Dataset Information

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

ABSTRACT: The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017-2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabies virus sequences from one sample were distinct from all positive samples processed at PABOL within two weeks, ruling out cross-contamination. Levels of rabies virus antigen and RNA were low in all brain structures tested, but were higher in brain stem and rostral spinal cord than in cerebellum, hippocampus or cortex. Taken together, the low level of rabies virus combined with higher abundance in more caudal brain structures suggest early infection. These cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR for low positive cases.

SUBMITTER: Dettinger L

PROVIDER: S-EPMC9504839 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Publications

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

Dettinger Lisa L Gigante Crystal M CM Sellard Maria M Seiders Melanie M Patel Puja P Orciari Lillian A LA Yager Pamela P Lute James J Regec Annette A Li Yu Y Xia Dongxiang D

Viruses 20220823 9

The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017-2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabie ...[more]

PMID: 36146650

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Project description:Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

Dataset Information

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

Publications

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

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