High levels of retroperitoneal fibromatosis (RF)-associated herpesvirus in RF lesions in macaques are associated with ORF73 LANA expression in spindleoid tumour cells

Abstract

Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; Human herpesvirus 8), the causative agent of KS, have been identified. In macaques, the RV1 lineage is represented by retroperitoneal fibromatosis (RF) herpesvirus (RFHV), the homologue of KSHV, whilst the RV2 lineage is represented by rhesus rhadinovirus (RRV), a more distantly related virus. Real-time quantitative PCR was used to estimate the loads of RV1 and RV2 rhadinoviruses in simian acquired immunodeficiency syndrome-associated RF (SAIDS-RF), a neoplasm of macaques with similarities to AIDS-associated KS. Both RV1 and RV2 rhadinoviruses were detected in macaques with RF. The RV1 loads were 220-to 4300-fold higher in RF tumours than in spleen, showing a strong tumour association (mean loads of 1 800 000 vs 2900 copies per 10 6 cells in tumours and spleen, respectively). In contrast, RV2 loads in the RF tumours were 100-fold lower than RV1 loads and showed similar levels in tumours and spleen (mean loads of 16 000 vs 24 000 copies per 10 6 cells, respectively). Immunostaining with antibodies reactive against RFHV ORF73 latency-associated nuclear antigen (LANA) showed intense nuclear staining of the spindleoid RF tumour cells. Correlation of viral load and the number of LANA-positive cells indicated that RF tumour cells contained multiple copies of the RFHV genome per cell. This pattern of infectivity is similar to that seen in KS tumours latently infected with KSHV. Our study demonstrates similarities in the biology of KSHV and RFHV and supports a role for RFHV in the aetiology of SAIDS-RF.

Next-Generation Sequence Analysis of the Genome of RFHVMn, the Macaque Homolog of Kaposi's Sarcoma (KS)-Associated Herpesvirus, from a KS-Like Tumor of a Pig-Tailed Macaque

“…We previously reported that macaque RF tumors are latently infected with RFHV, with several viral genomes per cell (17). This level of virus infection is similar to that seen in latent KSHV infections in KS tumor biopsy specimens and constitutes the highest level of RFHV that has been identified in infected macaque tissue.…”

“…This yielded an average depth of 3.3 reads per nucleotide. Since the oncostatin M gene and the macaque genome are diploid in each cell, the Illumina read data indicated that RFHV was present at 4.5 copies per cell, strongly correlating with our previous qPCR quantitation of 3.4 viral genomes per cell (17). We also mapped the Illumina reads onto the complete genome sequence of SRV-2, which we had detected previously in the RF tumor sample by PCR (41).…”

“…DNA was isolated from a portion of the tumor by digestion with proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. This DNA was found to contain 3.4 copies of the RFHVMn genome per cellular genome by real-time PCR (17). A library was constructed from the tumor DNA using the Illumina Genomic Sample DNA Preparation Kit according to the manufacturer's instructions.…”

“…We had previously shown by qPCR that this RF tumor contained 0.021 genome per cell of MneRV2, the pig-tailed macaque RV2 rhadinovirus homolog of RRV (17). This indicated that the RV2 virus was not present uniformly in the tumor cells and was probably present in infected lymphocytes infiltrating the tumor.…”

“…Based on these sequence and genetic similarities, RFHV was grouped with KSHV within the genus Rhadinovirus of gammaherpesviruses. Using quantitative-PCR (qPCR) techniques, high levels of RFHV genomic DNA were detected in RF tumors from different macaque species, with viral loads reaching several viral genomes per cell (16,17), corresponding to the viral load of latent KSHV in KS tumors (3). Furthermore, immunohistochemical studies revealed high levels of the RFHV ORF73 LANA homolog in the nuclei of the RF spindloid tumor cells, detected with a monoclonal antibody to KSHV LANA (16,18).…”