Kaposi Sarcoma in Mantled Guereza

We identified a novel Kaposi’s sarcoma herpesvirus–related rhadinovirus (Colobine gammaherpesvirus 1) in a mantled guereza (Colobus guereza kikuyensis). The animal had multiple oral tumors characterized by proliferation of latent nuclear antigen 1–positive spindle cells and was not co-infected with immunosuppressive simian viruses, suggesting that it had Kaposi sarcoma caused by this novel rhadinovirus.

well as tumorous tissue of the buccal area (upper lip and lower lip) were obtained for viral testing that included a pan herpesvirus nested PCR. Biopsy sites were closed intracutaneously by using a resorbable intracutaneous suture. The animal was treated with 0.2 mg/kg meloxicam (Metacam [5 mg/mL]; Boehringer Ingelheim Vetmedica GmbH, https://www.bi-vetmedica.com) and 10 mg/kg

Immunohistochemical Analysis
Immunohistochemical analysis was performed on paraffin-embedded sections by using the following primary antibodies: Ki67 antibody (monoclonal mouse antihuman Ki67 antigen,

Extraction of Total DNA from Samples
Total DNA was isolated from the indicated tissue samples by using the First-DNA All-Tissue Kit (GEN-IAL GmbH, https://www.gen-ial.de) according to the manufacturer's instructions. Concentration of extracted DNA was quantified by means of optical density (OD) measurements, and total DNA was adjusted to a concentration of 0.1 µg/µL. Subsequently, all DNA concentrations were verified by 3 OD measurements.

Qualitative PCR and Sequencing
Viral sequences were amplified by PCR from total DNA isolated from blood (in EDTA), a swab specimen obtained from cut surface of the masses, and tumorous tissues of the buccal area, upper lip, and lower lip. PCR amplification was performed by using previously reported pan erpesvirus PCR nested primer sets (1). The final volume of the PCR was 25 μL and contained the following components: 0.5 mmol/L MgCl2, 2.5% (vol/vol) dimethylsulfoxide, 0.2 mmol/L deoxynucleoside triphosphates, 1.2 μmol/L of each primer, 1× PCR buffer, and 0.25 μmol/L HotStarTaq DNA Polymerase (QIAGEN, https://www.qiagen.com). For amplification of the target sequences, samples were first heated to 95°C for 4.5 min; 55 cycles were then run with 1 cycle consisting of melting (20 s at 95°C), annealing (30 s at 46°C or 20 s at 46°C for nested PCR amplification), and extension (30 s at 72°C or 20 s at 72°C for nested PCR amplification).

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Samples were then incubated for 10 min at 72°C. PCR products were extracted from agarose gels after electrophoresis by using a commercial kit (NucleoSpin Gel and PCR Clean-Up; Macherey-Nagel, https://www.mn-net.com) according to the manufacturer's instructions and sequenced by using Sanger sequencing.

Quantitative PCR
For quantitative analysis of viral loads, primers and probes were designed that were specific for the polymerase gene. A GenScript (https://www.genscript.com) real-time PCR

Chip-Based Technology for Detection of Antibodies
For simultaneous detection of serum antibodies to herpes simplex viruses, simian immunodeficiency virus, simian retrovirus of type D, simian T-cell leukemia virus, measles virus, rhesus rhadinovirus, lymphocryptovirus, cytomegalovirus, and simian foamy virus, we used a commercial microarray multiplexing technology (Simian Panel E Kit; Intuitive Biosciences, http://intuitivebio.com). All steps were performed according to the manufacturer's instructions. Subsequently, microarrays were scanned and analyzed by using the image capture and analysis system AQ 1000 (Intuitive Biosciences).

Preparation of Antigens and Kaposi's Sarcoma Herpesvirus ELISA
ELISA coating material was prepared by induction of either empty iSLK cells or iSLK cells harboring Kaposi's sarcoma herpesvirus BAC16 with 2.5 mmol/L sodium butyrate and 1 µg/mL doxycycline for 2 days. Cells were then harvested by brief trypsinization and collected by centrifugation. Pelleted cells and debris were lysed in phosphate-buffered saline (PBS) containing