Robin W. Morgan

Marek's Disease Virus Encodes MicroRNAs That Map to meq and the Latency-Associated Transcript

ABSTRACT MicroRNAs (miRNAs) are a class of small (∼22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposis sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Mareks disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the meq oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The meq gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.

Induction of Host Gene Expression following Infection of Chicken Embryo Fibroblasts with Oncogenic Marek's Disease Virus

ABSTRACT Microarrays containing 1,126 nonredundant cDNAs selected from a chicken activated T-cell expressed sequence tag database (http://chickest.udel.edu ) were used to examine changes in host cell gene expression that accompany infection of chicken embryo fibroblasts (CEF) with Mareks disease virus (MDV). Host genes that were reproducibly induced by infection of CEF with the oncogenic RB1B strain of MDV included macrophage inflammatory protein, interferon response factor 1, interferon-inducible protein, quiescence-specific protein, thymic shared antigen 1, major histocompatibility complex (MHC) class I, MHC class II, β2-microglobulin, clusterin, interleukin-13 receptor alpha chain, ovotransferrin, a serine/threonine kinase, and avian leukosis virus subgroup J glycoprotein.

Deep Sequencing of Chicken microRNAs

BackgroundThe use of new, deep sequencing technologies has greatly accelerated microRNA discovery. We have applied this approach to the identification of chicken microRNAs and to the comparison of microRNAs in chicken embryo fibroblasts (CEF) infected with Mareks disease virus (MDV) to those present in uninfected CEF.ResultsWe obtained 125,463 high quality reads that showed an exact match to the chicken genome. The majority of the reads corresponded to previously annotated chicken microRNAs; however, the sequences of many potential novel microsRNAs were obtained. A comparison of the reads obtained in MDV-infected and uninfected CEF indicates that infection does not significantly perturb the expression profile of microRNAs. Frequently sequenced microRNAs include miR-221/222, which are thought to play a role in growth and proliferation. A number of microRNAs (e.g., let-7, miR-199a-1, 26a) are expressed at lower levels in MDV-induced tumors, highlighting the potential importance of this class of molecules in tumorigenesis.ConclusionDeep sequencing technology is highly suited for small RNA discovery. This approach is independent of comparative sequence analysis, which has been the primary method used to identify chicken microRNAs. Our results have confirmed the expression of many microRNAs identified by sequence similarity and identified a pool of candidate novel microRNAs.

Sequence Conservation and Differential Expression of Marek's Disease Virus MicroRNAs

ABSTRACT Mareks disease virus (MDV), a herpesvirus that causes a lymphoproliferative disorder in chickens, encodes a number of microRNAs derived primarily from two locations in the MDV genome. One cluster of microRNA genes flanks the meq oncogene, and a second cluster is found within the latency-associated transcript (LAT) region. The sequences of MDV microRNAs from a collection of field and reference strains with various levels of virulence were compared and found to be highly conserved. However, microRNAs from the meq cluster were detected at higher levels in lymphomas caused by a form of the virus designated very virulent plus (vv+; strain 615K, also known as T. King) than in those caused by a less virulent (very virulent [vv]) form (RB1B). For example, levels of mdv1-miR-M4, which shares a seed sequence with miR-155, a microRNA implicated in B-cell lymphoma, were threefold higher and levels of mdv1-miR-M2*/3p were more than sixfold higher in vv+ MDV-induced tumors than in vv MDV-induced tumors. In contrast, levels of the microRNAs from the LAT cluster were equivalent in tumors produced by vv and vv+ strains. Additionally, mdv1-miR-M4 is the MDV microRNA most highly expressed in tumors, where it accounts for 72% of all MDV microRNAs, as determined by deep sequencing. These data suggest that the meq cluster microRNAs play an important role in the pathogenicity of MDV.

Protection of chickens from Newcastle and Marek's diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein.

Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Mareks disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Mareks disease.

Transfection of chicken embryo fibroblasts with Marek's disease virus DNA.

Total DNA from Mareks disease virus (MDV)-infected chicken embryo fibroblasts was transfected into freshly plated secondary chicken embryo fibroblasts using calcium phosphate-mediated transfection. Transfection frequencies were dose-dependent and non-linear. The maximum transfection frequencies of nine MDV DNA preparations using 8-25 micrograms total DNA ranged from 45 to 898 plaques per calcium phosphate/DNA precipitate. Approximately 100-200 plaques per 60-mm tissue-culture dish using 1-5 micrograms total DNA from MDV-infected chicken embryo fibroblasts were typically obtained. Transfection was most efficient when the pH of the HEPES buffer was 7.0, no additional carrier DNA was added to the precipitates, and the cultures were exposed for 3 minutes to 15% buffered glycerol 4 hours after the addition of the calcium phosphate/DNA precipitates.

Stable Transfection of Reticuloendotheliosis Virus-Transformed Lymphoblastoid Cell Lines

Lymphoblastoid T cell lines were established by infection of chicken splenocytes with reticuloendotheliosis virus (REV). The target cells first were cultured in interleukin-containing conditioned medium or were stimulated by concanavalin A, or both. Most cell lines were T cells expressing CD3 and one of the T cell receptors, and all cell lines were positive for major histocompatibility complex (MHC) class II antigens. Several REV-transformed cell lines were stably transfected using electroporation with a selectable plasmid, pNL1, containing the neor gene. Transfected cell lines were selected using G418 and were maintained for periods up to 137 days. Transfected cell lines were susceptible to MHC class-I restricted lysis by cytotoxic T lymphocytes from REV-infected chickens.

Efficacy in Chickens of a Herpesvirus of Turkeys Recombinant Vaccine Containing the Fusion Gene of Newcastle Disease Virus: Onset of Protection and Effect of Maternal Antibodies

The onset of protection against Newcastle disease and the effect of maternal antibodies to Newcastle disease virus (NDV) and Mareks disease virus (MDV) on vaccine efficacy were determined following vaccination of chickens with a recombinant herpesvirus of turkeys (HVT) vaccine expressing the fusion (F) glycoprotein gene of NDV. Onset of protection following intra-abdominal administration of the recombinant HVT/F vaccine at 1 day of age and subsequent ocular challenge with the neurotropic velogenic Texas GB strain of NDV was determined to occur between days 14 and 21 post-vaccination (PV). Vaccination with the Hitchner B1 strain of NDV resulted in protection by day 6 PV, and vaccination with an inactivated NDV oil-emulsion vaccine induced protection by day 14 PV. One-day-old broiler-type chickens with maternal antibodies to both NDV and MDV and 1-day-old specific-pathogen-free (SPF) white leghorn chickens lacking maternal antibodies were vaccinated with the recombinant HVT/F vaccine or with control vaccines, challenged intra-abdominally with the very virulent RB1B strain of MDV on day 8 PV, and challenged with the Texas GB strain of NDV on day 29 PV. The HVT/F and NDV strain Hitchner B1 vaccines provided 73% and 80% protection, respectively, against NDV in broilers, whereas both vaccines resulted in 100% protection in SPF leghorns.

Avian herpesvirus as a live viral vector for the expression of heterologous antigens.

Control of Mareks disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.

MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific.

Many herpesviruses, including Mareks disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TR(L)), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96-100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function.