First molecular detection and characterization of Marek's disease virus in red-crowned cranes (Grus japonensis): a case report

Abstract

Background: Marek's disease virus (MDV) resides in the genus Mardivirus in the family Herpesviridae. MDV is a highly contagious virus that can cause neurological lesions, lymphocytic proliferation, immune suppression, and death in avian species, including Galliformes (chickens, quails, partridges, and pheasants), Strigiformes (owls), Anseriformes (ducks, geese, and swans), and Falconiformes (kestrels).

Case presentation: In 2015, two red-crowned cranes died in Nanjing (Jiangsu, China). It was determined that the birds were infected with Marek's disease virus by histopathological examination, polymerase chain reaction (PCR), gene sequencing and sequence analysis of tissue samples from two cranes. Gross lesions included diffuse nodules in the skin, muscle, liver, spleen, kidney, gizzard and heart, along with liver enlargement and gizzard mucosa hemorrhage. Histopathological assay showed that infiltrative lymphocytes and mitotic figures existed in liver and heart. The presence of MDV was confirmed by PCR. The sequence analysis of the Meq gene showed 100% identity with Md5, while the VP22 gene showed the highest homology with CVI988. Furthermore, the phylogenetic analysis of the VP22 and Meq genes suggested that the MDV (from cranes) belongs to MDV serotype 1.

Conclusion: We describe the first molecular detection of Marek's disease in red-crowned cranes based on the findings previously described. To our knowledge, this is also the first molecular identification of Marek's disease virus in the order Gruiformes and represents detection of a novel MDV strain.

Keywords: Clinical necropsy; Homology; Marek’s disease virus; PCR; Red-crowned crane.

Fig. 1

Clinical symptoms and pathological lesions. a-f, Nodules in the (a) skin, (b) muscle, (c) trachea, (d) liver, (e) gizzard, (f) heart; g, nodules in the vertical section of the liver; h, hemorrhage sites in the gizzard mucosa; i, left knee joint swelling

Fig. 2

Histopathological section (H&E staining) of the liver and heart. a and b, lymphomatous infiltration in the liver; c and d, lymphomatous infiltration in the heart. The arrow shows a range of leukocytes, including large lymphocytes, small lymphocytes lymphoblasts, and malignant cells with mitotic figure.

Fig. 3

Agarose gel electrophoresis of MDV from RT-PCR of the Meq and gB genes. a and c, Meq (347 bp, partial) was amplified from cDNAs and genomes of different tissues. b and d, gB (338 bp, partial) was amplified from cDNAs and genomes of different tissues. The feather sample was a homogenate of ground feathers and skin that contained feather follicle epithelium. The entire cell genome from Md5-infected CEF cells was used as a positive control. The cDNA from CEF cells was used as a negative control

Fig. 4

Amino acid sequence alignment of Meq (from crane). a, The full-length Meq gene (1020 bp) was amplified from the spleen genome. b, The amino acid (aa) sequences of Meq (339 aa) from cranes were aligned with 9 previously published MDV isolates (including CVI988, 814, GX0101, LMS, GA, RB-1B, Md11 and 648a). c, Substituted amino acids are listed, while deleted amino acids are denoted by strips in the alignment

Fig. 5

Amino acid sequence alignment of VP22 (from crane). a, The full-length VP22 gene (750 bp) was amplified from the liver genome. b, The amino acid sequence of VP22 (243 aa) from cranes was aligned with 9 previously published MDV isolates (including CVI988, 814, GX0101, LMS, GA, RB-1B, Md11 and 648a). Identical amino acids are denoted by strips in the alignment, and deleted amino acids are denoted by dots in the alignment

Fig. 6

Phylogenetic profile showing the relationships among MDV isolates based on a comparison of the (a) Meq gene and (b) VP22 gene. Phylogenetic analysis of MDV based on (a) Meq and (b) VP22 amino acid sequences. The tree was constructed using the neighbor-joining (N-J) analysis method in the MEGA 5.0 program with bootstrapping (1000)

Fig. 7

Amino acid sequence alignment of L-Meq (from crane). a, The full-length L-Meq gene (1197 bp) was amplified from spleen cDNA. b, The amino acid sequence of L-Meq (398 aa) from cranes was aligned with 11 previously published MDV isolates (including CVI988, 814, GX060167, MDCC-RP1, MDCC-MSB1, BC-1, GA, JM10, JM102, RM-1 and CU-2). Sequences were aligned using the DNAMAN software. c, Substituted amino acids are listed, while deleted amino acids are denoted by strips in the alignment