PEDV could hijack neonatal RBCs
To
detect whether PEDV binds to neonatal red blood cells (RBCs), neonatal
RBCs were isolated from newborn piglets and infected with PEDV strain ZJ
(MOI=0.1).
After
washing to remove unbound virus, cells were repeatedly frozen and thawed
and then detected by plaque assays. We found that the virus titer was
continuously decreased, until disappeared at 24 hpi (Fig 1A), indicating
that PEDV could bind to neonatal RBCs, but couldn’t survive for long.
Following, the levels of PEDV-N protein were gradually decreased after
infection (Fig 1B).
Although
the low level of PEDV-N protein was still detected at 24 hpi, the virus
had almost lost its viability and infectivity, speculating that despite
virus loss of activity, the residual viral structural protein could
continue to adhere on RBCs. Transmission electron microscopy (TEM)
observation visualized that single or clustered PEDV virion could be
internalized in neonatal RBCs (Fig 1C). These data suggested that PEDV
virion could hijack neonatal RBCs and maintain its viability and
infectivity for up to 12 h.
In
general,
a
blood circulation takes about 45 s for blood to circulate from the
heart, all around the body, and back to the heart again. Thus, once PEDV
hijacks neonatal RBCs, it is enough to spread all over the body.
PEDV
internalizes into RBCs via binding CD71 and clathrin-mediated
endocytosis
To
investigate whether the binding of PEDV to RBCs depends on RBCs from
different ages pigs, non-neonatal RBCs were isolated
from
40-day-old pigs and 120-day-old pigs and then infected with PEDV strain
ZJ (MOI=0.1).
We
observed that PEDV was more affinitive to neonatal RBCs than
non-neonatal RBCs (Fig 2A). In the blood circulation of newborn, there
were not only mature RBCs, but also a small amount of immature
CD71+ RBCs (Elahi, 2014). In agreement with previous
reports, we found that CD71 only expressed in neonatal RBCs, but none in
non-neonatal RBCs (Fig 2B). Moreover, our study has demonstrated that
CD71 was considered as a co-receptor for PEDV
infection
(unpublished).
To
investigate whether CD71 mediates PEDV binding neonatal RBCs, the
expression of CD71 was detected after 1 h to 24 h of infection.
The
protein levels of CD71 was continuously decreased during 1 h to 24 h of
PEDV infection (Fig 2C), due to PEDV invasion exhausting CD71 expression
in neonatal RBCs. Moreover, a blockade experiment was performed by using
an anti-CD71 antibody. After blocking CD71, the level of PEDV virions
binding to neonatal RBCs was decreased significantly (Fig 2D and 2E),
suggesting that CD71 acts as a receptor for PEDV infection and mediates
special binding of PEDV to neonatal
RBCs.
CD71
is employed to transport iron through clathrin-mediated endocytosis
(Gammella, Buratti, Cairo, & Recalcati, 2017). To verify whether PEDV
internalization into RBCs is mediated by
clathrin-mediated
endocytosis, an inhibitor
chlorpromazine
(CPZ) for preventing
clathrin
coated pit formation (Martinez, Cordo, & Candurra, 2007) were
pre-treated with RBCs. After inhibition with CPZ, the virus titer and
the level of PEDV-N protein in neonatal was decreased significantly (Fig
2F and 2G), implying that
PEDV
might be internalized in to RBCs through
clathrin-mediated
endocytosis. These data demonstrated that the presence of CD71 and
clathrin-mediated endocytosis promoted PEDV internalizing in neonatal
RBCs.