p.p1 pseudovirions could also be neutralized by sera

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 Complement proteins are present in the blood and in
various tissues in uncleaved, inactivated forms. Complement
action can be initiated by direct recognition of a microbial
invader by C1q (a component of C1) in the classical pathway,
or by recognition of cleaved C3b proteins in the alternative
pathway .Human
anti-sera neutralize hantavirus pseudovirions at titers comparable to PRNT

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To 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.Glycoprotein-specific antibodies neutralize pseudovirion infection

To 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. ResultsPseudovirion productionAndes (ANDV)
pseudovirions were prepared utilizing a vesicular stomatitis virus (VSV) vector
expressing the Renilla luciferase (rLuc) gene (Ray et al., 2010). VSV
pseudovirions with the VSV G gene provided in trans were used to transduce HEK
293T cells that had been transfected with plasmids expressing ANDV M segment.

After transduction, cells were incubated for 12-24h at 37 °C and
pseudovirion-containing media was collected, filtered, and the pH adjusted to
8.0. (Meda et al., 2012) Titration of pseudovirions

To 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.