In this study, we evaluated the effects of age, viral inoculum, route of infection, mosquito factors and immunocompetency on immunity against the WNV in the non-human primate model. We were unable to detect any clinical signs of the disease, despite the very advanced age of some of the animals, the use of CD8-depleted animals, and the very high dose of the virus inoculated. We confirmed prior reports that adult monkeys do not present with symptomatic WNV infection. More importantly, we here show that both aged and CD8-deficient non-human primates also resolve viral infection and fail to exhibit clinical symptoms of WNV disease when infected in a manner similar to the natural course of infection with a high dose of the virus. All animals displayed clear signs of contact with the virus, with measurable but quickly disappearing viral RNA, and/or by developing and maintaining humoral immune responses. In humans, only about 20% individuals develop symptoms, and 1
150 ends up with severe, potentially fatal, encephalitis. The incidence of encephalitis, however, rises to 10% and higher in humans over 65 
. One of the significant limitations in our study is the small cohort size, and it could be argued that even the combined 40 animals may have not been sufficient to reveal the few animals which may present with clinical symptoms. However, at a face value, if macaque susceptibility was similar to that in humans, we should have observed symptoms in at least 8 animals. The fact that we used a substantial number of old animals, as well as animals with drastically reduced adaptive cellular immunity (which in humans and mice serves as strong predictor of susceptibility), allows us to conclude that sensitivity of old macaques to WNV is low, certainly lower than in mice (note, that the same WNV stock, lethal for most old B6 mice at 1,000 pfu - corresponding to ~5×104
/kg - in our mouse studies 
was used in the present macaque study without producing any clinical symptoms in monkeys) or humans. Therefore, we conclude that macaques are unlikely to provide a good model of age-related susceptibility to WNV, but may be useful to elucidate mechanisms by which they rapidly resolve WNV infection. In that regard, it should be noted that the various approaches employed to elicit a clinical response, including increased viral inoculum and priming with SGE, resulted in either a more rapid resolution of viral RNA loads, or a complete lack thereof, likely reflecting robust innate responses. Indeed, even when animals exhibited viral genomes in both the blood and urine, multiple attempts to culture infectious virus from urine, whole blood, or plasma were unsuccessful. Attempts to enrich for the virus from urine using co-cultivation in Vero cells were also unsuccessful (not shown). This could potentially occur due to natural antibody, complement neutralization (no preliminary evidence was found for either), or by other mechanisms, whose investigation is outside of the scope of this study. Paradoxically, we have noted the longest viral persistence (albeit coupled with the inability to culture infectious virus) in Cohort 1 animals, which received the relatively lowest viral dose in the absence of SGE. We speculate that this is due to the fact that in all other cohorts either higher viral doses or the presence of SGE probably stimulated stronger innate immune response, which promptly neutralized the virus. Consistent with this possibility, we found lower Ab responses in animals immunized in the presence of SGE, than in those not receiving the extract ().
The humoral immune response in Cohort 1, which exhibited the most pronounced and longest viremia and WNV shedding, revealed a similar IgM response to Roehrig’s paper on persistence of IgM response in humans 
. The human IgM response usually coincides with the onset of clinical symptoms. Normally this response declines over 2–3 months but we saw persistence of these responses, sometimes lasting up to day 230, and suggesting persistence of antigen. Also of interest was the presence of a secondary IgM wave of response. This data is compelling in light of results showing persistent WNV infection in hamsters 
and findings of persistent IgM and non-culturable viral genomes shed in human urine 
collectively suggesting that WNV may not behave as a typical acute pathogen. Whether the prolonged anti-WNV Ab response is needed to control persistent virus in macaques or whether it merely represents a bystander phenomenon, with other mechanisms keeping the virus at bay, remains to be elucidated. Of note, we did not observe differences between adult and old animals in the intensity of Ab responses. This could be interpreted to mean that in monkeys either the entire immune system or perhaps just B cells do not undergo the process of age-related deterioration seen in rodents and humans. We do not believe this to be correct, because B and T cells in RM readily exhibit the landmarks of immune senescence, including conversion into memory cells 
, naïve cell reduction 
and reduced T-cell effector function and Ab production in response to vaccination 
and references therein]. Consistent with this, we observed a delay in T-cell proliferation in response to WNV infection, similar to the observations made in rodents 
Rather, we believe it likely that the interaction between the specific virus or category of viruses and/or the high viral dose used in all animals may have circumvented any age-related differences and had produced a robust humoral response. As mentioned above, we do not believe that adaptive responses were necessary, because robust limiting of viral loads in SGE-immunized RM caused full protection, which was accompanied by low Ab titers in these same animals.
Our primary hypothesis was that including animals of significantly advanced age and increasing dosage or including intravenous route of infection would elicit neurological symptoms. Instead both of these parameters correlated to rapid resolution of circulating viral RNA. Our second hypothesis, that more severe WNV disease can be induced by priming animals with mosquito SGE (as would occur in WNV-endemic areas) and remarkably, our third hypothesis, that deficient antiviral response in CD8-depleted and thymectomized CM, will also increase severity of WNV disease, were also proven incorrect. It was recently 
shown that increased mortality and reduced time to death occur when mice are pre-exposed to mosquito feeding. To mimic this paradigm we administered SGE in micro-subcutaneous injections but found instead enhanced protection, as evidence by the decreased viral load (including 20/20 aged and immunocompromised animals). This raises interesting questions on protection vs. exacerbation of the vector-borne disease. Our current study provides suggestive evidence of a heightened innate immune response including elevation of total monocyte counts (i.v. infection, not shown) and prolonged eosinophilia, in animals primed with SGE, however, these leads will require additional work to establish their importance. Indeed, understanding the mechanisms of protection at play in old and immunocompromised macaques against WNV could provide new therapeutic targets and strategies against this and other similar viral infection in the old age.