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 Moreover, our study has demonstrated that CD71 was considered as a co-receptor for PEDV infection (unpublished).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.