Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

Abstract

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.

Keywords: LN34 assay; diagnosis; formalin-fixed tissue; genetic typing; lyssavirus; rabies virus.

Figure 1

Immunohistochemical (IHC) staining for RABV antigen in fixed brain tissue from the Dominican Republic (DOM) patient (A18-2173). (A) RABV antigen detection at 400× magnification. (B) Neuronal cytoplasmic inclusions at 630× magnification. (C) Negative control brain at 400× magnification. Streptavidin–biotin complex staining method using rabbit antibodies against RABV nucleoprotein, signal development with AEC chromogen (magna red), and Gills hematoxylin counterstain.

Figure 2

Sequence alignment (115 bp) from LN34 amplicons link the human case A18-2173 (labelled in red) to the RABV strain (blue) circulating mainly in the Ouest Department of Haiti (HTI). Three groups of RABV were identified based on the sequence alignment: HTI contains two groups (blue and green) and DOM has three groups (blue, green, and purple).

Figure 3

Molecular phylogenetic analysis using 115 bp sequences generated from LN34 amplicons. (A) The Bayesian evolutionary analysis sampling tree (BEAST) showed that the human rabies case (A18_2173) grouped with samples from HTI. The major nodes are labelled with the posterior probabilities. * Posterior values > 0.5 are shown at each node. (B) The neighbor joining tree agreed with the BEAST tree for the major clades and showed strains with identical sequences. The selected nodes are labelled with bootstrap values. (C) The geographic location of samples from HTI and DOM. The blue star is the location where the patient was infected.