Experimental co-transmission of Simian Immunodeficiency Virus (SIV) and the macaque homologs of the Kaposi Sarcoma-Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV)

Abstract

Macaque RFHV and LCV are close homologs of human KSHV and EBV, respectively. No experimental model of RFHV has been developed due to the lack of a source of culturable infectious virus. Screening of macaques at the Washington National Primate Research Center detected RFHV in saliva of SIV-infected macaques from previous vaccine studies. A pilot experimental infection of two naïve juvenile pig-tailed macaques was initiated by inoculation of saliva from SIV-infected pig-tailed and cynomolgus macaque donors, which contained high levels of DNA (> 10(6) genomes/ml) of the respective species-specific RFHV strain. Both juvenile recipients developed SIV and RFHV infections with RFHV DNA detected transiently in saliva and/or PBMC around week 16 post-infection. One juvenile macaque was infected with the homologous RFHVMn from whole saliva of a pig-tailed donor, which had been inoculated into the cheek pouch. This animal became immunosuppressed, developing simian AIDS and was euthanized 23 weeks after inoculation. The levels of RFHV DNA in saliva and PBMC remained below the level of detection after week 17, showing no reactivation of the RFHVMn infection during the rapid development of AIDS. The other juvenile macaque was infected with the heterologous RFHVMf from i.v. inoculation of purified virions from saliva of a cynomolgus donor. The juvenile recipient remained immunocompetent, developing high levels of persistent anti-RFHV and -SIV antibodies. After the initial presence of RFHVMf DNA in saliva and PBMC decreased to undetectable levels by week 19, all attempts to reactivate the infection through additional inoculations, experimental infection with purified SRV-2 or SIV, or immunosuppressive treatments with cyclosporine or dexamethasone were unsuccessful. An heterologous LCV transmission was also detected in this recipient, characterized by continual high levels of LCVMf DNA from the cynomolgus donor in both saliva (> 10(6) genomes/ml) and PBMC (> 10(4) genomes/million cells), coupled with high levels of anti-LCV antibodies. The macaque was sacrificed 209 weeks after the initial inoculation. Low levels of LCVMf DNA were detected in salivary glands, tonsils and other lymphoid organs, while RFHVMf DNA was below the level of detection. These results show successful co-transmission of RFHV and LCV from saliva and demonstrate differential lytic activation of the different gammaherpesvirus lineages due to presumed differences in biology and tropism and control by the host immune system. Although this initial pilot transmission study utilized only two macaques, it provides the first evidence for experimental transmission of the macaque homolog of KSHV, setting the stage for larger transmission studies to examine the differential activation of rhadinovirus and lymphocryptovirus infections and the pathological effects of immunosuppression.

Fig 1. qPCR Screen for RV1 and RV2 rhadinovirus DNA in saliva.

Saliva was obtained from macaques at the WaNPRC undergoing routine health screening and tested for the presence of RV1 and RV2 rhadinovirus DNA. A) The highest RV1 and RV2 DNA levels detected during longitudinal screening. B) Correlation of RV1 and RV2 viral loads for the animals in panel A showing a positive level of DNA for either RV1 or RV2.

Fig 2. Longitudinal analysis of RV1 and RV2 rhadinovirus DNA in saliva of donor macaques.

The RV1 and RV2 DNA levels in longitudinal saliva samples of donor macaques A) cynomolgus macaque 01019, B) cynomolgus macaque 98039 and C) pig-tailed macaque A02241. Saliva collected on week 0 was used in the experimental transmission studies, as indicated. The level of LCV in the week 0 saliva of 01019, determined after the inoculation, is shown in panel A.

Fig 3. Timeline of experimental infections.

The outlines of the experimental infections and treatment is shown for the naïve juvenile pig-tailed macaques, which were initially inoculated with saliva on the same day. A) K04199 was inoculated with a purified preparation of saliva from the cynomolgus macaque 98039 i.v. and whole saliva from the pig-tailed macaque A02241 orally into the cheek pouches. K04199 also received a subsequent inoculation of saliva from the cynomolgus macaque 01019 i.v. to try to boost the infection. By week 23, K04199 developed clinical signs and hematological changes meeting the end-point criteria for AIDS protocols, and euthanasia was performed. B) M04203 was inoculated with a purified preparation of saliva from the cynomolgus macaque 01019 i.v. and whole saliva from the pig-tailed macaque A02241 orally into the cheek pouches. M04203 received a second iv inoculation of saliva from 01019 44 weeks after the initial infection to try to boost the infection. Subsequently, whole saliva from 01019 was administered to the cheek pouch at week 52. To activate rhadinovirus infections, M04203 was treated with daily injections of cyclosporine for 3 weeks (week 26–29) to induce immune suppression. The animal was subsequently infected with SRV-2B and SIVMne, which have previously been associated with rhadinovirus activation and associated pathologies. The animal received a course of dexamethasone treatment to induce immunosuppression and viral reactivation, which tapered down as indicated. The animal was euthanized at week 209.

Fig 4. Experimental rhadinovirus infection in juvenile macaque K04199.

The naïve juvenile pig-tailed macaque K04199 was inoculated intravenously with diluted, filtered saliva from the 98039 cynomolgus macaque donor (Saliva C) containing high levels of the RV1 rhadinovirus (RFHVMf), and was inoculated into the cheek pouches (cp) with whole saliva from the A02441 pig-tailed macaque (Saliva B) containing high levels of RFHVMn. Subsequently, K04199 was inoculated on week 12 with diluted, filtered saliva from the 01019 cynomolgus macaque donor (Saliva A) containing high levels of RFHVMf. Saliva and blood were collected weekly and/or bi-weekly. A) The RV1 genome levels were determined using the RV1 qPCR assay and the RV1 antibody levels were determined using the RV1 Luminex assay. B) the RV2 genome levels were determined using the RV2 qPCR assay and the RV2 antibody levels were determined using the RV2 Luminex assay. C) The SIV cDNA levels were determined using the SIV qPCR assay and the SIV antibody levels were determined using the SIV Luminex assay. D) The absolute number of cells in the lymphocyte subsets was determined, as indicated. K04199 developed clinical signs of SIV-induced AIDS and was terminated on week 23. MFI = mean fluorescent intensity.

Fig 5. Experimental rhadinovirus infection in juvenile macaque M04203.

The naïve juvenile pig-tailed macaque M04203 was inoculated intravenously (i.v.) with diluted filtered saliva from the 01019 cynomolgus macaque donor (Saliva “A”) containing high levels of RFHVMf and low levels of MfaLCV and was inoculated into the cheek pouches with whole saliva from pig-tailed macaque A02241 (Saliva “B”), which had high levels of RFHVMn, as indicated in the text. M04203 received a second iv inoculation of saliva from 01019 (Saliva “A”) 44 weeks after the initial infection. Subsequently, whole saliva from 01019 (Saliva “A”) was administered to the cheek pouch at week 52. To activate rhadinovirus infections, M04203 was treated with daily injections of cyclosporine for 3 weeks (week 26–29) to induce immune suppression, as indicated (green bar). The animal was subsequently infected with SRV-2B and SIVMne, which have previously been associated with rhadinovirus activation and associated pathologies (see text). Saliva and blood were collected weekly and/or bi-weekly. A) The RV1 genome levels were determined using the RV1 qPCR assay and the RV1 antibody levels were determined using the RV1 Luminex assay. B) the LCV genome levels were determined using the LCV qPCR assay and the LCV antibody levels were determined using the LCV Luminex assay. C) The SRV2 cDNA levels were determined using the SRV2 qPCR assay and the SRV2 antibody levels were determined using the SRV2 Luminex assay. D) The SIV cDNA levels were determined using the SIV qPCR assay and the SIV antibody levels were determined using the SIV Luminex assay.

Fig 6. Longitudinal analysis of lymphocyte subsets in M04203.

The absolute number of cells in lymphocyte subsets was determined from the blood samples in the experimental infection of M04103 shown in Fig 5.

Fig 7. Complete longitudinal analysis of the experimental infection of M04203.

The juvenile macaque M04203 was experimentally inoculated with saliva from the donor macaques as indicated in the legend to Fig 5. Blood and saliva samples were collected weekly and/or bi-weekly until the experiment was terminated 209 weeks after the initial saliva inoculation and M04203 was culled. At week 164, the animal received a course of dexamethasone treatment as indicated in the text and Fig 3. A) The RV1 genome levels were determined using the RV1 qPCR assay and the RV1 antibody levels were determined using the RV1 Luminex assay. B) the LCV genome levels were determined using the LCV qPCR assay and the LCV antibody levels were determined using the LCV Luminex assay. C) The absolute count of neutrophils, monocytes and lymphocytes was determined. D) the CD4/CD8 ratio was determined from the lymphocyte count.