Titration curves. Representative sera titrations are shown in Fig.

Titration of pseudovirionsTo titrate ANDV or VSV G pseudovirions expressing luciferase, we infected Vero E6 cells with serial dilutions and quantified infection by relative light units (RLUs). Basically to confirm that rLuc pseudovirion infection yielded signal in the linear range, cells were infected with 2-fold dilutions of pseudovirions stocks for 24 h before cell lysis and measurement by luminometer (Fig. 1A and 1B). Subsequent experiments were done at pseudovirion dilutions that resulted in RLUs between 2×106 and 4×106 in Vero E6 cells (1: 16), well within the linear range of the detection. Glycoprotein-specific antibodies neutralize pseudovirion infectionTo verify that infectivity was facilitated by ANDV glycoproteins and not by residual VSV G protein, pseudovirions were pretreated with either ANDV HPS convalescent patient antisera (immune sera) or with a neutralizing VSV G-specific antibody (I14). Infection of Vero E6 cells was reduced by more than 80% (IC80) when ANDV pseudovirions were pre-treated with  immune sera at 1:50 fold dilution, whereas VSV pseudovirions were not affected (Fig. 2). In contrast, VSV but not ANDV pseudovirions were neutralized after pre-treatment with the anti-VSV G monoclonal antibody (I14, 1:50). Thus, pseudovirion infection was mediated by the appropriate glycoproteins.Human anti-sera neutralize hantavirus pseudovirions at titers comparable to PRNT assaysTo determine if pseudovirions could also be neutralized by sera from ANDV HPS convalescent patients, we tested 60 sera samples (20 ANDV sera, 40 non-immune sera) for neutralizing activity against ANDV and VSV-G pseudovirions. ANDV positive sera were previously screened for virus neutralization titers by PRNT on Vero E6 cells (Hjelle); negative sera were screened by ELISA (Martinez). Pseudovirions were pre-incubated with 2-fold dilutions of sera prior to infecting Vero E6 cells and IC20 values calculated from the resulting neutralization curves. Representative sera titrations are shown in Fig. 3 and their corresponding IC80 and PRNT values reported in Table 1. We completed full titrations in triplicate for all sera.  At the highest sera concentration (1:50 dilution) non-immune sera were not able to neutralize ANDV pseudovirions, while all ANDV sera were able to completely neutralize infection.  ANDV sera Pseudovirion neutralization IC80 titers ranged from 1:400 to 1:6400. The respective geometric mean titer (GMTs) was 3.2492. In contrast, PRNT80 titers ranged from 1:100 to 1:3200, with a GMT of 2.978. These data show that neutralizing antibodies can be detected in immune human sera using this pseudovirions system at titers comparable to those used in PRNT assays.