Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek's Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1.

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.

Comparative Molecular Characterization of Three Gallid alphaherpesvirus Type 3 Strains 301B/1, HPRS24, and SB-1.

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.

Overexpression of cellular telomerase RNA enhances virus-induced cancer formation.

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.

History of the First-Generation Marek's Disease Vaccines: The Science and Little-Known Facts.

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.

Further observations on serotype 2 Marek's disease virus-induced enhancement of spontaneous avian leukosis virus-like bursal lymphomas in ALVA6 transgenic chickens.

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.

Detection and differentiation of CVI988 (Rispens vaccine) from other serotype 1 Marek's disease viruses.

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.

Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek's disease virus serotypes 1, 2, and 3.

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.

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

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.

A toll-like receptor 3 ligand enhances protective effects of vaccination against Marek's disease virus and hinders tumor development in chickens.

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.

Marek's disease virus expresses multiple UL44 (gC) variants through mRNA splicing that are all required for efficient horizontal transmission.

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.

Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

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.

Cloning of Gallid herpesvirus 3 (Marek's disease virus serotype-2) genome as infectious bacterial artificial chromosomes for analysis of viral gene functions.

Marek's disease virus serotype 2 (Gallid herpesvirus 3) is a non-pathogenic alphaherpesvirus belonging to the Mardivirus genus, used widely in live vaccines against Marek's disease. Although the complete genome sequence of the MDV-2 strain HPRS-24 has been published, very little is known about the gene functions. As a first step for carrying out functional genomic analysis of MDV-2, the full-length genome of the MDV-2 vaccine strain SB-1 was cloned as an infectious bacterial artificial chromosome (BAC) clone pSB-1. Virus reconstituted from the pSB-1 clone showed morphological and growth characteristics in cell culture very similar to the parent virus. Generation of SB-1 constructs deleted in glycoprotein E and viruses expressing Citrine-UL35 fusion protein by the application of different BAC mutagenesis techniques demonstrated the amenability of the pSB-1 clone for reverse genetics approaches to identify molecular determinants associated with different biological features of this virus. The generation of replication-competent infectious clones of SB-1, together with those of CVI988 and herpesvirus of turkey strains described previously, completes the portfolio of generating infectious BAC clones of the MD vaccine strains belonging to all the three serotypes, paving the way for the application of reverse genetics for functional analysis of immunogenic determinants of these vaccines as well as for developing novel recombinant vectors.

Functional homologies between avian and human alphaherpesvirus VP22 proteins in cell-to-cell spreading as revealed by a new cis-complementation assay.

VP22, encoded by the UL49 gene of Marek's disease virus (MDV), is indispensable for virus cell-to-cell spreading. We show herein that MDV UL49 can be functionally replaced with avian and human viral orthologs. Replacement of MDV VP22 with that of avian gallid herpesvirus 3 or herpesvirus of turkey, whose residue identity with MDV is close to 60%, resulted in 73 and 131% changes in viral spreading, respectively. In contrast, VP22 replacement with human herpes simplex virus type 1 resulted in 14% plaque formation. Therefore, heterologous avian and human VP22 proteins share sufficient structural homology to support MDV cell-to-cell spreading, albeit with different efficiencies.

Phylogeny and recombination history of gallid herpesvirus 2 (Marek's disease virus) genomes.

Phylogenetic analyses based on concatenated amino acid sequences from orthologous loci from eight genomes of alpha herpesviruses infecting birds provided strong support for the following hypotheses: (1) gallid HV3 is a sister taxon to gallid HV2 but gallid HV1 is not closely related to the other two chicken herpesviruses; (2) meleagrid HV1 is closer to both gallid HV2 and gallid HV3 than is gallid HV1; (3) within gallid HV2, the virulent GA genome forms an outgroup to both the avirulent CVI988 genome and the highly virulent Md5 and Md11 genomes. Analysis of the pattern of synonymous nucleotide substitution between orthologous genes shared by four complete genomes of gallid HV2 showed strong evidence of past events of homologous recombination that homogenized certain loci between genomes. Eight of these loci represented cases of loci homogenized between the CVI988, on the one hand, and the Md5 and Md11 genomes, on the other hand. Two others represented loci where the GA genome was homogenized with those of Md5 and Md11. The two loci (UL49.5 and RLORF12) that were homogenized among the virulent genomes GA, Md5, and Md11 are candidates for contributing to viral virulence.

Marek's disease virus type 2 (MDV-2)-encoded microRNAs show no sequence conservation with those encoded by MDV-1.

MicroRNAs (miRNAs) are increasingly being recognized as major regulators of gene expression in many organisms, including viruses. Among viruses, members of the family Herpesviridae account for the majority of the currently known virus-encoded miRNAs. The highly oncogenic Marek's disease virus type 1 (MDV-1), an avian herpesvirus, has recently been shown to encode eight miRNAs clustered in the MEQ and LAT regions of the viral genome. The genus Mardivirus, to which MDV-1 belongs, also includes the nononcogenic but antigenically related MDV-2. As MDV-1 and MDV-2 are evolutionarily very close, we sought to determine if MDV-2 also encodes miRNAs. For this, we cloned, sequenced, and analyzed a library of small RNAs from the lymphoblastoid cell line MSB-1, previously shown to be coinfected with both MDV-1 and MDV-2. Among the 5,099 small RNA sequences determined from the library, we identified 17 novel MDV-2-specific miRNAs. Out of these, 16 were clustered in a 4.2-kb long repeat region that encodes R-LORF2 to R-LORF5. The single miRNA outside the cluster was located in the short repeat region, within the C-terminal region of the ICP4 homolog. The expression of these miRNAs in MSB-1 cells and infected chicken embryo fibroblasts was further confirmed by Northern blotting analysis. The identification of miRNA clusters within the repeat regions of MDV-2 demonstrates conservation of the relative genomic positions of miRNA clusters in MDV-1 and MDV-2, despite the lack of sequence homology among the miRNAs of the two viruses. The identification of these novel miRNAs adds to the growing list of virus-encoded miRNAs.

Oncogenic Marek's disease viruses lacking the 132 base pair repeats can still be attenuated by serial in vitro cell culture passages.

Marek's disease virus (MDV) can be attenuated by serially passing the virus in cell culture. During cell culture passage, two copies of a 132 bp repeat are expanded to over 30 copies. We deleted the two copies of the 132 bp repeat region in a pathogenic MDV and demonstrated that the virus was still pathogenic. The pattern and frequency of tumors in the parental and mutant virus were the same. Early virus replication, and the appearance of persistent neurological disease were also similar between the parental and deleted virus. Nevertheless, wild-type MDV and the deletion virus could be attenuated by serial in vitro cell culture passages. Based upon analyzing the passage 40 viruses, attenuation of the MDV lacking the 132 bp repeats appears to occur in a manner that is analogous to the process occurring wild-type MDV attenuation. Whatever process is involved in the cell culture attenuation of MDV, the mechanism does not involve the 132 bp repeat region.

Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes.

The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6DeltaLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5DeltaLORF11 plaques were evident at 12-13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5DeltaLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5DeltaLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5DeltaLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5DeltaLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100x lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5DeltaLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.

Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product.

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype.

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.