Aoi Kurokawa

Marek’s disease virus (MDV) causes lymphomas and neurological disorders in chickens. Although vaccination largely controls outbreaks, highly virulent strains continue to emerge. The major MDV-encoded oncoprotein is Meq, functions as a transcription factor. Amino acid polymorphisms in Meq have been reported to influence virulence. Despite routine vaccination, MD still occurs sporadically in Japan. Japanese isolates harbour characteristic Meq polymorphisms, but their impact on MDV virulence remains unclear. We investigated the transcriptional regulation by Meq from Japanese isolates and evaluated the pathogenicity of recombinant MDV (rMDV) encoding Meq from the Japanese isolate Nr-c1. Nr-c1-Meq exhibited reduced transrepression and transactivation on viral gene promoters. An rMDV encoding Nr-c1-Meq (vNr-c1-Meq) induced lower mortality and tumourigenicity than an rMDV encoding Meq from the parental very virulent RB-1B strain (vRB-1B). vNr-c1-Meq did not cause visceral tumours or neurological disorders but resulted in distinct clinical signs, including open-mouth breathing. In lymphoid tissues from vNr-c1-Meq-infected chickens, a lower proportion of CD4 ⁺ T cells, the targets of MDV transformation, and lower viral loads were observed than those in vRB-1B-infected chickens. Histopathological examination revealed increased lymphocyte infiltration in bronchus-associated lymphoid tissues (BALT) in the vNr-c1-Meq group. Additionally, flow cytometric analysis showed elevated γδ T-cell proportions, which positively correlated with IFN- γ expression in vNr-c1-Meq-infected chickens and were linked to BALT hyperplasia. In comparison to the vRB-1B, vNr-c1-Meq infection resulted in attenuated disease progression and altered clinical signs. These findings suggest that Meq polymorphisms not only influence MD virulence but also clinical presentation.

Chicken lymphomas are generally classified into three virus-induced tumors: Marek’s disease (MD), lymphoid leukosis, and reticuloendotheliosis, with MD being the most common T-cell lymphoma in commercial poultry. In this study, we describe 23 cases of a novel cytotoxic T-cell lymphoma affecting layer chickens aged >1 year in Japan that is distinct from previously known chicken lymphomas. These cases were initially misidentified as MD during routine poultry inspection but were later confirmed as a distinct and novel form of lymphoma. The average incidence of this lymphoma was 0.016%. Histological analysis revealed that the tumors comprised small uniformly round cells that were CD3 ⁺ , CD4 ⁻ , and CD8 ⁺ , indicating a cytotoxic T-cell origin. Investigations using immunohistochemistry, in situ hybridization, and polymerase chain reaction ruled out the involvement of known tumor-inducing viruses in the development of these novel lymphomas. These findings confirm the existence of a novel lymphoma type in chickens and provide key histopathological and epidemiological data to aid in definitive diagnosis.

Marek’s disease virus (MDV) is the etiological agent of Marek’s disease (MD), a lymphoproliferative disorder in chickens. Polymorphisms in the MDV-encoded oncoprotein Meq are shared among field strains and correlate with their virulence. The attenuated vaccine strain CVI988 harbors unique amino acid polymorphisms in Meq, particularly at positions 71, 77, and 326. In this study, we investigated the impact of these polymorphisms on Meq protein function and MDV virulence. Reporter assays revealed that the substitutions, particularly A71S and K77E, markedly impaired the transcriptional regulatory activity of Meq. To evaluate their effect on virulence, we generated a recombinant MDV based on the very virulent RB-1B strain, encoding Meq with A71S and K77E substitutions (rRB-1B_Meq71/77). Chickens infected with rRB-1B_Meq71/77 developed neither clinical signs nor lymphomas. Flow cytometry revealed no expansion of infected cells in this group, but a marked increase in CD8⁺ T and γδ T cells during early infection. Histopathological analysis also confirmed the absence of MD-associated lesions. These findings demonstrate that the polymorphisms at positions 71 and 77 in the CVI988 strain are sufficient to abolish MDV virulence. This study provides insight into the molecular basis of MDV virulence and informs the strategy for the design of more effective vaccines.

Diagnóstico inmunohistoquímico de la enfermedad de Marek basado en la detección de Meq en 104 casos de campo de linfoma de pollo. La detección inmunohistoquímica del virus de la enfermedad de Marek-EcoRI-Q (Meq) en células tumorales es uno de los métodos más precisos para el diagnóstico definitivo de la enfermedad de Marek (EM). Anteriormente generamos varios anticuerpos monoclonales anti-Meq para inmunohistoquímica (IHC) para detectar Meq expresado en linfomas de Marek inducidos experimentalmente en secciones fijadas con formalina e incluidas en parafina. Aquí evaluamos la utilidad de los anticuerpos monoclonales anti-Meq y mezcla de tres anticuerpos monoclonales anti-Meq: 3A3-112, 5F7-82 y 6B5-128 para el diagnóstico inmunohistoquímico de Marek en 104 casos de campo de linfoma de pollo que ocurrió en Japón entre 1976 y 2023. Los resultados revelaron 100 casos de linfoma de células T, de los cuales 99 casos eran positivos a la presencia de Meq (Meq+) con la mezcla de anticuerpos. Se han identificado dos subtipos histológicos distintos en los tumores de la EM, lo que sugiere que el diagnóstico morfológico de la enfermedad Marek es complejo y enfatiza la necesidad de la detección IHC. Para el diagnóstico del linfoma de células B del pollo, se encontró que fue útil la detección del factor activador de células B perteneciente al receptor de la familia del factor de necrosis tumoral (BAFF-R) mediante IHC. Cuatro casos de linfoma de células B crecieron de manera consistente y uniforme y Meq fue indetectable. En conclusión, la detección del gene Meq mediante IHC es valiosa para el diagnóstico patológico de casos de la EM en el campo.

Providing a method to detect avian lymphocytes by immunohistochemistry (IHC) would be helpful for analyzing immune function and diagnosing diseases in birds. In this study, we comprehensively examined the immunohistochemical identification of avian T and B lymphocytes in formalin-fixed, paraffin-embedded tissues from 53 avian species across 15 orders, using eight commercially available lymphocyte markers. T lymphocytes from all 53 avian species tested were specifically detected by IHC using the anti-CD3 antibody (clone F7.2.38). The appropriate antibody for detecting avian B lymphocytes in IHC varied depending on the avian species. B lymphocytes were specifically labeled by IHC in 46 of 53 avian species (86.8%) using any of seven B cell markers. The anti-PAX5 antibody (clone SP34) immunohistochemically detected B lymphocytes from the majority of avian species (41 out of 53 species), excluding those in the orders Falconiformes (falcons) and Passeriformes (oscines). The anti-BAFF-R antibody (clone 2C4) proved suitable for detecting B lymphocytes in the orders Galliformes (landfowls) and Anseriformes (waterfowls) in IHC. Caution is advised when using the anti-BLA36 (clone A27-42) and two anti-CD20 (clone L26 and product No. PA5-16701) antibodies, which are commonly used as B cell markers in mammals, for detecting avian B lymphocytes. These antibodies reacted with cells located in both T and B cell areas in certain avian species. The anti-Bu-1a/b (clone AV20) and anti-CD79a (clone HM57) antibodies were found not to bind to B lymphocytes in various avian species in IHC.

Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.

Marek disease (MD) is a viral disease characterized by the development of lymphoma in poultry. Although morphologic confirmation of lymphoma is used to diagnose MD, immunohistochemical detection of MD virus- EcoRI-Q (Meq), which is a viral protein that is expressed exclusively in MD tumor cells, would further improve the accuracy of diagnosis. We developed monoclonal antibodies (mAbs) that specifically detect Meq by immunohistochemistry (IHC) using formalin-fixed, paraffin-embedded (FFPE) sections. We evaluated the sensitivity and specificity of 14 mAbs that we produced, using FFPE samples of MDCC-MSB1 cells, MD tumor tissues, and tissues of uninfected chickens. Four different antigen retrieval conditions were investigated. Thirteen mAbs reacted with Meq in FFPE sections, but immunohistochemical reactivity and specificity varied depending on the mAb and antigen retrieval condition; heat-induced antigen retrieval (HIAR) was more effective at detecting Meq than the other tested conditions. HIAR pH 9 tended to increase immunoreactivity and decrease specificity. Of the 5 mAbs that immunoreacted strongly with Meq without nonspecific reactions under the optimal antigen retrieval conditions, 3 mAbs (1C1-121, 3A3-112, 5F7-82) did not produce background staining of tumor or non-tumor tissues; 2 mAbs (2C5-11, 4A5-54) produced background staining. The mAb 6B5-128 reacted moderately with Meq without nonspecific reactions and background staining. The remaining mAbs showed weak immunoreactivity or problematic nonspecific reactions. Our results suggest that some of our developed mAbs can be used in IHC to detect Meq in FFPE sections with high specificity, and that the use of IHC may greatly improve the diagnosis of MD.