Emma L. Mohr

A rhesus macaque model of Asian-lineage Zika virus infection

Infection with Asian-lineage Zika virus (ZIKV) has been associated with Guillain–Barré syndrome and fetal abnormalities, but the underlying mechanisms remain poorly understood. Animal models of infection are thus urgently needed. Here we show that rhesus macaques are susceptible to infection by an Asian-lineage ZIKV closely related to strains currently circulating in the Americas. Following subcutaneous inoculation, ZIKV RNA is detected in plasma 1 day post infection (d.p.i.) in all animals (N=8, including 2 pregnant animals), and is also present in saliva, urine and cerebrospinal fluid. Non-pregnant and pregnant animals remain viremic for 21 days and for up to at least 57 days, respectively. Neutralizing antibodies are detected by 21 d.p.i. Rechallenge 10 weeks after the initial challenge results in no detectable virus replication, indicating protective immunity against homologous strains. Therefore, Asian-lineage ZIKV infection of rhesus macaques provides a relevant animal model for studying pathogenesis and evaluating potential interventions against human infection, including during pregnancy.

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques

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.

Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques

Background Zika 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 Findings Using 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/Significance An 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.

Oropharyngeal mucosal transmission of Zika virus in rhesus macaques

Zika virus is present in urine, saliva, tears, and breast milk, but the transmission risk associated with these body fluids is currently unknown. Here we evaluate the risk of Zika virus transmission through mucosal contact in rhesus macaques. Application of high-dose Zika virus directly to the tonsils of three rhesus macaques results in detectable plasma viremia in all animals by 2 days post-exposure; virus replication kinetics are similar to those observed in animals infected subcutaneously. Three additional macaques inoculated subcutaneously with Zika virus served as saliva donors to assess the transmission risk from contact with oral secretions from an infected individual. Seven naive animals repeatedly exposed to donor saliva via the conjunctivae, tonsils, or nostrils did not become infected. Our results suggest that there is a risk of Zika virus transmission via the mucosal route, but that the risk posed by oral secretions from individuals with a typical course of Zika virus infection is low.Zika virus (ZIKV) is present in body fluids, including saliva, but transmission risk through mucosal contact is not well known. Here, the authors show that oropharyngeal mucosal infection of macaques with a high ZIKV dose results in viremia, but that transmission risk from saliva of infected animals is low.

Infection via mosquito bite alters Zika virus tissue tropism and replication kinetics in rhesus macaques

Mouse and nonhuman primate models now serve as useful platforms to study Zika virus (ZIKV) pathogenesis, candidate therapies, and vaccines, but they rely on needle inoculation of virus: the effects of mosquito-borne infection on disease outcome have not been explored in these models. Here we show that infection via mosquito bite delays ZIKV replication to peak viral loads in rhesus macaques. Importantly, in mosquito-infected animals ZIKV tissue distribution was limited to hemolymphatic tissues, female reproductive tract tissues, kidney, and liver, potentially emulating key features of human ZIKV infections, most of which are characterized by mild or asymptomatic disease. Furthermore, deep sequencing analysis reveals that ZIKV populations in mosquito-infected monkeys show greater sequence heterogeneity and lower overall diversity than in needle-inoculated animals. This newly developed system will be valuable for studying ZIKV disease because it more closely mimics human infection by mosquito bite than needle-based inoculations.Vector saliva can affect infectivity and pathogenesis of vector-borne viruses, but this hasn’t been studied for Zika virus infection. Here, Dudley et al. show that mosquito-mediated Zika infection of macaques results in altered replication kinetics and greater sequence heterogeneity.

Ocular and uteroplacental pathology in a macaque pregnancy with congenital Zika virus infection.

Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.

A rhesus macaque model of Asia lineage Zika virus infection

Infection with Asian lineage Zika virus has been associated with Guillain-Barré syndrome and fetal abnormalities 1–4, but the mechanisms and risk factors for these outcomes remain unknown. Here we show that rhesus macaques are susceptible to infection by an Asian-lineage virus closely related to strains currently circulating in the Americas. Following subcutaneous inoculation, Zika virus RNA was detected in plasma one day post-infection (dpi) in all animals (N = 8, including 2 animals infected during the first trimester of pregnancy). Plasma viral loads peaked above 1 × 105 viral RNA copies/mL in seven of eight animals. Viral RNA was also present in saliva, urine, and cerebrospinal fluid (CSF), consistent with case reports from infected humans. Viral RNA was cleared from plasma and urine by 21 dpi in non-pregnant animals. In contrast, both pregnant animals remained viremic longer, up to 57 days. In all animals, infection was associated with transient increases in proliferating natural killer cells, CD8+ T cells, CD4+ T cells, and plasmablasts. Neutralizing antibodies were detected in all animals by 21 dpi. Rechallenge of three non-pregnant animals with the Asian-lineage Zika virus 10 weeks after the initial challenge resulted in no detectable virus replication, suggesting that primary Zika virus infection elicits protective immunity against homologous virus strains. These data establish that Asian-lineage Zika virus infection of rhesus macaques provides a relevant animal model for studying pathogenesis in pregnant and non-pregnant individuals and evaluating potential interventions against human infection, including during pregnancy.Infection with Asian lineage Zika virus has been associated with Guillain-Barre syndrome and fetal microcephaly 1-4. Here we show that rhesus macaques are susceptible to infection by an Asian lineage Zika virus isolate that shares more than 99% nucleotide identity with strains currently circulating in the Americas. Following subcutaneous inoculation, Zika virus RNA was detected in plasma one-day post infection (dpi) in all animals (N = 3). Plasma viral loads peaked above 1 x 106 viral RNA copies/mL in two of three animals. Viral RNA was also present in saliva, urine, and cerebrospinal fluid (CSF), consistent with case reports from infected humans. Zika virus RNA persisted in both plasma and urine at low levels for more than two weeks. Infection was associated with transient increases in proliferating natural killer cells, CD8+ and CD4+ T cells, and plasmablasts, suggesting pathogen sensing by the immune system. These data establish that Asian lineage Zika virus infection in rhesus macaques provides a relevant animal model for human infection. Furthermore, because fetal development is well characterized in rhesus macaques, infections in pregnant macaques will enable important studies of fetal defects associated with Zika virus.

Molecularly barcoded Zika virus libraries to probe in vivo evolutionary dynamics

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a “synthetic swarm” whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.

Miscarriage and stillbirth following maternal Zika virus infection in nonhuman primates

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.Zika virus infection during pregnancy is associated with an increased rate of fetal loss in nonhuman primates, as reported in this multicenter analysis.

Antibody responses to the Zika virus polyprotein in pregnant and non-pregnant macaques

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens. Author summary ZIKV has emerged as a vector-borne pathogen capable of causing serious illness in infected adults and congenital birth defects. The vulnerability of communities to future ZIKV outbreaks will depend, in part, on the prevalence and longevity of protective immunity, thought to be mediated principally by antibodies. We currently lack diagnostic assays able to differentiate ZIKV-specific antibodies from antibodies produced following infection with closely related DENV, and we do not know how long anti-ZIKV responses are detectable. Here we profile antibodies recognizing linear epitopes throughout the entire ZIKV polyprotein, and we profile cross-reactivity with the proteomes of other co-endemic arboviruses. We show that while ZIKV-specific antibody binding can be detected, these responses are generally weak and ephemeral, and false positives may arise through DENV infection. This may complicate efforts to discern ZIKV infection and to determine ZIKV seroprevalence using linear epitope-based assays. The method used in this study, however, has promise as a tool for profiling antibody responses for a broad array of neglected tropical diseases and other pathogens and in distinguishing serology of closely-related viruses.