Background: Clinical measurements commonly used to evaluate overall health of laboratory animals including complete blood count, serum chemistry, weight, and immunophenotyping, differ with respect to age, development, and environment. This report provides comprehensive clinical and immunological reference ranges for pediatric rhesus macaques over the first year of life. Methods: We collected and analyzed blood samples from 151 healthy rhesus macaques, aged 0-55 weeks, and compared mother-reared infants to two categories of nursery-reared infants; those on an active research protocol and those under derivation for the expanded specific-pathogen-free breeding colony. Hematology was performed on EDTA-anticoagulated blood using a Sysmex XT2000i, and serum clinical chemistry was performed using the Beckman AU480 chemistry analyzer. Immunophenotyping of whole blood was performed with immunofluorescence staining and subsequent flow cytometric analysis on a BD LSRFortessa. Plasma cytokine analysis was performed using a Millipore multiplex Luminex assay. Results: For hematological and chemistry measurements, pediatric reference ranges deviate largely from adults. Comparison of mother-reared and nursery-reared animals revealed that large differences depend on rearing conditions and diet. Significant differences found between two nursery-reared cohorts (research and colony animals) indicate large influences of experimental factors and anesthetic events on these parameters. Immune cells and cytokine responses presented with distinct patterns for infants depending on age, birth location, and rearing conditions. Conclusions: Our results illustrate how the immune system changed over time and that there was variability among pediatric age groups. Reference ranges of results reported here will support interpretations for how infection and treatment may skew common immune correlates used for assessment of pathology or protection in research studies as well as help veterinarians in the clinical care of infant non-human primates. We highlighted the importance of using age-specific reference comparisons for pediatric studies and reiterated the utility of rhesus macaques as a model for human studies. Given the rapid transformation that occurs in multiple tissue compartments after birth and cumulative exposures to antigens as individuals grow, a better understanding of immunological development and how this relates to timing of infection or vaccination will support optimal experimental designs for developing vaccines and treatment interventions.

Recent data in a nonhuman primate model showed that infants postnatally infected with Zika virus (ZIKV) were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. In this pilot study, we addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding antibodies remained detectable until viral infection at 5 months. Acute serum viremia was comparatively lower than adults infected with the same Brazilian isolate of ZIKV (n = 11 pregnant females, 4 males, and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute and our study lacks an important comparison with postnatally infected infants born to naïve dams, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.

Zika virus (ZIKV) infection is associated with congenital defects and pregnancy loss. Here, we found that 26% of nonhuman primates infected with Asian/American ZIKV in early gestation experienced fetal demise later in pregnancy despite showing few clinical signs of infection. Pregnancy loss due to asymptomatic ZIKV infection may therefore be a common but under-recognized adverse outcome related to maternal ZIKV infection.