Abstract
Human influenza viruses can be isolated efficiently from clinical samples using Madin-Darby canine kidney (MDCK) cells. However, this process is known to induce mutations in the virus as it adapts to this non-human cell-line. We performed a systematic study to record the pattern of MDCK-induced mutations observed across the whole influenza A/H3N2 genome. Seventy-seven clinical samples collected from 2009-2011 were included in the study. Two full influenza genomes were obtained for each sample: one from virus obtained directly from the clinical sample and one from the matching isolate cultured in MDCK cells. Comparison of the full-genome sequences obtained from each of these sources showed that 42% of the 77 isolates had acquired at least one MDCK-induced mutation. The presence or absence of these mutations was independent of viral load or sample origin (in-patients versus out-patients). Notably, all the five hemagglutinin missense mutations were observed at the hemaggutinin 1 domain only, particularly within or proximal to the receptor binding sites and antigenic site of the virus. Furthermore, 23% of the 77 isolates had undergone a MDCK-induced missense mutation, D151G/N, in the neuraminidase segment. This mutation has been found to be associated with reduced drug sensitivity towards the neuraminidase inhibitors and increased viral receptor binding efficiency to host cells. In contrast, none of the neuraminidase sequences obtained directly from the clinical samples contained the D151G/N mutation, suggesting that this mutation may be an indicator of MDCK culture-induced changes. These D151 mutations can confound the interpretation of the hemagglutination inhibition assay and neuraminidase inhibitor resistance results when these are based on MDCK isolates. Such isolates are currently in routine use in the WHO influenza vaccine and drug-resistance surveillance programs. Potential data interpretation miscalls can therefore be avoided by careful exclusion of such D151 mutants after further sequence analysis.
Highlights
Influenza viruses obtained from infected human host specimens can be isolated using several different cell-lines
The complete genome sequences of all the paired clinical and cultured influenza A/H3N2 (n= 77 direct from source + 77 Madin-Darby canine kidney (MDCK)-cultured = 154) were generated and subjected to an exhaustive phylogenetic analysis to screen for any artifacts and/or sequence mosaics that may have been induced by the sequencing and other laboratory methods or contaminants [18]
The results of this study demonstrate that in about 42% of clinical samples containing seasonal human influenza A/H3N2 virus, culturing in MDCK cells can lead to the emergence of variants carrying substitutions in most of the viral segments
Summary
Influenza viruses obtained from infected human host specimens can be isolated using several different cell-lines. A few reports of MDCK-induced mutations in individual gene segments have been published [12,14,16] These MDCK-induced mutations may have direct and significant impact on the data interpretation in studies related to viral molecular epidemiology [11], antigenicity and pathogenicity [7], and patterns of drug resistance [8,9,10,12,14]. For these studies, only the hemagglutinin (HA), neuraminidase (NA), and matrix protein (MP) genes were routinely sequenced [5,11,17]. An accurate characterization of the pattern of MDCK-induced mutations across the whole genome would improve the quality and accuracy of data interpretation of influenza virus mutation studies
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