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.

Isolation of multiple novel paramyxoviruses from pteropid bat urine

“…Cultures displaying syncytial CPE were screened using subfamily-or genus-specific primer sets following protocols described elsewhere (Tong et al, 2008). PCR products were sequenced directly for preliminary characterization of the viral genomic sequences.…”

“…MenPV was isolated from the 2009 Cedar Grove collection and reported previously . MenPV is another known bat rubulavirus, closely related to TioPV, and was found to be the aetiological agent of a disease in pigs in Phylogenetic analysis based on the 530 bp fragment of the large (L) gene obtained using pan-Paramyxovirinae primers (Tong et al, 2008), which is the most used sequence for paramyxovirus phylogeny studies (Drexler et al, 2012), indicated that three of the four newly isolated bat rubulaviruses clustered with the two previously identified Asian bat viruses, MenPV and TioPV. However, the fourth newly isolated virus, HerPV, was more closely related to the Achimota virus 2 isolated from bats in Ghana, Africa (Fig.…”

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes

“…Both degenerate 293 and non-degenerate primers for the PCR detection of reoviruses and paramyxoviruses failed to 294 identify this new virus. Of particular note, we were unable to detect Sunshine virus using the primer 295 sets published by Tong et al (2008). One of these primer sets (PAR-F1, PAR-F2 and PAR-R) was 296 designed to detect all the members of the subfamily Paramyxovirinae while another set (PNE-F1, 297 PNE-F2 and PNE-R) was designed to detect all the members of the subfamily Pneumovirinae.…”

Broadly Reactive Pan-Paramyxovirus Reverse Transcription Polymerase Chain Reaction and Sequence Analysis for the Detection of Canine Distemper Virus in a Case of Canine Meningoencephalitis of Unknown Etiology

“…Samples were tested by the panel of panparamyxovirus RT-PCR assays using a commercial 1-step RT-PCR kit. 22 13 buffer with a final concentration of 2.0 mM MgSO 4 , 20 units of RNase inhibitor, a 5-ml aliquot of RNA extracts, and 1 unit of an RT-Taq mix. c Water was added to achieve a final volume of 50 ml.…”

“…22 This group of paramyxovirus PCR primers collectively target highly conserved amino acid sequences within each of 2 subfamilies and each of 2 genera subgroups, based on the multiple alignments of 33 deduced, nonredundant paramyxovirus L polymerase genes. This broadly reactive RT-PCR, coupled with sequence analysis and IHC, identified CDV in this canine case of MUE.…”