PEDV-loaded RBCs transfusion induces typical PED symptoms
To determine whether PEDV could infect newborn piglets through hijacking
RBCs, an autotransfusion of PEDV-loaded RBCs were performed in newborn
piglets (Fig 3A). Newborn piglets were randomly assigned to 2 groups:
control group (transfusion with Dil-labeled
RBCs);
infection group (transfusion with PEDV-loaded and Dil-labeled RBCs). The
virus titer of transfused RBCs was counted at 3.2 ×
106 PFU/ 109 cells (Fig 3B). At 1
hpt (hour post-transfusion), the percent of Dil-labeled RBCs in
infection group was the same as that in control groups, but a small
amount of PEDV-loaded RBCs was detected in infection group (Fig 3C and
3E). However, at 48 hpt, the transfused PEDV-loaded RBCs in infection
group were vanished in the blood circulation (Fig 3C and 3E). The
newborn piglets in infection group appeared typical PED symptoms at 48
hpt, including severe watery diarrhea, dehydration and lethargy (Fig
3F).
Compared
with piglets in control group, the piglets in infection group revealed
multifocal to diffuse villous atrophy and destruction of intestinal
villous enterocytes through pathological observation (Fig 3G).
Furthermore,
a large amount of PEDV-positive cells was found in the intestinal villi
of the piglets transfused with PEDV-loaded RBCs through
immunofluorescence observation (Fig 3G). Subsequently, viral RNA levels
in different tissues were detected by quantitative reverse
transcription-polymerase chain reaction (qRT-PCR).
After
1 hpt, low levels of viral RNA were found in all tissues, and the viral
RNA content in the spleen was higher relatively (Fig 3H), suggesting
that the transfused RBCs could carry the virus and spread throughout the
body. Moreover, at 48 hpt, PEDV could already infect and colonize in
small intestine, and the peak vital RNA reached up 4.8 log10 in the
jejunum.
Further,
the PEDV-N protein was verified by Western blot and only a slight
immunoreactivity with PEDV-N protein was observed in spleen at 1 hpt,
while at 48 hpt, stronger levels were detected in jejunum and ileum (Fig
3I). Consequently, PEDV could colonize and infect small intestine of
neonatal piglets through transfusion with PEDV-loaded RBCs.
CD3+T cells acquire virus from RBCs by forming conjugation
Although
RBCs could carry PEDV to the whole body, the RBCs could not directly
contact the intestinal mucosa. Therefore, we speculated whether some
types of immune cells could recognize and capture the virus-loaded RBCs.
As ‘sentry’ cells in the blood, peripheral blood mononuclear cells
(PBMCs) play an important role in immune reactivity. To examine whether
PBMCs possess the ability to recognize and capture virus-loaded RBCs, a
co-culture model of RBCs and PBMCs was established (Fig 4A). After
co-culture with virus-loaded RBCs and PBMCs, the RBCs were removed by
ACK Lysis Buffer and the PBMCs were detected. The virus was detected in
PBMCs in only 1 h of co-cultrue by flow cytometry and Western blot (Fig
4B and 4C), suggesting that PEDV could transmit from RBCs to PBMCs. Our
previous study had indicated that CD3+ T cells in
PBMCs could capture PEDV from DCs and transfer the virus to intestinal
epithelial cells (IECs) (Li et al., 2018). We further investigated
whether CD3+ T cells could capture PEDV from RBCs (Fig
4D).
Compared to co-culture with normal RBCs and PBMCs, higher frequency of
CD3+ PEDV+ cells were present in
co-culture of virus-loaded RBCs and PBMCs (Fig 4E). As co-culture time
was prolonged, more CD3+ Dil+ cells
were increased,
suggesting
CD3+ T cell might form conjugation with RBCs to
capture PEDV (Fig 4F). Moreover, the conjugate structure was visualized
by TEM observation between the PEDV-loaded RBCs and
CD3+ T cells (Fig 5G), even though the virus transfer
was not visualized.
Nasal
capillary might be the entry of PEDV binding RBCs
Although
we have verified that transfusion with PEDV-loaded RBCs could cause
intestinal infection, the place where PEDV enter the blood of newborn
piglets to hijack RBCs is poorly defined.
Our
previous study verified that PEDV could cause typical diarrhea through
nasal spray and develop a transient NECs infection (Li
et al., 2018). Moreover, we found that numerous capillaries were
distributed under the nasal epithelium, and even many capillaries are
immediately adjacent to the nasal epithelium (Fig 5A). To further verify
whether these nasal capillaries might be the entry of PEDV binding RBCs,
a nasal spray challenge was performed in newborn piglets. After 12 h of
intranasal incubation with PEDV, PEDV-positive RBCs were found in the
capillaries adjacent to the nasal epithelial cells (NECs) by
immumohistochemical (IHC) observation (Fig 5B).
Nasal cavity is exposed in higher oxygen concentration condition than in
intestinal tissues (Carreau, El Hafny-Rahbi, Matejuk, Grillon, & Kieda,
2011; Elad, Wolf, & Keck, 2008). Moreover, oxygen concentration exerts
a significant effect on viral propagation and replication (Morinet,
Parent, Bergeron, Pillet, & Capron, 2015). To further speculate whether
the differences of oxygen concentration between nasal cavity and other
tissues exert an effect on PEDV binding to RBCs, an experiment of viral
infection in normoxic (20 % oxygen partial pressure,
pO2) or hypoxic (3 % pO2) condition was
performed.
Compared
in normoxic condition, RBCs exhibited lower affinity to PEDV in hypoxic
condition (Fig 5C and 5E). Considering that the nasal cavity is directly
connected to the environment air, the temperature is slightly lower than
other tissues (Elad et al., 2008). To further validate whether
temperature could affect PEDV binding to RBCs, RBCs were placed under
normoxic condition at 37 °C or 33 °C (the temperature of nasal cavity)
to culture and infected with PEDV, respectively. However, alteration
temperature exerted no effect on PEDV binding to RBCs (Fig 5d and 5E).
Therefore,
the relatively high oxygen condition in nasal cavity exerted a promoting
effect on PEDV binding to RBCs, rather than temperature. Although the
mechanism by which PEDV enter the blood and bind to RBC has been
unknown, the capillary adjacent to the NECs might allow the virus pass
through to bind to RBCs.