Elsevier

Journal of Clinical Virology

Volume 77, April 2016, Pages 21-28
Journal of Clinical Virology

Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

https://doi.org/10.1016/j.jcv.2016.01.015Get rights and content

Highlights

  • A variety of methods are used for enterovirus detection in tissue samples.

  • Their sensitivity vary and systematic comparisons have not been published.

  • RT-PCR, immunohistochemistry, in situ hybridization and proteomics were compared.

  • Proteomics and RT-PCR detected enteroviruses with the highest sensitivity.

  • Proteomics open new opportunities for enterovirus detection.

Abstract

Background

Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples.

Objectives

This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro.

Study design

A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10−1 to 10−8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR.

Results

RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10−8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10−7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10−6, while ISH detected the virus at dilutions of 10−4.

Conclusions

All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

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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