Identification of genetic evidence for dobrava virus spillover in rodents by nested reverse transcription (RT)-PCR and TaqMan RT-PCR - PubMed (original) (raw)

Identification of genetic evidence for dobrava virus spillover in rodents by nested reverse transcription (RT)-PCR and TaqMan RT-PCR

Manfred Weidmann et al. J Clin Microbiol. 2005 Feb.

Abstract

A survey of 158 rodents caught in the Czech Republic identified Dobrava virus sequences closely related to that of the Dobrava virus type strain in Apodemus sylvaticus and Mus musculus rodents. The identity of A. sylvaticus was unequivocally confirmed by random amplified polymorphic DNA analysis. The data seem to indicate hantavirus spillover from Apodemus flavicollis to other rodents.

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Figures

FIG. 1.

(Top panels) Comparison of the sensitivity of the nested PCR by use of the published protocol (left) and a touchdown protocol (right). The values above each lane indicate the number of transcribed RNA molecules tested. n, negative control. (Bottom panel) RAPD analysis of mouse tissues. RAPD analysis was done with primers OPA01 and OPA08 (Operon Technologies). The results obtained with primer OPA01 are shown. The patterns in lane 1 and lanes S are identical, as are the patterns in lane 2 and lanes F. The arrows indicate prominent bands specifically identifying the species with the RAPD pattern. Lanes: L, 100-bp DNA ladder; C, negative control; S, positive control for A. sylvaticus; F, positive control for A. flavicollis; M, positive control for A. microps; 1, sample 126 from which the DOBV sequence was amplified; 2, sample 133, a DOBV-negative sample from A. flavicollis.

FIG. 2.

(A) Phylogram rooted to DOBV strain East Slovakia-856-Aa. Sequences were aligned in reference to nucleotide positions 720 to 1041 of the DOBV-Af type strain (GenBank accession number L41916). Branch distances correspond to sequence divergence. The GenBank accession numbers of the sequences used for the phylogram are (respectively, from top to bottom) AJ578082, AJ578081, AF442623, AF442622, DVI410619, DVI410615, and AJ269554, L41916, AJ009773, AJ009775, AJ131673, AJ131672, AF085336, AF085337, AJ269550, and AJ269549). *, sequence obtained in this study from M. musculus; **, sequence obtained in this study from A. sylvaticus. Country abbreviations are according to the international traffic code and precede the strain designations. The percent identities of the sequences in the DOBV-Af group to the original DOBV type strain are as follows: Cz Boletice 126, 99.1%; Cz Boletice 11, 98.8%; SK Slovakia-400-Af, 94.7%; GR DOBV/Ano-Poroia/AfI9/1999, 95.7%; GR DOBV/Ano-Poroia/13Af/1999, 96%; RUS As-1/Goryachiy Klyuch-2000, 90.4%; RUS P-s1223/Krasnodar-2000, 90.4%.

FIG. 3.

(A) Subtree of the DOBV-Af group rooted to the DOBV-Af type strain aligned to a partial map of central Europe; (B) correlation of the geographic distance of the sites of isolation of the divergent DOBV-Af from strains the site of isolation of the Slovenian DOBV-Af type strain to the divergence of DOBV-Af isolates from the DOBV-Af type strain.

References

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Identification of genetic evidence for dobrava virus spillover in rodents by nested reverse transcription (RT)-PCR and TaqMan RT-PCR - PubMed (original) (raw)