Rapid molecular strategy for filovirus detection and characterization

Abstract

Filoviruses have the capacity to cause lethal outbreaks of hemorrhagic fever in primates. Here we present a simple consensus reverse transcription-PCR method for filovirus recognition and characterization and demonstrate its utility with all known filovirus strains. Phylogenetic assignment is achieved by automated web-based sequence analysis of amplification products.

FIG. 1.

Phylogenetic analysis of filoviral L-gene sequences. Forty filoviral RNA polymerase nucleotide sequences (regions comprising 593 nt) were aligned with CLUSTAL W. Phylogenetic analysis was performed by using the best model of nucleotide substitution according to Modeltest, HKY 85, with correction for gamma distribution = 0.3040, using the neighbor-joining method to reconstruct the phylogenetic tree (MEGA version 3.0 software package). Statistical significance was estimated by bootstrap analysis with 1,000 pseudoreplicate data sets. Strains are denoted by genus, species, and lineage. Black triangles indicate clinical samples detected and characterized in the present study. Open circles indicate virus strains used for assay validation. Black circles indicate virus isolates not previously sequenced in this 593-nt region.

FIG. 2.

Pairwise analysis of three virus sequences randomly chosen from the clinical samples obtained in the present study. A pairwise Needleman-Wunch (NW) score was calculated for MARV-226DRC00, MARV species MUSOKE lineage; MARV-ANGOLA-474, MARV species, MUSOKE lineage; and ZEBOV-ZAIRE 1995-5004, ZEBOV species, against all other members of the database. Graphs plot the 75th-to-25th percentile rage of the NW score (box), outliers (*), maximum (⟂︁), and minimum (⊥) NW score values for each of the three viruses with respect to individual filoviral lineages.