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Molecular Characterization of Oncogenic Genes, Pathotyping and Protectotyping of Marek?s Disease Virus

KrishiKosh

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Title Molecular Characterization of Oncogenic Genes, Pathotyping and Protectotyping of Marek?s Disease Virus
 
Creator Suresh, P.
 
Contributor Rajeswar, J. Johnson
 
Description A study was undertaken to isolate, characterize the oncogenic genes at molecular
level and to carryout pathotype and protectotype assay of serotype 1 Marek?s Disease Virus
(MDV) from the vaccinated poultry flocks which had the Marek?s Disease (MD) outbreak in
Tamil Nadu and Karnataka.
Eighty six blood, 54 organ and 42 feather follicle samples were collected from fifteen
farms. Blood samples were screened by PCR technique for serotype 1 specific 132 bprepeat
sequence and positive detection rate was 33.72 per cent. Organ samples from
respective 132 bp positive blood source alone subjected for isolation. Five representative
MDV serotype 1 strains were isolated from five out of 15 farms by using Duck embryo
fibroblast (DEF) culturing (33.33%). Three out of five isolates were free from Avian leukosis
virus (ALV) and Reticuloendotheliosis virus (REV) alone adapted in Chicken embryo
fibroblast (CEF) culture and produced typical cytopathic effect (CPE) (MDV serotype 1
Plaques) after 2-4 passages. The recovery rate was 100, 20 and 20 per cent from
lymphocyte, organ (Spleen) and feather follicle samples respectively. Isolates recovered
from organ and feather follicles were not considered as different ones from isolates of
lymphocyte source in the same farm. Isolates were named as Ind/TN/11/01, Ind/KA/12/02
and Ind/ TN/12/03.
Oncogenes viz., Meq, pp38 and vIL8 were amplified and sequenced from each one
of the CEF adapted isolates by PCR. Amplified product size was 1081 bp, 1006 bp and 887
bp for Meq, pp38 and vIL8 genes respectively.
Homology analysis showed that the homology of the nucleotide sequences within the
three local isolates were 99.5-99.7, 99.3-99.8 and 99.8-100 per cent for Meq, pp38 and vIL8
genes respectively. Homology between MDVs isolated in this study and other isolates of this
area reported by earlier workers were 98.7-98.9 and 79.9-98.4 per cent for Meq and pp38
genes respectively. However there were no sequencing reports of vIL8 gene in GenBank by
earlier workers of this area. The isolates were shown to have a homology of 99.1-99.8, 97.3-
98.3 and 94.6-95.1 per cent with various isolates of China for Meq, pp38, and vIL8 genes
respectively and 98.5 - 99.2, 98.4 - 100 and 96.6 - 99.5 per cent with isolates of Europe and
USA for Meq, pp38 and vIL8 genes respectively.
Alignment analysis of the nucleotide sequences showed that nucleotide mutations at
five different positions in Meq gene, two different positions in pp38 gene and three different
positions in vIL8 gene displayed perfect regularity in MDVs circulating in southern part of
India, which could be considered as features of field MDVs prevalent in this area recently. In
addition, the mutation in Meq gene at positions 251, 260 and 437 were unique and coincides
with very virulent strains from China GX0101, GXY2 and Hungarian strain ATE. The
mutation at positions 283 and 300 were unique and coincides only with very virulent strain
ATE of Hungary. There was a single nucleotide mutation at position 155 (A to T), 369 (A to
C), 462 (C to A) and 548 (C to T) in the isolate Ind/TN/12/03 which showed very virulent
character in pathotyping assay. The mutation at position 477 in pp38 gene was unique and
coincides with very virulent strains from China and USA including XJ 03, TQ20 and Md5. At
position 265, the very virulent plus strains including 584 A, 648 A and very virulent strain
Md5 possess a point mutation G to C which is different from very virulent RB1B strain and
isolates studied.
Phylogenetic analysis of Meq, pp38 and vIL8 protein sequences revealed that field
MDVs in this area evolved independently but have similarities with very virulent strains of
China and primary oncogene Meq has got more similarities with very virulent Hungarian
strain.
In pathotyping trial, birds inoculated with field isolate Ind/TN/12/03 were highly
depressive in body weight (75.34?3.04 g 15 dpi) and relative bursal weight (1.64?0.06 at 15
dpi) when compared to those inoculated with the other two isolates (Ind/TN/11/01 and
Ind/KA/12/02) and uninoculated control (bwt 111.33?1.30 g & b/b 4.33?0.1115 dpi).
Incidence of Early Mortality Syndrome (53%) and Lymphoma (86%) induced by
Ind/TN/12/03 was comparable with very virulent strains published elsewhere. The
percentage of MD incidence induced by Ind/TN/12/03 was 57.5 and 25 per cent in
monovalent and bivalent vaccine inoculated birds respectively compared to uninoculated
control (100%) in protectotyping assay. Based on the above findings in pathotyping and
protectotyping experimental trials isolate Ind/TN/12/03 was designated as very virulent MDV
and other two isolates were considered as virulent MDVs.
 
Date 2016-07-26T16:01:43Z
2016-07-26T16:01:43Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/69967
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University