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Real-Time RT-PCR for the Detection of Lyssavirus Species.

Deubelbeiss A, Zahno ML, Zanoni M, Bruegger D, Zanoni R - J Vet Med (2014)

Bottom Line: For the detection of species, seven probes were developed.Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown.Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and Immunology, 3012 Berne, Switzerland.

ABSTRACT
The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic tree with 543 bp fragments of N. Phylogenetic tree obtained with MEGA5 software [35]. The length of branches (horizontal lines) corresponds to phylogenetic distance between different sequences (scale bar in substitutions per site). Numbers proximal to nodes indicate bootstrap confidence of subjacent groups. Lyssavirus strains used in this study are depicted in red. Sequences are given with GenBank or own designation followed by the two-letter country code and a two-letter code for the source species as follows: Bt, bat; Ca, cat; Ci, African civet; Dg, dog; Fx, fox; Hu, human; Jc, jackal; Ra, raccoon; Rd, raccoon dog; Ro, rodent; Sh, shrew; Sk, skunk, except for Pa, cSAD, and CV, which are standing for Pasteur strain, Street Alabama Dufferin strain, and challenge virus standard, respectively. The currently used abbreviations for species are shown next to the tree. RABV, rabies virus; ABLV, Australian bat lyssavirus; WCBV, West Caucasian bat virus; IKOV, Ikoma virus; LLEBV, Lleida bat lyssavirus; MOKV, Mokola virus; SHIBV, Shimoni bat virus; LBV, Lagos bat virus; KHUV, Khujand virus; BBLV, Bokeloh bat lyssavirus; ARAV, Aravan virus; DUVV, Duvenhage virus; EBLV, European bat lyssavirus; IRKV, Irkut virus.
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fig4: Phylogenetic tree with 543 bp fragments of N. Phylogenetic tree obtained with MEGA5 software [35]. The length of branches (horizontal lines) corresponds to phylogenetic distance between different sequences (scale bar in substitutions per site). Numbers proximal to nodes indicate bootstrap confidence of subjacent groups. Lyssavirus strains used in this study are depicted in red. Sequences are given with GenBank or own designation followed by the two-letter country code and a two-letter code for the source species as follows: Bt, bat; Ca, cat; Ci, African civet; Dg, dog; Fx, fox; Hu, human; Jc, jackal; Ra, raccoon; Rd, raccoon dog; Ro, rodent; Sh, shrew; Sk, skunk, except for Pa, cSAD, and CV, which are standing for Pasteur strain, Street Alabama Dufferin strain, and challenge virus standard, respectively. The currently used abbreviations for species are shown next to the tree. RABV, rabies virus; ABLV, Australian bat lyssavirus; WCBV, West Caucasian bat virus; IKOV, Ikoma virus; LLEBV, Lleida bat lyssavirus; MOKV, Mokola virus; SHIBV, Shimoni bat virus; LBV, Lagos bat virus; KHUV, Khujand virus; BBLV, Bokeloh bat lyssavirus; ARAV, Aravan virus; DUVV, Duvenhage virus; EBLV, European bat lyssavirus; IRKV, Irkut virus.

Mentions: The 543 bp amplification products of the nucleoprotein gene obtained in the heminested PCR reactions were sequenced (Table 1) and analysed phylogenetically with additional sequences retrieved from GenBank representing all known lyssaviruses. The similarity of the 543 bp nucleotide sequences of the nucleoprotein gene (positions 74–616 according to the Pasteur virus genome) among the lyssaviruses included ranged from 64.6% to 90.1% (Hamming distance). Similarity at the amino acid level was 68.0–95%. The resulting phylogenetic tree obtained by the neighbor-joining method implemented in the MEGA5 software is presented in Figure 4. All known genotypes were resolved with high bootstrap confidence with our samples grouping expectedly.


Real-Time RT-PCR for the Detection of Lyssavirus Species.

Deubelbeiss A, Zahno ML, Zanoni M, Bruegger D, Zanoni R - J Vet Med (2014)

Phylogenetic tree with 543 bp fragments of N. Phylogenetic tree obtained with MEGA5 software [35]. The length of branches (horizontal lines) corresponds to phylogenetic distance between different sequences (scale bar in substitutions per site). Numbers proximal to nodes indicate bootstrap confidence of subjacent groups. Lyssavirus strains used in this study are depicted in red. Sequences are given with GenBank or own designation followed by the two-letter country code and a two-letter code for the source species as follows: Bt, bat; Ca, cat; Ci, African civet; Dg, dog; Fx, fox; Hu, human; Jc, jackal; Ra, raccoon; Rd, raccoon dog; Ro, rodent; Sh, shrew; Sk, skunk, except for Pa, cSAD, and CV, which are standing for Pasteur strain, Street Alabama Dufferin strain, and challenge virus standard, respectively. The currently used abbreviations for species are shown next to the tree. RABV, rabies virus; ABLV, Australian bat lyssavirus; WCBV, West Caucasian bat virus; IKOV, Ikoma virus; LLEBV, Lleida bat lyssavirus; MOKV, Mokola virus; SHIBV, Shimoni bat virus; LBV, Lagos bat virus; KHUV, Khujand virus; BBLV, Bokeloh bat lyssavirus; ARAV, Aravan virus; DUVV, Duvenhage virus; EBLV, European bat lyssavirus; IRKV, Irkut virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Phylogenetic tree with 543 bp fragments of N. Phylogenetic tree obtained with MEGA5 software [35]. The length of branches (horizontal lines) corresponds to phylogenetic distance between different sequences (scale bar in substitutions per site). Numbers proximal to nodes indicate bootstrap confidence of subjacent groups. Lyssavirus strains used in this study are depicted in red. Sequences are given with GenBank or own designation followed by the two-letter country code and a two-letter code for the source species as follows: Bt, bat; Ca, cat; Ci, African civet; Dg, dog; Fx, fox; Hu, human; Jc, jackal; Ra, raccoon; Rd, raccoon dog; Ro, rodent; Sh, shrew; Sk, skunk, except for Pa, cSAD, and CV, which are standing for Pasteur strain, Street Alabama Dufferin strain, and challenge virus standard, respectively. The currently used abbreviations for species are shown next to the tree. RABV, rabies virus; ABLV, Australian bat lyssavirus; WCBV, West Caucasian bat virus; IKOV, Ikoma virus; LLEBV, Lleida bat lyssavirus; MOKV, Mokola virus; SHIBV, Shimoni bat virus; LBV, Lagos bat virus; KHUV, Khujand virus; BBLV, Bokeloh bat lyssavirus; ARAV, Aravan virus; DUVV, Duvenhage virus; EBLV, European bat lyssavirus; IRKV, Irkut virus.
Mentions: The 543 bp amplification products of the nucleoprotein gene obtained in the heminested PCR reactions were sequenced (Table 1) and analysed phylogenetically with additional sequences retrieved from GenBank representing all known lyssaviruses. The similarity of the 543 bp nucleotide sequences of the nucleoprotein gene (positions 74–616 according to the Pasteur virus genome) among the lyssaviruses included ranged from 64.6% to 90.1% (Hamming distance). Similarity at the amino acid level was 68.0–95%. The resulting phylogenetic tree obtained by the neighbor-joining method implemented in the MEGA5 software is presented in Figure 4. All known genotypes were resolved with high bootstrap confidence with our samples grouping expectedly.

Bottom Line: For the detection of species, seven probes were developed.Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown.Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and Immunology, 3012 Berne, Switzerland.

ABSTRACT
The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

No MeSH data available.


Related in: MedlinePlus