Detection of Neutralizing Antibodies against Zika Virus in Wild Nonhuman Primates in Rwanda

Zika virus (ZIKV) belongs to the family Flaviviridae, genus Flavivirus, is mainly transmitted by Aedes spp. mosquitoes (Song et al. 2017), and has both an urban and sylvatic (forest) cycle. The sylvatic cycle involves nonhuman primates (NHPs) and forest-based mosquitoes (Valentine et al. 2019). Zika virus was first isolated from a rhesus macaque (Macaca mulatta) in Uganda (Dick et al. 1952; McCrae and Kirya 1982) and in mosquitoes. It was then isolated in vervet monkey (Chlorocebus aethiops), patas monkey (Erythrocebus patas), and Aedes sp. mosquitoes from Senegal (Valentine et al. 2019). Several serosurveys have reported antibodies against ZIKV in a wide range of NHPs across Africa, from countries including Uganda, Senegal, Nigeria, Gambia, Zambia, and Tanzania (Buechler et al. 2017; Valentine et al. 2019; Wastika et al. 2019); however, most of the assays used did not differentiate ZIKV from other flaviviruses that also have sylvatic cycles (Valentine et al. 2019). To contribute to the available knowledge and understand the role of NHPs in maintaining a ZIKV sylvatic cycle, we tested for specific ZIKV neutralizing antibodies in blood samples from a range of wild NHPs from Rwandan national parks.

The characteristics of Rwanda's national parks differ by region and animal population. In the north, Volcanoes National Park is dominated by eastern mountain gorillas (Gorilla beringei beringei) and golden monkeys (Cercopithecus mitis kandti). In the southwest, Nyungwe National Park is a tropical forest and habitat for chimpanzees (Chimpanzees pan troglodytes shweinfuthii). The eastern part of the country is a savanna region with Akagera National Park, olive baboons (Papio anubis), and vervet monkeys (Chlorocebus pygerythrus) are mainly distributed in this park.

We obtained 97 archived NHP sera from the biological resource center (Bio-Bank) in Kigali, Rwanda. Samples had been collected from habituated and unhabituated animals in Volcanoes, Nyungwe, and Akagera National Parks, either during veterinary interventions (2006–16) or as part of wildlife disease surveillance (2012–16). For each NHP sample, the habitat of origin of animal, animal species, location of the animal when found, sex, age, and health status was documented.

We tested all sera for antibodies against ZIKV with an optimized microneutralization enzyme-linked immunosorbent assay (MNT-ELISA); this incorporates an ELISA in the procedure to define the end-point neutralizing titer (Vorndam and Beltran 2002). Briefly, cell culture plates were prepared by seeding 96-well plates with Vero cells at 2×10⁵ cells/mL in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich Pty Ltd, Darmstadt, Germany) at 100 µL/well and incubated at 37 C, 10% CO₂ for 1–2 d until the monolayer achieved 75% confluence. Afterwards, 50 µL of each serum sample were diluted 1:20 in DMEM and inactivated at 56 C for 30 min; 220 µL of the diluted serum were then dispensed in microtiter well plates in triplicates and a further twofold serial dilution was done from 1:20 up to 1:1280. An equal volume of ZIKV MR-766 strain (110 µL) was added at a concentration of 2.0×10⁴ plaque-forming units/mL as previously determined by plaque assay. The serum–virus mixture was incubated for 2 h at room temperature, inoculated onto the Vero cells, then incubated at 37 C, 10% CO₂ for 3 d. The cells were fixed with an ethanol–methanol mixture then the ELISA was carried out. An endpoint titer <40 indicated the absence of anti-ZIKV neutralizing antibodies (negative sample); an endpoint titer >40 indicated the presence of anti-ZIKV virus-neutralizing antibodies (positive sample).

The 97 NHP sera were from five chimpanzees (0.5 mL/vial), 55 gorillas (0.5 mL/vial), seven golden monkeys, and seven unidentified monkey species (0.25 mL/vial), 20 vervet monkeys (0.25 mL/vial), and three olive baboons (0.25 mL/vial; Fig. 1). The NHPs varied in age, sex, and health status (Table 1).

Of the 97 samples, a single sample from a male adult olive baboon, collected April 2012, Akagera National Park, tested positive for ZIKV neutralizing antibodies with an MNT_>640. The other NHPs (including two further olive baboons) from the same park tested negative.

Our detection of specific neutralizing antibodies against ZIKV in a wild olive baboon suggests a possible role of NHPs in a sylvatic cycle in Rwanda. Akagera National Park, at an altitude of over 1,300 m, is a savannah complex ecosystem with open grassland and wetlands. More than half of the sampled NHPs (n=55) were mountain gorillas that spend most of their time above 2,225–2,805 m (Weber and Vedder 1983; Akayezu et al. 2019). The Centers for Disease Control estimate the maximum altitude for sustainable Aedes aegyptii (the vector) presence to be about 1,800 m (Watts et al. 2017). The main ZIKV vectors have been documented to occur dominantly in the eastern region of Rwanda (Seruyange et al. 2017). Antibodies against ZIKV have been found in a wide variety of NHPs (Valentine et al. 2019), and animal models suggest wide susceptibility (Li et al. 2016; Hirsch et al. 2017). Furthermore, Seruyange et al. (2018) provided evidence of ZIKV neutralizing antibodies among human blood donors in the eastern region of Rwanda (Seruyange et al., 2018). Serological evidence of ZIKV infection was previously reported in olive baboon and a vervet monkey during a 2012 yellow fever risk assessment survey in Rwanda (Demanou et al. 2014); however, other NHP species tested in our study were not included during that survey. It remains unclear if the negative results in other NHPs represent the absence of the vector or unfavorable ecological conditions for ZIKV. Because most NHPs in the monkey category live for a short period and ZIKV transmission occurs in waves when favorable environmental conditions promote vector and virus propagation, many individuals of these species may not be exposed during the period of low transmission.

Our study had several limitations. We tested a limited number of sera, in a convenient sample. Cross reactivity of neutralizing antibodies of ZIKV-related flaviviruses, mainly yellow fever virus and dengue fever virus, is a possibility, but one strength of our study is the specificity of the microneutralization test using the ZIKV MR-766 strain.

The detectable neutralizing antibodies against ZIKV in an olive baboon suggested exposure of NHPs to ZIKV in the eastern part of Rwanda. Continued surveillance and further research on humans, animals, and vectors, especially at boundaries of urban and forest areas, is needed to detect active infections and to understand the role of wildlife in Zika virus transmission.

The authors acknowledge the support by the Rwanda Development Board (RDB), US Centers for Disease Control and Prevention at Kenya Medical Research Institute (CDC-KEMRI), and The Emerging Pandemic Threats 1 program-USAID (EPT1-USAID)/PREDICT. Special acknowledgment to the team of gorilla doctors and conservation program at RDB for facilitating access to the biobank of nonhuman primates.