qRT-PCR analysis of SIV-RNA showed that all compartments of the monkeys were viral RNA-positive during the acute phase of infection. The kinetics of viral RNA expression during this time was characterized by a period of undetectable RNA levels (below 60 copies/ml, triplicate samples), followed by a peak of viral RNA production, and then a set point nadir in the first 5 weeks after infection. The largest amount of viral RNA in the saliva, feces and urine was observed during the first 4 weeks of viral replication, coincident with the rise and fall of viral RNA levels observed in the peripheral blood ().
SIV RNA levels in various compartments of rhesus monkeys during the first 90 days following infection
The median peak in viral RNA levels in the blood of these five monkeys (day 14 after infection) was 107 vRNA copies/ml of plasma in the peripheral blood, 105 vRNA copies/ml for both saliva and feces and 103 vRNA copies/ml in the urine. SIV RNA reached detectable levels in the peripheral blood as early as day 3 following infection. Viral RNA was detected in saliva in two of the five animals by day 10 and all animals by day 14. Both feces and urine of all animals had readily detectable levels of viral RNA by day 14. However, both feces and urine viral RNA levels fell to undetectable levels by 4 weeks postinfection.
Viral RNA was detected in the peripheral blood and saliva of the monkeys over a prolonged period of time. Saliva viral RNA levels varied in each monkey, consistent with the plasma viral RNA set point values. During the postacute phase of infection (after day 35), viral RNA levels in saliva were diminished significantly. Occasional positive saliva samples were, however, observed in animals with high set point plasma viral RNA levels.
To determine the total amount of detectable viral RNA in each of the sampled compartments, we calculated the total viral burden during acute viremia by area under the curve analysis (AUC). We found a significant difference (P = 0.003) between the AUC values in these monkeys in the peripheral blood and the other compartments. Blood plasma RNA levels reached 108 viral RNA copies/ml whereas the other compartments were found to be, on average, two orders of magnitude lower. We found that the levels of viral RNA did not differ significantly in saliva, feces or urine ().
SIV burden in various compartments during primary infection
In view of the established importance of plasma RNA levels for clinical disease progression in HIV-infected individuals, we were interested in exploring the association between this value and viral RNA levels in each of the other monitored compartments in these monkeys. No direct correlation was observed between plasma viral RNA levels and viral RNA levels measured in saliva, feces or urine (). Interestingly, however, these data demonstrate that viral RNA is detectable in the saliva, feces and urine only when plasma viral loads are in excess of 104 RNA copies/ml in the blood. This observation suggests that total body viral RNA levels must surpass a threshold prior to their detection in saliva, feces and urine. The viral RNA in these compartments may even represent ‘spillover’ from the blood.
Association between SIV RNA levels in blood plasma and other compartments
To complement these studies, we were also interested in exploring the utility of assessing viral RNA levels in an easily and inexpensively stored format. We, therefore, evaluated the use of saliva and whole blood spotting onto Whatman 903 cards for monitoring and quantification of viral RNA. Dried sample spotting obviates the need for sample processing and requires low sample volumes. Moreover, control experiments assessing viral RNA extracted from dried blood spots (DBSs) up to 6 months after spotting showed that this method allowed for stable room temperature sample storage (data not shown). Similar results regarding the stability of DBS specimens have also been reported by other groups [11
]. Although the risk of carrying over contamination from cutting devices is low [14
], we chose to avoid this altogether and cut out the entire dried sample spot with a 2 mm border. This also provided the benefit of an exact sample volume for extraction (i.e., 50 μl) resulting in very low sample-to-sample variation in viral RNA quantification.
In contrast to other sampling methods, the monitoring of viral RNA levels from dried saliva spots would allow the rapid and simple screening of large cohorts of vaccinees. Therefore, we monitored SIV RNA levels using dried saliva spots () and detected viral RNA as early as day 14 in three of the five animals, in all five of the animals on day 17 and in four of the 5 animals on day 21. We then compared these values to those obtained from the monitoring of frozen saliva samples. The association of spotted saliva viral RNA levels with the viral RNA levels obtained from frozen saliva was modest (r = 0.6253, P < 0.0001) (.). These results likely reflect not only the diminished total viral RNA levels present in this compartment, but also the smaller sample volumes used in saliva spotting (50 μl) than in the monitoring of frozen samples (200 μl).
Monitoring of SIV RNA dried saliva spots during the first 90 days following infection
In contrast, dried blood spotting provided a robust method to evaluate both SIV-infection status and viral RNA levels. The longitudinal monitoring of SIV RNA levels from dried blood spotting from these monkeys is shown in . In this format, SIV RNA was detectable in four of the five animals by day 7 and all of the five animals by day 10. The median viral load as measured from dried blood spotting was 0.2 log lower than values obtained from matched samples of processed frozen plasma (). Importantly, the dried blood spotting based monitoring had a dynamic range comparable to monitoring in frozen plasma, from 103 to more than 108 viral RNA copies/ml of plasma. Moreover, the correlation of DBS viral load versus plasma viral load measurements was quite good () (r = 0.8319, P < 0.0001).
Monitoring of SIV RNA in dried blood spots during the first 90 days following infection
Therefore, between days 10 and 28 following infection, when plasma viral RNA levels were at least 104 copies/ml in all evaluated monkeys, we found that all blood spots were RNA positive. By comparison, 80% of saliva, 70% of feces and 50% of urine samples were SIV viral RNA-positive during this period. Detection of SIV RNA as dried saliva spots was approximately half (44%) compared with screening methods using fresh frozen saliva.
The detection of SIV RNA after the acute phase peak of viremia (i.e. set point) dropped to near or completely undetectable in all compartments with the exception of the peripheral blood. During this period, dried blood spotting based methods were capable of detecting SIV acquisition in 90% of samples. The 10% of samples that scored negative were from a single animal that strongly controlled set point viremia. This animal routinely had peripheral blood plasma viral RNA levels of less than 500 copies/ml after day 50 of infection.