Longitudinal Patterns of Viremia and Oral Shedding of Rhesus Rhadinovirus and Retroperitoneal Fibromatosis Herpesviruses in Age-Structured Captive Breeding Populations of Rhesus Macaques (Macaca mulatta)

Abstract

Rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), 2 closely related γ2 herpesviruses, are endemic in breeding populations of rhesus macaques at our institution. We previously reported significantly different prevalence levels, suggesting the transmission dynamics of RRV and RFHV differ with regard to viral shedding and infectivity. We designed a longitudinal study to further examine the previously observed differences between RRV and RFHV prevalence and the potential influence of age, season, and housing location on the same 90 rhesus macaques previously studied. Virus- and host-genome–specific real-time PCR assays were used to determine viral loads for both RRV and RFHV in blood and saliva samples collected at 6 time points over an 18-mo period. Proportions of positive animals and viral load in blood and saliva were compared between and within viruses by age group, location, and season by using 2-part longitudinal modeling with Bayesian inferences. Our results demonstrate that age and season are significant determinants, with age as the most significant factor analyzed, of viremia and oral shedding for both RRV and RFHV, and these pathogens exhibit distinctly different patterns of viremia and oral shedding over time within a single population.

Rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV) are 2 closely related γ2 herpesviruses that are endemic in many captive macaque populations.^5,6,12,42,45 Both RRV and RFHV are macaque homologs of human herpesvirus 8, also known as Kaposi sarcoma-associated herpesvirus (KSHV). Recognition of histologic similarities between KS lesions in humans and retroperitoneal fibromatosis lesions in macaques led to the identification of RFHV.²⁵ Retroperitoneal fibromatosis is characterized as an aggressive proliferation of highly vascular fibrous tissue subjacent to the peritoneum covering the ileocecal junction and associated lymph nodes.³⁶ A less common cutaneous form has also been recognized.³⁸ Although it has not been fully sequenced, RFHV appears genetically more similar to KSHV than does RRV.^11,24 RFHV subsequently was classified with KSHV as a γ2 rhadinovirus of the RV1 lineage.^11,25

RRV was first isolated from 3 rhesus macaques maintained in captivity at the New England Primate Research Center.¹² One of those isolates (H26-95) has been fully sequenced and found to show homology to both KSHV and RFHV.^7,12,30,34 A second isolation of RRV was made at the Oregon National Primate Research Center from an SIV-infected animal with a lymphoproliferative disorder.³² RRV has been classified as a γ2 rhadinovirus of the RV2 lineage. RRV has been associated with development of persistent lymphadenopathy and B cell hyperplasia in animals coinfected with SIV.^23,27,32,45 To date, these 2 isolates represent the only complete RRV genomes sequenced, thereby limiting knowledge of sequence variations in populations of macaques.^12,32,33

Typical of most herpesviruses, both RRV and RFHV establish latent infections in immunocompetent hosts after primary infection.^2,13 Immunosuppression leads to lytic replication and disease manifestations.^38,8,31 We previously reported that RRV and RFHV are both endemic in breeding colonies of captive rhesus macaques at our institution, and RRV–RFHV coinfection is common.⁴² The implications of coinfection with regard to potential viral interactions and host immune responses to 2 closely related viruses have not been considered adequately in developing nonhuman primate models of human rhadinovirus infection. In addition, RRV is also one of the persistent viral infections targeted for elimination from some SPF rhesus breeding colonies.¹⁸ Understanding the natural history of RRV and RFHV may enable improvement of management practices to exclude these viruses from SPF populations.^18,21,43

In this longitudinal study, we examined changes in prevalence of detectable viremia and oral shedding for RRV and RFHV. We also assessed changes in viral loads of RRV and RFHV in blood and saliva over time and determined the influence of selected host and environmental determinants, specifically age, breeding corral location, and season, in the same group of rhesus macaques as was the focus of a previous cross-sectional study.⁴²

Materials and Methods

Animals.

A total of 90 rhesus macaques (Macaca mulatta) ranging in age from 7 mo to 21 y were selected randomly from 3 outdoor half-acre breeding corrals for sequential sampling over 18 mo, as published previously.⁴² All monkeys housed in breeding corrals at the California National Primate Research Center are examined quarterly, and the collection of heparinized whole blood and saliva samples for rhadinovirus testing was incorporated into this routine examination schedule. The facility is AAALAC-accredited, and all animals were maintained in accordance with provisions of the Animal Welfare Act³ and the Guide for the Care and Use of Laboratory Animals.¹⁶ All aspects of this study were approved by the IACUC.

Sampling scheme.

We randomly selected 3 of 14 nonSPF breeding corrals (nos. 8, 13, and 15) for longitudinal sampling.⁴² Corrals 8 and 13 house both Indian and a few Indian–Chinese macaques, whereas corral 15 houses only Indian macaques. The prevalences of other possibly confounding viral infections in the study population were estimated by using a multiplex microbead immunoassay for antibody detection; in addition real-time PCR results were available in cases where confirmatory testing was required. The results were as follows: simian betaretrovirus, 4% antibody-indeterminate and 0% PCR-positive; simian T-cell leukemia virus, 3% antibody-positive; B virus, 29% antibody-positive; SIV, 0% antibody-positive; and simian foamy virus, 99% antibody-positive.^17,41 The levels of possible confounding viral coinfections were consistent with levels observed throughout the facility breeding colony and were not further analyzed in the current study. Systematic sampling methods were used to calculate the number of macaques to be sampled from each of the 3 corrals to estimate the prevalence of viremia and oral shedding with an error-bound of 10%.²⁹ As described previously, all macaques included in the study were antibody-positive for rhadinovirus according to an RRV immunofluoresence assay.⁴² Systematic sampling was applied to each corral independently to maintain the randomness of this technique. All conventional breeding corrals are maintained by using identical husbandry practices, thereby allowing extrapolation of the findings of this study to all nonSPF breeding corrals at the facility.

Macaques that were relocated out of these 3 breeding populations during the study period were considered lost to follow-up. The strategy of multiple imputation in sequential order was used to account for these losses and maintain statistical power and allowed inclusion of the partial data collected on animals lost to follow up.^26,46 A default estimate of 50% prevalence of RRV and RFHV was used in the calculation of sample size for each of the 3 corrals individually, thereby yielding the largest calculated sample size for each corral to accommodate losses to follow-up, and a correction for small sample populations (n < 120) was performed as part of the sample size calculation.³¹ Each of the 3 corrals was independently sampled once a quarter over an 18-mo period (6 time points). The number of macaques sampled from each corral is summarized in Table 1. Some animals were lost to follow up throughout the course of this study; Table 1 illustrates which sampling groups from which corral were effected by those losses.

Table 1.

Distribution of macaques by age group and corral at each of the 6 time points

		Time point
Age group (y)	Corral	1	2	3	4	5	6
0.6–1.5	8	3	3	3	3	3	2
	13	2	2	2	2	2	2
	15	5	5	5	5	5	5
1.6–1.9	8	5	3	3	2	2	2
	13	23	23	22	20	20	19
	15	1	1	1	1	1	1
2.0–2.9	8	6	6	6	6	5	5
	13	7	7	7	7	7	7
	15	2	2	2	2	2	2
3.0–4.9	8	8	7	7	7	5	3
	13	0	0	0	0	0	0
	15	1	1	1	1	1	1
5.0–8.9	8	9	9	9	9	9	8
	13	0	0	0	0	0	0
	15	4	3	2	2	2	2
Older than 9.0	8	8	8	6	6	6	6
	13	1	1	0	0	0	0
	15	5	5	5	5	5	5
Total		90	86	81	78	75	70

Samples.

Samples of heparinized blood and saliva swabs were collected from each animal at each time point as described previously.⁴² Briefly, blood samples were collected into vacuum phlebotomy tubes containing sodium heparin (catalog no. 367961, Becton Dickenson, Franklin Lakes, NJ). Blood samples were centrifuged at approximately 1000 × g for 15 min; plasma was removed, aliquoted and stored at −20 °C for future antibody testing. Saliva was collected by using commercial collection swabs (catalog no. GD1000, Salivary Diagnostic Systems Brooklyn, NY). DNA was extracted from 200 µL each saliva and buffy coat sample by using spin columns (catalog no. 159914, Qiagen, Valencia, CA).

PCR.

Real-time PCR was used for the detection of RRV- and RFHV-specific DNA in heparinized blood and saliva samples.^5,9,42 The oncostatin M gene (OSM) was used as the housekeeping gene control for this assay as described previously;⁴² the OSM real-time PCR assay allows for relative quantification of viral genomes detected per number of host cellular genomes in blood samples (viral load).¹⁰ Because both cell-associated and -free virus are detected in saliva samples, viral load was calculated per volume of DNA sample tested. Samples initially were tested in duplicate. Samples were considered virus-positive when amplification was seen in both of the initial 2 tests and negative if no amplification was seen in either of the initial 2 tests. Samples with discrepant test results were retested in duplicate, for a total of 4 PCR tests completed. Amplification was considered an ‘indeterminate’ test result if any of the 4 PCR replicates were discordant, (that is, if 1, 2, or 3 of the 4 PCR tests were considered indeterminate). Because one of the main goals of this study was optimizing removal of these viruses from SPF colonies, indeterminate test results were treated as virus-positive in the statistical analysis. A rhadinovirus immunofluorescent assay was completed on all macaques at the first time point, as described in the previous cross-sectional study.⁴² All animals tested positive for rhadinovirus antibodies, but this assay cannot distinguish between RRV and RFHV antibodies. Further antibody testing was not completed; however, if assays that discriminate between RRV and RFHV become available, testing could be completed at that time.

Calculation of viral load.

The previously established standard curves for OSM-, RRV-, and RFHV-specific real-time PCR reactions were used to determine the numbers of viral and cellular genomes detected in each sample.⁴² A panel of positive and negative control samples (some of known plasmid dilutions) were tested on each plate to validate the use of the previous standard curves. Samples were tested in duplicate, and the cycle threshold values obtained were averaged to determine the number of cellular genomes tested. Determination of the number of cellular genomes analyzed and comparison with the viral genome number from standard curves allows for calculation of the viral genome copy number per number of cellular genomes in blood samples.⁵ Because much of the virus detected in saliva samples is cell-free, viral load for saliva samples was calculated as viral copies per volume of DNA tested.

Statistical analysis.

Prior to analysis, data were stratified by age, corral location, and season. Age effect was studied by separating animals into 6 age groups based on the age of the animal at the start of the longitudinal study: age group 1, 0.6 through 1.5 y; age group 2, 1.6 through 1.9 y; age group 3, 2.0 through 2.9 y; age group 4, 3.0 through 4.9 y; age group 5, 5.0 through 8.9 y; and age group 6, 9 y and older (Table 1). Corral location effects were studied by comparing results obtained from each of the 3 corrals (nos. 8, 13, and 15) sampled. Seasonal effects were studied by comparing results obtained from samples collected in winter (January, February, and March), spring (April, May, and June), summer (July, August, and September), and fall (October, November, and December).

We first analyzed the effects of season, corral location, and age on the proportions of macaques PCR-positive for RRV or RFHV (or both) in both blood and saliva samples. For this proportional analysis, a logistic regression model with random intercepts was fitted by using the MIXED and GLIMMIX procedures in the Statistical Analysis System software (SAS 9.1, SAS Institute, Cary, NC). Bonferroni correction was applied to correct for multiple comparisons of samples over time and across age or season groups.^38,29,28,44

We then analyzed the effects of season, corral location, and age on both the proportions of macaques that were PCR-positive and the viral loads in both blood and saliva for PCR-positive macaques. For this analysis, Bayesian modeling by Markov Chain Monte Carlo algorithms was performed by using 2 software packages: WinBUGS 1.4.3 (MRC Biostatistics Unit, Cambridge, UK) and R 2.8.1 (Lucent Technologies, Murray Hill, NJ).^20,22,14,35 A 2-part model based on Markov Chain Monte Carlo algorithms was developed by using the WinBUGS platform for joint modeling of both the binomial viral proportion data and the semicontinuous viral load data for each virus in positive samples.²² By ‘semicontinuous,’ we mean that viral load data are a mixture of zeros (virus-negative) and positive numbers (virus-positive). This model also accommodated missing values in our data due to animals lost to follow up. The joint model (that is, 2-part model) consisted of a logistic regression model with random effects for proportion (binary) data and a conditional Poisson regression with random effects to describe the viral load data. A total of 20 animals were lost to follow up over the 18-mo study, leaving missing values in both proportion and viral load data. To accommodate the missing values, the multiple partial imputation method was applied. First, 5 sets of data were generated where missing values of proportion data were imputed by using the logistic regression model with random effects. The Gibbs sampling process for creating imputations was used in generating 5 different complete data sets with imputed values representing missing data points (with 20,000 iterations between imputations).^15,26,46 Then we fitted the 2-part model by using the MCMC method in WinBugs to create 100,000 simulations. Finally, the 5 versions of estimates of the parameters in the models were combined by using Rubin rule to generate overall conclusions.^26,46 Findings were considered statistically significant if the P values were 0.05 or less.

The figures generated in this study are based on both the model predictors as well as observed data (age, corral location, season). To estimate copy numbers, the fitted model was used with observed data in the sample for age, cage, and season. Therefore, the figures represent what was predicted by a combination of the model and the observed data, which may differ slightly from those observed in the study itself because of the sampling scheme used. The figures only demonstrate the sampled data; the conclusions based on the parameters of the fitted model tell us the true inferences regarding the target population. All data presented in figures summarize the means of the overall 500,000 iterations performed in the statistical model based on the 480 data points collected from the original 90 animals studied

Results

The age distribution and number of macaques sampled over all 6 time points are presented in Table 1. Of the 90 animals selected for longitudinal sampling, 20 were lost to follow up during the course of this study. Viremia and salivary shedding of RRV and RFHV was intermittent, and coinfections were common. Tables 2 and 3 illustrate the percentages of macaques that shed either RRV and RFHV or both at all time points. Patterns of salivary shedding and viremia for each virus over all 6 time points are shown in Figures 1 through 7. All data presented in figures summarize the means of the 500,000 iterations performed in statistical modeling based on the 480 data points collected from the original 90 animals studied. Our conclusions are based on the parameters of the fitted models, whereas the figures only demonstrate the sampled data. Few macaques from the oldest group (9 y and older) were PCR-positive, but the findings were statistically significant (P = 0.0056).

Table 2.

PCR results for RRV and RFHV in blood and saliva macaques over all 6 time points

		RFHV
		Blood + Saliva+	Blood + Saliva –	Blood – Saliva +	Blood – Saliva –
RRV	Blood + Saliva +	3.93%	1.65%	12.60%	19.42%
	Blood + Saliva –	1.45%	1.65%	4.13%	18.18%
	Blood – Saliva +	0.83%	0.21%	2.89%	8.26%
	Blood – Saliva –	1.45%	0.21%	5.17%	17.98%

Data are given as the proportion of all samples tested.

Table 3.

Samples (no. [%]) PCR-positive for RRV and RFHV by season and age group over all 6 time points

			Age group (y)
			0.6–1.9	1.6–1.9	2.0–2.9	3.0–4.9	5.0–8.9	older than 9.0		Total
RRV	blood	fall	15 (5)	15 (5)	16 (5)	11 (4)	15 (5)	6 (2)		78 (26)
		spring	18 (6)	35 (12)	16 (5)	10 (3)	10 (3)	7 (2)		96 (32)
		summer	21 (7)	21 (7)	8 (3)	7 (2)	12 (4)	3 (1)		72 (24)
		winter	14 (5)	29 (10)	8 (3)	5 (2)	1 (0.3)	0 (0)		57 (19)
									Total	233
RRV	saliva	fall	9 (4)	10 (4)	7 (3)	5 (2)	4 (2)	2 (1)		37 (16)
		spring	10 (4)	28 (12)	13 (6)	10 (4)	7 (3)	4 (2)		72 (31)
		summer	20 (9)	19 (8)	10 (4)	11 (5)	12 (5)	1 (0.4)		73 (31)
		winter	13 (6)	31 (13)	4 (2)	3 (1)	1 (0.4)	1 (0.4)		53 (23)
									Total	235
RFHV	blood	fall	4 (7)	1 (2)	0 (0)	0 (0)	3 (6)	0 (0)		8 (15)
		spring	4 (7)	5 (9)	2 (4)	1 (2)	0 (0)	2 (4)		14 (26)
		summer	4 (7)	7 (13)	0 (0)	2 (4)	3 (6)	0 (0)		16 (30)
		winter	4 (7)	6 (11)	0 (0)	2 (4)	4 (7)	0 (0)		16 (30)
									Total	54
RFHV	saliva	fall	4 (3)	2 (1)	3 (2)	5 (3)	8 (5)	5 (3)		27 (17)
		spring	9 (6)	13 (8)	7 (4)	9 (6)	6 (4)	5 (3)		49 (31)
		summer	11 (7)	11 (7)	4 (3)	9 (6)	14 (9)	1 (1)		50 (31)
		winter	7 (4)	14 (9)	2 (1)	4 (3)	6 (4)	0 (0)		33 (21)
									Total	159

Figure 1.

Numbers of macaques with repeated positive test results for RRV and RFHV in blood and saliva over all 6 time points. Partial data for animals lost to follow up were included in this graph.

Figure 7.

Viral load (mean ± SEM) of RRV and RFHV in (A–C) blood or (D–F) saliva according to the statistical modeling of the 480 data points collected by corral (A and D, corral 8; B and E, corral 13; C and F, corral 15) and age group.

Figure 3.

Viral load (mean ± SEM) of RRV and RFHV in (A) blood and (B) saliva from statistical modeling by season.

Figure 6.

Viral load (mean ± SEM) of RRV and RFHV in (A–C) blood or (D–F) saliva according to the statistical modeling of the 480 data points collected by corral (A and D, corral 8; B and E, corral 13; C and F, corral 15) and season.

Age.

The mean proportions of PCR-positive results by age category for both blood and saliva samples over all time points are summarized in Figure 2. All macaques in which RFHV infections were detected were found to be coinfected with RRV at 1 or more of the 6 time points. However, numerous RRV infections were identified in the absence of RFHV infection. For RRV, a peak mean prevalence for both viremia and salivary shedding was found in young animals (age group 2, 1.6 through 1.9 y), whereas no such peak was observed for RFHV. In contrast, peak mean salivary shedding of RFHV was found in animals older than 5 y of age (age groups 5 and 6).

Figure 2.

Proportions (mean ± 1 SD) of macaques with RRV and RFHV in blood and saliva by (A) season and (B) age group from statistical modeling.

Figures 2 and 4 through 7 illustrate the differences of both prevalence (proportion PCR-positive) and viral load values for RRV and RFHV by age group. Age was a significant (MIXED analysis, P < 0.0001) factor affecting the proportion of macaques that were PCR-positive for RRV in blood samples. In general, younger animals were significantly more likely to be PCR positive for RRV in blood than older animals at all time points. A higher proportion of animals younger than 1.6 y (age group 1) were PCR-positive for RRV in blood compared with animals older than 5 y of age (age groups 5 and 6; MIXED analysis: Bayesian P = 0.02597 and P = 0.00615, respectively; Bonferroni-adjusted: P = 0.0087 and P = 0.0013, respectively). Macaques 1.6 through 1.9 y old (age group 2) had the highest prevalence of RRV viremia across all time points and were significantly more likely to be PCR-positive in blood than were animals in age groups 4 through 6 (MIXED analysis: Bayesian P = 0.00804, P = 0.00366, and P = 0.00117, respectively; Bonferroni-adjusted: P = 0.0009, P < 0.0001, and P < 0.0001, respectively). In addition, significantly fewer macaques older than 9 y were PCR-positive for RRV in blood compared with animals younger than 3 y (MIXED analysis: Bayesian P = 0.01287; Bonferroni-adjusted: P = 0.0056).

Figure 4.

Viral load (mean ± SEM) of RRV and RFHV in (A) blood and (B) saliva from statistical modeling by age group.

Age was another significant determinant of viral load of RRV in blood. Across all age groups, older macaques had a lower viral load in blood than did younger animals. Macaques in age groups 1 through 3 had higher viral loads than did animals in age groups 5 (5 to 8.9 y; Bayesian P = 0.00841, P = 0.01357, and P = 0.00343, respectively) and 6 (older than 9 y; Bayesian P = 0.00036, P = 0.01356, and P = 0.00343, respectively). In addition, animals older than 9 y had lower copy numbers of RRV in blood than did animals between 5 and 9 y of age (Bayesian P = 0.02436).

Age was a significant (MIXED analysis, P < 0.0001) factor affecting the proportion of macaques PCR-positive for RRV in saliva samples: younger animals were more likely to be shedding virus in oral secretions than were older animals. Rhesus younger than 1.6 y (age group 1) were less likely to be shedding RRV in saliva than were macaques 1.6 through 1.9 y old (age group 2; Bayesian P = 0.00580) but were more likely to be shedding RRV in saliva compared with animals older than 5 y (age groups 5 and 6; Bayesian P = 0.00961 and P = 0.00471, respectively). Animals in age group 2 (1.6 through 1.9 y) were more likely to be PCR-positive for RRV in saliva than were macaques in age groups 4 through 6 (Bayesian P = 0.00367, P = 0.00001, and P = 0.00001, respectively). Macaques in age group 3 (2.0 through 2.9 y) were more likely to be PCR-positive for RRV in saliva than were animals in age groups 5 (Bayesian P = 0.01546) and 6 (Bayesian P = 0.00787), as were animals in age group 4 (Bayesian P = 0.048 and P = 0.0206, respectively).

Significant age-associated differences also were found for RRV viral loads in saliva. The highest RRV viral load in saliva occurred in age group 2 animals (1.6 through 1.9 y). Older animals (age groups 5 and 6) had lower viral loads in saliva than did those in age group 2 (Bayesian P = 0.00723 and P = 0.00720, respectively).

No statistically significant differences in prevalence among age groups were found in animals PCR positive for RFHV in blood. In contrast to RRV, viral loads for RFHV in blood were found to be significantly higher in the oldest animals (age group 6) than for age groups 1 and 3 to 6 (Bayesian P = 0.02315; P = 0.0455; P = 0.02445; P = 0.00596; respectively). Due to a large standard error, the viral loads for RFHV in blood for age groups 2 and 6 were not significantly different (Bayesian P = 0.8).

Similar to RFHV findings in blood samples, no statistically significant differences in the prevalence levels of RFHV in saliva were noted by age or season. Viral loads for RFHV in saliva were significantly lower in age group 6 compared with age group 1 macaques (Bayesian P = 0.00550).

Season.

Significant seasonal effects on viremia and oral shedding were observed for both RRV and RFHV (Figures 2, 4 through 7).

A higher proportion of macaques were PCR-positive for RRV in blood in the spring than during winter months (Bayesian P = 0.02572). Although more animals were viremic in spring, viral loads were lower in spring than in winter, fall, or summer months (Bayesian P = 0.00154, P < 0.00001, and P = 0.00049, respectively). Summer yielded higher copy numbers of RRV in blood compared with winter months (Bayesian P = 0.03714).

Similar seasonal effects were found for detection of RRV in saliva. Fewer macaques were PCR-positive for RRV in saliva during the fall than in spring, summer (MIXED analysis, Bayesian P = 0.00333 and P < 0.00001, respectively; Bonferroni-adjusted: P = 0.0220 and P < 0.0001, respectively), and winter (Bayesian P = 0.01033) months. Viral copy numbers for RRV in saliva were lower in fall than summer (Bayesian P = 0.0004). In addition, viral copy numbers for RRV in saliva were higher in summer than winter months (Bayesian P = 0.03418).

Seasonal effects were also evident for RFHV. Higher copy numbers of RFHV in blood occurred in fall than spring months (Bayesian P = 0.01242). However, animals sampled in fall had lower copy numbers of RFHV in saliva than they did in winter, spring, and summer months (Bayesian P = 0.00032, P = 0.00420, and P = 0.00841, respectively).

Corral location.

Corral location was identified as a significant determinant of RRV in blood, as illustrated in Figures 5 through 7. Lower copy numbers of RRV in blood were found in corral 13 compared with corral 8 (Bayesian P = 0.03997). In contrast, RFHV copy numbers in blood were higher in corral 15 than corral 8 (Bayesian P = 0.01844), and corral 15 had a higher proportion of macaques PCR-positive for RFHV in saliva than did corral 8 (MIXED analysis, Bayesian P = 0.00031; Bonferroni-adjusted, P < 0.0001). Compared with corral 15, corral 13 had a lower proportion of animals PCR-positive for RFHV in saliva (Bayesian P = 0.00362) over all time points. However, macaques in corrals 13 and 15 had lower copy numbers of RFHV in saliva than did those in corral 8 (Bayesian P = 0.00889 and P = 0.00266, respectively).

Figure 5.

Proportions (mean ± 1 SD) of macaques with RRV and RFHV in blood and saliva according to the statistical modeling of the 480 data points collected by corral (A and D, corral 8; B and E, corral 13; C and F, corral 15) and (A–C) season or (D–F) age group.

Discussion

Age was a significant determinate of RRV proportions and viral loads. Patterns of viral load mirrored what was observed for RRV proportions in blood and saliva samples, with younger animals showing higher viral load values. Age likewise was a determinate of RFHV proportions and viral load but in a different manner for both blood and saliva samples: higher proportions of older rather than younger animals were PCR-positive for RFHV in saliva. Viral loads of RFHV in blood samples were similar to those for RRV, with higher loads in macaques younger than 2 y compared with older animals. The viral load of RFHV in saliva samples had a slightly different pattern, with macaques between 3 and 5 y of age having the highest viral load detected.

Significant differences were observed for RRV and RFHV by using season as the determinate, although further sampling may be required to understand these findings. The proportions of macaques PCR-positive for RRV in blood and saliva were similar during winter and summer seasons but had large differences in spring and fall seasons, with blood samples always giving a higher proportion of positive macaques, compared with saliva samples. In contrast to findings for RRV, higher proportions of macaques were PCR-positive for RFHV in saliva compared with blood for all seasons. In addition, lower viral loads of RRV in saliva were found in fall compared with summer months. The summer season had the highest proportion of animals PCR-positive for RFHV in saliva. Little seasonal effect was observed in viral loads for RFHV in both blood and saliva samples.

Contrary to our initial hypothesis, corral location was found to be a significant factor affecting the proportions of macaques PCR-positive for RFHV in both blood and saliva. In addition, corral location affected RRV viral loads in blood. Statistically significant difference between corrals 8 and 15 led to further analysis of possible confounding variables. Among many factors that were not included this analysis but that might explain the observed differences in prevalence are population density and host genetics. RFHV prevalence was inversely related to population density: corral 8 had the highest population density but the lowest proportion of animals PCR-positive for the virus in both blood and saliva samples. RFHV was shed in saliva at the highest levels in corral 15, the least dense population tested. Further analysis of husbandry records showed that 7 of the 18 macaques sampled from corral 15 had been acquired from another primate research facility more then 5 y before this study was initiated (1999), whereas all of the other animals included in this study were born at the current facility, perhaps accounting for unexpected host genetic differences. The significant differences in viral prevalence could be related to infection with different viral strains, differences in overall viral exposure, population dynamics due to the differences in population densities, or host genetic differences, all of which highlight important areas for future investigation in the development and use of this primate model of human KSHV infection. All of the studied macaque populations are maintained by using identical husbandry practices, and the current study was not equipped to address the many possible explanations listed. The large number of animals lost to follow up in this study and the small number of samples collected limit the conclusions that can be drawn from this study. The inclusion of indeterminate test results in this study may have increased the number of false positives included in the statistical analysis. While one of the main goals of this study is maintenance of SPF colonies which requires overly cautious evaluation of diagnostic tests this may lead to an overestimation of the prevalence of both RRV and RFHV in this population. However, the statistical methods allowed greater examination of patterns observed in this limited sampling study and highlight areas for further study.

Both RRV and RFHV are endemic in the breeding population of rhesus macaques at the current facility. These 2 γ2 herpesviruses demonstrated significantly different patterns of infection, viremia, and shedding. Macaques sampled in this study were more likely to be PCR-positive for RRV in blood but for RFHV in saliva. Our results have demonstrated that shedding of both RRV and RFHV in oral secretions is intermittent. The previously reported higher prevalence of RRV than RFHV⁴² perhaps is explained in part by the fewer animals shedding RFHV than RRV as well as the lower amounts of RFHV shed compared with RRV. Age was the most significant determinate of RRV and RFHV proportion and viral load, but season was a significant determinant as well. Location was associated with unexpected significant differences, and whether these are actual differences and not artifacts of the limited numbers of animals sampled needs to be confirmed. Similar to the pattern observed for the simian betaherpesvirus rhesus cytomegalovirus, rhesus macaques are infected at a very young age, which is in contrast to the pattern observed for the simian alphaherpesvirus, B virus (Cercopithecine herpesvirus 1), for which the peak of seroconversion occurs around 3 y of age, corresponding to the onset of sexual maturity in rhesus macaques.^19,39,40 Additional viral coinfections (simian betaretrovirus, simian T-cell leukemia virus, and others) to RRV and RFHV were not analyzed in the current study and are confounding variables that should be studied further. RRV and RFHV are both endemic viral pathogens of research macaque populations, and a better understanding of their natural history and epidemiology, both as individual infections and coinfections, will contribute to refinement of this nonhuman primate model of human KSHV.