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Detection of myxoma viruses encoding a defective M135R gene from clinical cases of myxomatosis; possible implications for the role of the M135R protein as a virulence factor.

Belsham GJ, Polacek C, Breum SØ, Larsen LE, Bøtner A - Virol. J. (2010)

Bottom Line: An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007.Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor.This frame-shift mutation results in expression of a greatly truncated product.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark. grbe@vet.dtu.dk

ABSTRACT

Background: Myxoma virus is a member of the Poxviridae and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy.

Results: An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain (in the genes M009L, M036L and M148R) were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature.

Conclusions: It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis.

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Evidence for a frame-shift mutation within the M135R gene from Danish samples of myxoma virus. Panel (a). The nucleotide sequence of the myxoma virus M135R gene, plus flanking sequences, is shown. Primers M135Rfor and M135Rrev (see Table 2), indicated in bold italics, were used to amplify by PCR the M135R gene, together with some flanking sequences at both termini, and these fragments were sequenced directly. The myxoma virus sequences included in primers M135RXFOR and M135RAPAREV (Table 2) for amplifying the coding sequence for M135R are underlined. The initiation and termination codons within these primers are indicated in bold capitals. The region of the gene in which a frame-shift mutation was present in certain virus samples is indicated within a rectangle. Panels (b) and (c.) Sequence traces obtained by analysis of a wt M135R gene sequence (as in the myxoma vaccine, panel (b)) and the mutant (+G) form (panel (c)) found in the majority of Danish clinical samples in 2007. The region shown corresponds to the portion of the sequence contained within the rectangle in panel (a). The region of the M135R gene which is predicted to be translated in a different reading frame, downstream of the insertion of a G nucleotide, is indicated in red in panel (a). The termination codons (TAA) for the wt and mutant M135R gene products are indicated in bold capitals.
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Figure 2: Evidence for a frame-shift mutation within the M135R gene from Danish samples of myxoma virus. Panel (a). The nucleotide sequence of the myxoma virus M135R gene, plus flanking sequences, is shown. Primers M135Rfor and M135Rrev (see Table 2), indicated in bold italics, were used to amplify by PCR the M135R gene, together with some flanking sequences at both termini, and these fragments were sequenced directly. The myxoma virus sequences included in primers M135RXFOR and M135RAPAREV (Table 2) for amplifying the coding sequence for M135R are underlined. The initiation and termination codons within these primers are indicated in bold capitals. The region of the gene in which a frame-shift mutation was present in certain virus samples is indicated within a rectangle. Panels (b) and (c.) Sequence traces obtained by analysis of a wt M135R gene sequence (as in the myxoma vaccine, panel (b)) and the mutant (+G) form (panel (c)) found in the majority of Danish clinical samples in 2007. The region shown corresponds to the portion of the sequence contained within the rectangle in panel (a). The region of the M135R gene which is predicted to be translated in a different reading frame, downstream of the insertion of a G nucleotide, is indicated in red in panel (a). The termination codons (TAA) for the wt and mutant M135R gene products are indicated in bold capitals.

Mentions: The region of the myxoma virus M029L gene amplified in the diagnostic qPCR assay is quite short (119 bp). In order to obtain some indication of whether all the cases of myxomatosis in Denmark during 2007 were caused by the same strain of virus, the sequence of a larger region of the genome, from 13 selected samples, was examined. For this purpose, the M135R gene was chosen as a target since it had recently been reported to be an important virulence factor [8]. The DNA preparations from these different clinical samples (which had each been shown to contain high levels of myxoma virus DNA using the qPCR assay described above) plus samples from a myxoma vaccine (Cunivak) and a tissue culture grown sample of myxoma virus were used as the template in standard PCRs to amplify the gene encoding the M135R protein. Each of the tested clinical samples, as well as the vaccine and the cell culture grown virus, generated the expected product in this analysis, supporting the specificity of the qPCR assay which had targeted a completely different region of the genome. The amplified products (ca.650 bp) were then sequenced using the primers used for the PCR reaction (see Figure 2, panel a). The PCR products obtained from the commercial myxoma vaccine and from three of the clinical samples (which each originated from Lolland in south-eastern Denmark, e.g. sample 8770, see Table 3) had sequences which are identical to the published myxoma virus M135R gene sequence [4]. A sample obtained from an archived cell culture grown virus had a single nt substitution (C to T) which encoded a change of amino acid residue 141 from His (H) to Tyr (Y). However, unexpectedly, the ten other recent Danish samples analysed, predominantly obtained from different locations in Eastern Denmark around Copenhagen, each had the same single nucleotide insertion within the M135R coding sequence compared to the published sequence. This mutation resulted in a run of 5 G's being expanded to 6 G's and this insertion was very clear in the original sequence traces (see Figure 2, panels b and c). This sequence change results in a shift in reading frame after the translation of amino acid residue 19 (out of a total of 178 residues for the wt protein). The frame-shift is predicted to result in protein synthesis termination after a total of 40 codons have been translated. The C-terminal region of this aberrant product is predicted to be comprised of amino acid residues decoded in a different reading frame from the wt protein (see Figure 2, panel a).


Detection of myxoma viruses encoding a defective M135R gene from clinical cases of myxomatosis; possible implications for the role of the M135R protein as a virulence factor.

Belsham GJ, Polacek C, Breum SØ, Larsen LE, Bøtner A - Virol. J. (2010)

Evidence for a frame-shift mutation within the M135R gene from Danish samples of myxoma virus. Panel (a). The nucleotide sequence of the myxoma virus M135R gene, plus flanking sequences, is shown. Primers M135Rfor and M135Rrev (see Table 2), indicated in bold italics, were used to amplify by PCR the M135R gene, together with some flanking sequences at both termini, and these fragments were sequenced directly. The myxoma virus sequences included in primers M135RXFOR and M135RAPAREV (Table 2) for amplifying the coding sequence for M135R are underlined. The initiation and termination codons within these primers are indicated in bold capitals. The region of the gene in which a frame-shift mutation was present in certain virus samples is indicated within a rectangle. Panels (b) and (c.) Sequence traces obtained by analysis of a wt M135R gene sequence (as in the myxoma vaccine, panel (b)) and the mutant (+G) form (panel (c)) found in the majority of Danish clinical samples in 2007. The region shown corresponds to the portion of the sequence contained within the rectangle in panel (a). The region of the M135R gene which is predicted to be translated in a different reading frame, downstream of the insertion of a G nucleotide, is indicated in red in panel (a). The termination codons (TAA) for the wt and mutant M135R gene products are indicated in bold capitals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2820016&req=5

Figure 2: Evidence for a frame-shift mutation within the M135R gene from Danish samples of myxoma virus. Panel (a). The nucleotide sequence of the myxoma virus M135R gene, plus flanking sequences, is shown. Primers M135Rfor and M135Rrev (see Table 2), indicated in bold italics, were used to amplify by PCR the M135R gene, together with some flanking sequences at both termini, and these fragments were sequenced directly. The myxoma virus sequences included in primers M135RXFOR and M135RAPAREV (Table 2) for amplifying the coding sequence for M135R are underlined. The initiation and termination codons within these primers are indicated in bold capitals. The region of the gene in which a frame-shift mutation was present in certain virus samples is indicated within a rectangle. Panels (b) and (c.) Sequence traces obtained by analysis of a wt M135R gene sequence (as in the myxoma vaccine, panel (b)) and the mutant (+G) form (panel (c)) found in the majority of Danish clinical samples in 2007. The region shown corresponds to the portion of the sequence contained within the rectangle in panel (a). The region of the M135R gene which is predicted to be translated in a different reading frame, downstream of the insertion of a G nucleotide, is indicated in red in panel (a). The termination codons (TAA) for the wt and mutant M135R gene products are indicated in bold capitals.
Mentions: The region of the myxoma virus M029L gene amplified in the diagnostic qPCR assay is quite short (119 bp). In order to obtain some indication of whether all the cases of myxomatosis in Denmark during 2007 were caused by the same strain of virus, the sequence of a larger region of the genome, from 13 selected samples, was examined. For this purpose, the M135R gene was chosen as a target since it had recently been reported to be an important virulence factor [8]. The DNA preparations from these different clinical samples (which had each been shown to contain high levels of myxoma virus DNA using the qPCR assay described above) plus samples from a myxoma vaccine (Cunivak) and a tissue culture grown sample of myxoma virus were used as the template in standard PCRs to amplify the gene encoding the M135R protein. Each of the tested clinical samples, as well as the vaccine and the cell culture grown virus, generated the expected product in this analysis, supporting the specificity of the qPCR assay which had targeted a completely different region of the genome. The amplified products (ca.650 bp) were then sequenced using the primers used for the PCR reaction (see Figure 2, panel a). The PCR products obtained from the commercial myxoma vaccine and from three of the clinical samples (which each originated from Lolland in south-eastern Denmark, e.g. sample 8770, see Table 3) had sequences which are identical to the published myxoma virus M135R gene sequence [4]. A sample obtained from an archived cell culture grown virus had a single nt substitution (C to T) which encoded a change of amino acid residue 141 from His (H) to Tyr (Y). However, unexpectedly, the ten other recent Danish samples analysed, predominantly obtained from different locations in Eastern Denmark around Copenhagen, each had the same single nucleotide insertion within the M135R coding sequence compared to the published sequence. This mutation resulted in a run of 5 G's being expanded to 6 G's and this insertion was very clear in the original sequence traces (see Figure 2, panels b and c). This sequence change results in a shift in reading frame after the translation of amino acid residue 19 (out of a total of 178 residues for the wt protein). The frame-shift is predicted to result in protein synthesis termination after a total of 40 codons have been translated. The C-terminal region of this aberrant product is predicted to be comprised of amino acid residues decoded in a different reading frame from the wt protein (see Figure 2, panel a).

Bottom Line: An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007.Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor.This frame-shift mutation results in expression of a greatly truncated product.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark. grbe@vet.dtu.dk

ABSTRACT

Background: Myxoma virus is a member of the Poxviridae and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy.

Results: An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain (in the genes M009L, M036L and M148R) were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature.

Conclusions: It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis.

Show MeSH
Related in: MedlinePlus