RESUMEN
The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.
Asunto(s)
Pollos , Herpesvirus Gallináceo 2 , Enfermedad de Marek , Enfermedades de las Aves de Corral , Animales , Enfermedad de Marek/virología , Enfermedad de Marek/epidemiología , Pollos/virología , Egipto/epidemiología , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/epidemiología , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/aislamiento & purificación , Secuencias Repetidas Terminales , Virus de la Reticuloendoteliosis/genética , Virus de la Reticuloendoteliosis/aislamiento & purificación , Integración Viral , Genoma ViralRESUMEN
Marek's disease is a significant illness in chickens and a potential threat to the poultry industry worldwide. Marek's disease virus (MDV) causes immunosuppression and lymphoproliferative disease in chickens, but the turkey is an unusual host for the virus, and tumors caused by MDV in turkeys are unique. This study sampled 15 asymptomatic commercial turkey flocks (five spleens from each flock) at slaughter. Gallid alphaherpesvirus 2 (GaHV-2) was identified by PCR of spleen samples of two flocks. A phylogenetic analysis of the Meq gene was also performed. Sequencing and phylogenetic analysis revealed that the turkey GaHV-2 had genetic similarity with GaHV-2 strains recently detected in the Iranian commercial layer and breeder turkey flocks. This is the first time MDV has been detected in turkey flocks of Iran, and therefore, further assays including experimental inoculation to demonstrate pathotype characteristics in vivo are needed.
Nota de investigación- Virus de la enfermedad de Marek en parvadas comerciales de pavos en Irán. La enfermedad de Marek es una enfermedad importante de los pollos y una amenaza potencial para la industria avícola en todo el mundo. El virus de la enfermedad de Marek (MDV) causa inmunosupresión y una enfermedad linfoproliferativa en pollos, pero el pavo es un huésped inusual para este virus y los tumores causados por el virus de la enfermedad de Marek en pavos son únicos. En este estudio se recolectaron muestras de 15 lotes de pavos comerciales asintomáticos (cinco bazos de cada lote) en el momento del sacrificio. El Gallid alphaherpesvirus 2 (GaHV-2) fue identificado por PCR de muestras de bazo de dos parvadas. También se realizó un análisis filogenético del gene Meq. La secuenciación y el análisis filogenético revelaron que el GaHV-2 de pavo tenía una similitud genética con las cepas de GaHV-2 detectadas recientemente en parvadas de postura comerciales y de pavos reproductores iraníes. Esta es la primera vez que se detecta el virus de Marek en parvadas de pavos de Irán y, por lo tanto, se necesitan más ensayos, incluida la inoculación experimental, para demostrar las características del patotipo in vivo.
Asunto(s)
Herpesvirus Gallináceo 2 , Enfermedad de Marek , Filogenia , Enfermedades de las Aves de Corral , Pavos , Animales , Enfermedad de Marek/virología , Enfermedad de Marek/epidemiología , Irán/epidemiología , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/aislamiento & purificación , Herpesvirus Gallináceo 2/clasificación , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/epidemiologíaRESUMEN
Marek's disease (MD) is one of the most significant neoplastic diseases of poultry caused by Marek's disease virus (MDV), an oncogenic avian herpesvirus which is responsible for great economic losses to the poultry industry worldwide. MD is being manifested as an acute disease with lymphomas in multiple visceral organs. In the present study, an outbreak of MD was investigated in one of the poultry farms from Andhra Pradesh, India. The gross lesions in the affected birds included lymphomas in different visceral organs like liver, spleen, proventriculus, heart and ovaries. Histopathology revealed presence of uniform lymphoblastoid cell infiltration typical of MD. The isolation of the virus was carried out in duck embryo fibroblast cells. After three blind passages, the cell cultures revealed plaque formation typical of MDV. Further confirmation of the virus was carried out by PCR targeting 132 bp repeats of serotype1 MDV and the oncogenes Meq and vIL8 were amplified and sequenced. The nucleotide and phylogenetic analysis of the virus confirmed the virus as virulent serotype 1 MDV. The present outbreak suggests the need for change in the vaccination regimen of MD vaccination with appropriate serotype 1 MD vaccines in Indian poultry flocks as the HVT and bivalent vaccines are unable to protect the flocks against virulent MDV.
Asunto(s)
Brotes de Enfermedades/veterinaria , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/epidemiología , Enfermedades de las Aves de Corral/epidemiología , Aves de Corral , Animales , Brotes de Enfermedades/prevención & control , India/epidemiología , Enfermedad de Marek/virología , Reacción en Cadena de la Polimerasa/veterinaria , Enfermedades de las Aves de Corral/virología , Vacunación/veterinariaRESUMEN
Marek's disease virus (MDV) encodes a basic-leucine zipper (BZIP) protein, Meq, which is considered the major MDV oncoprotein. It has been reported that the oncogenicity of Meq is associated with its interaction with C-terminal binding protein 1 (CtBP), which is also an interaction partner of Epstein-Barr virus encoded EBNA3A and EBNA3C oncoproteins. Since both EBNA3C and CtBP interact with histone deacetylase 1 (HDAC1) and HDAC2, we examined whether Meq shares this interaction with chicken HDAC1 (chHDAC1) and chHDAC2. Using confocal microscopy analysis, we show that Meq co-localizes with chHDAC1 and chHDAC2 in the nuclei of MDV lymphoblastoid tumor cells. In addition, immunoprecipitation assays demonstrate that Meq interacts with chHDAC1 and chHDAC2 in transfected cells and MDV lymphoblastoid tumor cells. Using deletion mutants, interaction domains were mapped to the N-terminal dimerization domain of chHDAC1 and chHDAC2, and the BZIP domain of Meq. Our results further demonstrate that this interaction mediates the degradation of chHDAC1 and chHDAC2 via the proteasome dependent pathway. In addition, our results show that Meq also induces the reduction of global ubiquitinated proteins through a proteasome dependent pathway. In conclusion, our results provide evidence that Meq interacts with chHDAC1 and chHDAC2, and induces their proteasome dependent degradation.
Asunto(s)
Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Linfoma/patología , Proteínas Oncogénicas Virales/metabolismo , Enfermedades de las Aves de Corral/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Pollos , Herpesvirus Gallináceo 2/aislamiento & purificación , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Renales/virología , Linfoma/metabolismo , Linfoma/virología , Enfermedad de Marek/complicaciones , Enfermedad de Marek/metabolismo , Enfermedad de Marek/patología , Enfermedad de Marek/virología , Proteínas Oncogénicas Virales/genética , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/virología , ProteolisisRESUMEN
Marek's disease (MD) is an important disease of avian species and a potential threat to the poultry industry worldwide. In this study, 16 dead commercial chickens from flocks with suspected MD were necropsied immediately after death. Pathological findings were compatible with MD, and gallid alphaherpesvirus 2 was identified in PCR of spleen samples. Virus isolation was performed in primary cell culture, and partial sequencing of the meq gene of the isolate revealed >99% nucleotide sequence identity to virulent and very virulent plus strains from a number of European countries, placing it in the same subclade of clade III as two virulent Italian strains and a very virulent plus Polish strain as well as virulent strains of geese and ducks. The data reported here indicate that a virulent strain of Marek's disease virus is circulating in Turkey and has not been stopped by the current national vaccination programme.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/virología , Aves de Corral/virología , Animales , Secuencia de Bases/genética , Células Cultivadas , Pollos/virología , Patos/virología , Gansos/virología , Italia , Filogenia , Polonia , Enfermedades de las Aves de Corral/virología , Turquía , Virulencia/genéticaRESUMEN
General diagnosis of poultry viruses primarily relies on detection of viruses in samples, but many farms are located in remote areas requiring logistic transportation. Filter paper cards are a useful technology that offer an alternative for collecting and preserving samples without hazardous exposure. The goal of this study was to compare three filter papers: the Flinders Technology Associates filter (FTA®) card, dried blood spot (DBS) card and qualitative filter paper (FP) grade 2 to collect poultry samples. In particular, we have used Newcastle disease virus (NDV) to evaluate safety and a Marek's disease virus (MDV) attenuated vaccine (CVI988) to evaluate stability of viral DNA. This experiment was divided into two parts. The first part was to determine the DNA stability and detection limit of CVI988 in samples collected in different paper supports after four storage times (3, 7, 14 and 30 days post spot). The second part was to determine the safety of papers by evaluating the viral inactivation efficacy using NDV as a representative virus. Results showed that all papers could preserve CVI988 DNA at all times, with a detection limit of 0.5â PFU/5â µl for FTA® and DBS cards, and 5â PFU/5â µl for FP. Our results showed that the NDV remained viable and infectious on the DBS card and FP, while no viable virus was detected on the FTA® card, suggesting that the FTA® card was safest to use. Therefore, the use of the DBS card and FP for infectious sample collection should be discouraged and reconsidered. RESEARCH HIGHLIGHTS The detection limits of the FTA® card, DBS card and FP for CVI988 detection were 0.5, 0.5 and 5â PFU/5â µl, respectively. All three filter papers could preserve viral DNA for at least 30 days of post spot. The DBS card and FP are not suitable for collecting NDV samples, which is one of the major economical threats for the poultry industry worldwide.
Asunto(s)
Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/virología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Enfermedades de las Aves de Corral/virología , Manejo de Especímenes/veterinaria , Animales , ADN Viral/genética , Herpesvirus Gallináceo 2/genética , Límite de Detección , Virus de la Enfermedad de Newcastle/genética , Aves de Corral , Inactivación de VirusRESUMEN
BACKGROUND: Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection on the intestinal microflora of chickens. RESULTS: A 29-week commercial layer flock positive for avian leukosis virus-J (ALV-J), Marek's disease virus (MDV) and avian reticuloendotheliosis virus (REV) was selected, and fresh fecal samples were collected and examined for the composition of the gut microflora by Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The operational taxonomic units (OTUs) of the fecal microbiota differentiated the chickens infected with only ALV-J and those coinfected with ALV-J and MDV or REV from infection-negative chickens. The enrichment and diversity of cloacal microflora in chickens infected with ALV-J alone were slightly different from those in the infection-negative chickens. However, the diversity of cloacal microflora was significantly increased in chickens coinfected with both ALV-J and MDV or REV. CONCLUSIONS: The intestinal microbiota was more strongly disturbed in chickens after coinfection with ALV-J and MDV or REV than after infection with ALV-J alone, and there may be underlying mechanisms by which the capacity for the stabilization of the intestinal flora was impaired due to viral infection and tumorigenesis.
Asunto(s)
Bacterias/clasificación , Coinfección/veterinaria , Microbioma Gastrointestinal , Enfermedades de las Aves de Corral/virología , Animales , Leucosis Aviar/virología , Virus de la Leucosis Aviar/aislamiento & purificación , Bacterias/genética , Bacterias/aislamiento & purificación , Pollos , Heces/microbiología , Femenino , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/microbiología , ARN Ribosómico 16S , Virus de la Reticuloendoteliosis/aislamiento & purificación , Infecciones por Retroviridae/veterinaria , Infecciones Tumorales por Virus/veterinariaRESUMEN
(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek's disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480)H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Herpesvirus Gallináceo 2/enzimología , Enfermedad de Marek/virología , Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismo , Proteínas Virales/metabolismo , Animales , Enzimas Desubicuitinizantes/genética , Herpesvirus Gallináceo 2/aislamiento & purificación , Humanos , Especificidad por Sustrato , Ubiquitinación , Proteínas Virales/genéticaRESUMEN
Marek's disease (MD) is a lymphoproliferative disease important to the poultry industry worldwide; it is caused by Gallid alphaherpesvirus 2 (GaHV-2). The virulence of GaHV-2 isolates has shifted over the years from mild to virulent, very virulent and very virulent +. Nowadays the disease is controlled by vaccination, but field strains of increased virulence are emerging worldwide. Economic losses due to MD are mostly associated with its acute form, characterized by visceral lymphomas. The present study aimed to molecularly classify a group of 13 GaHV-2 strains detected in vaccinated Italian commercial chicken flocks during acute MD outbreaks, and to scrutinize the ability of predicting GaHV-2 virulence, according to the meq gene sequence. The full-length meq genes were amplified, and the obtained amino acid (aa) sequences were analysed, focusing mainly on the number of stretches of four proline molecules (PPPP) within the transactivation domain. Phylogenetic analysis was carried out with the Maximum Likelihood method using the obtained aa sequences, and the sequences of Italian strains detected in backyard flocks and of selected strains retrieved from GenBank. All the analysed strains showed 100% sequence identity in the meq gene, which encodes a Meq protein of 339 aa. The Meq protein includes four PPPP motifs in the transactivation domain and an interruption of a PPPP motif due to a proline-to-serine substitution at position 218. These features are typically encountered in highly virulent isolates. Phylogenetic analysis revealed that the analysed strains belonged to a cluster that includes high-virulence GaHV-2 strains detected in Italian backyard flocks and a hypervirulent Polish strain. Our results support the hypothesis that the virulence of field isolates can be suggested by meq aa sequence analysis.
Asunto(s)
Pollos/virología , Herpesvirus Gallináceo 2/clasificación , Enfermedad de Marek/virología , Proteínas Oncogénicas Virales/genética , Enfermedades de las Aves de Corral/virología , Secuencia de Aminoácidos , Animales , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/aislamiento & purificación , Italia/epidemiología , Enfermedad de Marek/epidemiología , Filogenia , Enfermedades de las Aves de Corral/epidemiología , Análisis de Secuencia de Proteína/veterinaria , Virulencia/genéticaRESUMEN
The prevalence of Marek's disease (MD) caused by Gallid herpesvirus-2 (GaHV-2) has been increasing in chickens in China despite universal vaccination. Among the possible reasons for this trend, of Reticuloendotheliosis virus (REV) contamination in vaccines could lead to co-infection and reduce the vaccine efficacy. Here, we report the epidemiological findings of our continuous surveillance of MD, and an examination of the effects of REV and/or GaHV-2 co-infection. A total of 1230 samples were collected between 2011 and 2015 from 305 flocks covering many of the chicken-raising regions of China. Among these, 606 samples were determined to be GaHV-2-positive, 13.0% of which were found to be co-infected with REV from 18.8% of the flocks. One GaHV-2 strain (HS/1412), a REV strain (HS/1412R), and a GaHV-2 and REV-co-infected strain (HS/1412â¯GR) were isolated from different chickens of a GaHV-2 and REV co-infected flock. Pathogenicity tests showed that HS/1412 and HS/1412â¯GR caused disease in all chickens and that HS/1412R induced morbidity in 84.6% of the infected chickens. HS/1412â¯GR induced 100% mortality and 76.9% tumor formation, which were significantly higher frequencies than those observed with strain HS/1412 (38.5% and 15.4%, respectively) and HS/1412R (0% and 0%). These results indicate that co-infection with GaHV-2 and REV might explain the persistent, sporadic outbreaks of neoplastic disease in some commercial flocks, resulting in a significant economic burden to the poultry industry of China.
Asunto(s)
Pollos , Coinfección/veterinaria , Enfermedad de Marek/complicaciones , Neoplasias/veterinaria , Enfermedades de las Aves de Corral/virología , Infecciones por Retroviridae/veterinaria , Animales , China/epidemiología , Coinfección/epidemiología , Coinfección/virología , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/epidemiología , Neoplasias/epidemiología , Neoplasias/virología , Virus de la Reticuloendoteliosis/aislamiento & purificación , Infecciones por Retroviridae/complicaciones , Infecciones por Retroviridae/epidemiología , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/epidemiología , Infecciones Tumorales por Virus/veterinaria , Infecciones Tumorales por Virus/virologíaRESUMEN
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 (nâ¯=â¯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 (Pâ¯>â¯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.
Asunto(s)
Pollos , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/aislamiento & purificación , Herpesvirus Gallináceo 2/aislamiento & purificación , Herpesvirus Gallináceo 3/aislamiento & purificación , Enfermedad de Marek/prevención & control , Animales , ADN Viral/genética , Polvo , Femenino , Genoma Viral , Infecciones por Herpesviridae/prevención & control , Vivienda para Animales , Vacunación/veterinaria , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
Marek's disease virus (MDV) is the most well-cited example of vaccine-driven virulence evolution. MDV induces a lymphoproliferative disease in chickens, which is currently controlled by widespread vaccination of flocks. Unfortunately, Marek's disease (MD) vaccines, while effective in preventing tumours, do not prevent viral replication and mutation, which has been hypothesized as the major driving force for increased MDV virulence of field strains during the past 40 years in US commercial flocks. To limit future virulence increases, there is interest in characterizing MDV strain genomes collected over the years and associating genetic variations with variation in virulence. In this study, we characterized 70 MDV genomes with known virulence by complete or targeted DNA sequencing, and identified genetic variants that showed association with virulence. Our results revealed a number of MDV genes as would be expected for a complex trait. In addition, phylogenetic analysis revealed a clear separation of strains that varied by virulence. Interestingly, high virulence isolates from the same farms persisted over years despite eradication attempts, which has implications on control efforts. Given the growing ability to bioengineer the MDV genome, it should be feasible to experimentally test whether these individual variants influence virulence markers alone or combinations. Once validated, these markers may provide an alternative to live bird testing for evaluating virulence of new MDV field strains.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/patogenicidad , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/virología , Proteínas Virales/genética , Animales , Pollos , Femenino , Genoma Viral , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/aislamiento & purificación , Masculino , Filogenia , Estados Unidos , Proteínas Virales/metabolismo , VirulenciaRESUMEN
VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek's disease virus (MDV), the Mardivirus prototype; it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159-162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Herpesvirus Gallináceo 2/aislamiento & purificación , Histonas/metabolismo , Enfermedad de Marek/virología , Dominios Proteicos , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular , Pollos , ADN Viral/análisis , ADN Viral/genética , Regulación Viral de la Expresión Génica , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/crecimiento & desarrollo , Mardivirus/genética , Mardivirus/aislamiento & purificación , Análisis de Secuencia de Proteína , Proteínas Virales/genética , Proteínas Estructurales Virales , Replicación ViralRESUMEN
Marek's disease (MD) is one of the most important neoplastic diseases of poultry caused by Marek's disease virus (MDV), an oncogenic avian herpes virus, which is responsible for great economic losses to the poultry industry worldwide. Inspite of the usage of HVT and bivalent vaccines in the poultry flocks, MD continues to be aâ¯major threat to the poultry industry in India. In the present study, MD outbreaks were reported in poultry farms from different regions of Andhra Pradesh, India. The postmortem examination of dead birds showed presence of lymphomas in different visceral organs suggestive of virulent oncogenic MDVs. Histopathological examination revealed infiltration of pleomorphic lymphoblastoid cells typical of MD. The blood and tissue samples were collected and PCR was standardized targeting aâ¯132 bp tandem repeat region specific for serotype-1 MD viruses. Further, the characterization of the oncogenes i.e. Meq and viral interleukin 8 (vIL-8) was carried out by PCR and nucleotide sequencing. The sequence analysis of Meq gene of different clinical cases from India revealed >99 % homology with RB1B (very virulent) and GA (virulent) strains and that of vIL-8 gene showed >99 % identity with virulent strains LS and LMS. Phylogenetic analysis of oncogenes was carried out with other available sequences in the GenBank. Finally, we conclude that MDV strains obtained in the present outbreaks in India could be designated as virulent or very virulent pathotypes based on nucleotide, amino acid and phylogenetic analysis of the viruses.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/virología , Proteínas Oncogénicas/genética , Filogenia , Enfermedades de las Aves de Corral/virología , Proteínas Virales/genética , Animales , Pollos , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/aislamiento & purificación , Herpesvirus Gallináceo 2/patogenicidad , India , Enfermedad de Marek/patología , Enfermedades de las Aves de Corral/patología , VirulenciaRESUMEN
Marek's disease has brought enormous loss in chicken production worldwide and the increasing virulence of Marek's disease virus (MDV) became a severe problem. To better understand the genetic basis underlying, a Chinese MDV strain HNGS101 isolated from immunized chickens was sequenced. Phylogenetic analysis implied that HNGS101 showed more relatedness to Eurasian strains than GaHV-2 circulating in North America. Recombination networks analysis showed the evidence of recombination among MDV strains, and several recombination events in the UL and US region were found. Further analysis indicated that the HNGS101 strain seemed to be generated by the recombination of the earliest Eurasian strains and North American strains in the US region, which may be responsible for the MD outbreaks in China. In summary, this study demonstrates recombination events among MDV strains [corrected], which may shed light on the mechanism of virulence enhancement.
Asunto(s)
Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/virología , Filogenia , Enfermedades de las Aves de Corral/virología , Animales , Pollos , China/epidemiología , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/epidemiología , Epidemiología Molecular , Enfermedades de las Aves de Corral/epidemiología , Recombinación GenéticaRESUMEN
Marek's disease virus is a herpesvirus of chickens that costs the worldwide poultry industry more than US$1 billion annually. Two generations of Marek's disease vaccines have shown reduced efficacy over the last half century due to evolution of the virus. Understanding where the virus is present may give insight into whether continued reductions in efficacy are likely. We conducted a 3-yr surveillance study to assess the prevalence of Marek's disease virus on commercial poultry farms, determine the effect of various factors on virus prevalence, and document virus dynamics in broiler chicken houses over short (weeks) and long (years) timescales. We extracted DNA from dust samples collected from commercial chicken and egg production facilities in Pennsylvania, USA. Quantitative PCR was used to assess wild-type virus detectability and concentration. Using data from 1018 dust samples with Bayesian generalized linear mixed effects models, we determined the factors that correlated with virus prevalence across farms. Maximum likelihood and autocorrelation function estimation on 3727 additional dust samples were used to document and characterize virus concentrations within houses over time. Overall, wild-type virus was detectable at least once on 36 of 104 farms at rates that varied substantially between farms. Virus was detected in one of three broiler-breeder operations (companies), four of five broiler operations, and three of five egg layer operations. Marek's disease virus detectability differed by production type, bird age, day of the year, operation (company), farm, house, flock, and sample. Operation (company) was the most important factor, accounting for between 12% and 63.4% of the variation in virus detectability. Within individual houses, virus concentration often dropped below detectable levels and reemerged later. These data characterize Marek's disease virus dynamics, which are potentially important to the evolution of the virus.
Asunto(s)
Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/virología , Vigilancia de Guardia/veterinaria , Crianza de Animales Domésticos/economía , Animales , Pollos , Granjas , Genotipo , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/economía , Enfermedad de Marek/epidemiología , Pennsylvania , Enfermedades de las Aves de Corral/economía , Enfermedades de las Aves de Corral/epidemiologíaRESUMEN
The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay's applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.
Asunto(s)
Pollos/virología , Herpesvirus Gallináceo 2/inmunología , Herpesvirus Gallináceo 2/aislamiento & purificación , Vacunas contra la Enfermedad de Marek/inmunología , Enfermedad de Marek/diagnóstico , Enfermedades de las Aves de Corral/diagnóstico , Animales , Pollos/inmunología , Cartilla de ADN/genética , Sondas de ADN , Plumas/virología , Herpesvirus Gallináceo 2/genética , Inmunización/veterinaria , Enfermedad de Marek/prevención & control , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y EspecificidadRESUMEN
Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease (MD), a lymphoproliferative disease in chickens. Understanding of MDV gene function advanced significantly following the cloning of the MDV genome as either a series of overlapping cosmids or as a bacterial artificial chromosome (BAC), both of which could produce viable MDV. The objectives of this study were to compare multiple virulent MDV BAC clones using the Avian Disease and Oncology Laboratory's pathotyping assay, and to demonstrate the use of these clones as standardized reagents for a modified pathotyping assay by other laboratories. To date, MDV BAC clones have been produced for at least 10 MDV strains from all three serotypes including several virulent serotype 1 strains. We determined that MDV BAC clones exist for each virulent pathotype, despite the fact that these clones are not always equal in virulence to their corresponding parental strains. One clone from each pathotype was further evaluated in commercial specific-pathogen-free (SPF) chickens and found suitable for use in assays such as best-fit pathotyping, although results were variable based on the source of the SPF birds. The benefits of using BAC clones, which include easy shipping, ability to more easily manipulate, and long-term ability to use at a low passage level, are likely to result in the use of BAC clones as standard reagents for MD research. The use of the defined set of clones should allow side-by-side comparison, allowing researchers to better interpret results produced in different laboratories using different MDV field strains. Furthermore, a modified best-fit pathotyping assay has been proposed using these clones and reduced bird numbers.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Técnicas Genéticas , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/virología , Patología Molecular/métodos , Enfermedades de las Aves de Corral/virología , Animales , Pollos , Técnicas Genéticas/normas , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/patogenicidad , Enfermedad de Marek/patología , Enfermedades de las Aves de Corral/patología , VirulenciaRESUMEN
Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.
Asunto(s)
Pollos/virología , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/aislamiento & purificación , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/virología , Animales , Células Cultivadas , Embrión de Pollo , Colombia , ADN Viral , Femenino , Fibroblastos/virología , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/patogenicidad , Enfermedad de Marek/patología , Filogenia , Enfermedades de las Aves de Corral/patología , Neuropatía Ciática/veterinaria , Neuropatía Ciática/virología , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Serotipificación , Timo/virologíaRESUMEN
Gallid herpesvirus 2 (GaHV-2) causes malignant lymphomas in chickens (Marek's disease, MD). Although MD is controlled through vaccination efforts, field isolates of GaHV-2 have increased in virulence worldwide and even cause MD in vaccinated chickens. GaHV-2 strains are classified into four categories (mild, virulent, very virulent and very virulent +) based on the virulence exhibited in experimental infection in unvaccinated or MD-vaccinated susceptible chickens. Although MD cases are sporadically reported in Japan, the recent field strains of GaHV-2 in Japan have not been characterized. During isolation of recent field strains by using primary chicken kidney cell cultures, a method classically used for GaHV-2 isolation, vaccine strains were simultaneously isolated. Therefore, it is necessary to separate vaccine strains to characterize the virulence and pathogenicity of the GaHV-2 strains currently distributed in Japan. In this study, we prepared cell suspensions from the spleens of MD-symptomatic chickens, inoculated day-old-chicks and isolated GaHV-2 strains by primary chicken kidney cell cultures at 2-3 weeks post inoculation. The isolated strains were passaged several times on chicken embryo fibroblast cells, and PCR analysis revealed that the isolated strains were not contaminated with vaccine strains. Moreover, the contaminant vaccine strains were completely removed by the purification of plaques observed in chicken kidney cells. These procedures are necessary to isolate GaHV-2 field strains from vaccine strains in order to carry out future studies to characterize these strains and glean insights into GaHV-2 virulence and pathogenicity.