Dataset Information

A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

ABSTRACT: Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), a fatal disease of boid snakes with an economic and ecological impact, as it affects both captive and wild constrictor snakes. The clinical picture of BIBD is highly variable but often only limited. Intracytoplasmic inclusion bodies (IB), which develop in most cell types including blood cells, are the pathognomonic hallmark of BIBD; their detection represents the diagnostic gold standard of the disease. However, IBs are not consistently present in clinically healthy reptarenavirus carriers, which can, if undetected, lead to and maintain the spread of the disease within and between snake populations. Sensitive viral detection tools are required for screening and control purposes; however, the genetic diversity of reptarenaviruses hampers the reverse transcription (RT) PCR-based diagnostics. Here, we describe a multiplex RT-PCR approach for the molecular diagnosis of reptarenavirus infection in blood samples. The method allows the detection of a wide range of reptarenaviruses with the detection limit reaching 40 copies per microliter of blood. Using 245 blood samples with a reference RT-PCR result, we show that the technique performs as well as the segment-specific RT-PCRs in our earlier studies. It can identify virus carriers and serve to limit reptarenavirus spreading in captive snake collections.

SUBMITTER: Baggio F

PROVIDER: S-EPMC10747477 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

Baggio Francesca F Hetzel Udo U Prähauser Barbara B Dervas Eva E Michalopoulou Eleni E Thiele Tanja T Kipar Anja A Hepojoki Jussi J

Viruses 20231125 12

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), a fatal disease of boid snakes with an economic and ecological impact, as it affects both captive and wild constrictor snakes. The clinical picture of BIBD is highly variable but often only limited. Intracytoplasmic inclusion bodies (IB), which develop in most cell types including blood cells, are the pathognomonic hallmark of BIBD; their detection represents the diagnostic gold standard of the disease. However, IBs are not consistently ...[more]

PMID: 38140554

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Project description:Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well serotyping assay in near future for rapid identification of dengue suspected sample screening.

Project description:Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log?? cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

Dataset Information

A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

Publications

A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

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