A single-chain variable fragment antibody exerts anti-nervous necrosis virus activity by irreversible binding

Nervous necrosis virus (NNV), causing almost 100% mortality among infected larvae and juvenile fish, is severely affecting the aquaculture industry. Although various vaccines against NNV were studied, they can hardly provoke strong enough immunity in fry with immature immune systems. To eliminate the infection possibility during fertilization, we applied an anti-NNV strategy by neutralization based on a single-chain variable fragment (ScFv) antibody, named Sc3F9, against orange-spotted grouper nervous necrosis virus (OGNNV). Sc3F9 binds to the shell domain of viral capsid protein (CP) with a dissociation constant (K-D) of 48.6 nM by interacting with two discontinuous target sites on CP fragments of G(122)-L-128 and L-141-A(148). The binding capacity was enhanced by prokaryotic expressing double copies of Sc3F9 (DSc3F9) in tandem, and the augmented avidity (K-D = 29.5 nM) with irreversible binding to OGNNV was stable even in high salinity, indicating the wide application potential in seawater. DSc3F9 exhibited great recognition breadth within the Betanodavirus genus to bind all NNV serotypes in working concentration (40 mu g/mL) and distinguished these three serotypes by different levels of binding capacity in low concentration (10 mu g/mL). DSc3F9 blocked virus-like particle entry by destructing its virion as well as strongly neutralized OGNNV with an average half-maximal inhibitory concentration (IC50) value of 0.64 mu M. Moreover, DSc3F9 efficiently eliminated the possible NNV infection during fertilization by washing grouper eggs before artificial insemination without reducing the fertility rate and hatchability. Thus, our results not only obtained a strong anti-NNV candidate but also provided an approach to NNV decontamination, especially in obtaining NNV-free grouper eggs.