Sensitive and broadly reactive reverse transcription-PCR assays to detect novel paramyxoviruses

Abstract

We have developed a set of reverse transcription-PCR assays for the detection and identification of known and novel paramyxoviruses in clinical specimens. Primers were designed from the conserved motifs of the polymerase pol gene sequences to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. The consensus-degenerate hybrid oligonucleotide primer design and seminested or nested PCR assay design were used to enhance the breadth of reactivity and sensitivity of the respective assays. Using expressed RNA and 10-fold dilution series of virus-infected tissue culture isolates from different members of the family or genera, these assays were able to detect on average between 100 and 500 copies of template RNA. The assays were specific to the respective group of genera or subfamily viruses. This set of primers enhances our ability to look for novel viruses in outbreaks and diseases of unknown etiology.

FIG. 1.

Similarity plots of the aligned paramyxoviruses' genomes. The plots were obtained using an in-house program based on multiple alignments of viral genomes from 29 different strains in the Paramyxoviridae (A), 22 different strains in the Paramyxovirinae (B), and 7 different strains in the Pneumovirinae (C). The identity percentage score given on the y axis was calculated based on the exact-match percentage with a window of 25 nucleotide positions, and the window was progressively moved across the alignment in 1-nucleotide-position steps. The x axis shows the first position of the window in the multiple alignments of the viral genomes.

FIG. 2.

Amplification of RNAs from 25 different viral members in the subfamily Paramyxovirinae by one-step RT-PCR using the pan-PAR-F1/PAR-R primer pair (A) or the pan-PAR-F2/PAR-R primer pair (B). Viral names are abbreviated as shown in Table 1.

FIG. 3.

Amplification of RNAs from 14 different viral members in the genera of Henipavirus, Morbillivirus, and Respirovirus and two unclassified viral members in the subfamily Paramyxovirinae by one-step RT-PCR using the pan RES-MOR-HEN-F1/RES-MOR-HEN-R primer pair (A) or the pan RES-MOR-HEN-F2/RES-MOR-HEN-R primer pair (B). Each viral name is abbreviated as shown in Table 1.

FIG. 4.

Gel electrophoresis of amplification products of a one-step RT-PCR assay against RNA from eight different members of Avulavirus and Rubulavirus genera and one previously unclassified member of the subfamily Paramyxovirinae. The pan AVU-RUB-F1/AVU-RUB-R primer pair (A) or the pan AVU-RUB-F2/AVU-RUB-R primer pair (B) was used. Each virus gives an appropriately sized band and is identified by its abbreviation as shown in Table 1.

FIG. 5.

Gel electrophoresis of amplification products of one-step RT-PCR assays against RNA from seven different members of the subfamily Pneumovirinae. (A) Pan PNE-F1/PNE-R primer pair. (B) Pan PNE-F2/PNE-R primer pair. Each virus gives an appropriately sized band and is identified by its abbreviation as shown in Table 1.

FIG. 6.

Gel electrophoresis of amplification products with subfamily and genus group seminested RT-PCR assays showing the appropriately sized positive band for the positive control material for the respective RT-PCR assay and no appropriately sized band for negative-control material or a pool of RNA from other common respiratory pathogens, as described in Materials and Methods. The seminested RT-PCR assays are pan-PAR (Paramyxovirinae subfamily), pan RES-MOR-HEN (group of Respirovirus, Morbillivirus, and Henipavirus genera), pan-AVU-RUB (group of Avulavirus and Rubulavirus genera), and pan-PNE (Pneumovirinae subfamily). Lanes: 1, positive control; 2, negative control (water); 3, blank; 4, pool of RNA from other common respiratory pathogens.

FIG. 7.

Improved detection sensitivity by seminested RT-PCR and by genus subgroup primers with less degeneracy. (A) Seminested RT-PCR amplification of RNA extracted from 10-fold serial dilution of mumps virus stock using the pan-AVU-RUB-F1/AVU-RUB-R primer pair (1st run) and the pan-AVU-RUB-F2/AVU-RUB-R primer pair (2nd run). (B) Seminested RT-PCR amplification of RNA extracted from 10-fold serial dilution of mumps virus stock using the pan-PAR-F1/PAR-R primer pair (1st run) and the pan-PAR-F2/PAR-R primer pair (2nd run).