SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

Differences in neuroinvasion and protective innate immune pathways between encephalitic California Serogroup orthobunyaviruses.

The California serogroup (CSG) of Orthobunyaviruses comprises several members capable of causing neuroinvasive disease in humans, including La Crosse orthobunyavirus (LACV), Jamestown Canyon orthobunyavirus (JCV), and Inkoo orthobunyavirus (INKV). Despite being genetically and serologically closely related, their disease incidences and pathogenesis in humans and mice differ. We have previously shown that following intraperitoneal inoculation of weanling mice, LACV was highly pathogenic while JCV and INKV were not. To determine why there were differences, we examined the ability of these viruses to invade the CNS and compared the host innate immune responses that regulated viral pathogenesis. We found that LACV was always neuroinvasive, which correlated with its high level of neuroinvasive disease. Interestingly, JCV was not neuroinvasive in any mice, while INKV was neuroinvasive in most mice. The type I interferon (IFN) response was critical for protecting mice from both JCV and INKV disease, although in the periphery JCV induced little IFN expression, while INKV induced high IFN expression. Despite their differing neuroinvasive abilities, JCV and INKV shared innate signaling components required for protection. The presence of either cytoplasmic Rig-I-Like Receptor signaling or endosomal Toll-Like Receptor signaling was sufficient to protect mice from JCV or INKV, however, inhibition of both pathways rendered mice highly susceptible to neurological disease. Comparison of IFN and IFN-stimulated gene (ISG) responses to INKV in the brains of resistant wild type (WT) mice and susceptible immune knockout mice showed similar IFN responses in the brain, but WT mice had higher ISG responses, suggesting induction of key ISGs in the brain is critical for protection of mice from INKV. Overall, these results show that the CSG viruses differ in neuroinvasiveness, which can be independent from their neuropathogenicity. The type I IFN response was crucial for protecting mice from CSG virus-induced neurological disease, however, the exact correlates of protection appear to vary between CSG viruses.

Placental Myeloid Cells Protect against Zika Virus Vertical Transmission in a Rag1-Deficient Mouse Model.

The ability of Zika virus (ZIKV) to cross the placenta and infect the fetus is a key mechanism by which ZIKV causes microcephaly. How the virus crosses the placenta and the role of the immune response in this process remain unclear. In the current study, we examined how ZIKV infection affected innate immune cells within the placenta and fetus and whether these cells influenced virus vertical transmission (VTx). We found myeloid cells were elevated in the placenta of pregnant ZIKV-infected Rag1-/- mice treated with an anti-IFNAR Ab, primarily at the end of pregnancy as well as transiently in the fetus several days before birth. These cells, which included maternal monocyte/macrophages, neutrophils, and fetal myeloid cells contained viral RNA and infectious virus, suggesting they may be infected and contributing to viral replication and VTx. However, depletion of monocyte/macrophage myeloid cells from the dam during ZIKV infection resulted in increased ZIKV infection in the fetus. Myeloid cells in the fetus were not depleted in this experiment, likely because of an inability of liposome particles containing the cytotoxic drug to cross the placenta. Thus, the increased virus infection in the fetus was not the result of an impaired fetal myeloid response or breakdown of the placental barrier. Collectively, these data suggest that monocyte/macrophage myeloid cells in the placenta play a significant role in inhibiting ZIKV VTx to the fetus, possibly through phagocytosis of virus or virus-infected cells.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death.

BACKGROUND: A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6-8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. METHODS: To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. RESULTS: BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. CONCLUSIONS: These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

Cutting Edge: CCR2 Is Not Required for Ly6Chi Monocyte Egress from the Bone Marrow but Is Necessary for Migration within the Brain in La Crosse Virus Encephalitis.

Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2-/- mice following infection. Interestingly, CCR2 was required for iMO trafficking from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain but was essential for trafficking within the brain parenchyma. Analysis of other orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2 requirement for iMO egress to the blood is not universal for all viruses.

Differences in Neuropathogenesis of Encephalitic California Serogroup Viruses.

The California serogroup of orthobunyaviruses comprises a group of mosquitoborne viruses, including La Crosse (LACV), snowshoe hare (SSHV), Tahyna (TAHV), Jamestown Canyon (JCV), and Inkoo (INKV) viruses, that cause neurologic disease in humans of differing ages with varying incidences. To determine how the pathogenesis of these viruses differs, we compared their ability to induce disease in mice and replicate and induce cell death in vitro. In mice, LACV, TAHV, and SSHV induced neurologic disease after intraperitoneal and intranasal inoculation, and JCV induced disease only after intranasal inoculation. INKV rarely induced disease, which correlated with less viral antigen in the brain than the other viruses. In vitro, all viruses replicated to high titers; however, LACV, SSHV, and TAHV induced high cell death, whereas JCV and INKV did not. Results demonstrated that CSG viruses differ in neuropathogenesis in vitro and in vivo, which correlates with the differences in pathogenesis reported in humans.

Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons.

BACKGROUND: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. METHODS: Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. RESULTS: Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. CONCLUSIONS: These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.

Adaptive Immune Responses to Zika Virus Are Important for Controlling Virus Infection and Preventing Infection in Brain and Testes.

The recent association between Zika virus (ZIKV) and neurologic complications, including Guillain-Barré syndrome in adults and CNS abnormalities in fetuses, highlights the importance in understanding the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response, suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show in this article, although wild-type C57BL/6 mice mount cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by Abs in Rag1-/- mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Ig-treated Rag1-/- mice or wild-type mice treated with anti-type I IFNR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared with nonpregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in the control of ZIKV infection and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.

Using immunocompromised mice to identify mechanisms of Zika virus transmission and pathogenesis.

Zika virus (ZIKV) is responsible for a recent global epidemic that has been associated with congenital brain malformations in fetuses and with Guillain-Barré syndrome in adults. Within the last 2 years, a major effort has been made to develop murine models to study the mechanism of viral transmission, pathogenesis and the host immune response. Here, we discuss the findings from these models regarding the role that the innate and adaptive immune responses have in controlling ZIKV infection and pathogenesis. Additionally, we examine how innate and adaptive immune responses influence sexual and vertical transmission of ZIKV infection as well as how these responses can influence the ability of ZIKV to cross the placenta and to induce damage in the developing brain.

Salmonella Meningitis Associated with Monocyte Infiltration in Mice.

In the current study, we examined the ability of Salmonella enterica serovar Typhimurium to infect the central nervous system and cause meningitis following the natural route of infection in mice. C57BL/6J mice are extremely susceptible to systemic infection by Salmonella Typhimurium because of loss-of-function mutations in Nramp1 (SLC11A1), a phagosomal membrane protein that controls iron export from vacuoles and inhibits Salmonella growth in macrophages. Therefore, we assessed the ability of Salmonella to disseminate to the central nervous system (CNS) after oral infection in C57BL/6J mice expressing either wild-type (resistant) or mutant (susceptible) alleles of Nramp1. In both strains, oral infection resulted in focal meningitis and ventriculitis with recruitment of inflammatory monocytes to the CNS. In susceptible Nramp1-/- mice, there was a direct correlation between bacteremia and the number of bacteria in the brain, which was not observed in resistant Nramp1+/+ mice. A small percentage of Nramp1+/+ mice developed severe ataxia, which was associated with high bacterial loads in the CNS as well as clear histopathology of necrotizing vasculitis and hemorrhage in the brain. Thus, Nramp1 is not essential for Salmonella entry into the CNS or neuroinflammation, but may influence the mechanisms of CNS entry as well as the severity of meningitis.

Lymphocytes have a role in protection, but not in pathogenesis, during La Crosse Virus infection in mice.

BACKGROUND: La Crosse Virus (LACV) is a primary cause of pediatric viral encephalitis in the USA and can result in severe clinical outcomes. Almost all cases of LACV encephalitis occur in children 16 years or younger, indicating an age-related susceptibility. This susceptibility is recapitulated in a mouse model where weanling (3 weeks old or younger) mice are susceptible to LACV-induced disease, and adults (greater than 6 weeks) are resistant. Disease in mice and humans is associated with infiltrating leukocytes to the CNS. However, what cell types are infiltrating into the brain during virus infection and how these cells influence pathogenesis remain unknown. METHODS: In the current study, we analyzed lymphocytes recruited to the CNS during LACV-infection in clinical mice, using flow cytometry. We analyzed the contribution of these lymphocytes to LACV pathogenesis in weanling mice using knockout mice or antibody depletion. Additionally, we studied at the potential role of these lymphocytes in preventing LACV neurological disease in resistant adult mice. RESULTS: In susceptible weanling mice, disease was associated with infiltrating lymphocytes in the CNS, including NK cells, CD4 T cells, and CD8 T cells. Surprisingly, depletion of these cells did not impact neurological disease, suggesting these cells do not contribute to virus-mediated damage. In contrast, in disease-resistant adult animals, depletion of both CD4 T cells and CD8 T cells or depletion of B cells increased neurological disease, with higher levels of virus in the brain. CONCLUSIONS: Our current results indicate that lymphocytes do not influence neurological disease in young mice, but they have a critical role protecting adult animals from LACV pathogenesis. Although LACV is an acute virus infection, these studies indicate that the innate immune response in adults is not sufficient for protection and that components of the adaptive immune response are necessary to prevent virus from invading the CNS.

SARM1, Not MyD88, Mediates TLR7/TLR9-Induced Apoptosis in Neurons.

Neuronal apoptosis is a key aspect of many different neurologic diseases, but the mechanisms remain unresolved. Recent studies have suggested a mechanism of innate immune-induced neuronal apoptosis through the stimulation of endosomal TLRs in neurons. TLRs are stimulated both by pathogen-associated molecular patterns as well as by damage-associated molecular patterns, including microRNAs released by damaged neurons. In the present study, we identified the mechanism responsible for TLR7/TLR9-mediated neuronal apoptosis. TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required the TLR adaptor molecule SARM1, which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation in neurites and TLR-induced apoptosis. These studies identify a non-MyD88 pathway of TLR7/ TLR9 signaling in neurons and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage.

Age-dependent myeloid dendritic cell responses mediate resistance to la crosse virus-induced neurological disease.

UNLABELLED: La Crosse virus (LACV) is the major cause of pediatric viral encephalitis in the United States; however, the mechanisms responsible for age-related susceptibility in the pediatric population are not well understood. Our current studies in a mouse model of LACV infection indicated that differences in myeloid dendritic cell (mDC) responses between weanling and adult mice accounted for susceptibility to LACV-induced neurological disease. We found that type I interferon (IFN) responses were significantly stronger in adult than in weanling mice. Production of these IFNs required both endosomal Toll-like receptors (TLRs) and cytoplasmic RIG-I-like receptors (RLRs). Surprisingly, IFN expression was not dependent on plasmacytoid DCs (pDCs) but rather was dependent on mDCs, which were found in greater number and induced stronger IFN responses in adults than in weanlings. Inhibition of these IFN responses in adults resulted in susceptibility to LACV-induced neurological disease, whereas postinfection treatment with type I IFN provided protection in young mice. These studies provide a definitive mechanism for age-related susceptibility to LACV encephalitis, where mDCs in young mice are insufficiently activated to control peripheral virus replication, thereby allowing virus to persist and eventually cause central nervous system (CNS) disease. IMPORTANCE: La Crosse virus (LACV) is the primary cause of pediatric viral encephalitis in the United States. Although the virus infects both adults and children, over 80% of the reported neurological disease cases are in children. To understand why LACV causes neurological disease primarily in young animals, we used a mouse model where weanling mice, but not adult mice, develop neurological disease following virus infection. We found that an early immune response cell type, myeloid dendritic cells, was critical for protection in adult animals and that these cells were reduced in young animals. Activation of these cells during virus infection or after treatment with type I interferon in young animals provided protection from LACV. Thus, this study demonstrates a reason for susceptibility to LACV infection in young animals and shows that early therapeutic treatment in young animals can prevent neurological disease.

Capillaries in the olfactory bulb but not the cortex are highly susceptible to virus-induced vascular leak and promote viral neuroinvasion.

Viral neuroinvasion is a critical step in the pathogenesis of viral encephalitis. Multiple mechanisms of neuroinvasion have been identified, but their relative contribution to central nervous system (CNS) infection remains unclear for many viruses. In this study, we examined neuroinvasion of the mosquito-borne bunyavirus La Crosse (LACV), the leading cause of pediatric viral encephalitis in the USA. We found that the olfactory bulb (OB) and tract were the initial areas of CNS virus infection in mice. Removal of the OB reduced the incidence of LACV-induced disease demonstrating the importance of this area to neuroinvasion. However, we determined that infection of the OB was not due to axonal transport of virus from olfactory sensory neurons as ablation of these cells did not affect viral pathogenesis. Instead, we found that OB capillaries were compromised allowing leakage of virus-sized particles into the brain. Analysis of OB capillaries demonstrated specific alterations in cytoskeletal and Rho GTPase protein expression not observed in capillaries from other brain areas such as the cortex where leakage did not occur. Collectively, these findings indicate that LACV neuroinvasion occurs through hematogenous spread in specific brain regions where capillaries are prone to virus-induced activation such as the OB. Capillaries in these areas may be "hot spots" that are more susceptible to neuroinvasion not only for LACV, but other neurovirulent viruses as well.

C-C motif chemokine receptor 2 and 7 synergistically control inflammatory monocyte recruitment but the infecting virus dictates monocyte function in the brain.

Inflammatory monocytes (iMO) are recruited from the bone marrow to the brain during viral encephalitis. C-C motif chemokine receptor (CCR) 2 deficiency substantially reduces iMO recruitment for most, but not all encephalitic viruses. Here we show CCR7 acts synergistically with CCR2 to control this process. Following Herpes simplex virus type-1 (HSV-1), or La Crosse virus (LACV) infection, we find iMO proportions are reduced by approximately half in either Ccr2 or Ccr7 knockout mice compared to control mice. However, Ccr2/Ccr7 double knockouts eliminate iMO recruitment following infection with either virus, indicating these receptors together control iMO recruitment. We also find that LACV induces a more robust iMO recruitment than HSV-1. However, unlike iMOs in HSV-1 infection, LACV-recruited iMOs do not influence neurological disease development. LACV-induced iMOs have higher expression of proinflammatory and proapoptotic but reduced mitotic, phagocytic and phagolysosomal transcripts compared to HSV-1-induced iMOs. Thus, virus-specific activation of iMOs affects their recruitment, activation, and function.

Hyaluronan anchored to activated CD44 on central nervous system vascular endothelial cells promotes lymphocyte extravasation in experimental autoimmune encephalomyelitis.

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 µm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis.

One of the key events in viral encephalitis is the ability of virus to enter the central nervous system (CNS). Several encephalitic viruses, including La Crosse Virus (LACV), primarily induce encephalitis in children, but not adults. This phenomenon is also observed in LACV mouse models, where the virus gains access to the CNS of weanling animals through vascular leakage of brain microvessels, likely through brain capillary endothelial cells (BCECs). To examine age and region-specific regulatory factors of vascular leakage, we used genome-wide transcriptomics and targeted siRNA screening to identify genes whose suppression affected viral pathogenesis in BCECs. Further analysis of two of these gene products, Connexin43 (Cx43/Gja1) and EphrinA2 (Efna2), showed a substantial effect on LACV pathogenesis. Induction of Cx43 by 4-phenylbutyric acid (4-PBA) inhibited neurological disease in weanling mice, while Efna2 deficiency increased disease in adult mice. Thus, we show that Efna2 and Cx43 expressed by BCECs are key mediators of LACV-induced neuroinvasion and neurological disease.

Zika virus diversity in mice is maintained during early vertical transmission from placenta to fetus, but reduced in fetal bodies and brains at late stages of infection.

Since emerging in French Polynesia and Brazil in the 2010s, Zika virus (ZIKV) has been associated with fetal congenital disease. Previous studies have compared ancestral and epidemic ZIKV strains to identify strain differences that may contribute to vertical transmission and fetal disease. However, within-host diversity in ZIKV populations during vertical transmission has not been well studied. Here, we used the established anti-interferon treated Rag1-/- mouse model of ZIKV vertical transmission to compare genomic variation within ZIKV populations in matched placentas, fetal bodies, and fetal brains via RNASeq. At early stages of vertical transmission, the ZIKV populations in the matched placentas and fetal bodies were similar. Most ZIKV single nucleotide variants were present in both tissues, indicating little to no restriction in transmission of ZIKV variants from placenta to fetus. In contrast, at later stages of fetal infection there was a sharp reduction in ZIKV diversity in fetal bodies and fetal brains. All fetal brain ZIKV populations were comprised of one of two haplotypes, containing either a single variant or three variants together, as largely homogenous populations. In most cases, the dominant haplotype present in the fetal brain was also the dominant haplotype present in the matched fetal body. However, in two of ten fetal brains the dominant ZIKV haplotype was undetectable or present at low frequencies in the matched placenta and fetal body ZIKV populations, suggesting evidence of a strict selective bottleneck and possible selection for certain variants during neuroinvasion of ZIKV into fetal brains.

Zika virus vertical transmission in interferon receptor1-antagonized Rag1-/- mice results in postnatal brain abnormalities and clinical disease.

The mechanisms by which vertically transmitted Zika virus (ZIKV) causes postnatal brain development abnormalities and congenital disease remain poorly understood. Here, we optimized the established anti-IFNAR1 treated, Rag1-/- (AIR) mouse model of ZIKV infection to examine the consequence of vertical transmission on neonate survival and postnatal brain development. We found that modulating the infectious dose and the frequency of anti-IFNAR1 treatment of pregnant mice (termed AIRlow mice) prolonged neonatal survival allowing for pathogenesis studies of brain tissues at critical postnatal time points. Postnatal AIRlow mice all had chronic ZIKV infection in the brain that was associated with decreased cortical thickness and cerebellar volume, increased gliosis, and higher levels of cell death in many brain areas including cortex, hippocampus and cerebellum when compared to controls. Interestingly, despite active infection and brain abnormalities, the neurodevelopmental program remained active in AIRlow mice as indicated by elevated mRNA expression of critical neurodevelopmental genes in the brain and enlargement of neural-progenitor rich regions of the cerebellum at a developmental time point analogous to birth in humans. Nevertheless, around the developmental time point when the brain is fully populated by neurons, AIRlow mice developed neurologic disease associated with persistent ZIKV infection in the brain, gliosis, and increased cell death. Together, these data show that vertically transmitted ZIKV infection in the brain of postnatal AIRlow mice strongly influences brain development resulting in structural abnormalities and cell death in multiple regions of the brain.

Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain.

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c-/-) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c-/- mice.