Laboratoire de Virologie et Barrière d'Espèces, UR 086, INRA, Centre de recherches de Tours, 37380 Nouzilly, France
Correspondence
S. Laurent
slaurent{at}tours.inra.fr
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ABSTRACT |
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MAIN TEXT |
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Very little is known about the molecular evolution of MDV. Herpesviruses, like other DNA viruses, have low mutation rates (estimated at 3x108 substitutions per site per year) (McGeoch et al., 2000), and single-nucleotide polymorphism (SNP) has been shown to be a useful tool for the study of genetic polymorphism in herpesviruses (Faga et al., 2001
; Franti et al., 1998
).
To assess the variability of MDV over 17 years (19821999), we carried out a retrospective molecular epidemiological study to characterize 68 French MDV isolates obtained from field cases and compare them with reference strains from the National Center for Biotechnology Information (NCBI) database. All isolates are denoted by the year of isolation followed by their specific number. Fifty-one chicken samples corresponding to paraffin-embedded samples of retrospective MD cases (198398) were confirmed by histological analysis. Forty-eight of these samples originated from eight French regions located in the western part of France, with 20 originating from the region of Pays de la Loire. The remaining three samples (83-2, 90-5, 94-1) originated from the Ivory Coast (Africa), although the chickens were imported as chicks from French breeding companies. The vaccination status of all these chickens was unknown. Fourteen other samples were collected in 1999, originating exclusively from Pays de la Loire, and were blood samples. The vaccination status was known: eight had been vaccinated with CVI-988 (99-2, 99-8, 99-12, 99-15, 99-13, 99-16, 99-17, 99-18) and the remaining five had not been vaccinated. Finally, three samples were obtained from turkeys with clinical signs of MDV infection. Two of these were obtained as part of the paraffin-embedded sample collection (82-T and 91-T) and the remaining sample was a blood sample dating from 1999 (99-T). Total DNA was also extracted from a Poulvac Marek CVI vaccine dose (Fort Dodge) and the MDV-established lymphoblastoid cell lines MSB1 (Akiyama et al., 1973) and PA5 (Fragnet et al., 2003
).
DNA was extracted from cell pellets of MDV cell lines, the vaccine dose, whole blood samples (50 µl) and tissue fragments as described by Fragnet et al. (2003). Tissue fragments of paraffin-embedded blocks were previously rehydrated as described by Oud et al. (1986)
. Two target genes specific for MDV-serotype 1 were amplified by nested PCR. The first target corresponded to part of the glycoprotein D (gD) gene described previously (Laurent et al., 2001
). The gD gene is located in the short unique region of the genome and has been identified as non-essential for the oncogenicity and horizontal transmission of MDV (Anderson et al., 1998
). The second target gene selected was the Meq gene, located in the long terminal repeat region, and encodes a bZIP protein of the Fos/Jun family that is involved in MDV oncogenesis (Liu & Kung, 2000
). The Meq gene fragment was amplified with the following primers: Meq F 5'-GAGAAGGCGGGCACGGTACAGGTG-3' with Meq R 5'-AACCGGAGCAATGTGGAGCGTTAG-3' for the first round of PCR and Meq NF 5'-GAGATGTCTCAGGAGCCAGAGCC-3' with Meq NR 5'-GGAGGTTCAGGAACGGGATCGTGCG-3' for the second round of nested PCR. The PCR conditions were as described previously (Laurent et al., 2001
). Direct sequencing of gel-purified DNA was performed twice with independent DNA templates obtained from separate PCRs.
The 68 gD and Meq sequences amplified from French field samples were aligned with sequences of MDV reference strains and cell lines (Figs 1 and 2). A high degree of conservation was observed for both genes, with a maximum divergence between pairs of isolates of 2 %. Moreover, despite their difference in function and location in the viral genome, the two genes displayed similar levels of variability, with nucleotide substitutions occurring at eight of the 203 positions (3·9 %) within the gD gene and at nine of the 301 positions (3 %) within the Meq gene.
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For the Meq gene, six alleles (IVI) were defined according to the five SNPs identified (Fig. 2). The two MDV tumour cell lines PA5 and MSB1 presented the specific feature of having a second mutation in codons already affected by SNPs (codons 77 and 80, respectively), confirming that these sites are hot-spot mutation regions. Our target sequence corresponded to the region encoding part of the N-terminal region of the MEQ protein which encompassed the leucine-zipper motif and the basic region containing the major nuclear localization signals (NLS) and nucleolar functions (NoLs) (Liu & Kung, 2000
). Three SNPs were identified in the basic region and may consequently affect NLS or NoLs function. The other three SNPs were in the leucine-zipper motif, but none of these were residues defining this motif. It would be of interest to assess the differences in NoLs and NLS functions of the various alleles.
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Most of the French isolates clustered in allele II, which did not include any of the reference MDV strains of foreign origin present in the database. Furthermore, strains RB1B (Schat et al., 1982), GA (Eidson & Schmittle, 1968
) and MD5 (Witter et al., 1980
) originating from the USA had allele III; the eight strains of Chinese origin fell into two other specific allele groups, I and VI; and the European strains, CVI988 of Dutch origin (Rispens et al., 1972
) and the PA5 cell line from chickens infected with strain HPRS16 (Purchase & Biggs, 1967
) of British origin, both had allele IV. It would be of interest to extend this study to MDV strains from other countries, to evaluate potential clustering as a function of geographical origin.
Theoretically, given that avian sequences displayed three and six alleles for the gD and Meq genes, respectively, 18 different combinations were possible. Only nine combinations (C1C9) were observed, and 85 % of French field samples corresponded to three of these combinations: C1, C2 and C3 (Table 1). Furthermore, 60·5 % of the samples, including the three turkey samples, were grouped in a single combination (C1) where no reference MDV strain was represented. The six minor combinations (C4C9) each corresponded to only zero to three French samples. No avian field sample was found in the C9 group, which contained the CVI-988 strain, showing that even in CVI-988-vaccinated chickens only virulent MDV were detected. PA5 was also associated with the C9 combination, but differs from CVI-988 by two other specific mutations in the Meq sequence (Fig. 2
). Hence the PA5 and CVI-988 strains can be differentiated. This finding could be important for the development of a specific system for detecting the CVI-988 vaccine virus in chicks after vaccination, in order to monitor correct administration of the vaccine.
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Received 5 November 2003;
accepted 6 February 2004.
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