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.