Virus detection and typing methods

4.2.1 RT-PCR based methods

4.2.1.1 Viral RNA extraction method

Viral RNA was extracted from a 10% stool suspension (Report I), rectal swab solution (Report II), serum (Reports II and III), plasma (Report III) and cell culture supernatant (Reports I, II and III). Extractions were done from 140µl of the samples using a silica column based RNA extraction kit according to the manufacturer’s protocol (QIAamp viral RNA kit, Qiagen, Hilden, Germany) (Reports I, II and III).

A subgroup of samples in Report I was also selected for nucleic acid extraction by MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, Mannheim, Germany) using automated nucleic acid extraction robot according to the manufacturer’s protocol (MagNA Pure LC Instrument, Roche, Mannheim, Germany) (Table 4).

Table 4. Summary of sample materials, virus detection methods and statistical methods used in the publications.

Study

Report I

(PCR inhibitors)

Report II (DAISY)

Report III (DIPP)

Report IV (VirDiab) Sample type Stool

suspension

Serum, rectal swab

Serum, plasma Serum

HLA typing not analyzed analyzed analyzed analyzed

Detection method

RT-PCR RT-PCR ELISA (IgM,

IgG, IgA)

RT-PCR Neutralization antibody assay

RNA extraction method

Viral RNA Kit Total nucleic acid extraction

kit

Viral RNA Kit Viral RNA Kit n.a.

Statistical method

Wilcoxon test Cox regression Monte Carlo permutation test

Conditional logistic regression

Mann Whitney test

Conditional logistic regression

n.a. = not applicable

4.2.1.2 Virus detection using RT-PCR methods

Enterovirus detection from stool suspension (Report I), rectal swab solution (Report II), serum (Reports II and III), plasma (Report III) and cell culture supernatant (Report I, II and III) was done using enterovirus specific two step RT-PCR. This enterovirus-specific method amplifies a 115bp long fragment of enteroviral 5’UTR, which was lineary quantified using a liquid hybridization assay [158]. RT-PCR amplification was done using a mixture of RT-buffer (Promega, Madison, WI, USA), 0,5mM dNPT (Pharmacia Biotech, Uppsal, Sweden), 4U of RNase inhibitor (Promega), 50pmol (4-) reverse transcriptase enzyme (Promega) and 10µl of the RNA template. The total volume of reaction was 40µl and reactions were carried out at 37^oC for 60min. A parallel reaction was also performed with the addition of 0,5%

BSA (Report I). The PCR reactions were performed using 0,2mM dNTP, 1U of DyNazyme DNA polymerase (Finnzymes, Espoo, Finland), 0,2µM of enterovirus-specific primers (4- and 636+bio). The total reaction volume was 100µl. PCR amplification started with denaturation of cDNA and primers at 94^oC for 3min, followed by 40 cycles at 94^oC for 30s, 53^oC for 45s and at 72^oC for 1 min. The final extension was done at 72^oC for 7min [158]. A parallel reaction was also performed in Report I with the addition of 0,5% BSA in the PCR mixture.

The PCR amplicons were quantified by a liquid-phase hybridization assay using europium labelled probes (PerkinElmer, Turku, Finland). A biotinylated PCR product was first allowed to bind to a microtiter well, coated with streptavidin. After denaturation of the PCR amplicon, the well was washed to remove the free-floating DNA strand. Detection of single strand DNA was performed with a europium labelled probe specific for enterovirus sequence (PerkinElmer Wallack, Turku, Finland). Finally, the europium fluorescence was quantified in a time-resolved manner by a Victor multilabel counter (PerkinElmer Vallack, Turku, Finland) [158].

The cut-off limit for a positive sample was set to the level corresponding five times the signal of the negative control (water sample). All positive samples were re-tested and finally a sample was interpreted as a positive sample if a minimum two out of three tests were positive. All test runs included one virus positive control and two virus negative control samples. The samples were blinded without knowing the case-control status of the child.

The SFV was used as a standard RNA template for monitoring PCR inhibitors in stool samples (Report I). A specific primer pair was designed for amplification 201bp long region of the 5’ noncoding region of SFV. RT-PCR and PCR reactions were performed parallel with and without BSA. PCR amplicons were quantified using a

samarium labeled SFV specific probe. This method with primer and probe sequences is described more detailed in Report I.

4.2.1.3 Sequencing analysis

PCR amplicons were purified using a MinElute Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. Further extracted amplicons were sequenced with primers used in PCR (BigDye v. 3.1. Applied Biosystems) by an automated sequencer (Applied biosystems) (Report II).

4.2.2 Plaque reduction neutralization assay

The plaque reduction neutralization test was used in Report IV for quantification of neutralization antibody titers against CBV 1-6 ATCC prototypes in the VirDiab samples. The CBV1 and CBV3 antibodies were also measured against wild-type virus strains isolated from Finland. The test serum was spiked with 100 pfu of the virus and incubated for 1 h at 37°C followed by overnight incubation at RT. This mixture was pipetted on a monolayer of green monkey kidney cells (GMK) on six-well plates in a plaque assay medium containing the Minimal Essential Medium (MEM) supplemented with 1% fetal bovine serum, 40U/ml Penicillin-Streptomycin, 0,0023% Glucose, 1*L-Glutamine, 1,5mM MgCl2 and 1,5mM Carboxymethyl Cellulose (HEPES). Both virus positive and negative control wells were included in all test runs. The number of plaques was counted after incubation at 37°C for 48h.

Sera were tested using 1/4 and 1/16 dilutions and the cut-off limit for a seropositive sample was more than 80% reductions of the plaques. The antibody positive series were titered for higher dilutions against ATCC CBV strains (titers 1/64, 1/256 and 1/1024). These analyses were conducted using samples with blinded identity.

4.2.3 ELISA assay

Enterovirus antibodies were measured from a subset of samples of Report II using an enzyme-linked immunosorbent assay (ELISA). These samples were collected between the years 1993-2004. Capture ELISA (EIA) was used to measure IgM antibodies against a cocktail of purified and heat treated CBV3, CAV16 and E11 viruses. In addition, IgG and IgA class antibodies were tested using a purified and

heat treated CBV4 virus and a synthetic enterovirus peptide antigen KEVPALTVETGAT-C as antigens in indirect ELISA. This synthetic peptide is common for various enterovirus serotypes. In all mentioned ELISA assays the definition of infection was based on a two-fold or higher increase in the antibody level between two consecutive samples, when reaching a signal-to-background ratio of three. The analyses were done blinded without knowing the case-control status of the child [89].