Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10–12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

BackgroundZika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV.Methodology/Principal FindingsUsing our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form.Conclusions/SignificanceAn effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains.

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated. Author summaryMaternal ZIKV infection in pregnancy is associated with severe fetal anomalies, including microcephaly. It has been shown that infection manifests differently in pregnancy than in the non-pregnant state, with prolonged maternal viremia. ZIKV is spread by mosquitos and through sexual contact and since its first detection in early 2015, has become endemic to the Americas. While much has been learned from studying infected human pregnancies, there are still many questions concerning transmission of ZIKV from mother to fetus. Investigating ZIKV infection in non-human primates could help answer these questions due to similarities in the immune system, and the tissues separating the fetus from the mother during pregnancy. Our study serves to model ZIKV transmission in early and late pregnancy, as well as study the effects of this infection on the fetus and mother at these different times in pregnancy. The data collected provides an important insight on ZIKV in pregnancy where the pregnancies have been monitored throughout the entire infection period until term, and suggests that vertical transmission may be very efficient, although severe fetal outcomes are uncommon.

words; 150 word max)Zika virus (ZIKV) isolates are genetically diverse, but belong to two recognized lineages, termed "African" and "Asian." Asian ZIKV infection during pregnancy causes fetal abnormalities including microcephaly. Developing an effective preventative Zika virus vaccine that protects pregnant women is essential for minimizing fetal abnormalities; at least 18 groups are developing ZIKV vaccines (Hayden, 2016). The genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, and the degree to which immunity against one ZIKV strain could provide protection against another is unknown. Here we show that rhesus macaques infected with East African ZIKV strain MR766 are completely protected from subsequent infection with heterologous Asian ZIKV. MR766 is more genetically divergent from all known Asian ZIKV strains than Asian ZIKV strains are from one another. Therefore, ZIKV strain selection is unlikely to compromise vaccine effectiveness. Highlights (85 characters max per bullet point)• African Zika virus (ZIKV) strain MR766 productively infects macaques (68 characters)• Immunity elicited by MR766 protects macaques against heterologous Asian ZIKV (77 characters)• In vivo restoration of a putative N-linked glycosylation site in MR766 (70 characters)• Immunogen selection is unlikely to adversely affect the breadth of vaccine protection (85 characters) eTOC (52 words; 80 words max) An effective Zika virus vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses are primed by infection with East African ZIKV are completely protected from reinfection with heterologous Asian ZIKV. Any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains.

Captive-bred rhesus macaques of Indian origin represent one of the most important large animal models for infectious disease, solid organ transplantation and stem cell research. There is a dearth of information defining hematopoietic development, including neutrophil leukocyte differentiation in this species using multicolor flow cytometry. In the current study we sought to identify cell surface markers that delineate neutrophil progenitor populations with characteristic immunophenotypes. We defined four different post-mitotic populations based on their CD11b and CD87 expression pattern, and further refined their immunophenotypes using CD32, CD64, lactoferrin and myeloperoxidase as antigenic markers. The four subsets contained myelocyte, metamyelocyte, band, and segmented neutrophil populations. We compared our flow cytometry-based classification with the classical nuclear morphology-based classification. We found overlap of immunological phenotype between populations of different nuclear morphology and identified phenotypically different subsets within populations of similar nuclear morphology. We assessed the responsiveness of these populations to stimulatory signals such as LPS, fMPL or PMA, and demonstrated significant differences between human and rhesus macaque neutrophil progenitors. In this study we provided evidence for species-specific features of granulopoiesis that ultimately manifested in the divergent immunophenotypes of the fully differentiated segmented neutrophils of humans and rhesus macaques. Additionally, we found functional markers that can be used to accurately quantify neutrophil progenitors by flow cytometry. While these markers do not coincide with the classical nuclear-morphology-based grading they enable us to perform functional studies monitoring immunophenotypic markers.