Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells
Section snippets
Background
Enterovirus1 (EV) infections are common in all age groups. They are usually asymptomatic or cause only mild respiratory symptoms, but can also lead to more severe illness including hand, foot and mouth disease, myocarditis, meningitis, encephalitis, pancreatitis, systemic infection in newborns and paralysis. EV infections may also play a role in the pathogenesis of chronic diseases such as dilated cardiomyopathy [1], chronic fatigue syndrome [2] and type 1 diabetes2
Objectives
The aim of the study was to evaluate the relative sensitivities of proteomics, ISH, IHC and RT-PCR methods to detect one common EV, Coxsackievirus B14 (CVB1), using human A549 cells diluted to contain differing ratios of uninfected to in vitro EV-infected cells.
Preparation of EV-infected cell arrays
Human A549 alveolar basal epithelial cells were grown in monolayers in Nutrient Mixture F-12Ham, N 6658 (Sigma–Aldrich®) medium in T175 bottles and infected with CVB1, ATCC strain (10–15 MOI). The infection was stopped at four different time points (1 h, 2 h, 4 h, and 6 h post infection) to obtain a series of infected cells representing different stages of viral replication cycle. The cells from different time points were mechanically detached, pooled and washed with the growth medium. The cells
RT-PCR
Viral RNA was detected by RT-PCR in all samples although the semi-nested method was most sensitive. This yielded a positive signal from even the most dilute sample (10−8) whereas the real-time RT-PCR method gave a positive signal in the second most dilute sample (10−7). Ct values from real-time RT-PCR experiments with different dilutions of infected cells are shown in Supplementary Table 2.
Proteomics
MS-based targeted LC/MRM/MS/MS assay focused on the CVB1 2C protein peptide SVATNLIGR and the peptide
Discussion
The present study provides important information to guide the selection of assays capable of optimally detect EVs in infected cells. Although the conditions prevailing in mammalian cells infected in vitro do not completely resemble those in clinical tissue samples, the results provide a firm indication of the sensitivity and specificity of each method.
All methods tested were able to detect CVB1 with good sensitivity. However, depending on the method used, the detection limit varied and RT-PCR
Conflict of interest
HH is a minor (<5%) shareholder and a member of the Board in Vactech Ltd., which develops vaccines and diagnostic assays for picornavirus infections.
Funding
None
Ethical approval
Not required
Acknowledgements
This study was supported by JDRF grants for the nPOD-Virus Group, JDRF 25-2012-516 and JDRF 25-2012-770, the Diabetes Research Foundation in Finland, the Sigrid Juselius Foundation, the Academy of Finland and the European Commission (Persistent Virus Infection in Diabetes Network [PEVNET], Frame Programme 7, Contract No. 261441). Additional support was from a Diabetes Research Wellness Foundation Non-Clinical Research Fellowship and, since 2014, a JDRF Career Development Award (
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