2.8 Focus reduction neutralization assay (FRNT)
The cross-neutralizing activity against G1 and G2 PEDV strains between
the anti-G1-NTD and –G2-NTD serum was evaluated using a focus reduction
neutralization test (FRNT) with minor modifications (Vaigya et al.,
2010). Initially, 500 μl of
infection
medium-diluted virus stock solution (200 pfu/ml) was mixed with equal
volume of twofold serial infection medium-diluted either the polyclonal
antisera or the
control
syngeneic sera, and then incubated
for
1 h at 37ºC. The mixture was added to confluent Vero cells in 6-well
plates, which were initially washed twice with infection medium, then
incubated for 2 h at room temperature to allow adsorption of the virus.
After incubation, the medium was removed. The infected cells were
overlaid with 1% sodium carboxymethyl cellulose that was dissolved
infection medium and incubated for 36 h. The overlaid medium was removed
and the cells were fixed as in IFA, and incubated with anti-G1-NTD or
–G2-NTD serum for 1 h at 37 ºC. After being washed three times with
PBS, the monolayer cells were incubated with HRP-conjugated goat
anti-mouse IgG (1:1000) for 1 h at 37 ºC. Finally, virus-specific foci
were positively stained with 3, 3 - diaminobenzidine (DAB) and counted
under microscope. Serum neutralization titers were expressed as the
reciprocals of the highest serum dilution resulting in reduction of 50%
focus forming unit.