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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

Alignment of probes RABVD1-P (a) and LysGT6-P (b) with a RABV strain isolated from a raccoon dog from Poland. Matches in nondegenerated positions are displayed as dots. Matches in wobbled positions are highlighted in yellow. Mismatches are highlighted in turquoise.
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fig3: Alignment of probes RABVD1-P (a) and LysGT6-P (b) with a RABV strain isolated from a raccoon dog from Poland. Matches in nondegenerated positions are displayed as dots. Matches in wobbled positions are highlighted in yellow. Mismatches are highlighted in turquoise.

Mentions: A total of 31 lyssaviruses (19 identified as classical RABV, 7 as EBLV-1, 4 as EBLV-2, and 1 as DUVV) were used to test the diagnostic performance of the PCR (Table 1). All viruses used were detected with the expected band size in the hnRT-PCR technique (Table 4). The specificity of the amplified product was confirmed as RABV, EBLV-1, EBLV-2, or DUVV by sequencing in each case. Host GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was detected in all of the cell culture supernatants and mouse brain suspensions as internal control, indicating that sample material was present and RNA isolation, reverse transcription, and amplification were not inhibited. All samples were also tested positive by the real-time RT-PCR assay (Table 4). Sendai virus as internal control for inhibition was run in parallel using the same cycling conditions. In the real-time RT-PCR (NDWD) with an annealing temperature of 50°C, some samples were detected with more than one probe. With the exception of the sample derived from a raccoon dog from Poland, the probe for the homologous genotype always yielded the lowest CT-value. In this exceptional case, the probe for the detection of genotype 6 was slightly lower than the one for genotype 1 (15.0 versus 16.1; Table 4). Comparison of these two probes with the sequence of the rabies variant in question showed 1 and 2 mismatches with the RABVD1-P and LysGT6-P probe, respectively (Figure 3). Duvenhage virus, also designated as genotype 4, was detectable with the probe adapted for genotype 6 in spite of 3 mismatches (LysGT6-P). The probe LysGT6-P was able to detect synthetic DNA fragments of BBLV, ARAV, and IRKV, although containing one or two mismatches, respectively. The synthetic DNA fragments of KHUV, ABLV, LBV, MOKV, SHIBV, and WCBV, which exhibited from four (ABLV/KHUV) up to ten (WCBV) mismatches to the best fitting probe for GT1, GT6, or GT5, respectively, were all detected with the corresponding species-specific new probe (Table 2). 105.2 copies of the synthetic DNA were detected at CT-values of 27.9–33.5 for ABLV, MOKV, SHIBV, and WCBV (not shown). The efficiency of detection of KHUV, which showed an atypical amplification plot at the highest number of 1010.2 copies of the synthetic DNA, and that of LBV was lower with CT-values of 40.0 and 40.5 at a copy number of 105.2, respectively (not shown). Multiplexing of combinations of these probes (ivvLBV-P (FAM: 6-carboxyfluorescein), ivvKhujand-P (YY: Yakima Yellow), and ivvSBLV2-P (Cy5: Cyanine 5), as well as ivvWCBV-P (FAM), ivvMok-P (YY), and ivvABLV2n-P (Cy5)) as for the species 1, 5, and 6 was not successful.


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

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

Alignment of probes RABVD1-P (a) and LysGT6-P (b) with a RABV strain isolated from a raccoon dog from Poland. Matches in nondegenerated positions are displayed as dots. Matches in wobbled positions are highlighted in yellow. Mismatches are highlighted in turquoise.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4590848&req=5

fig3: Alignment of probes RABVD1-P (a) and LysGT6-P (b) with a RABV strain isolated from a raccoon dog from Poland. Matches in nondegenerated positions are displayed as dots. Matches in wobbled positions are highlighted in yellow. Mismatches are highlighted in turquoise.
Mentions: A total of 31 lyssaviruses (19 identified as classical RABV, 7 as EBLV-1, 4 as EBLV-2, and 1 as DUVV) were used to test the diagnostic performance of the PCR (Table 1). All viruses used were detected with the expected band size in the hnRT-PCR technique (Table 4). The specificity of the amplified product was confirmed as RABV, EBLV-1, EBLV-2, or DUVV by sequencing in each case. Host GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was detected in all of the cell culture supernatants and mouse brain suspensions as internal control, indicating that sample material was present and RNA isolation, reverse transcription, and amplification were not inhibited. All samples were also tested positive by the real-time RT-PCR assay (Table 4). Sendai virus as internal control for inhibition was run in parallel using the same cycling conditions. In the real-time RT-PCR (NDWD) with an annealing temperature of 50°C, some samples were detected with more than one probe. With the exception of the sample derived from a raccoon dog from Poland, the probe for the homologous genotype always yielded the lowest CT-value. In this exceptional case, the probe for the detection of genotype 6 was slightly lower than the one for genotype 1 (15.0 versus 16.1; Table 4). Comparison of these two probes with the sequence of the rabies variant in question showed 1 and 2 mismatches with the RABVD1-P and LysGT6-P probe, respectively (Figure 3). Duvenhage virus, also designated as genotype 4, was detectable with the probe adapted for genotype 6 in spite of 3 mismatches (LysGT6-P). The probe LysGT6-P was able to detect synthetic DNA fragments of BBLV, ARAV, and IRKV, although containing one or two mismatches, respectively. The synthetic DNA fragments of KHUV, ABLV, LBV, MOKV, SHIBV, and WCBV, which exhibited from four (ABLV/KHUV) up to ten (WCBV) mismatches to the best fitting probe for GT1, GT6, or GT5, respectively, were all detected with the corresponding species-specific new probe (Table 2). 105.2 copies of the synthetic DNA were detected at CT-values of 27.9–33.5 for ABLV, MOKV, SHIBV, and WCBV (not shown). The efficiency of detection of KHUV, which showed an atypical amplification plot at the highest number of 1010.2 copies of the synthetic DNA, and that of LBV was lower with CT-values of 40.0 and 40.5 at a copy number of 105.2, respectively (not shown). Multiplexing of combinations of these probes (ivvLBV-P (FAM: 6-carboxyfluorescein), ivvKhujand-P (YY: Yakima Yellow), and ivvSBLV2-P (Cy5: Cyanine 5), as well as ivvWCBV-P (FAM), ivvMok-P (YY), and ivvABLV2n-P (Cy5)) as for the species 1, 5, and 6 was not successful.

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