Comparative Susceptibility of Madin-Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens.

Matthew Suderman,Neil Pople,Tamiru N. Alkie,Davor Ojkic,Mariko Moniwa,Andre Broes,Yohannes Berhane

doi:10.3390/v13122346

Abstract

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Highlights

Swine-origin influenza A viruses (IAV-S) cause a respiratory disease in pigs which is often complicated by infection with secondary bacterial respiratory pathogens [1]
The objective of the current study was to compare the susceptibility of conventional Madin–Darby canine kidney (MDCK) cell line and MDCK-derived cell lines for the primary isolation of IAV-S from various clinical specimens such as nasal swabs, lung samples and oral fluids that were collected from Canadian swine herds
All cell culture lysate supernatants were tested for IAV matrix gene using real-time RT-PCR to detect viral RNA and samples were confirmed positive for virus isolation when cycle threshold (Ct) values from lysates were lower than the original corresponding specimens

Summary

Introduction

Swine-origin influenza A viruses (IAV-S) cause a respiratory disease in pigs which is often complicated by infection with secondary bacterial respiratory pathogens [1]. Three subtypes of IAV-S, namely H1N1, H1N2 and H3N2 viruses have been isolated from pigs worldwide [3,4] and continue to evolve genetically and antigenically in swine herds [2,3]. The first evidence of isolation of influenza A viruses (IAVs) from pigs dates back to 1930 [5]. Since embryonating chicken eggs (ECE) and mammalian and avian origin primary and continuous cells have been used for the recovery of IAV-S from pigs [6,7,8,9,10,11,12]. While virus isolation in embryonated chicken eggs and many molecular techniques for IAV-S detection are available, isolation and characterization of IAV-S in continuous cell culture systems has some advantages and additional benefits [13]

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