (HVT-LT/SB-1). Además, la vacunación in ovo con HVT/SB-1 o HVT-LT/SB-1 parecen acelerar la maduración inmune, con patrones de expresión que sugieren una migración acelerada de células T y células asesinas naturales en el bazo. Finalmente, la vacuna HVT/SB-1 dio como resultado una inducción coordinada de IL-12p40 y una regulación a la baja de supresores de las señales de citocinas 1 y 3, indicativas de los patrones clásicos de macrófagos 1 y células cooperadoras tipo 1.


Assuntos
Imunidade Adaptativa/genética , Herpesvirus Meleagrídeo 1/imunologia , Herpesvirus Galináceo 3/imunologia , Imunidade Inata/genética , Vacinas contra Doença de Marek/imunologia , Transcrição Gênica , Vacinas Virais/imunologia , Animais , Embrião de Galinha , Fibroblastos , Doença de Marek/imunologia
5.
J Virol ; 93(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31554689

RESUMO

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.


Assuntos
Vírus da Leucose Aviária/isolamento & purificação , Leucose Aviária/complicações , Leucose Aviária/virologia , Coinfecção/virologia , Linfoma/complicações , Linfoma/virologia , Doença de Marek/complicações , Doenças das Aves Domésticas/virologia , Sequência de Aminoácidos , Animais , Vírus da Leucose Aviária/genética , Galinhas/virologia , Suscetibilidade a Doenças , Regulação Viral da Expressão Gênica , Genótipo , Herpesvirus Galináceo 3 , Incidência , Doença de Marek/virologia , Vacinas contra Doença de Marek , Análise de Sequência de DNA , Transdução de Sinais , Transcriptoma , Vacinação , Vacinas Virais
6.
Vet Microbiol ; 236: 108393, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31500729

RESUMO

Monitoring of Marek's disease virus (MDV) and infectious laryngotracheitis virus (ILTV) genome using poultry dust can be useful to monitor on-farm vaccination protocols but there are no set guidelines for collection of this sample type. This study assessed different dust collection methods for MDV and ILTV detection in a vaccinated layer flock (n = 1700) from day-old to 50 weeks of age. Birds were vaccinated against MDV at day-old, and ILTV by drinking water at week 6 and eye drop at week 12. Dust samples were collected weekly by settle plates (1-3 plates/15 m2) or by scraping surfaces in the poultry shed and tested for ILTV and MDV genomic copies (GC) by PCR. ILTV GC were detected 4 weeks post water vaccination, peaked at weeks 12-14 and became mostly undetectable after week 18. MDV was detected in dust on week 1, peaked at weeks 3-6, declined 3 logs by week 26 and remained detectable at this level until week 50. There was no difference in the detection rates of ILTV and MDV collected from settle plates in different locations of the shed (P > 0.10). There was no difference between settle plate and scraped samples in ILTV GC load but higher MDV GC were found in scraped samples. The settle plate method appears to reflect the current level of vaccine virus in the flock while the scrape method likely represents a cumulative record of shedding. Assessment of viral GC in dust samples is a good candidate for a practical method of estimating successful vaccine administration.


Assuntos
Galinhas , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/isolamento & purificação , Herpesvirus Galináceo 2/isolamento & purificação , Herpesvirus Galináceo 3/isolamento & purificação , Doença de Marek/prevenção & controle , Animais , DNA Viral/genética , Poeira , Feminino , Genoma Viral , Infecções por Herpesviridae/prevenção & controle , Abrigo para Animais , Vacinação/veterinária , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
7.
Oncogene ; 38(10): 1778-1786, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30846849

RESUMO

The telomerase RNA subunit (TR) is overexpressed in many tumors; however, the contribution of TR in cancer formation remains elusive. The most frequent clinically diagnosed cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a TR (vTR) that plays an important role in virus-induced tumorigenesis and shares 88% sequence identity with its cellular homologue. To determine if the cellular TR possesses pro-oncogenic activity, we replaced vTR with the cellular homologue in the virus genome. Insertion of cellular TR resulted in a strong overexpression in virus infected cells, while virus replication was not affected. Strikingly, cellular TR promoted tumor formation as efficient as vTR, while tumorigenesis was severely impaired in the absence of vTR. Our data provide the first evidence that overexpression of cellular TR can contribute to tumor formation in vivo using this natural virus-host model for herpesvirus-induced oncogenesis.


Assuntos
Transformação Celular Neoplásica/genética , Herpesvirus Galináceo 3/fisiologia , Doença de Marek/virologia , RNA/genética , Telomerase/genética , Animais , Células Cultivadas , Embrião de Galinha , Feminino , Herpesvirus Galináceo 3/genética , Masculino , Doença de Marek/enzimologia , RNA/metabolismo , Telomerase/metabolismo , Replicação Viral
8.
Virology ; 522: 1-12, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29979959

RESUMO

Marek's disease (MD) is a pathology of chickens associated with paralysis, immune suppression, and the rapid formation of T-cell lymphomas. MD is caused by the herpesvirus, Marek's disease virus (MDV). We examined endoplasmic reticulum (ER) stress and the activation of unfolded protein response (UPR) pathways during MDV infection of cells in culture and lymphocytes in vivo. MDV strains activate the UPR as measured by increased mRNA expression of GRP78/BiP with concomitant XBP1 splicing and induction of its target gene, EDEM1. Cell culture replication of virulent, but not vaccine MDVs, activated the UPR at late in infection. Pathotype-associated UPR activation was induced to a greater level by a vv + MDV. Discrete UPR activation was observed during MDV in vivo infection, with the level of UPR modulation being affected by the MDV oncoprotein Meq. Finally, ATF6 was found to be activated in vv + MDV-induced primary lymphomas, suggesting a possible role in tumor progression.


Assuntos
Herpesvirus Galináceo 3/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Doença de Marek/patologia , Doenças das Aves Domésticas/patologia , Resposta a Proteínas não Dobradas , Animais , Células Cultivadas , Galinhas , Perfilação da Expressão Gênica , Linfócitos/patologia , Linfócitos/virologia
9.
Avian Dis ; 60(4): 715-724, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27902902

RESUMO

Shortly after the isolation of Marek's disease (MD) herpesvirus (MDV) in the late 1960s vaccines were developed in England, the United States, and The Netherlands. Biggs and associates at the Houghton Poultry Research Station (HPRS) in England attenuated HPRS-16, the first cell-culture-isolated MDV strain, by passaging HPRS-16 in chick kidney cells. Although HPRS-16/Att was the first commercially available vaccine, it never became widely used and was soon replaced by the FC126 strain of herpesvirus of turkeys (HVT) vaccine developed by Witter and associates at the Regional Poultry Research Laboratory (now Avian Disease and Oncology Laboratory [ADOL]) in East Lansing, MI. Ironically, Kawamura et al. isolated a herpesvirus from kidney cell cultures from turkeys in 1969 but never realized its potential as a vaccine against MD. Rispens of the Central Veterinary Institute (CVI) developed the third vaccine. His associate, Maas, had found commercial flocks of chickens with MDV antibodies but without MD. Subsequently, Rispens isolated a very low pathogenic strain from hen number 988 from his MD antibody-positive flock, which was free of avian leukosis virus and clinical MD. This isolate became the CVI-988 vaccine used mostly in The Netherlands. During the late 1970s, HVT was no longer fully protective against some new emerging field strains. The addition of SB-1, isolated by Schat and Calnek, to HVT improved protection against the emerging very virulent strains. In the 1990s CVI-988 became the worldwide vaccine gold standard. This review will present data from published papers and personal communications providing additional information about the exciting 15-yr period after the isolation of MDV to the development of the different vaccines.


Assuntos
Vacinas contra Doença de Marek/história , Vacinas contra Doença de Marek/imunologia , Doença de Marek/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Animais , Galinhas , Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 2/imunologia , Herpesvirus Galináceo 3/genética , Herpesvirus Galináceo 3/imunologia , História do Século XX , História do Século XXI , Doença de Marek/história , Doença de Marek/imunologia , Doença de Marek/virologia , Vacinas contra Doença de Marek/administração & dosagem , Vacinas contra Doença de Marek/genética , Doenças das Aves Domésticas/história , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia
10.
Avian Pathol ; 44(1): 23-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25407937

RESUMO

Breeders of the 2009 generation of Avian Disease and Oncology Laboratory transgenic chicken line ALVA6, known to be resistant to infection with subgroups A and E avian leukosis virus (ALV), were vaccinated at hatch with a trivalent Marek's disease (MD) vaccine containing serotypes 1, 2, and 3 Marek's disease virus (MDV) and were maintained under pathogen-free conditions from the day of hatch until 75 weeks of age. Spontaneous ALV-like bursal lymphomas, also termed lymphoid leukosis (LL)-like lymphomas, were detected in 7% of the ALVA6 breeders. There was no evidence of infection with exogenous and endogenous ALV as determined by virus isolation tests of plasma and tumour tissue homogenates. For the next three generations, serotype 2 MDV was eliminated from the trivalent MD vaccine used. Results show, for the first time, that removal of serotype 2 MDV from MD vaccines eliminated spontaneous LL-like lymphomas within 50 to 72 weeks of age for at least three consecutive generations. Two experiments were also conducted to determine the influence of in ovo vaccination with serotype 2 MD vaccines on enhancement of spontaneous LL-like lymphomas in ALVA6 chickens. Chickens from the 2012 generation were each inoculated in ovo or at hatch with 5000 plaque-forming units of serotype 2 MDV. Results indicate that by 50 weeks of age the incidence of spontaneous LL-like lymphomas in chickens inoculated in ovo with serotype 2 MDV was comparable with that in chickens inoculated with virus at hatch, suggesting that the augmentation effect of serotype 2 MDV is independent of age of vaccination.


Assuntos
Animais Geneticamente Modificados/genética , Bolsa de Fabricius/patologia , Galinhas , Herpesvirus Galináceo 3/patogenicidade , Linfoma/veterinária , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Animais , Vírus da Leucose Aviária/imunologia , Bolsa de Fabricius/virologia , Herpesvirus Galináceo 3/genética , Herpesvirus Galináceo 3/imunologia , Linfoma/patologia , Linfoma/virologia , Reação em Cadeia da Polimerase , Especificidade da Espécie , Vacinas Virais/genética , Vacinas Virais/uso terapêutico
11.
Avian Dis ; 58(2): 232-43, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25055627

RESUMO

The serotype 1 Marek's disease virus (MDV) is the causative agent for Marek's disease (MD), a lymphoproliferative disease of chickens of great concern to the poultry industry. CVI988 (Rispens vaccine), an attenuated serotype 1 MDV, is currently the most efficacious commercially available vaccine for preventing MD. However, it is difficult to detect and differentiate CVI988 when other serotype 1 MDVs are present. To facilitate the detection of CVI988, we developed two sets of primers for a mismatch amplification mutation assay (MAMA) PCR that targeted the single nulceotide polymorphism associated with the H19 epitope of the phosphorylated protein 38 gene. The PCR was very specific. One primer set (oncogenic primers) amplified DNA from 15 different serotype 1 MDVs except CVI988. The other primer set (CVI988 primers) amplified DNA from CVI988 but not from any of the other 15 serotype 1 MDVs. A real-time PCR assay was developed using MAMA primers, and specificity and sensitivity was evaluated in vitro and in vivo. Mixtures of plasmids (CVI988 plasmid and oncogenic plasmid) at various concentrations were used to evaluate the sensitivity/specificity of MAMA primers in vitro. Both primer setswere able to amplify as little as one copy of their respective plasmid. Oncogenic primers were highly specific and only amplified CVI988 plasmid when the concentration of oncogenic plasmid was very low (1 X 10(1)) and CVI988 plasmid was very high (1 X 10(6)). Specificity of CVI988 primers was not as high because they could amplify oncogenic plasmids when the concentration of CVI988 plasmid was 1 x 10(3) and the concentration of oncogenic 1 x 10(2). Validation of MAMA primers in in vivo samples demonstrated that oncogenic primers can be used for both early diagnosis of MD in feather pulp (FP) samples collected at 3 wk of age and confirmation of MD diagnosis in tumors. CVI988 primers could be used to monitor CVI988 vaccination in samples with a low load of oncogenic MDV DNA (latently infected samples or negative) but not in samples with a high load of oncogenic MDV DNA (tumors). Our results suggest that monitoring CVI988 vaccination in FP samples collected at 1 wk of age ensures the specificity of the CVI988 primers.


Assuntos
Galinhas , Herpesvirus Galináceo 2/imunologia , Herpesvirus Galináceo 3/imunologia , Vacinas contra Doença de Marek/imunologia , Doença de Marek/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Embrião de Galinha , Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 3/genética , Doença de Marek/imunologia , Vacinas contra Doença de Marek/genética , Polimorfismo de Nucleotídeo Único , Doenças das Aves Domésticas/imunologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia
12.
Poult Sci ; 93(3): 550-5, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24604847

RESUMO

The Toll-like receptor (TLR) signaling pathway is one of the innate immune defense mechanisms against pathogens in vertebrates and invertebrates. However, the role of TLR in non-MHC genetic resistance or susceptibility to Marek's disease (MD) in the chicken is yet to be elucidated. Chicken embryo fibroblast (CEF) cells from MD susceptible and resistant lines were infected either with Marek's disease virus (MDV) or treated with polyionosinic-polycytidylic acid, a synthetic analog of dsRNA, and the expression of TLR and pro-inflammatory cytokines was studied at 8 and 36 h posttreatment by quantitative reverse transcriptase PCR. Findings of the present study reveal that MDV infection and polyionosinic-polycytidylic acid treatment significantly elevated the mRNA expression of TLR3, IL6, and IL8 in both susceptible and resistant lines. Furthermore, basal expression levels in uninfected CEF for TLR3, TLR7, and IL8 genes were significantly higher in resistant chickens compared with those of susceptible chickens. Our results suggest that TLR3 together with pro-inflammatory cytokines may play a significant role in genetic resistance to MD.


Assuntos
Proteínas Aviárias/genética , Galinhas , Citocinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Doença de Marek/genética , Doenças das Aves Domésticas/genética , Receptores Toll-Like/genética , Animais , Proteínas Aviárias/metabolismo , Embrião de Galinha , Citocinas/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/veterinária , Fibroblastos , Herpesvirus Galináceo 3/fisiologia , Doença de Marek/imunologia , Doença de Marek/virologia , Poli I-C/administração & dosagem , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Transdução de Sinais , Receptores Toll-Like/metabolismo
13.
Avian Dis ; 57(2 Suppl): 340-50, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23901745

RESUMO

Marek's disease virus (MDV) is a highly contagious virus that induces T-lymphoma in chicken. This viral infection still circulates in poultry flocks despite the use of vaccines. With the emergence of new virulent strains in the field over time, MDV remains a serious threat to the poultry industry. More than 40 yr after MDV identification as a herpesvirus, the visualization and purification of fully enveloped infectious particles remain a challenge for biologists. The various strategies used to detect such hidden particles by electron microscopy are reviewed herein. It is now generally accepted that the production of cell-free virions only occurs in the feather follicle epithelium and is associated with viral, cellular, or both molecular determinants expressed in this tissue. This tissue is considered the only source of efficient virus shedding into the environment and therefore the origin of successful transmission in birds. In other avian tissues or permissive cell cultures, MDV replication only leads to a very low number of intracellular enveloped virions. In the absence of detectable extracellular enveloped virions in cell culture, the nature of the transmitted infectious material and its mechanisms of spread from cell to cell remain to be deciphered. An attempt is made to bring together the current knowledge on MDV morphogenesis and spread, and new approaches that could help understand MDV morphogenesis are discussed.


Assuntos
Herpesvirus Meleagrídeo 1/ultraestrutura , Herpesvirus Galináceo 2/ultraestrutura , Herpesvirus Galináceo 3/ultraestrutura , Doença de Marek/transmissão , Doenças das Aves Domésticas/transmissão , Animais , Herpesvirus Meleagrídeo 1/crescimento & desenvolvimento , Herpesvirus Galináceo 2/crescimento & desenvolvimento , Herpesvirus Galináceo 3/crescimento & desenvolvimento , Doença de Marek/virologia , Morfogênese , Aves Domésticas , Doenças das Aves Domésticas/virologia
14.
Avian Dis ; 57(2 Suppl): 539-43, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23901773

RESUMO

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.


Assuntos
Galinhas , Herpesvirus Meleagrídeo 1/genética , Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 3/genética , Doença de Marek/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Herpesvirus Meleagrídeo 1/metabolismo , Herpesvirus Galináceo 2/metabolismo , Herpesvirus Galináceo 3/metabolismo , Doença de Marek/genética , Técnicas de Diagnóstico Molecular/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Sensibilidade e Especificidade , Análise de Sequência de DNA/veterinária
15.
Avian Dis ; 57(2 Suppl): 544-54, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23901774

RESUMO

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5-20 ng/microl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24-600 VCN/mg for MDV-1, 48-1200 VCN/mg for MDV-2, and 182-4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1.


Assuntos
Galinhas , Herpesvirus Meleagrídeo 1/genética , Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 3/genética , Doença de Marek/virologia , Proteínas Oncogênicas Virais/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Criação de Animais Domésticos , Animais , Poeira/análise , Herpesvirus Meleagrídeo 1/metabolismo , Herpesvirus Galináceo 2/metabolismo , Herpesvirus Galináceo 3/metabolismo , Doença de Marek/genética , Proteínas Oncogênicas Virais/metabolismo , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reprodutibilidade dos Testes , Estações do Ano , Sensibilidade e Especificidade , Baço/virologia , Vitória
16.
Res Vet Sci ; 94(3): 496-503, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23164636

RESUMO

Cell-mediated cytotoxic responses are critical for control of Marek's disease virus (MDV) infection and tumour development. However, the mechanisms of virus clearance mediated by cytotoxic responses in the bursa of Fabricius of chickens during MDV infection are not fully understood. In this study, the host cytotoxic responses during MDV infection in the bursa were investigated by examining the expression of genes in the cell lysis pathways. Partial up-regulation existed in the expression of the important cytolytic molecule granzyme A (GzmA), Fas, NK lysin and DNA repair enzyme Ape1, whereas little or no expression appeared in other cytolytic molecules, including perforin (PFN) and Fas ligand (FasL), and molecules involved in DNA repair and apoptosis in the bursa during MDV infection. These results suggest that less sustained cytotoxic activities are generated in the bursa of MDV-infected chickens. The findings of this study provide a more detailed insight into the host cytotoxic responses to MDV infection.


Assuntos
Bolsa de Fabricius/metabolismo , Herpesvirus Galináceo 3/imunologia , Doença de Marek/metabolismo , Animais , Apoptose/imunologia , Western Blotting/veterinária , Bolsa de Fabricius/imunologia , Bolsa de Fabricius/fisiopatologia , Galinhas/imunologia , Galinhas/metabolismo , Reparo do DNA/imunologia , Proteína Ligante Fas/metabolismo , Regulação da Expressão Gênica , Herpesvirus Galináceo 3/fisiologia , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Doença de Marek/imunologia , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Replicação Viral , Receptor fas/metabolismo
17.
Avian Pathol ; 41(1): 69-75, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22845323

RESUMO

Several highly efficacious vaccines are currently available for control of Marek's disease, a lymphoproliferative disease in chickens. However, these vaccines are unable to prevent infection with Marek's disease virus (MDV) in vaccinated birds. This leads to shedding of virulent MDV from feather follicle epithelium and skin epithelial cells of vaccinated and infected chickens. The objective of the present study was to study the interactions between a vaccine strain (CVI988/Rispens) and a very virulent strain of MDV (RB1B) in feathers. We examined genome load and replication of CVI988 and MDV-RB1B strains at various time points post infection. Moreover, we evaluated cytokine expression in feathers as indicators of immunity generated in response to vaccines against MDV. Analysis of feathers collected between 4 and 21 days post infection (d.p.i.) revealed a steady level of CVI988 genome load in the presence or absence of RB1B. Infection with MDV resulted in a significant increase in RB1B genome load peaking at 14 d.p.i. Importantly, vaccination with CVI988 resulted in a significant reduction in accumulation of MDV-RB1B in feathers. RB1B genome accumulation in feather tips was associated with increased expression of interferon-α at 14 d.p.i. and interferon-Sγ at earlier time points, 4 and 7 d.p.i. compared with 10 and 14 d.p.i. Interleukin-10 and interleukin-6 were up-regulated at 14 d.p.i. in the infected groups. This study expands our understanding of the dynamics of replication of vaccine and virulent MDV strains in the feathers and illuminates mechanisms associated with immunity to Marek's disease.


Assuntos
Galinhas , Plumas/virologia , Herpesvirus Galináceo 3/patogenicidade , Vacinas contra Doença de Marek/farmacologia , Doença de Marek/imunologia , Doença de Marek/prevenção & controle , Replicação Viral/efeitos dos fármacos , Animais , Primers do DNA/genética , Interferon-alfa/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Carga Viral/efeitos dos fármacos , Carga Viral/veterinária , Virulência , Replicação Viral/fisiologia
18.
Viral Immunol ; 25(5): 394-401, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22857262

RESUMO

Marek's disease (MD) is caused by Marek's disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.


Assuntos
Herpesvirus Meleagrídeo 1/imunologia , Herpesvirus Galináceo 3/imunologia , Vacinas contra Doença de Marek/administração & dosagem , Vacinas contra Doença de Marek/imunologia , Doença de Marek/imunologia , Doença de Marek/prevenção & controle , Poli I-C/administração & dosagem , Receptor 3 Toll-Like/imunologia , Animais , Galinhas , Herpesvirus Meleagrídeo 1/genética , Herpesvirus Galináceo 3/genética , Herpesvirus Galináceo 3/patogenicidade , Interferon gama/análise , Interleucina-10/análise , Receptor 3 Toll-Like/metabolismo , Vacinação/veterinária , Carga Viral
19.
J Virol ; 86(15): 7896-906, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22593168

RESUMO

Marek's disease (MD) is a devastating oncogenic viral disease of chickens caused by Gallid herpesvirus 2, or MD virus (MDV). MDV glycoprotein C (gC) is encoded by the alphaherpesvirus UL44 homolog and is essential for the horizontal transmission of MDV (K. W. Jarosinski and N. Osterrieder, J. Virol. 84:7911-7916, 2010). Alphaherpesvirus gC proteins are type 1 membrane proteins and are generally anchored in cellular membranes and the virion envelope by a short transmembrane domain. However, the majority of MDV gC is secreted in vitro, although secondary-structure analyses predict a carboxy-terminal transmembrane domain. In this report, two alternative mRNA splice variants were identified by reverse transcription (RT)-PCR analyses, and the encoded proteins were predicted to specify premature stop codons that would lead to gC proteins that lack the transmembrane domain. Based on the size of the intron removed for each UL44 (gC) transcript, they were termed gC104 and gC145. Recombinant MDV viruses were generated in which only full-length viral gC (vgCfull), gC104 (vgC104), or gC145 (vgC145) was expressed. Predictably, gCfull was expressed predominantly as a membrane-associated protein, while both gC104 and gC145 were secreted, suggesting that the dominant gC variants expressed in vitro are the spliced variants. In experimentally infected chickens, the expression of each of the gC variants individually did not alter replication or disease induction. However, horizontal transmission was reduced compared to that of wild-type or revertant viruses when the expression of only a single gC was allowed, indicating that all three forms of gC are required for the efficient transmission of MDV in chickens.


Assuntos
Processamento Alternativo , Regulação Viral da Expressão Gênica , Herpesvirus Galináceo 3/metabolismo , Proteínas Virais/biossíntese , Animais , Antígenos Virais/biossíntese , Antígenos Virais/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/virologia , Embrião de Galinha , Galinhas/metabolismo , Galinhas/virologia , Herpesvirus Galináceo 3/genética , Herpesvirus Galináceo 3/patogenicidade , Doença de Marek/genética , Doença de Marek/metabolismo , Doença de Marek/transmissão , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas Virais/genética
20.
J Virol Methods ; 183(2): 196-200, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22575575

RESUMO

A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.


Assuntos
Galinhas/virologia , Genes Virais , Herpesvirus Galináceo 3/genética , Doença de Marek/virologia , Técnicas de Amplificação de Ácido Nucleico , Animais , DNA Viral/genética , DNA Viral/isolamento & purificação , Herpesvirus Galináceo 3/isolamento & purificação , Limite de Detecção , Doença de Marek/diagnóstico , Técnicas de Diagnóstico Molecular
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