Evidence of natural Zika virus infection in neotropical non-human primates in Brazil

Abstract

In Africa, Old World Primates are involved in the maintenance of sylvatic circulation of ZIKV. However, in Brazil, the hosts for the sylvatic cycle remain unknown. We hypothesized that free-living NHPs might play a role in urban/periurban ZIKV dynamics, thus we undertook an NHP ZIKV investigation in two cities in Brazil. We identified ZIKV-positive NHPs and sequences obtained were phylogenetically related to the American lineage of ZIKV. Additionally, we inoculated four C. penicillata with ZIKV and our results demonstrated that marmosets had a sustained viremia. The natural and experimental infection of NHPs with ZIKV, support the hypothesis that NHPs may be a vertebrate host in the maintainance of ZIKV transmission/circulation in urban tropical settings. Further studies are needed to understand the role they may play in maintaining the urban cycle of the ZIKV and how they may be a conduit in establishing an enzootic transmission cycle in tropical Latin America.

Figure 1

Geoprocessing map of the NHPs and mosquitoes captured in the Vila Toninho neighborhood. (A) Schematic representation of the area where mosquitoes are regularly collected in the Vila Toninho neighborhood. The hatched area represents the area where there is no specimen collection. The blue dots represent the collection points of the mosquitoes and the quantity of specimen collected. (B) Schematic representation of the collection points of the nine NHP found dead. The NHPs identified by ID PR 17-05, PR 17-15, PR 17–22, PR 17–23, PR 17–27 were analyzed and tested positive for ZIKV in one or more tissue samples and are represented by a red triangle. The black triangles represent the NHPs collected but not tested. (C) Satellite image of the Vila Toninho neighborhood. The boundary of the neighborhood is marked in white. Vegetation cover area can be seen in green surrounding the neighborhood. (D) Overlap of the area of the animals and mosquitoes collection. The ZIKV-positive PR 17–27 is overlapping with a ZIKV-positive Ae. aegypti mosquito pool. (E) Overlap of the areas of animas and mosquito collections with the presence of the DENV-positive Ae. aegypti mosquitoes (Vila Toninho satellite image by Google Earth Pro 7.3.1.4507 (64-bit) software. URL https://www.google.com/maps/@−20.84677,−49.34063,5682 m/data = !3m1!1e3). Map data: Google, 2018 DigitalGlobe.

Figure 2

Molecular Phylogenetic analysis of Zika virus by the Maximum Likelihood method. The four strains obtained from NHPs (marmosets) are highlighted in red. Bootstrap values above 90% are shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.7699)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 52.6922% sites). The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. There were a total of 10269 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

Figure 3

Viremia measurement in experimentally ZIKV-infected Callithrix penicilata collected from day −1 until 19 dpi. One-step qRT-PCR was used to measure semi quantitatively the ZIKV RNA loads in the serum of four animals at indicated days p.i. and represented as viral RNA copies per mL of sample standard curve. The curve was obtained from a standard sample with known titer after serial dilutions (5 × 10¹ to 5 × 10⁶ copies/mL) on the plasma of the non-infected marmosets.Values are expressed by RNA genome copies per mL for all the infected marmosets. Viremia was detected in the serum of marmosets 1, 3 and 4 on day 2 p.i. and in all infected marmosets on day 3 p.i. The figure shows that viremia increased on day 5 p.i. when compared to other evaluated days for all the infected marmosets. p.i.: post infection. NHP: non-human primates. Day −1: day before the infection.

Figure 4

ZIKV infection in Callithrix penicilata alters total leukocyte counts. The animals were followed for 28 days. The data represents the results obtained of a pooled sample from four marmosets prior to infection (day 0) and in different days post infection (dpi). The infection altered total leukocyte count in blood inducing leukocytosis 7 dpi and 21 dpi in a two-wave fashion. Results are presented as counts/mm³ per ml of blood.

Figure 5

ZIKV infection in Callithrix penicilata alters hematocrit levels. The animals were followed for 28 days. The data represents the results obtained of a pooled sample from four marmosets prior to infection (day 0) and on different days post infection (dpi). No differences were observed in the hematocrit levels during the course of the infection. Results are presented as hematocrit percentage.

Figure 6

IgG titers in Callithrix penicilata after ZIKV infection. To evaluate the adaptive immune response after ZIKV infection, plasma samples were evaluated on days 0 (prior to infection) and 2,3,4,5,8,9,12,15,19 and 60 d.p.i. by an indirect ELISA. The IgG titers peaked from day 9 to 12 p.i. in the serum of marmosets 1,2 and 3 and on day 15 p.i. in the serum of marmoset 2 when compared to the other evaluated days. IgG titers were detectable until day 19 p.i. in all marmosets.