Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan;13(1):71-82.
doi: 10.1111/irv.12613. Epub 2018 Oct 22.

A newly developed tetraplex real-time RT-PCR for simultaneous screening of influenza virus types A, B, C and D

Dinah Henritzi  1 , Bernd Hoffmann  1 , Silke Wacheck  2 , Stefan Pesch  2 , Georg Herrler  3 , Martin Beer  1 , Timm C Harder  1
Affiliations

A newly developed tetraplex real-time RT-PCR for simultaneous screening of influenza virus types A, B, C and D

Dinah Henritzi et al. Influenza Other Respir Viruses. 2019 Jan.

Abstract

Background: Human- or avian-to-swine transmissions have founded several autonomously circulating influenza A virus (IAV) lineages in swine populations that cause economically important respiratory disease. Little is known on other human influenza virus types, like B (IBV) and C (ICV) in European swine, and of the recently detected novel animal influenza virus type D (IDV).

Objectives: Development of a cost-effective diagnostic tool for large-scale surveillance programmes targeting all four influenza virus types.

Methods: An influenza ABCD tetraplex real-time RT-PCR (RT-qPCR) was developed in the frame of this study. A selection of reference virus strains and more than 4000 porcine samples from a passive IAV surveillance programme in European swine with acute respiratory disease were examined.

Results: Two IBV, a single IDV but no ICV infections were identified by tetraplex RT-qPCR. IBV and IDV results were confirmed by conventional RT-PCR and partial sequence analysis.

Conclusions: The tetraplex RT-qPCR proved fit for purpose as a sensitive, specific and high-throughput tool to study influenza virus transmission at the human-animal interface. Complementing close-meshed active virological and serological surveillance is required to better understand the true incidence and prevalence of influenza virus type B, C and D infections in swine.

Keywords: European surveillance; influenza virus types A, B, C and D; multiplex RT-qPCR; swine.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Collection sites of the field samples. Maps were calculated based on latitude and longitude data with the online tool Microreact (http://www.edge.microreact.org/).41 Part A shows all collection sites distributed over Middle Europe, and Part B shows the location of the positive samples (orange: IBV; green: IDV)
Figure 2
Figure 2
Analysis of the detection limit of the ABCD tetraplex RT‐qPCR based on the mean value of triplicate serial 10‐fold dilutions of transcribed viral RNA ranging from 108 to 1 copies/reaction (A1‐D1). Linear regression analysis revealed correlation coefficients (R2) and slope values (A2‐D2). A, IAV, FAM signal; B, IBV, ROX signal; C, ICV, Cy5 signal; D, IDV, HEX signal
Figure 3
Figure 3
Analysis of the sensitivity of the ABCD tetraplex RT‐qPCR in simulated co‐infections. IAV‐positive swine nasal samples with cq‐values of 20, 25 and 30, and IAV‐negative swine samples and PBS were mixed with RNA of either IBV (A), ICV (B) or IDV (C) each of Cq = 30. Mixtures were analysed three times in the RT‐qPCR
Figure 4
Figure 4
Comparison of the IAV cq‐values from the initial (monoplex‐) IAV screening and the cq‐values of the IAV (BCD)‐tetraplex RT‐qPCR. First screening was done within a week of sample receipt; thereafter, RNA was stored at −80°C up to 26 months. Cut‐off for the ABCD tetraplex RT‐qPCR was set at cq 40
See this image and copyright information in PMC

References

    1. Rajao DS, Anderson TK, Gauger PC, Vincent AL. Pathogenesis and vaccination of influenza A virus in swine. Curr Top Microbiol Immunol. 2014;385:307‐326. - PubMed
    1. Vincent A, Awada L, Brown I, et al. Review of influenza A virus in swine worldwide: a call for increased surveillance and research. Zoonoses and Public Health. 2014a;61(1):4‐17. - PubMed
    1. Vincent AL, Lager KM, Anderson TK. A brief introduction to influenza A virus in swine. Methods Mol Biol. 2014b;1161:243‐258. - PubMed
    1. Pleschka S. Overview of influenza viruses. Curr Top Microbiol Immunol. 2013;370:1‐20. - PubMed
    1. Tong S, Zhu X, Li Y, et al. New world bats harbor diverse influenza A viruses. PLoS Pathog. 2013;9(10):e1003657. - PMC - PubMed

Publication types

MeSH terms

Associated data