Molecular characterization and phylogenetic analysis of a virulent Marek’s disease virus field strain in broiler chickens in Japan

<p>Marek’s disease is a lymphoproliferative disease causing a serious threat in poultry production. Field strains of Marek’s disease virus (MDVs) are continuously re-emerging, causing great economical losses to the poultry industry worldwide in spite of the intensive vaccination and restrictive management policy used. Histopathological and molecular characterizations of MDVs are essential for monitoring the changes of viruses and evaluating the effectiveness of existing vaccines. During 2016, 190 visceral tumour tissues representing 30 vaccinated chicken flocks from the Gifu prefecture, Japan, were analysed. A pathological examination revealed the presence of lymphoproliferative lesions in the visceral organs. Polymerase chain reaction screening of tissue specimens using specific primers for avian leucosis virus, reticuloendotheliosis virus, and MDV was positive only for MDV. The polymerase chain reaction products of meq, pp38, virus-induced IL-8 homology, and glycoprotein MDV genes were sequenced and used for homology, phylogenetic, and similarity level analysis with the published reference of MDVs in the database. The results revealed high similarity between the field isolates, vv and vv+ strains of MDV from the USA and China. Several point mutations in the nucleotide sequence of the field isolates and their deduced amino acid sequences were detected in those genes. The present molecular analyses indicated that nucleotide and amino acid changes could be valuable criteria for differentiation and determination of the pathogenicity and oncogenicity of MDVs according to the Avian Disease and Oncology Laboratory pathotyping in vivo studies. Furthermore, the results suggest that development of a new vaccine must be considered to overcome this devastating avian oncogenic viral disease.</p

Alignment of the VP1 nucleotide sequence of Egyptian strains and reference strains shows several mutations in new strain and vaccinal strains.

Alignment of the VP1 nucleotide sequence of Egyptian strains and reference strains shows several mutations in new strain and vaccinal strains.</p

Alignment of VP1 amino acids sequence of Egyptian strains and reference strains shows several points of mutations in new strain and vaccinal strains.

Alignment of VP1 amino acids sequence of Egyptian strains and reference strains shows several points of mutations in new strain and vaccinal strains.</p

Histopathological lesions in FMDV naturally infected animals.

(a) Vesicles formations (arrows) with elevation of superficial epithelium in the stratified squamous epithelium of dermis, H&E, X200. (b) Vesicular stomatitis of the dental pad of infected calf characterized by leukocytic infiltrations of mucosal and submucosal layer of cornified epithelial tissue (arrows), H&E, X200. (c) Zenker,s necrosis (arrows) of muscular layers of stratified squamus epithelim with edema inbetween (arrowheads) of cattle, H&E, X200. (d) Mild myocarditis of the myocardium of buffalo with mild lymphocytic cell aggregations (arrows), H&E, X400. (e) Moderate myocarditis of the myocardium of buffalo calf (less than 2 Y) with moderate lymphocytic cell aggregations (arrows) and mild myocardial muscle necrosis (arrowheads), H&E, X400. (f) Severe non- suppurative myocarditis of heart of calf with a significant number of lymphocytic cell aggregations (arrows) with complete lysis of necrosed muscle fibers (arrowheads) and replacement of this muscles by a large number of inflammatory cells (arrows). The black star, referring to the myocardial muscle that still normal not necrosed, H&E, X400.</p

FMDV sequences reference strains published in GeneBank.

FMDV sequences reference strains published in GeneBank.</p

Alignment of the VP1 nucleotide sequence of Egyptian strains and reference strains shows several mutations in new strain and vaccinal strains.

Alignment of the VP1 nucleotide sequence of Egyptian strains and reference strains shows several mutations in new strain and vaccinal strains.</p

Fig 5 -

(a) liver of buffalo calf, lymphocytic cell aggregations around central hepatic veniules (arrows) with hydropic degeneration (arrowheads) of hepatic cells, H&E, X400. (b) Mild hydropic degeneration of hepatic cells (arrows) with mild coagulative necrosis of hepatic cells (arrowheads), H&E, X400. (c) The intestine of FMDV infected buffalo showing catarrhal enteritis with leukocytic cell infiltratons of the intestinal villi (arrows), H&E, X100. (d) Severe lymphocytic enteritis (arrows) with degeneration of intestinal villi, H&E, X200.</p

A map showing some recent FMD outbreaks in certain Egyptian governorates.

A map showing some recent FMD outbreaks in certain Egyptian governorates.</p

Histopathological lesions of FMDV infected animals.

(a) Necrosis of myocardial fibers (arrows) of a heart of adult cattle with moderate myocarditis (arrowheads), H&E, X200. (b) Degeneration and necrosis of myocardial muscles (arrows) surrounded by inflammatory cells, H&E, X400. (c) Normal lung of a buffalo showing normal alveoli, H&E, X100. (d) purulent pneumonia of a lung with diffuse infiltration of alveoli by neutrophils and lymphocytic cells (arrows), with serohemorrhagic and fibrinous pneumonia (stars), H&E, X100. (e) Congestion of perivascular capillary (arrowheads) by RBCs and leukocytic cells (arrowheads) with serohemorrhagic and fibrinous pneumonia (stars), H&E, X400. (f) lung alveoli filled by exudative inflammation (black star) containing significant number of leukocytic cells, H&E, X400.</p

Clinical and gross pathological lesions in calves, cattle and buffalo of FMDV naturally infected animals.

(a) Heavy salivation of infected cattle. (b) Hemorrhagic area of sub mucosa of oral commissure of a calf (arrows). (c) Ulcer formation of dental pad with secondary bacterial infection. (d) Marked whitish necrosed areas all over the heart (black star). (e) A longitudinal section in the heart ventricle and interventricular septum, showing yellowish to whitish streaking of myocardial necrosis (black star)"tiger heart". (f) cardial hemorrhage alover the heart atrium, ventricles and papillary muscles (arrows). (g) Focal ulcerative lesions of ruminal pillars (arrows).</p