Discussion
As it causes devastating losses to the swine industry, coronavirus PEDV
has aroused wide mounting concern worldwide (Choudhury, Dastjerdi,
Doyle, Frossard, & Steinbach, 2016; C. Lee, 2016). PEDV infection
mainly cause diarrhea, dehydration and even death in suckling piglets.
Although several reports has indicated that piglets developed viremia
after PEDV infection (Chen et al., 2016; Jung et al., 2014), the
mechanism of how PEDV spreads in the blood remains unclear. Our previous
study has indicated that PEDV could be carried by CD3+T cells to enter the blood (Li et al.,
2018).
In addition to CD3+ T cells, could PEDV bind to other
cell types to facilitate transmission? In the present study, we
demonstrated that the coronavirus PEDV could hijack RBCs and spread in
the blood circulation, causing intestine infection.
Red
blood cells are the most abundant cell in the bloodstream, serving for
oxygen transport. Despite of loss of nuclei and organelles, RBCs still
express numerous cell surface receptors to contact with the exogenous
agents in the blood, performing several additional immunological
functions (Baum, Ward, & Conway, 2002). However,
several receptors are employed by pathogens for binding RBCs to spread
in the blood. The most notorious pathogen is the malaria parasite, which
infects RBCs by binding to receptors on the cell surface, such asPlasmodium vivax and Plasmodium knowlesi attaching Duffy
Ag receptor for chemokines (DARC) (Boddey & Cowman, 2013; Horuk et al.,
1993), and Plasmodium falciparum adhering
glycophorin A (GYPA) (Farrow et al., 2011). Moreover, RBCs are
considered as possible vehicles for virus transmission, such as HIV-1
(Beck et al., 2009), Dengue virus (Sutherland et al., 2016) and West
Nile virus (WNV) (Rios, Daniel, Chancey, Hewlett, & Stramer, 2007). In
this study, we verified that PEDV could bind to RBCs and maintain its
viability and infectivity for 12 h.
Mounting researches have indicated that there is a small amount of
immature RBCs in the circulation of fetuses and newborns, containing a
nucleus and cell surface transferrin receptor (CD71) (Elahi, 2014,
2019). The CD71+ RBCs are enriched during pregnancy or
newborns, and have distinctive effect on suppressing innate immune
responses (Delyea et al., 2018; Dunsmore et al., 2017; Elahi, 2014;
Elahi et al., 2013).
Similar
to previous researches, CD71 was only present in the RBCs of newborn
piglets, but not of 40-120 days old piglets. As a transmembrane receptor
for
guaranteeing
iron (Gammella et al., 2017), CD71 is also considered as a preferred
entry for human pathogenic arenaviruses (Abraham, Corbett, Farzan, Choe,
& Harrison, 2010; Radoshitzky et al., 2011), hepatitis C virus (HCV)
(Martin & Uprichard, 2013) and malaria parasite (Gruszczyk, Huang, et
al., 2018; Gruszczyk, Kanjee, et al., 2018). Moreover, our previous
study has proved that CD71 was considered as a co-receptor for
Transmissible gastroenteritis virus (TGEV) (Zhang, Hu, Yuan, & Yang,
2018) and PEDV infection (unpublished), which could be caught by spike
protein of PEDV. In our study, the presence of CD71 exerted a
significant effect on promoting PEDV adhering neonatal RBCs. CD71 is
employed to internalize iron through
clathrin-mediated
endocytosis. Meanwhile, clathrin-mediated endocytosis also played an
important role in facilitating PEDV internalizing into RBCs, just as
HIV-1, Influenza and Ebola virus (EBOV) invade target cells
(Aleksandrowicz et al., 2011; Mercer & Helenius, 2009). Therefore, PEDV
could exploit CD71 and clathrin-mediated endocytosis to internalize in
neonatal RBCs.
Through
autotransfusion with PEDV-loaded RBCs,
we
found that PEDV could cause intestinal infection in newborn piglets.
Until
now, blood transfusions are still the route of transmission of many
virus, as is the case for HIV-1 (D. Lee, 2006), Hepatitis E Virus
(Izopet, 2018), DENV (Pozzetto, Memmi, & Garraud, 2015), HCV (Schuch,
Thimme, & Hofmann, 2015) and WNV (David & Abraham, 2016).
The
essential characteristics of transmission through blood transfusion are
that the virus could survive or maintain viability in the blood or blood
components and cause infection through the blood vessels route (Glynn et
al., 2013). Although coronavirus PEDV persists in RBCs and maintains its
viability and infectivity for a relatively short time (about 12 h), but
it is enough for PEDV to hijack RBCs to the target tissues.
After
1 h of transfusion, PEDV appeared in the spleen, which is the only
lymphoid organ that filters the blood. Macrophage in the spleen could
phagocytose RBCs that bind to pathogens (Minasyan, 2016), such as RBCs
infected by Plasmodium (de Back et al., 2014). However, after 48
h, transfusion with PEDV-loaded RBCs caused intestine infection and PEDV
could colonize in intestinal epithelial cells. PEDV could hitchhike RBCs
to reach the small intestine and colonize in IECs (Li et al., 2018).
Since RBCs are thought to be not able to penetrate the vascular
endothelium under normal physiological condition, we speculated that the
RBCs could be recognized and transferred the virus to certain types of
immune cells in the blood.
As
a sentry in the peripheral blood, peripheral blood mononuclear cells
(PBMCs) play a very important role in immune response against both
infectious invaders. After co-culture, we verified that PEDV could
transmit from RBCs to PBMCs within a short time. Further,
CD3+T cells were determined as the main cells types in PBMCs for capturing
virus, and could form conjugation to acquire PEDV from RBCs within 1 h.
Moreover, PEDV could continue to hitchhike CD3+ T
cells and migrate to the intestine, causing typical diarrhea (Li et al.,
2018). Several reports have hypothesized that RBCs might be exploited by
pathogen as a “Trojan horse” to carry them to the target cells (Baum
et al., 2002; Beck et al., 2009). This would be similar with HIV-1
binding to DCs, which could
transfer the virus to the susceptible cells in the lymph nodes (Harman,
Kim, Nasr, Sandgren, & Cameron, 2013; Manches, Frleta, & Bhardwaj,
2014). However, the detailed mechanism of PEDV transfer from RBCs to T
cells needs to be validated.
In general, RBCs circulate in the
blood vessels, and are difficult to directly contact with pathogens from
outside. The previous study in our group have indicated that PEDV could
infect piglets through airborne transmission and develop a transient
nasal epithelium infection (Li et al., 2018). Moreover, numerous
capillaries are distributed under the nasal epithelial cells (NECs), and
some are even adjacent to the NECs. In addition, the permeability of the
capillaries in the lamina propria of the nasal mucosa was very high than
that of other tissues (Watanabe, Saito, Watanabe, & Mizuhira, 1980).
Through intranasal incubation with PEDV, PEDV-loaded RBCs were found in
the
capillary
adjacent to the NECs. Although the mechanism by which PEDV could enter
the nasal capillary after infection with NECs has poorly been clarified,
nasal capillary might be the possible entry for PEDV binding RBCs.
Further, the nasal cavity is a contacting room between respiratory
system and environment air. Cells in nasal cavity are exposed in
relatively high oxygen condition with 19-21 % oxygen partial pressure
(pO2),
but are exposed in lower oxygen condition with 6-8 % pO2 (Carreau et
al., 2011). And increasing evidences have indicated that oxygen
concentration exerted significant effect on virus infection (Ebbesen &
Zachar, 1998; Mazzon et al., 2013; Rastelli et al., 2016). In the
present study, the relatively high oxygen concentration promoted the
affinity of PEDV to RBCs. Besides, the temperature is in nasal cavity is
cooler than in other tissues, with an average temperature of 33 °C
(Bailey, Casey, Pawar, & Garcia, 2017; Forero et al., 2017), but
reducing temperature had no effect on PEDV binding RBCs. Therefore, the
relatively higher oxygen concentration in nasal cavity might be a
promoting factor for PEDV binding RBCs.
In
summary, our study has demonstrated for the first time that the
coronavirus PEDV could hijack RBCs and spread throughout the body,
causing intestine infection. PEDV could bind and be endocytosed into
neonatal RBCs through CD71 and clathrin-mediated endocytosis (Fig 6).
Subsequently,
PEDV-loaded RBCs could be recognized and transferred the virus to
CD3+ T cells in PBMCs by form conjugation. Moreover,
the capillary adjacent to the nasal epithelium was speculated as the
entry for PEDV binding RBCs. Our studies put forward a new insight into
the role of RBCs in coronavirus PEDV as potential cells for virus
transmission, which might be helpful to reveal the transmission and
pathogenesis of viruses with the same characteristics, even though the
detailed mechanisms remain to be clarified.