Biology subjects

Koenig, M. R.

Publications and source records attributed to Koenig, M. R..

7 recordsLinked to original sources

bioRxiv2023

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 with RT-qPCR, in situ hybridization for ZIKV RNA, 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 post-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 vertical ZIKV transmission through the fetal membranes. Authors SummaryZika virus (ZIKV) can be vertically transmitted from mother to fetus during pregnancy resulting in adverse pregnancy outcomes. For vertical transmission to occur, ZIKV must overcome the barriers of the maternal-fetal interface, yet the exact pathway ZIKV takes remains undefined. The maternal-fetal interface consists of the maternal decidua, the placenta, and the fetal membranes. ZIKV could reach the fetus through the placenta if it can infect the layer of cells that are directly exposed to maternal blood. ZIKV could also reach the fetus by infecting the decidua and then the adjacent fetal membranes. To determine the pathways of ZIKV vertical transmission, we infected pregnant macaques and evaluated ZIKV burden in the maternal-fetal interface and fetus shortly after maternal infection. The pattern of infection observed suggests that ZIKV vertically transmits through the fetal membranes, not the placenta. This finding is significant because it challenges the assumption that vertical transmission occurs exclusively across the placenta. By including the fetal membranes in our models of vertical transmission, we can more accurately determine which pathogens can be vertically transmitted. Ultimately, this study demonstrates that fetal membranes are an essential barrier to pathogens that warrant further investigation.

pathology↗

bioRxiv2022

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

BackgroundCongenital 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 FindingsTo evaluate the potential for 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; however, we did find an increase in the occurrence of decidual vasculitis and necrosis in ZIKV-exposed pregnancies compared to gestational-age-matched controls. Conclusions/SignificanceThis study demonstrates that African-lineage ZIKV, like Asian-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. Author SummaryZika virus infection during pregnancy can result in adverse pregnancy outcomes including birth defects and miscarriage. There are two distinct genetic backgrounds of Zika virus: Asian-lineage and African-lineage. Currently, only Asian-lineage Zika virus is causally associated with adverse pregnancy outcomes in people. However, experimental studies have shown that African-lineage Zika virus can infect the fetus during pregnancy and cause adverse outcomes. Adverse pregnancy outcomes may not be associated with infection in people until there is an outbreak in a naive population. Thus, as African-lineage Zika virus continues to spread globally, the risk that it may pose to pregnant people remains a public health concern. In this study, we demonstrate that African-lineage Zika virus can be transmitted from the mother to the fetus during pregnancy. This study is significant because we used rhesus macaques, an animal that shares many key elements of Zika virus infection in pregnant people. This study is also significant because we inoculated pregnant macaques with a very small amount of virus, suggesting that fetal infection reported in previously published macaque studies is not limited to high-dose inoculation.

pathology↗

bioRxiv2020

Derivation of macaque trophoblast stem cells

AbstractNonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSC) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first trimester placental villous cytotrophoblasts followed by culture in TSC medium to "reprogram" the cells to a proliferative state. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was >4,000-fold higher in ST culture media compared to TSC media. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and macaque placental nonclassical MHC class I molecule, Mamu-AG. TSC differentiation to extravillous trophoblasts (EVTs) with or without the ALK-5 inhibitor A83-01 resulted in differing morphologies but similar expression of Mamu-AG and CD56 as assessed by flow cytometry, hence further refinement of relevant EVT markers is needed. Our preliminary characterization of macaque TSCs suggests that these cells represent a proliferative, self-renewing TSC population capable of differentiating to STs in vitro thereby establishing an experimental model of primate placentation.

developmental biology↗

bioRxiv2019

Pharyngeal motor cortex grey matter abnormalities and retinal photoreceptor layer dysfunction in macaques exposed to Zika virus in utero

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.

microbiology↗

bioRxiv2019

Long-term protection of rhesus macaques from Zika virus reinfection

By the end of the 2016 Zika virus (ZIKV) outbreak, it is estimated that there were up to 100 million infections in the Americas. In approximately one in seven infants born to mothers infected during pregnancy, ZIKV has been linked to microcephaly, developmental delays, or other congenital disorders collectively known as congenital Zika syndrome (CZS). Guillain-Barre syndrome (GBS) in ZIKV infected adults. It is a global health priority to develop a vaccine against ZIKV that elicits long-lasting immunity, however, the durability of immunity to ZIKV is unknown. Previous studies in mice and nonhuman primates have been crucial in vaccine development but have not defined the duration of immunity generated by ZIKV infection. In this study, we rechallenged five rhesus macaques with ZIKV two years after a primary ZIKV infection. We show that primary ZIKV infection generates high titers of neutralizing antibodies (nAbs) that protect from detectable plasma viremia following rechallenge and persist for at least 27 months. While additional longitudinal studies are necessary with longer time frames, this study establishes a new experimentally defined minimal length of protective ZIKV immunity.\n\nAuthor SummaryZIKV emerged as a vector-borne pathogen capable of causing illness in infected adults and congenital birth defects in infants born to mothers infected during pregnancy. Despite the drop in ZIKV cases since the 2015-16 epidemic, questions concerning the prevalence and longevity of protective immunity have left vulnerable communities fearful that they may become the center of next ZIKV outbreak. While pre-existing herd immunity in regions of past outbreaks may dampen the potential for future outbreaks to occur, we currently do not know the longevity of protective immunity to ZIKV after a person becomes infected. Here, we establish a new experimentally defined minimal length of protective ZIKV immunity. We show that five rhesus macaques initially infected with ZIKV two years prior to rechallenge elicit a durable immune response that protected from detectable plasma viremia. While this work establishes a new minimal length of protective immunity, additional studies are necessary to define the maximum length of protective immunity following ZIKV infection.

immunology↗

bioRxiv2019

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-Barre 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.\n\nAuthor summaryPre-existing immunity to one of the four DENV serotypes is known to increase the risk of severe disease upon secondary infection with a different serotype. Due to the antigenic similarities between ZIKV and DENV, it has been proposed that these viruses could interact in a similar fashion. Data from in vitro experiments and murine models suggests that pre-existing immunity to one virus could either enhance or protect against infection with the other. These somewhat contradictory findings highlight the need for immune competent animal models for understanding the role of cross-reactive antibodies in flavivirus pathogenesis. We examined secondary ZIKV or DENV infections in rhesus and cynomolgus macaques that had previously been infected with the other virus. We assessed the outcomes of secondary ZIKV or DENV infections by quantifying vRNA loads, clinical and laboratory parameters, body temperature, and weight for each cohort of animals and compared them with control animals. These comparisons demonstrated that within a year of primary infection, secondary infections with either ZIKV or DENV were similar to primary infections and were not associated with enhancement or reduction in severity of disease based on the outcomes that we assessed.

pathology↗

bioRxiv2017

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 barcode clonotypes 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 64 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.\n\nAuthor summaryUnderstanding the complex dynamics of Zika virus (ZIKV) infection during pregnancy and during transmission to and from vertebrate host and mosquito vector is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and reservoir establishment. We sought to develop a virus model system for use in nonhuman primates and mosquitoes that allows for the genetic discrimination of molecularly cloned viruses. This \"synthetic swarm\" of viruses incorporates a molecular barcode that allows for tracking and monitoring individual viral lineages during infection. Here we infected rhesus macaques with this virus to study the dynamics of ZIKV infection in nonhuman primates as well as during mosquito infection/transmission. We found that the proportions of individual barcoded viruses remained relatively stable during acute infection in pregnant and nonpregnant animals. However, in a pregnant animal, the complexity of the virus population declined precipitously 8 days following infection, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques, and remained low for the subsequent duration of viremia.

microbiology↗