Experimental infection of immunocompetent macaques.
A rhadinovirus was isolated from PBMC of a pig-tailed macaque (animal 19545). This virus was called PRV19545 for PRV isolate 19545, to distinguish it from RRV isolates. Virus stocks of PRV19545 were prepared as described previously for RRV (21
). Sequencing of the R1 gene (15
) and gB gene (1
) revealed PRV19545 to be closely related to but distinct from RRV isolate 26-95 (21
). Sequencing of full-length gB genes from nine rhadinovirus isolates from three species of macaques has recently revealed all to be rather closely related, with up to 7.2% divergence at the amino acid level (1
). However, phylogenetic analysis demonstrated that the rhadinovirus isolates grouped according to species of origin, not primate facility of origin (1
). These analyses suggested that RRV and PRV are closely related but distinct viruses. RRV26-95 and PRV19545 were inoculated intravenously into a total of 11 macaque monkeys in the absence of concurrent SIV infection (Table ). Both rhesus and pig-tailed macaques were used for these experimental inoculations in order to examine the effects of same- versus cross-species infection on pathogenic potential (Table ). Two of the rhesus monkeys that were inoculated with PRV were antibody positive to RRV at the time of inoculation. The other nine recipient monkeys were antibody negative. Blood samples were obtained at periodic intervals after inoculation and used for measurement of antibody responses and virus recovery.
All monkeys inoculated with RRV seroconverted to positive anti-RRV antibody status within the early weeks after inoculation (Fig. A). The anti-RRV antibodies have persisted at high levels for as long as we have followed the animals. The three antibody-negative macaques that were inoculated with PRV seroconverted to positive anti-PRV antibody status within the early weeks after inoculation (Fig. B). The anti-PRV antibodies have also persisted at high levels for as long as we have followed the animals. Animals infected with RRV or PRV made antibodies that reacted strongly with both RRV and PRV antigens. Serologic cross-reactivity between RRV and PRV was extensive, with a tendency toward slightly increased reactivity to the homologous virus (data not shown). These results demonstrate consistent, persistent infection of naive macaques by RRV and PRV. The two RRV-antibody-positive rhesus monkeys (190-96 and 195-96) that were inoculated with PRV exhibited an increase in antibody reactivity to PRV following inoculation (Fig. C), suggesting a possible take of PRV in rhesus monkeys already infected with RRV.
FIG. 1 Antibody responses following experimental inoculation with RRV or PRV. (A) Antibody response to RRV by ELISA in macaque monkeys inoculated with RRV. Mm, M. mulatta; Mn, M. nemestrina. (B) Antibody response to PRV by ELISA in macaque monkeys inoculated (more ...)
Experimental infection of macaque monkeys with PRV and RRV was also demonstrated by virus recovery from PBMC using rhesus monkey fibroblast cultures. RRV or PRV was recovered from the majority of inoculated animals at two or more time points. Attempts to recover rhadinovirus from RRV-seronegative animals in control experiments have repeatedly failed. RRV or PRV was recovered at one or more time points from 11 of the 11 monkeys used in these studies. We also attempted to roughly quantitate the numbers of infectious cells in PBMC by performing RRV and PRV recoveries with serial threefold dilutions of cells starting at 106 PBMC in duplicate. Representative results from one set of monkeys inoculated at the same time with RRV are shown in Fig. . RRV and PRV loads in this assay appeared to peak 1 to 4 weeks following the inoculation. The highest loads reached a numerical score of 5, which corresponds to virus recovery with 12,345 PBMC (Fig. ). gB sequences from virus recovered from 190-96 and 195-96 that were already RRV positive when PRV19545 was inoculated did not correspond to the PRV19545 gB sequence (data not shown).
FIG. 2 Semiquantitative recovery of RRV from PBMC of inoculated monkeys. The numbers of infectious cells in PBMC were quantitated as described in Materials and Methods. Code for PBMC load: 0, virus was not recovered even when 106 PBMC were used; 1, virus was (more ...)
Five of 11 animals had a febrile reaction, as defined by an increase from baseline measurements of >2.0°C or absolute temperature >104.0°C at any time point. Fever was recognized as early as 48 h following inoculation and persisted for up to 14 days. Of the two rhesus macaques seropositive to RRV prior to inoculation (Mm 190-96 and 195-96), neither became febrile. Of the animals that became febrile, two were rhesus macaques inoculated with RRV (same species), two were rhesus macaques inoculated with PRV (cross species), and one was a pig-tailed macaque inoculated with RRV (cross species).
The complete blood counts for each animal included leukocyte count, absolute and relative counts of lymphocytes, neutrophils, eosinophils, basophils, and monocytes, hematocrit, platelet count, erythrocyte count, hemoglobin, mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration. Values remained within normal limits in all animals postinoculation. While there were minor fluctuations, there were no consistent changes in any of these values postinoculation. This was also true when the group was examined as a whole and when subgroups (RRV versus PRV, seropositive versus seronegative or M. mulatta M. nemestrina) were examined individually.
Skin and oral biopsies showed no unusual features. There were no clinically apparent cutaneous manifestations, and these animals remained healthy and free of disease throughout the 437 days of follow-up.
Rhadinovirus-associated lymphadenopathy in immunocompetent SIV-negative macaques.
Clinically evident lymphadenopathy was detected in 8 of the 11 animals as soon as 2 weeks after rhadinovirus inoculation. Microscopic morphologic features were similar regardless of the rhadinovirus inoculum. Histologically, the lymphadenopathy was characterized at 2 weeks by marked paracortical lymphocytic hyperplasia which effaced normal architecture (Fig. A). This paracortical expansion was accompanied by an abundance of small arborizing vessels lined by hypertrophied and hyperplastic endothelium (Fig. B). The expanded paracortex contained increased numbers of immunoblasts, mitotic figures, and histiocytes and moderate numbers of small lymphocytes. Immunostaining revealed an increase in both CD20-positive B lymphocytes and CD3-positive T lymphocytes within the expanded paracortical zone. Dispersed within this diffuse paracortical expansion were occasional regions of follicular hyperplasia. Mantle zones were variably developed, and occasional follicles were confluent. An increased number of small blood vessels were found between the developing follicles.
FIG. 3 Morphologic features associated with experimental inoculation with RRV. (A) Marked paracortical lymphocytic hyperplasia 2 weeks following RRV inoculation in an immunocompetent rhesus macaque. Hematoxylin-and-eosin (H&E) stain; magnification, ×53. (more ...)
Lymphadenopathy was absent in the two rhesus macaques that were seropositive at the time of PRV inoculation, 190-96 and 195-96. Histologically these animals lacked the marked vascular changes present in most of the other animals. Mild to moderate paracortical expansion was present in animal 195-96.
The paracortical expansion in animals with lymphadenopathy was less pronounced by 4 weeks postinoculation and had been replaced by extensive follicular hyperplasia. The most severe follicular hyperplasia was seen in rhesus macaques 380-96, 282-96, and 266-97, all of which received PRV. In two animals (380-96 and 266-97), follicular hyperplasia had completely effaced the normal architecture of the medulla and cortex at 4 weeks after infection (Fig. D). Regions of vascular hyperplasia were still evident in the paracortex and surrounding follicles. Rhesus macaques 190-96 and 195-96 lacked any follicular hyperplasia.
By 12 weeks postinoculation, clinically recognized lymphadenopathy had resolved in all of the eight monkeys in which lymphadenopathy was seen. In all animals, there were increased numbers of involuted follicles characterized by small germinal centers and the presence of periodic acid-Schiff stain-positive material. These hyalinized follicles were occasionally penetrated by a single small blood vessel and less commonly surrounded by layers of loosely concentric lymphocytes (Fig. E and F). These follicular changes, although nonspecific, are unusual in normal animals and share features with the hyaline-vascular variant of Castleman’s disease in humans. In animals in which enlargement of the peripheral lymph nodes was still present, there was a combination of follicular hyperplasia and continued paracortical expansion with vascular hyperplasia.
Rhadinovirus infection of immunodeficient macaques.
Six rhesus monkeys that had been previously infected with SIV were also inoculated with PRV or RRV (Table ). Three of these six were already antibody positive to RRV at the time of PRV inoculation (Table ). Four additional rhesus monkeys that were RRV negative and SIV negative were coinoculated with RRV plus SIV or PRV plus SIV (Table ).
Prior infection with SIV appeared to result in weaker and/or delayed antibody responses to RRV or PRV (Fig. and ). Rhesus monkeys 196-94, 181-90, and 229-91 were all SIV infected and RRV negative at the time of RRV or PRV inoculation, and all had weaker or delayed antibody responses to RRV or PRV (Table , Fig. , and Fig. ). Coinoculation with RRV and SIV or PRV and SIV also appeared to weaken or delay the antibody response to PRV or RRV in most animals (Fig. ). Despite the marginal or absent anti-PRV antibody response in coinoculated monkeys 84-96 and 91-96, PRV was recovered from PBMC on repeated occasions following the PRV-plus-SIV inoculation, and thus these animals were clearly infected by the PRV inoculation.
Antibody responses to PRV following PRV inoculation into SIV-infected M. mulatta (Mm) monkeys.
Antibody responses to RRV or PRV in M. mulatta (Mm) monkeys coinoculated with SIV and RRV or SIV and PRV.
Interpretation of the lymph node changes in immunodeficient macaques was confounded by preexisting pathology in the lymph nodes, including follicular hyperplasia and dysplasia, findings common in SIV-infected macaques. Clinically there was enlargement of lymph nodes (lymphadenopathy) relative to the preinoculation state in all animals following rhadinovirus inoculation, and this enlargement was most severe in animals rhesus monkeys 229-91 and 168-94. In immunodeficient RRV-negative rhesus macaques (229-91, 181-90, and 196-94) inoculated with rhadinovirus, histologic changes included paracortical expansion and vascular hyperplasia accompanied by florid follicular hyperplasia. In seropositive animals, paracortical expansion was seen to a lesser degree and there was exacerbation of follicular hyperplasia. The specificity of these changes is unknown.
Of the six rhesus macaques that were previously infected with SIV and subsequently inoculated with RRV or PRV, four have died. Three of these four PRV/RRV-inoculated rhesus macaques that were previously infected with SIV died with thrombosis, vascular hypertrophy, and nonsuppurative vasculitis of pulmonary vessels. Aseptic proliferative endocarditis was present in two cases (Fig. G). These changes are characteristic of SIV arteriopathy, a condition of unknown etiology recognized commonly in SIV-infected macaques (12
). Pulmonary lymphocytic infiltrates were present in the three animals with arteriopathy and accompanied by renal lymphoid infiltrates in two cases. These changes, indicative of a lymphoproliferative disorder recognized in SIV-infected macaques (11
), are of unknown etiology and a common morphologic feature found at necropsy.
Animals coinoculated with SIVmac251 and PRV or RRV developed a clinical lymphadenopathy by 2 weeks postinoculation. Histologic features in these animals were similar to those seen in 8 of the 11 immunologically normal macaques described above, including extensive vascular hyperplasia and paracortical expansion. The degree of vascular hyperplasia within lymph nodes was more florid than that seen in immunologically normal animals and persisted in three animals (rhesus macaques 121-96, 91-96, and 84-96) at 12 weeks postinoculation.
Monkeys coinoculated with SIV and RRV or SIV and PRV also appeared to have an attenuated antibody response to SIV (Fig. ). Coinfected animals not only had weaker antibody responses but also had shorter mean survival times than 40 SIVmac251-infected controls studied previously (155 days versus 560 days; P < 0.019). Coinoculated monkeys developed SIV RNA loads in plasma that were about 10-fold higher than those found in historical controls inoculated with the same stock of SIVmac251 only (Fig. ). Differences at weeks 1, 2, 4, and 12 were statistically significant by the Mann-Whitney rank sum test (P = 0.020 to 0.048). Animals that were coinfected with SIV and RRV or PRV at the same time died with a variety of opportunistic infections characteristic of simian AIDS, including those caused by Cryptosporidium parvum, P. carinii, and enteropathogenic Escherichia coli (Table ). Pulmonary lymphocytic infiltrates were present in two of four animals. In contrast to the animals previously infected with SIV and then inoculated with rhadinovirus, pulmonary vascular lesions were not identified in any of these four animals (Table ).
Anti-SIV antibody responses in M. mulatta (Mm) monkeys inoculated with SIV and RRV, SIV and PRV, or SIV alone. conj, conjugate.
SIV RNA loads in plasma in monkeys inoculated with SIV alone, or SIV and RRV, or SIV and PRV. Numbers in parentheses refer to numbers of animals used for determining the average for each data point.
TABLE 2 Summary of survival and necropsy findings in SIV-infected rhesus macaques inoculated with RRV andPRV Detection of RRV in tissues and blood.
Lymph node, oral mucosa, skin, and PBMC were obtained at 2, 4, and 12 weeks postinoculation and tested for the presence of RRV by PCR (Table ). Viral DNA was amplified from all three tissues and from PBMC. Samples from RRV-inoculated, SIV-negative animals were PCR positive for RRV sequences by 2 weeks postinoculation in lymph node (four of four), oral mucosa (two of four), skin (one of four), and PBMC (one of three). Virus persisted in the tissues to at least 12 weeks postinoculation, as demonstrated by PCR in lymph node (three of four), skin (two of four), and PBMC (three of four). Fewer tissues were positive for RRV by PCR in two animals that were coinoculated with RRV and SIVmac251.
TABLE 3 Detection by PCR of RRV in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following experimentalinoculation
To determine the cell type infected in peripheral blood, we performed PCR using sorted cells from PBMC. PBMC were harvested from pig-tailed macaque 270-97 at 2 weeks after inoculation with RRV. Cells were stained for CD20, CD8, CD4, and CD14 and were sorted by flow cytometry. DNA was isolated from the sorted, pelleted cells, and PCR was performed to determine the primary cell type infected with RRV. CD20+ cells were positive at 15,000 cells per reaction and remained positive down to a dilution of 3,200 cells per reaction (Fig. ). CD8+ cells were positive at 15,000 cells per reaction and were negative at dilutions of 7,500 cells or less. CD4+ and CD14+ cells were PCR negative for RRV. This dilution analysis indicated that the CD20+ B lymphocyte is the primary cell type infected with RRV in M. nemestrina.
RRV DNA in sorted cells. Products of PCR for RRV DNA in sorted cells were visualized by ethidium bromide staining following agarose gel electrophoresis. MW, molecular weight; FACS, fluorescence-activated cell sorting.