Classification of Marek's disease viruses according to pathotype: Philosophy and methodology

El concepto de patotipo en la enfermedad de Marek (MD) data probablemente de finales de los 1950s cuando se reconoció una forma más virulenta de enfermedad Benton y Cover, 1957). Las distinciones entre las diferentes cepas de virus de MD (MDV) fueron aún mayores al describirse el patotipo vv a principios de los ochenta y el vv+ en los noventa. La designación de patotipo refleja propiedades biológicas importantes que se correlacionan con la capacidad de romper la inmunidad maternal en el campo. A pesar de ello, los métodos de clasificación de los diferentes patotipos en varios laboratorios no han sido uniformes, lo cual ha impedido una comparación crítica de los resultados. El método utilizado en el Avian Disease and Oncology Laboratory (ADOL) se basa en la inducción de lesiones linfoproliferativas en pollos vacunados. Este método ha sido utilizado para clasificar más de 45 aislados y es la base para la clasificación actual de los patotipos de cepas de MDV. Las limitaciones de este método son varias: necesidad de un tipo específico de pollos (15x7 ab+), uso de un gran número de animales y de un método estadístico para comparar las respuestas lesionales con las de las cepas control JM/102W y Md5. Debido a estas limitaciones no ha sido y no es probablemente usado en otros laboratorios. La comparación en el patotipado puede ser mejorada mediante la comparación de aislados de campo con cepas prototipo como las JM/102W, Md5 y 648A (American Type Culture Collection) o sus equivalentes. Los datos pueden ser generados mediante diferentes procedimientos in vivo que miden la inducción de tumores, enfermedad neurològica (por lesiones neoplásicas o no neoplásicas), o únicamente por criterios no neoplásicos (como el peso de los órganos linfoides o la replicación vírica). Los métodos basados en criterios neoplásicos, especialmente cuando son generados en pollos inmunizados de MD, probablemente se correlacionarán mejor con el método del ADOL y serán más relevantes en cuanto a la evolución de los virus de MD en el campo. En base a los datos de diferentes experimentos, se propone una modificación del método ADOL que utiliza menos animales y puede ser llevado a cabo en pollos SPF comerciales. El método modificado se basa en una comparación con el que mejor clasifica las cepas prototipo, y se espera que de resultados en general comparables con el método original. Otros criterios alternativos (ver abajo) también se evalúan como métodos primarios de patotipificación o como adjuntos a otros métodos de patotipificación. Se presentan las ventajas y desventajas de estos métodos alternativos.The concept of pathotype in Marek's disease (MD) probably dates from the recognition of a more virulent form of the disease in the late 1950s (Benton & Cover, 1957). Distinctions between MD virus strains were further expanded with the description of the vv pathotype in the early 1980s and of the vv + pathotype in the 1990s. Pathotype designations reflect important biological properties that correlate with the break-through of vaccinal immunity in the field. However, pathotyping methods applied by various laboratories have not been uniform, preventing critical comparison of results. Better uniformity of pathotyping procedures is desirable. The Avian Disease and Oncology Laboratory (ADOL) method is based on induction of lymphoproliferative lesions in vaccinated chickens. This method has been used to pathotype more than 45 isolates and is the basis for the current pathotype classification of MD virus strains. Its limitations include requirements for a specific type of chickens (15 x 7 ab+), large numbers of animals, and a statistical method to compare lesion responses to those of JM/102W and Md5 control strains. Because of these limitations, it has not been and is not likely to be used in other laboratories. Comparability in pathotyping can be improved by the comparison of field isolates with standard prototype strains such as JM/102W, Md5 and 648A (American Type Culture Collection) or their equivalents. Data may be generated by different in vivo procedures that measure tumour induction, neurological disease (both neoplastic and non-neoplastic lesions), or solely non-neoplastic criteria (such as lymphoid organ weights or virus replication). Methods based on neoplastic criteria, especially when generated in MD-immunized chickens, will probably correlate most closely with that of the ADOL method and be most relevant to evolution of MD virus in the field. Based on data from several trials, a modification of the ADOL method that utilizes fewer chickens and can be conducted with commercial specific pathogen free strains is proposed. The modified method is based on "best fit" comparisons with prototype strains, and is expected to provide results generally comparable with the original method. A variety of other alternative criteria (see earlier) are also evaluated both for primary pathotyping and as adjuncts to other pathotyping methods. Advantages and disadvantages of alternative methods are presented.Facultad de Ciencias Veterinaria

Classification of Marek's disease viruses according to pathotype: Philosophy and methodology

El concepto de patotipo en la enfermedad de Marek (MD) data probablemente de finales de los 1950s cuando se reconoció una forma más virulenta de enfermedad Benton y Cover, 1957). Las distinciones entre las diferentes cepas de virus de MD (MDV) fueron aún mayores al describirse el patotipo vv a principios de los ochenta y el vv+ en los noventa. La designación de patotipo refleja propiedades biológicas importantes que se correlacionan con la capacidad de romper la inmunidad maternal en el campo. A pesar de ello, los métodos de clasificación de los diferentes patotipos en varios laboratorios no han sido uniformes, lo cual ha impedido una comparación crítica de los resultados. El método utilizado en el Avian Disease and Oncology Laboratory (ADOL) se basa en la inducción de lesiones linfoproliferativas en pollos vacunados. Este método ha sido utilizado para clasificar más de 45 aislados y es la base para la clasificación actual de los patotipos de cepas de MDV. Las limitaciones de este método son varias: necesidad de un tipo específico de pollos (15x7 ab+), uso de un gran número de animales y de un método estadístico para comparar las respuestas lesionales con las de las cepas control JM/102W y Md5. Debido a estas limitaciones no ha sido y no es probablemente usado en otros laboratorios. La comparación en el patotipado puede ser mejorada mediante la comparación de aislados de campo con cepas prototipo como las JM/102W, Md5 y 648A (American Type Culture Collection) o sus equivalentes. Los datos pueden ser generados mediante diferentes procedimientos in vivo que miden la inducción de tumores, enfermedad neurològica (por lesiones neoplásicas o no neoplásicas), o únicamente por criterios no neoplásicos (como el peso de los órganos linfoides o la replicación vírica). Los métodos basados en criterios neoplásicos, especialmente cuando son generados en pollos inmunizados de MD, probablemente se correlacionarán mejor con el método del ADOL y serán más relevantes en cuanto a la evolución de los virus de MD en el campo. En base a los datos de diferentes experimentos, se propone una modificación del método ADOL que utiliza menos animales y puede ser llevado a cabo en pollos SPF comerciales. El método modificado se basa en una comparación con el que mejor clasifica las cepas prototipo, y se espera que de resultados en general comparables con el método original. Otros criterios alternativos (ver abajo) también se evalúan como métodos primarios de patotipificación o como adjuntos a otros métodos de patotipificación. Se presentan las ventajas y desventajas de estos métodos alternativos.The concept of pathotype in Marek's disease (MD) probably dates from the recognition of a more virulent form of the disease in the late 1950s (Benton & Cover, 1957). Distinctions between MD virus strains were further expanded with the description of the vv pathotype in the early 1980s and of the vv + pathotype in the 1990s. Pathotype designations reflect important biological properties that correlate with the break-through of vaccinal immunity in the field. However, pathotyping methods applied by various laboratories have not been uniform, preventing critical comparison of results. Better uniformity of pathotyping procedures is desirable. The Avian Disease and Oncology Laboratory (ADOL) method is based on induction of lymphoproliferative lesions in vaccinated chickens. This method has been used to pathotype more than 45 isolates and is the basis for the current pathotype classification of MD virus strains. Its limitations include requirements for a specific type of chickens (15 x 7 ab+), large numbers of animals, and a statistical method to compare lesion responses to those of JM/102W and Md5 control strains. Because of these limitations, it has not been and is not likely to be used in other laboratories. Comparability in pathotyping can be improved by the comparison of field isolates with standard prototype strains such as JM/102W, Md5 and 648A (American Type Culture Collection) or their equivalents. Data may be generated by different in vivo procedures that measure tumour induction, neurological disease (both neoplastic and non-neoplastic lesions), or solely non-neoplastic criteria (such as lymphoid organ weights or virus replication). Methods based on neoplastic criteria, especially when generated in MD-immunized chickens, will probably correlate most closely with that of the ADOL method and be most relevant to evolution of MD virus in the field. Based on data from several trials, a modification of the ADOL method that utilizes fewer chickens and can be conducted with commercial specific pathogen free strains is proposed. The modified method is based on "best fit" comparisons with prototype strains, and is expected to provide results generally comparable with the original method. A variety of other alternative criteria (see earlier) are also evaluated both for primary pathotyping and as adjuncts to other pathotyping methods. Advantages and disadvantages of alternative methods are presented.Facultad de Ciencias Veterinaria

Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation

Some herpesviruses, particularly lymphotropic viruses such as Marek's disease virus (MDV) and human herpesvirus 6 (HHV-6), integrate their DNA into host chromosomes. MDV and HHV-6, among other herpesviruses, harbor telomeric repeats (TMRs) identical to host telomeres at either end of their linear genomes. Using MDV as a natural virus-host model, we show that herpesvirus TMRs facilitate viral genome integration into host telomeres and that integration is important for establishment of latency and lymphoma formation. Integration into host telomeres also aids in reactivation from the quiescent state of infection. Our results and the presence of TMRs in many herpesviruses suggest that integration mediated by viral TMRs is a conserved mechanism, which ensures faithful virus genome maintenance in host cells during cell division and allows efficient mobilization of dormant viral genomes. This finding is of particular importance as reactivation is critical for virus spread between susceptible individuals and is necessary for continued herpesvirus evolution and survival

Herpesvirus Telomerase RNA(vTR)-Dependent Lymphoma Formation Does Not Require Interaction of vTR with Telomerase Reverse Transcriptase (TERT)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)n. Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity

Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines

Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma