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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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Effect of the frame-shift mutation on expression of the M135R gene product. Panel (a). CAT-M135R fusion genes were constructed to demonstrate the effect of the +G insertion within the M135R gene on protein expression. Plasmids containing either the wt (pCATM135Rwt) or the frame-shift variant (pCATM135R+G) of the M135R sequence were constructed. The expected products from the plasmids containing just the CAT open reading frame (ORF) (i), the CAT ORF fused to the wt M135R ORF (ii) and CAT fused to the mutant M135R+G sequence (iii) are illustrated. Panel (b). The plasmids shown in panel (a) were transfected into BHK cells that had been infected with the recombinant vaccinia virus vTF7-3 which expresses the T7 RNA polymerase. Cell extracts were prepared 20 h later and analysed by SDS-PAGE and immunoblotting using rabbit anti-CAT antibodies. Detection was achieved using peroxidase-labelled anti-rabbit IgG and chemiluminescence reagents with a BioRad Chemidoc XRS. Lane 1, pGEMCATBX (without a termination codon at the end of CAT but translation terminates within the vector sequence); lane 2, pGEMCATBS (with a termination codon, see panel (a)); lanes 3 and 4, plasmids (pCATM135Rwt) containing the fused CAT and wt M135R ORFs (n.b., there is no termination codon at the end of the CAT sequence); lanes 5 and 6, plasmids (pCATM135Rwt+G) containing the fused CAT and mutant M135R+G sequences and lane 7, No DNA (negative control). The migration of molecular weight markers is indicated. The products corresponding to those expected from constructs (i), (ii) and (iii) shown in panel (a) are marked.
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Figure 3: Effect of the frame-shift mutation on expression of the M135R gene product. Panel (a). CAT-M135R fusion genes were constructed to demonstrate the effect of the +G insertion within the M135R gene on protein expression. Plasmids containing either the wt (pCATM135Rwt) or the frame-shift variant (pCATM135R+G) of the M135R sequence were constructed. The expected products from the plasmids containing just the CAT open reading frame (ORF) (i), the CAT ORF fused to the wt M135R ORF (ii) and CAT fused to the mutant M135R+G sequence (iii) are illustrated. Panel (b). The plasmids shown in panel (a) were transfected into BHK cells that had been infected with the recombinant vaccinia virus vTF7-3 which expresses the T7 RNA polymerase. Cell extracts were prepared 20 h later and analysed by SDS-PAGE and immunoblotting using rabbit anti-CAT antibodies. Detection was achieved using peroxidase-labelled anti-rabbit IgG and chemiluminescence reagents with a BioRad Chemidoc XRS. Lane 1, pGEMCATBX (without a termination codon at the end of CAT but translation terminates within the vector sequence); lane 2, pGEMCATBS (with a termination codon, see panel (a)); lanes 3 and 4, plasmids (pCATM135Rwt) containing the fused CAT and wt M135R ORFs (n.b., there is no termination codon at the end of the CAT sequence); lanes 5 and 6, plasmids (pCATM135Rwt+G) containing the fused CAT and mutant M135R+G sequences and lane 7, No DNA (negative control). The migration of molecular weight markers is indicated. The products corresponding to those expected from constructs (i), (ii) and (iii) shown in panel (a) are marked.

Mentions: On the basis of the sequencing data, it was apparent that DNA fragments containing the insertion of a single nucleotide within the M135R coding sequence should encode a very different protein than the wt sequence (see Figure 2, panel a). In order to confirm that this single nucleotide insertion was not a sequencing or PCR artefact, new independent PCR products derived from the viral DNA preparations were made using different primers (M135RXFOR and M135RAPAREV, see Table 2). Sequencing of these new amplicons confirmed the presence of the single G insertion in the majority of the Danish field samples analysed (data not shown). These primers were also designed to allow the fusion of these amplified myxoma virus sequences downstream of the CAT coding sequence (lacking a termination codon, as in pGEMCATBX, see Figure 3) in order to express CAT-M135R fusion proteins.


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)

Effect of the frame-shift mutation on expression of the M135R gene product. Panel (a). CAT-M135R fusion genes were constructed to demonstrate the effect of the +G insertion within the M135R gene on protein expression. Plasmids containing either the wt (pCATM135Rwt) or the frame-shift variant (pCATM135R+G) of the M135R sequence were constructed. The expected products from the plasmids containing just the CAT open reading frame (ORF) (i), the CAT ORF fused to the wt M135R ORF (ii) and CAT fused to the mutant M135R+G sequence (iii) are illustrated. Panel (b). The plasmids shown in panel (a) were transfected into BHK cells that had been infected with the recombinant vaccinia virus vTF7-3 which expresses the T7 RNA polymerase. Cell extracts were prepared 20 h later and analysed by SDS-PAGE and immunoblotting using rabbit anti-CAT antibodies. Detection was achieved using peroxidase-labelled anti-rabbit IgG and chemiluminescence reagents with a BioRad Chemidoc XRS. Lane 1, pGEMCATBX (without a termination codon at the end of CAT but translation terminates within the vector sequence); lane 2, pGEMCATBS (with a termination codon, see panel (a)); lanes 3 and 4, plasmids (pCATM135Rwt) containing the fused CAT and wt M135R ORFs (n.b., there is no termination codon at the end of the CAT sequence); lanes 5 and 6, plasmids (pCATM135Rwt+G) containing the fused CAT and mutant M135R+G sequences and lane 7, No DNA (negative control). The migration of molecular weight markers is indicated. The products corresponding to those expected from constructs (i), (ii) and (iii) shown in panel (a) are marked.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Effect of the frame-shift mutation on expression of the M135R gene product. Panel (a). CAT-M135R fusion genes were constructed to demonstrate the effect of the +G insertion within the M135R gene on protein expression. Plasmids containing either the wt (pCATM135Rwt) or the frame-shift variant (pCATM135R+G) of the M135R sequence were constructed. The expected products from the plasmids containing just the CAT open reading frame (ORF) (i), the CAT ORF fused to the wt M135R ORF (ii) and CAT fused to the mutant M135R+G sequence (iii) are illustrated. Panel (b). The plasmids shown in panel (a) were transfected into BHK cells that had been infected with the recombinant vaccinia virus vTF7-3 which expresses the T7 RNA polymerase. Cell extracts were prepared 20 h later and analysed by SDS-PAGE and immunoblotting using rabbit anti-CAT antibodies. Detection was achieved using peroxidase-labelled anti-rabbit IgG and chemiluminescence reagents with a BioRad Chemidoc XRS. Lane 1, pGEMCATBX (without a termination codon at the end of CAT but translation terminates within the vector sequence); lane 2, pGEMCATBS (with a termination codon, see panel (a)); lanes 3 and 4, plasmids (pCATM135Rwt) containing the fused CAT and wt M135R ORFs (n.b., there is no termination codon at the end of the CAT sequence); lanes 5 and 6, plasmids (pCATM135Rwt+G) containing the fused CAT and mutant M135R+G sequences and lane 7, No DNA (negative control). The migration of molecular weight markers is indicated. The products corresponding to those expected from constructs (i), (ii) and (iii) shown in panel (a) are marked.
Mentions: On the basis of the sequencing data, it was apparent that DNA fragments containing the insertion of a single nucleotide within the M135R coding sequence should encode a very different protein than the wt sequence (see Figure 2, panel a). In order to confirm that this single nucleotide insertion was not a sequencing or PCR artefact, new independent PCR products derived from the viral DNA preparations were made using different primers (M135RXFOR and M135RAPAREV, see Table 2). Sequencing of these new amplicons confirmed the presence of the single G insertion in the majority of the Danish field samples analysed (data not shown). These primers were also designed to allow the fusion of these amplified myxoma virus sequences downstream of the CAT coding sequence (lacking a termination codon, as in pGEMCATBX, see Figure 3) in order to express CAT-M135R fusion proteins.

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