Porcine epidemic diarrhea virus

nsp12 protein sequences were derived from available CoV ORF1ab sequences. These sequences are comprised of HKU19 (YP_005352862.1), porcine deltacoronavirus (AMN91620.1), HKU11 (YP_002308478.1), transmissible gastroenteritis virus (P0C6Y5.1), 229E-related bat CoV (APD51497.1), HuCoV-NL63 (AVA26872.1), porcine epidemic diarrhea virus (AKJ21970.1), HKU8 (YP_001718610.1), HKU22 (AHB63507.1), infectious bronchitis virus (AAS00078.1), turkey CoV (ACV87277.1), HuCoV-OC43 (YP_009555238.1), murine hepatitis virus (YP_209229.2), HuCoV-HKU1 (ARB07596.1), MERS-CoV (ATG84853.1), HKU4 (ABN10847.1), SARS-CoV (AAP33696.1), and HKU9 (AVP25405.1). These sequences were aligned with Clustal Omega^{53 (link)}. A subset of these aligned sequences is shown in Supplementary Fig. 3. The full alignment was used as input for ConSurf^{54 (link)} to generate the images in Fig. 2.

All OCT images were obtained by the Heidelberg Spectralis spectral domain OCT (SD-OCT) (Heidelberg Engineering, Heidelberg, Germany). The following scan patterns were performed on both eyes and centered on the fovea: 20 × 15 degree raster scans consisting of 19 high-speed line scans. IRF, SRF, PED, VMA, and PEDT were determined from the baseline OCT images and PEDH and PEDW at baseline were measured by two ophthalmologists blinded to the group assignments. The distance was taken between the inner surface of the Bruch’s membrane and the outer surface of the RPE. The PED area of each B-scan was measured using ImageJ (an open-source software developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The summation of the whole PED area in each volume scan was used as an index for PEDV (Fig. 1). The two ophthalmologists performed triplicate measurements respectively and recorded the average value. PED was not counted if there was not at the fovea.Fig. 1

Flow chart of measuring PEDV using ImageJ. Step 1, take out the PED scan that needs to be measured; Step 2, circle the outline of the PED; Step 3, measure the circled area. This is the PED area of a B-scan. The summation of the whole PED area in each volume scan was used as an index for PEDV. PEDV the volume of PED

PED is a prominent feature of the AMD process, manifesting as an anatomical separation of retinal pigment epithelium (RPE) from the underlying Bruch layer [14 (link)]. PEDs were classified as either serous, fibrovascular, drusenoid, or hemorrhagic (Fig. 2). Drusenoid PEDs are composed of one or more drusen or soft drusen. Serous PEDs usually consists mainly of hyporeflective material. Fibrovascular PEDs can be seen as a hyperreflective material adhered to the outer PED layer. Hemorrhagic PEDs show a mixture of hyporeflective and hyperreflective material [15 (link)].Fig. 2

Imaging SD-OCT. A Serous PED; B Fibrovascular PED; C Drusenoid PED; D Hemorrhagic PED. PED pigment epithelial detachment

DMEM containing 100 μg/ml streptomycin and 100 units/ml penicillin was used for culturing PEDV on Vero cells. To explore the importance of glucose and glutamine metabolism in PEDV replication, 12-well plates confluent with a monolayer of Vero cells were seeded and infected with PEDV. The effect of metabolites on PEDV infection was analyzed using normal DMEM, glucose-free DMEM, and DMEM supplemented with glucose and lactate. The effects of glutamine on PEDV were analyzed using glutamine-free DMEM and DMEM supplemented with glutamine. The PEDV GD/HZ/2016, CV777 and TB strains of PEDV available in our laboratory were used for this study.