Mpox virus (MPXV) vertical transmission and fetal demise in a pregnant rhesus macaque model.

Infection with clade I Mpox virus (MPXV) results in adverse pregnancy outcomes, yet the potential for vertical transmission resulting in fetal harm with clade IIb MPXV, the clade that is currently circulating in the Western Hemisphere, remains unknown. We established a rhesus macaque model of vertical MPXV transmission with early gestation inoculation. Three pregnant rhesus macaques were inoculated intradermally with 1.5 × 10^5 plaque forming units (PFU) of clade IIb MPXV near gestational day (GD) 30 and animals were monitored for viremia and maternal and fetal well-being. Animals were euthanized to collect tissues at 5, 14, or 25 days post-inoculation (dpi). Tissues were evaluated for viral DNA (vDNA) loads, infectious virus titers, histopathology, MPXV mRNA and protein localization, as well as MPXV protein co-localization with placental cells including, Hofbauer cells, mesenchymal stromal cells, endothelial cells, and trophoblasts. vDNA was detected in maternal blood and skin lesions by 5 dpi. Lack of fetal heartbeat was observed at 14 or 25 dpi for two dams indicating fetal demise; the third dam developed significant vaginal bleeding at 5 dpi and was deemed an impending miscarriage. vDNA was detected in placental and fetal tissue in both fetal demise cases. MPXV localized to placental villi by ISH and IHC. Clade IIb MPXV infection in pregnant rhesus macaques results in vertical transmission to the fetus and adverse pregnancy outcomes, like clade I MPXV. Further studies are needed to determine whether antiviral therapy with tecovirimat will prevent vertical transmission and improve pregnancy outcomes. One Sentence Summary: Clade IIb Mpox virus infection of pregnant rhesus macaques results in vertical transmission from mother to fetus and adverse pregnancy outcomes.

Control of maternal Zika virus infection during pregnancy is associated with lower antibody titers in a macaque model.

Introduction: Zika virus (ZIKV) infection during pregnancy results in a spectrum of birth defects and neurodevelopmental deficits in prenatally exposed infants, with no clear understanding of why some pregnancies are more severely affected. Differential control of maternal ZIKV infection may explain the spectrum of adverse outcomes. Methods: Here, we investigated whether the magnitude and breadth of the maternal ZIKV-specific antibody response is associated with better virologic control using a rhesus macaque model of prenatal ZIKV infection. We inoculated 18 dams with an Asian-lineage ZIKV isolate (PRVABC59) at 30-45 gestational days. Plasma vRNA and infectious virus kinetics were determined over the course of pregnancy, as well as vRNA burden in the maternal-fetal interface (MFI) at delivery. Binding and neutralizing antibody assays were performed to determine the magnitude of the ZIKV-specific IgM and IgG antibody responses throughout pregnancy, along with peptide microarray assays to define the breadth of linear ZIKV epitopes recognized. Results: Dams with better virologic control (n= 9) cleared detectable infectious virus and vRNA from the plasma by 7 days post-infection (DPI) and had a lower vRNA burden in the MFI at delivery. In comparison, dams with worse virologic control (n= 9) still cleared detectable infectious virus from the plasma by 7 DPI but had vRNA that persisted longer, and had higher vRNA burden in the MFI at delivery. The magnitudes of the ZIKV-specific antibody responses were significantly lower in the dams with better virologic control, suggesting that higher antibody titers are not associated with better control of ZIKV infection. Additionally, the breadth of the ZIKV linear epitopes recognized did not differ between the dams with better and worse control of ZIKV infection. Discussion: Thus, the magnitude and breadth of the maternal antibody responses do not seem to impact maternal virologic control. This may be because control of maternal infection is determined in the first 7 DPI, when detectable infectious virus is present and before robust antibody responses are generated. However, the presence of higher ZIKV-specific antibody titers in dams with worse virologic control suggests that these could be used as a biomarker of poor maternal control of infection and should be explored further.

Vertical transmission of African-lineage Zika virus through the fetal membranes in a rhesus macaque (Macaca mulatta) model.

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes.

Infection of the maternal-fetal interface and vertical transmission following low-dose inoculation of pregnant rhesus macaques (Macaca mulatta) with an African-lineage Zika virus.

BACKGROUND: Congenital Zika virus (ZIKV) infection can result in birth defects, including malformations in the fetal brain and visual system. There are two distinct genetic lineages of ZIKV: African and Asian. Asian-lineage ZIKVs have been associated with adverse pregnancy outcomes in humans; however, recent evidence from experimental models suggests that African-lineage viruses can also be vertically transmitted and cause fetal harm. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the pathway of vertical transmission of African-lineage ZIKV, we inoculated nine pregnant rhesus macaques (Macaca mulatta) subcutaneously with 44 plaque-forming units of a ZIKV strain from Senegal, (ZIKV-DAK). Dams were inoculated either at gestational day 30 or 45. Following maternal inoculation, pregnancies were surgically terminated seven or 14 days later and fetal and maternal-fetal interface tissues were collected and evaluated. Infection in the dams was evaluated via plasma viremia and neutralizing antibody titers pre- and post- ZIKV inoculation. All dams became productively infected and developed strong neutralizing antibody responses. ZIKV RNA was detected in maternal-fetal interface tissues (placenta, decidua, and fetal membranes) by RT-qPCR and in situ hybridization. In situ hybridization detected ZIKV predominantly in the decidua and revealed that the fetal membranes may play a role in ZIKV vertical transmission. Infectious ZIKV was detected in the amniotic fluid of three pregnancies and one fetus had ZIKV RNA detected in multiple tissues. No significant pathology was observed in any fetus; and ZIKV did not have a substantial effect on the placenta. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that a very low dose of African-lineage ZIKV can be vertically transmitted to the macaque fetus during pregnancy. The low inoculating dose used in this study suggests a low minimal infectious dose for rhesus macaques. Vertical transmission with a low dose in macaques further supports the high epidemic potential of African ZIKV strains.

Frequent first-trimester pregnancy loss in rhesus macaques infected with African-lineage Zika virus.

In the 2016 Zika virus (ZIKV) pandemic, a previously unrecognized risk of birth defects surfaced in babies whose mothers were infected with Asian-lineage ZIKV during pregnancy. Less is known about the impacts of gestational African-lineage ZIKV infections. Given high human immunodeficiency virus (HIV) burdens in regions where African-lineage ZIKV circulates, we evaluated whether pregnant rhesus macaques infected with simian immunodeficiency virus (SIV) have a higher risk of African-lineage ZIKV-associated birth defects. Remarkably, in both SIV+ and SIV- animals, ZIKV infection early in the first trimester caused a high incidence (78%) of spontaneous pregnancy loss within 20 days. These findings suggest a significant risk for early pregnancy loss associated with African-lineage ZIKV infection and provide the first consistent ZIKV-associated phenotype in macaques for testing medical countermeasures.

Quantification of early gait development: Expanding the application of Catwalk technology to an infant rhesus macaque model.

BACKGROUND: Understanding gait development is essential for identifying motor impairments in neurodevelopmental disorders. Defining typical gait development in a rhesus macaque model is critical prior to characterizing abnormal gait. The goal of this study was to 1) explore the feasibility of using the Noldus Catwalk to assess gait in infant rhesus macaques and 2) provide preliminary normative data of gait development during the first month of life. NEW METHOD: The Noldus Catwalk was used to assess gait speed, dynamic and static paw measurements, and interlimb coordination in twelve infant rhesus macaques at 14, 21, and 28 days of age. All macaque runs were labeled as a diagonal or non-diagonal walking pattern. RESULTS: Infant rhesus macaques primarily used a diagonal (mature) walking pattern as early as 14 days of life. Ten infant rhesus macaques (83.3%) were able to successfully walk across the Noldus Catwalk at 28 days of life. Limited differences in gait parameters were observed between timepoints because of the variability within the group at 14, 21, and 28 days. COMPARISON WITH EXISTING METHODS: No prior gait analysis system has been used to provide objective quantification of gait parameters for infant macaques. CONCLUSIONS: The Catwalk system can be utilized to quantify gait in infant rhesus macaques less than 28 days old. Future applications to infant rhesus macaques could provide a better understanding of gait development and early differences within various neurodevelopmental disorders.

Fetal loss in pregnant rhesus macaques infected with high-dose African-lineage Zika virus.

Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4-8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques exposed to 1x104 PFU (low dose) of African-lineage ZIKV at gestational day (GD) 45. Here, we exposed eight pregnant rhesus macaques to 1x108 PFU (high dose) of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this exposure system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose exposure to African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy loss could be strain-specific.

Human immune globulin treatment controls Zika viremia in pregnant rhesus macaques.

There are currently no approved drugs to treat Zika virus (ZIKV) infection during pregnancy. Hyperimmune globulin products such as VARIZIG and WinRho are FDA-approved to treat conditions during pregnancy such as Varicella Zoster virus infection and Rh-incompatibility. We administered ZIKV-specific human immune globulin as a treatment in pregnant rhesus macaques one day after subcutaneous ZIKV infection. All animals controlled ZIKV viremia following the treatment and generated robust levels of anti-Zika virus antibodies in their blood. No adverse fetal or infant outcomes were identified in the treated animals, yet the placebo control treated animals also did not have signs related to congenital Zika syndrome (CZS). Human immune globulin may be a viable prophylaxis and treatment option for ZIKV infection during pregnancy, however, more studies are required to fully assess the impact of this treatment to prevent CZS.

Special Issue "Pediatric Viral Infection Long-Term Consequences".

This Special Issue was focused on advancing our understanding of the long-term consequences of pediatric viral infections [...].

Neonatal Development in Prenatally Zika Virus-Exposed Infant Macaques with Dengue Immunity.

Infants exposed to Zika virus (ZIKV) prenatally may develop birth defects, developmental deficits, or remain asymptomatic. It is unclear why some infants are more affected than others, although enhancement of maternal ZIKV infection via immunity to an antigenically similar virus, dengue virus (DENV), may play a role. We hypothesized that DENV immunity may worsen prenatal ZIKV infection and developmental deficits in offspring. We utilized a translational macaque model to examine how maternal DENV immunity influences ZIKV-exposed infant macaque neurodevelopment in the first month of life. We inoculated eight macaques with prior DENV infection with ZIKV, five macaques with ZIKV, and four macaques with saline. DENV/ZIKV-exposed infants had significantly worse visual orientation skills than ZIKV-exposed infants whose mothers were DENV-naive, with no differences in motor, sensory or state control development. ZIKV infection characteristics and pregnancy outcomes did not individually differ between dams with and without DENV immunity, but when multiple factors were combined in a multivariate model, maternal DENV immunity combined with ZIKV infection characteristics and pregnancy parameters predicted select developmental outcomes. We demonstrate that maternal DENV immunity exacerbates visual orientation and tracking deficits in ZIKV-exposed infant macaques, suggesting that human studies should evaluate how maternal DENV immunity impacts long-term neurodevelopment.

Previous exposure to dengue virus is associated with increased Zika virus burden at the maternal-fetal interface in rhesus macaques.

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.

African-Lineage Zika Virus Replication Dynamics and Maternal-Fetal Interface Infection in Pregnant Rhesus Macaques.

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The viruses responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in vitro and in vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titers and caused more-severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here, we infected four pregnant rhesus macaques with a low-passage-number strain of African-lineage ZIKV and compared its pathogenesis to those for a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-inoculated controls. The viral replication kinetics for the two experimental groups were not significantly different, and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery (1 to 1.5 weeks prior to full term) in either group. However, a significantly higher burden of ZIKV viral RNA (vRNA) was found in the maternal-fetal interface tissues of the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV of any genetic lineage poses a threat to pregnant individuals and their infants. IMPORTANCE ZIKV was first identified in 1947 in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015 to 2016. In its most recent update, the WHO stated that improved understanding of African-lineage ZIKV pathogenesis during pregnancy must be a priority. The recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here, we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational nonhuman primate model. We show that African-lineage isolates replicate with kinetics similar to those of Asian-lineage isolates and can infect the placenta. However, there was no evidence of more-severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to pregnant individuals and their infants and the need for epidemiological and translational in vivo studies with African-lineage ZIKV.

Quantitative definition of neurobehavior, vision, hearing and brain volumes in macaques congenitally exposed to Zika virus.

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design.

Primary infection with dengue or Zika virus does not affect the severity of heterologous secondary infection in macaques.

Zika virus (ZIKV) and dengue virus (DENV) are genetically and antigenically related flaviviruses that now co-circulate in much of the tropical and subtropical world. The rapid emergence of ZIKV in the Americas in 2015 and 2016, and its recent associations with Guillain-Barré syndrome, birth defects, and fetal loss have led to the hypothesis that DENV infection induces cross-reactive antibodies that influence the severity of secondary ZIKV infections. It has also been proposed that pre-existing ZIKV immunity could affect DENV pathogenesis. We examined outcomes of secondary ZIKV infections in three rhesus and fifteen cynomolgus macaques, as well as secondary DENV-2 infections in three additional rhesus macaques up to a year post-primary ZIKV infection. Although cross-binding antibodies were detected prior to secondary infection for all animals and cross-neutralizing antibodies were detected for some animals, previous DENV or ZIKV infection had no apparent effect on the clinical course of heterotypic secondary infections in these animals. All animals had asymptomatic infections and, when compared to controls, did not have significantly perturbed hematological parameters. Rhesus macaques infected with DENV-2 approximately one year after primary ZIKV infection had higher vRNA loads in plasma when compared with serum vRNA loads from ZIKV-naive animals infected with DENV-2, but a differential effect of sample type could not be ruled out. In cynomolgus macaques, the serotype of primary DENV infection did not affect the outcome of secondary ZIKV infection.

Using Macaques to Address Critical Questions in Zika Virus Research.

Zika virus (ZIKV) and nonhuman primates have been inextricably linked since the virus was first discovered in a sentinel rhesus macaque in Uganda in 1947. Soon after ZIKV was epidemiologically associated with birth defects in Brazil late in 2015, researchers capitalized on the fact that rhesus macaques are commonly used to model viral immunity and pathogenesis, quickly establishing macaque models for ZIKV infection. Within months, the susceptibility of pregnant macaques to experimental ZIKV challenge and ZIKV-associated abnormalities in fetuses was confirmed. This review discusses key unanswered questions in ZIKV immunity and in the pathogenesis of thecongenital Zika virus syndrome. We focus on those questions that can be best addressed in pregnant nonhuman primates and lessons learned from developing macaque models for ZIKV amid an active epidemic.

Modeling Zika Virus-Associated Birth Defects in Nonhuman Primates.

In utero infection with Zika virus (ZIKV) during pregnancy can lead to the development of birth defects and postnatal deficits. A nonhuman primate (NHP) model of congenital ZIKV infection can help fill the gaps in knowledge where tissue studies are required to define viral pathogenesis and identify targets for therapeutic intervention. This model system has already identified critical features of ZIKV pathogenesis in congenital infection. Before translating these NHP studies to human clinical trials, we must understand the similarities and differences between human and NHP fetal immune system development, neural development, and infant assessment tools. Because of the overall similarity between fetal and infant development in humans and NHPs, this NHP model can complement human clinical trials by defining immune correlates of protection and evaluating therapeutic interventions.

Antibody responses to Zika virus proteins 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.

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.

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.