A strand-specific real-time quantitative RT-PCR assay for distinguishing the genomic and antigenomic RNAs of Rift Valley fever phlebovirus

Breanna Tercero, Kaori Terasaki, Keisuke Nakagawa, Krishna Narayanan, Shinji Makino

Research output: Contribution to journalArticle

Abstract

Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral ‘tag’ sequence at the 5’ end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay. We used this assay system to examine the kinetics of intracellular accumulation of genomic and antigenomic viral RNAs in mammalian cells infected with the MP-12 strain of RVFV. The genomic RNA copy numbers, for all three viral RNA segments, were higher than that of their corresponding antigenomic RNAs throughout the time-course of infection, with a notable exception, wherein the M segment genomic and antigenomic RNAs exhibited similar copy numbers at specific times post-infection. Overall, this assay system could be a useful tool to gain an insight into the mechanisms of RNA replication and packaging in RVFV.

Original languageEnglish (US)
Article number113701
JournalJournal of Virological Methods
Volume272
DOIs
StatePublished - Oct 1 2019

Fingerprint

Phlebovirus
Rift Valley Fever
Reverse Transcription
RNA
Polymerase Chain Reaction
Viral RNA
Complementary DNA
Product Packaging
Infection
Real-Time Polymerase Chain Reaction
Genome

Keywords

  • Rift Valley fever phlebovirus
  • RNA replication
  • Strand-specific quantitative PCR

ASJC Scopus subject areas

  • Virology

Cite this

A strand-specific real-time quantitative RT-PCR assay for distinguishing the genomic and antigenomic RNAs of Rift Valley fever phlebovirus. / Tercero, Breanna; Terasaki, Kaori; Nakagawa, Keisuke; Narayanan, Krishna; Makino, Shinji.

In: Journal of Virological Methods, Vol. 272, 113701, 01.10.2019.

Research output: Contribution to journalArticle

@article{e223ec05426e4f89bab2458718b22066,
title = "A strand-specific real-time quantitative RT-PCR assay for distinguishing the genomic and antigenomic RNAs of Rift Valley fever phlebovirus",
abstract = "Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral ‘tag’ sequence at the 5’ end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay. We used this assay system to examine the kinetics of intracellular accumulation of genomic and antigenomic viral RNAs in mammalian cells infected with the MP-12 strain of RVFV. The genomic RNA copy numbers, for all three viral RNA segments, were higher than that of their corresponding antigenomic RNAs throughout the time-course of infection, with a notable exception, wherein the M segment genomic and antigenomic RNAs exhibited similar copy numbers at specific times post-infection. Overall, this assay system could be a useful tool to gain an insight into the mechanisms of RNA replication and packaging in RVFV.",
keywords = "Rift Valley fever phlebovirus, RNA replication, Strand-specific quantitative PCR",
author = "Breanna Tercero and Kaori Terasaki and Keisuke Nakagawa and Krishna Narayanan and Shinji Makino",
year = "2019",
month = "10",
day = "1",
doi = "10.1016/j.jviromet.2019.113701",
language = "English (US)",
volume = "272",
journal = "Journal of Virological Methods",
issn = "0166-0934",
publisher = "Elsevier",

}

TY - JOUR

T1 - A strand-specific real-time quantitative RT-PCR assay for distinguishing the genomic and antigenomic RNAs of Rift Valley fever phlebovirus

AU - Tercero, Breanna

AU - Terasaki, Kaori

AU - Nakagawa, Keisuke

AU - Narayanan, Krishna

AU - Makino, Shinji

PY - 2019/10/1

Y1 - 2019/10/1

N2 - Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral ‘tag’ sequence at the 5’ end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay. We used this assay system to examine the kinetics of intracellular accumulation of genomic and antigenomic viral RNAs in mammalian cells infected with the MP-12 strain of RVFV. The genomic RNA copy numbers, for all three viral RNA segments, were higher than that of their corresponding antigenomic RNAs throughout the time-course of infection, with a notable exception, wherein the M segment genomic and antigenomic RNAs exhibited similar copy numbers at specific times post-infection. Overall, this assay system could be a useful tool to gain an insight into the mechanisms of RNA replication and packaging in RVFV.

AB - Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral ‘tag’ sequence at the 5’ end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay. We used this assay system to examine the kinetics of intracellular accumulation of genomic and antigenomic viral RNAs in mammalian cells infected with the MP-12 strain of RVFV. The genomic RNA copy numbers, for all three viral RNA segments, were higher than that of their corresponding antigenomic RNAs throughout the time-course of infection, with a notable exception, wherein the M segment genomic and antigenomic RNAs exhibited similar copy numbers at specific times post-infection. Overall, this assay system could be a useful tool to gain an insight into the mechanisms of RNA replication and packaging in RVFV.

KW - Rift Valley fever phlebovirus

KW - RNA replication

KW - Strand-specific quantitative PCR

UR - http://www.scopus.com/inward/record.url?scp=85069704745&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85069704745&partnerID=8YFLogxK

U2 - 10.1016/j.jviromet.2019.113701

DO - 10.1016/j.jviromet.2019.113701

M3 - Article

AN - SCOPUS:85069704745

VL - 272

JO - Journal of Virological Methods

JF - Journal of Virological Methods

SN - 0166-0934

M1 - 113701

ER -