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case_images: Comparative visualization of Kaposi Sarcoma (KS) from the Ocular Surface and Control Samples: (A) Macroscopic view of the ocular surface affected by KS. (B) Histological examination at low magnification (X4) of the KS lesion, stained with hematoxylin and eosin. (C) Detailed histological view at higher magnification (X20) derived from Image panel (B). (D) Immunohistochemical (IHC) analysis. LANA positivity in the ocular KS lesion, utilizing Abcam's Rat anti-LANA primary antibody. (E) IHC control for LANA with a KS-negative skin biopsy. (F) Magnified view emphasizing endothelial cell markers CD31 with positive IHC staining in the ocular KS lesion. (G) IHC depiction revealing EBNA1 positivity in the KS lesion, employing Abcam's mouse anti-EBNA1 primary antibody. The image exhibits both diffuse and punctate nuclear staining patterns.(H) IHC analysis using Abcam's mouse anti-EBNA1 primary antibody, indicating EBNA1 absence in a skin biopsy without KS. (I) Mouse isotype control for IHC. (J) IHC representation highlighting EBNA1 positivity in the ocular KS lesion, using Invitrogen's mouse anti-EBNA1 primary antibody. The staining presents a punctate nuclear pattern. (K) IHC examination employing Invitrogen's mouse anti-EBNA1 primary antibody, demonstrating the absence of EBNA1 in a skin biopsy without KS. (L) IHC analysis with the CD20 antibody, confirming the lack of B cells in the KS lesion. All digital microscopic images of the stained slides were captured using the MoticEasyScan Pro 6 scanner (Motic, USA) and analyzed using the Motic DSAssistant VM 3.0 software. For the journal presentation, images were cropped to a 500x500 pixel resolution using Microsoft's Paint 3D software.
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case_images: Upon clinical examination, a red fleshy lesion was observed, primarily located at the nasal limbus, involving the bulbar conjunctiva, caruncle, cornea, and upper and lower fornix ( Figure 1A ). Two independent histopathologists, PJ and FM, confirmed the diagnosis of conjunctiva KS after analyzing hematoxylin and eosin (H&E) FFPE-stained tissue sections ( Figures 1B, C ). The lesion section was examined with immunohistochemistry (IHC) targeting Kaposi sarcoma-associated herpesvirus- encoded latency-associated nuclear antigen (LANA) protein using an anti-LANA Rat monoclonal antibody LN53 (Ab4103, Abcam, USA) at a 1/200 dilution ( Figure 1D ), to confirm the KS diagnosis. Supplement Figures 2 ) and KSHV-negative skin tissue ( Figure 1E ). Since endothelial cells are the targeted cells infected by KSHV, IHC against endothelial cell markers CD-31 ( Figure 1F ) was performed and shown to be positive in this KS tissue. Since B lymphocytes are the major cell type targeted for EBV infection, the ocular KS tissue was stained for human CD20 (anti-Human CD20cy, mouse monoclonal antibody [L26] (M0755, Dako, USA) at a 1/200 dilution) by IHC. No B lymphocytes were observed among the intratumoral infiltrating leucocytes ( Figure 1L ). Our results demonstrated the presence of EBV infection in the tumor cells using both anti-EBV nuclear antigen/EBNA1 antibodies from Abcam and Invitrogen ( Figures 1G, J , respectively). The mouse isotype control showed no EBNA1 staining ( Figure 1I ). Invitrogen ( Supplement Figure 1C ). In contrast, both anti-EBNA1 antibodies from Abcam and Invitrogen were tested negative in the normal non-KS skin tissue ( Figures 1H, K , respectively) and negative control BC3 (EBV negative) cell line ( Supplement Figures 1B, D ). Notably, the EBNA1 antibody from Abcam displayed two clear staining patterns in both IHC ( Figure 1G ) and IFA ( Figure 4C ): a diffuse signal and punctate nuclear staining. The EBNA1 antibodies from Invitrogen showed a more distinct punctate staining in both IHC ( Figure 1J ) and IFA ( Figure 4I ). Supplementary Figures 1F ,  3B, E ). Similarly, when the BC3 (EBV negative, KSHV positive) cell line was stained with anti-EBNA1 antibodies from both Abcam ( Supplementary Figures 1B ,  3H ) and Invitrogen ( Supplementary Figures 1D ,  3K ), no cross-reactivity was observed.
case_images: Visualization of Epstein Barr Virus (EBV)-encoded RNAs in control and Kaposi Sarcoma (KS) cells via RNAscope In-Situ Hybridization (ISH): (A) Akata cell line infected with EBV. The presence of EBV-encoded small RNAs (EBER-RNAscope) is evident from the red dots, highlighted by the arrow. (B) EBV-negative Akata 31 cell line. The absence of EBER with RNAscope is observed, indicating no EBV presence. (C) KS lesion from the ocular surface. Red dots and the arrow point to the EBER RNAscope positive cell, confirming the presence of EBV. (D) Skin biopsy without KS. The absence of red dots indicates EBER-RNAscope negativity, indicating no EBV presence. All digital microscopic images of the stained slides were captured using the MoticEasyScan Pro 6 scanner (Motic, USA) and analyzed using the Motic DSAssistant VM 3.0 software. For the journal presentation, images were cropped to a 500x500 pixel resolution using Microsoft's Paint 3D software.
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case_images: Supplement Figures 2 ) and KSHV-negative skin tissue ( Figure 1E ). The presence of EBV in the ocular KS tissue was further confirmed with RNAscope ISH ( Figure 2C ) to detect the presence of EBV-encoded small RNAs (EBER) (EBER1 probe, 310271, ACDbio, USA) according to the manufacturer's protocol ( Figure 2 ). Specificity of the EBER RNAscope ISH was demonstrated with positive signal only in the Akata cell line (EBV positive) ( Figure 2A ), and no signals were detected in the control AKATA31 (EBV negative) cell line ( Figure 2B ) and the EBV negative skin tissue ( Figure 2D ).
case_images: DNA Polymerase Chain Reaction (PCR) analysis of Kaposi Sarcoma (KS) tumor from the ocular surface: DNA was extracted from frozen tissue samples of the KS tumor for PCR analysis. (A) PCR detecting Kaposi sarcoma-associated herpesvirus (KSHV) using Latency-Associated Nuclear Antigen (LANA) primers. Case: Patient sample demonstrating KSHV positivity. Positive Control: BC3 cell line, known to be infected solely by KSHV. Negative Control: Water. (B) PCR detecting Epstein Barr virus (EBV) using Epstein Barr virus Nuclear Antigen-1 (EBNA1) primers. Case: Patient sample showing EBV positivity. Positive Control: Akata cell line, known to be infected solely by EBV. Negative Controls: Akata31 cell line, normal skin, and water.
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case_images: Figure 3A ). Other negative controls included skin tissue without KS and water ( Figure 3B ). Our PCR results confirmed the presence of both KSHV and EBV viral DNA in this ocular KS tissue, as shown in  Figures 3A, B , respectively. Supplementary Figures 1F ,  3B, E ). Similarly, when the BC3 (EBV negative, KSHV positive) cell line was stained with anti-EBNA1 antibodies from both Abcam ( Supplementary Figures 1B ,  3H ) and Invitrogen ( Supplementary Figures 1D ,  3K ), no cross-reactivity was observed.
case_images: Immunofluorescence Assay (IFA) showing the colocalization of the Kaposi sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen (LANA) protein and Epstein Barr virus's nuclear antigen 1 (EBNA1) with endothelial cell markers, specifically CD31. (A-F) (Using EBNA1 from Abcam): (A) Nuclear staining with DAPI. (B) Overlay image of DAPI and LANA, denoting the presence of KSHV within the cell nucleus. (C) Overlay image of DAPI and EBNA1 (Abcam antibody), indicating the presence of EBV within the cell nucleus. (D) Overlay image of DAPI and CD31, emphasizing that the tumor cells exhibit endothelial cell markers. (E) Tri-overlay of A, B, and C, illustrating the colocalization of EBNA1 and LANA within tumor cells. (F) Quad-overlay of A, B, C, and D, highlighting the colocalization of EBNA1 and LANA in CD31-positive tumor cells. (G-L) (Using EBNA1 from Invitrogen): (G) Nuclear staining with DAPI. (H) Overlay image of DAPI and LANA, denoting the presence of KSHV within the cell nucleus. (I) Overlay image of DAPI and EBNA1 (Invitrogen antibody), indicating the presence of EBV within the cell nucleus. (J) Overlay image of DAPI and CD31, emphasizing that the tumor cells exhibit endothelial cell markers. (K) Tri-overlay of G, H, and I, illustrating the colocalization of EBNA1 and LANA within tumor cells. (L) Quad-overlay of G, H, I, and J, highlighting the colocalization of EBNA1 and LANA in CD31-positive tumor cells. All images were captured at 40X magnification with a KEYENCE microscope and analyzed using the KEYENCE BZ-X800 Analyzer. For journal presentation, images were cropped to a 300x300 pixel resolution using Microsoft's Paint 3D software.
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case_images: To determine whether the ocular KS tumor cells are co-infected by both KSHV and EBV ( Figure 4 ), multi-colored immunofluorescence assays (IFA) were used to detect the co-localization of both KSHV LANA and EBV EBNA1 proteins in the tumor cells according to previously published methodology. Alexa Fluor donkey anti-mouse IgG and anti-rat IgG from Invitrogen were used as secondary antibodies. As shown in  Figures 4C, I  (Abcam and Invitrogen, respectively), the anti-EBNA1 antibody indicated the presence of EBNA1 proteins (red signals) within the nucleus of the tumor cells, where LANA ( Figures 4B, H ) proteins (green signals) were also detected. Similarly, the Abcam and Invitrogen anti-EBNA1 antibodies also detected the presence of EBNA1 proteins within the nucleus of LANA-positive cells ( Figures 4E, K , respectively). Notably, the EBNA1 antibody from Abcam displayed two clear staining patterns in both IHC ( Figure 1G ) and IFA ( Figure 4C ): a diffuse signal and punctate nuclear staining. The EBNA1 antibodies from Invitrogen showed a more distinct punctate staining in both IHC ( Figure 1J ) and IFA ( Figure 4I ). Since our human CD20 IHC staining showed a lack of B lymphocytes within the ocular KS tissue, we stained the tumor tissue against human endothelial cell markers CD31 ( Figures 4D, J ), using IFA to identify the cell type co-infected with EBV and KSHV. Co-staining of CD31 with either Abcam anti-EBNA1 ( Figure 4F ) or Invitrogen ( Supplement Figure 4L ) indicated that the EBV-infected cells are also endothelial cells, same as the KSHV-infected cell type as demonstrated by the co-staining of CD31 with LANA ( Figures 4F, L ). To further validate the absence of "bleed through" signals during multi-colored IFA, we stained slides exclusively with either anti-EBNA1 alone ( Supplementary Figures 4B, F ) or anti-LANA alone ( Supplementary Figures 4J ), followed by scanning the slide at channels used for both EBNA1 (AF647, red color) and LANA (AF488, green color) ( Supplementary Figures 4C, G, K ) so that both color channels were displayed separately to verify that signals from one color were not bleeding into the other channel.