Establishment and optimization of a rapid and convenient viral RNA transcript copy reduction neutralization test (VcRNT) for quantification of hantaan orthohantavirus (HTNV) neutralizing antibodies

Rodent-borne orthohantavirus causes severe hemorrhagic fever worldwide, with hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus cardiopulmonary syndrome (HCPS) in the Amrerica. In East Asia, Hantaan orthohantavirus (HTNV) is the main pathogen responsible for severe HFRS, with a case fatality rate up to 10 % with no specific treatment available. The antisera or neutralizing antibody (NAb) is able to block virus infection, however, the traditional NAb titer measuring based on focus reduction neutralization test (FRNT) is quite labour-extensive and takes 7–10 days. This study aims to shorten the measuring time of NAb neutralization efficiency by 1–2 days based on quantitative RT-PCR. For this purpose, we developed an in vitro transcripted viral RNA standard and generated a viral RNA copy number standard curve. Using this standard curve, we compared the HTNV propagation kinetics between viral RNA copy numbers and secreted infectious virion. The detection limit and suitable timeframe and condition for qRT-PCR based viral RNA copy numbers measuring was also determined. In addition, when applying this method to measuring the NAb neutralization efficiency of HFRS convalescent serum samples, we could obtain the NAb neutralization efficiency within 1 or 2 days. Furthermore, this method was also nicely correlated with the FRNT - based NAb measurement. To conclude, we established a rapid and convenient viral RNA transcripts copy reduction neutralization test (VcRNT) to measure NAb neutralization efficiency that could finish within 1 or 2 days, and provided a reliable and efficient alternative for FRNT.