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The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Significance The COVID-19 pandemic and the ever-evolving variants of SARS-CoV-2 are taking a toll on human health. Despite the successful rollout of vaccines, effective therapies are still urgently needed. Our studies here showing that Nsp1 selectively blocks translation of host but not viral proteins by proper coordination of its N- and C-terminal domains to advance our understanding on SARS-CoV-2 pathogenesis. Our finding that stem-loop 1, a highly conserved sequence in the SARS-CoV-2 5′ UTR, is necessary and sufficient for bypassing Nsp1-mediated shutdown led to the design of antisense oligonucleotides targeting this sequence that make viral translation susceptible to Nsp1 shutdown, interfere with viral replication, and protect SARS-CoV-2–infected mice. This strategy of turning SARS-CoV-2’s own virulence against itself could be harnessed therapeutically.

Abstract Background COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood. Methods We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue. Results We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2. Conclusions Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

Global public health infrastructure is unprepared for emerging pathogen epidemics, in part because diagnostic tests are not developed in advance. The recent Zika, Ebola, and SARS-CoV-2 virus epidemics are cases in point. We demonstrate here that multicolored gold nanoparticles, when coupled to cross-reactive monoclonal antibody pairs generated from a single immunization regimen, can be used to create multiple diagnostics that specifically detect and distinguish related viruses. The multiplex approach for specific detection centers on immunochromatography with pairs of antibody-conjugated red and blue gold nanoparticles, coupled with clustering algorithms to detect and distinguish related pathogens. Cross-reactive antibodies were used to develop rapid tests for i) Dengue virus serotypes 1-4, ii) Zika virus, iii) Ebola and Marburg viruses, and iv) SARS-CoV and SARS-CoV-2 viruses. Multiplexed rapid antigen tests based on multicolored nanoparticles and cross-reactive antibodies and can be developed prospectively at low cost to improve preparedness for epidemic outbreaks.

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity.

© 2020 Bosch et al. Background Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1–4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiat-ing DENV serotypes and none are currently commercially available. Methodology/Principle findings We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitiv-ity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 sero-typing were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07–100%. Significance Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer conven

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity.

© 2020 Bosch et al. Background Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1–4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiat-ing DENV serotypes and none are currently commercially available. Methodology/Principle findings We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitiv-ity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 sero-typing were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07–100%. Significance Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer conven

Copyright © 2020 The Authors, Alzheimer’s disease (AD) is a neurodegenerative disorder that causes cognitive decline, memory loss, and inability to perform everyday functions. Hallmark features of AD—including generation of amyloid plaques, neurofibrillary tangles, gliosis, and inflammation in the brain—are well defined; however, the cause of the disease remains elusive. Growing evidence implicates pathogens in AD development, with herpes simplex virus type I (HSV-1) gaining increasing attention as a potential causative agent. Here, we describe a multidisciplinary approach to produce physiologically relevant human tissues to study AD using human-induced neural stem cells (hiNSCs) and HSV-1 infection in a 3D bioengineered brain model. We report a herpes-induced tissue model of AD that mimics human disease with multicellular amyloid plaque–like formations, gliosis, neuroinflammation, and decreased functionality, completely in the absence of any exogenous mediators of AD. This model will allow for future studies to identify potential downstream drug targets for treating this devastating disease.

Copyright © 2019 American Chemical Society. Across zoonotic pathogens, RNA viruses are responsible for disproportionate levels of human disease, suffering, and death. Neurotropic RNA viruses (e.g., rabies, Japanese and Eastern Equine Encephalitis, Ebola, West Nile, Powassan) infect the brain and spinal cord, causing meningitis, encephalitis, microcephaly, and Guillain-Barré syndrome. Mechanistic data explaining the molecular mechanisms of these diseases are lacking, and the enclosure of the central nervous system and the associated meninges in bone complicates access for diagnosis, clinical treatment, and research. Here, we discuss new tissue models, imaging methods, and molecular techniques that are changing research aimed at understanding the pathogenesis of neurotropic RNA viruses.

Copyright © 2019 American Chemical Society. Across zoonotic pathogens, RNA viruses are responsible for disproportionate levels of human disease, suffering, and death. Neurotropic RNA viruses (e.g., rabies, Japanese and Eastern Equine Encephalitis, Ebola, West Nile, Powassan) infect the brain and spinal cord, causing meningitis, encephalitis, microcephaly, and Guillain-Barré syndrome. Mechanistic data explaining the molecular mechanisms of these diseases are lacking, and the enclosure of the central nervous system and the associated meninges in bone complicates access for diagnosis, clinical treatment, and research. Here, we discuss new tissue models, imaging methods, and molecular techniques that are changing research aimed at understanding the pathogenesis of neurotropic RNA viruses., U.S. Public Health Service (Award U19AI131135)

Brain organoids grown from human pluripotent stem cells self-organize into cytoarchitectures resembling the developing human brain. These three-dimensional models offer an unprecedented opportunity to study human brain development and dysfunction. Characterization currently sacrifices spatial information for single-cell or histological analysis leaving whole-tissue analysis mostly unexplored. Here, we present the SCOUT pipeline for automated multiscale comparative analysis of intact cerebral organoids. Our integrated technology platform can rapidly clear, label, and image intact organoids. Algorithmic- and convolutional neural network-based image analysis extract hundreds of features characterizing molecular, cellular, spatial, cytoarchitectural, and organoid-wide properties from fluorescence microscopy datasets. Comprehensive analysis of 46 intact organoids and ~ 100 million cells reveals quantitative multiscale “phenotypes" for organoid development, culture protocols and Zika virus infection. SCOUT provides a much-needed framework for comparative analysis of emerging 3D in vitro models using fluorescence microscopy.

Rapid diagnostic tests (point-of-care devices) are critical components of informed patient care and public health monitoring (surveillance applications). We propose that among the many rapid diagnostics platforms that have been tested or are in development, lateral flow immunoassays and synthetic biology-based diagnostics (including CRISPR-based diagnostics) represent the best overall options given their ease of use, scalability for manufacturing, sensitivity, and specificity. This review describes the identification of lateral flow immunoassay monoclonal antibody pairs that detect and distinguish between closely related pathogens and that are used in combination with functionalized multicolored nanoparticles and computational methods to deconvolute data. We also highlight the promise of synthetic biology-based diagnostic tests, which use synthetic genetic circuits that activate upon recognition of a pathogen-associated nucleic acid sequence, and discuss how the combined or parallel use of lateral flow immunoassays and synthetic biology tools may represent the future of scalable rapid diagnostics.

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015–2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms., NIH (Grants AI-100190, 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201)

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen., U.S. Public Health Service (award AI100190)

Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.

Lateral flow assays (LFAs) are highly attractive for point-of-care (POC) diagnostics for infectious disease, food safety, and many other medical uses. The unique optical, electronic, and chemical properties that arise from the nanostructured and material characteristics of nanoparticles provide an opportunity to increase LFA sensitivity and impart novel capabilities. However, interfacing to nanomaterials in complex biological environments is challenging and can result in undesirable side effects such as non-specific adsorption, protein denaturation, and steric hindrance. These issues are even more acute in LFAs where there are many different types of inorganic–biological interfaces, often of a complex nature. Therefore, the unique properties of nanomaterials for LFAs must be exploited in a way that addresses these interface challenges. LFAs for infectious disease, food safety, and many other applications have been enhanced by nanotechnology. However, interface effects in LFAs are much more complicated, which is problematic because POC devices must be robust, simple, and easy to use. To fully utilize the unique properties of nanotechnology, these interface issues must be understood, controlled, and also leveraged., NIH NIAID (AI100190)

Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention. Keywords: Zika virus; neural progenitors; CRISPR screen; fetal CNS infection; human pluri; potent stem cells, National Institutes of Health (U.S.) (Grant R01 MH104610), National Institutes of Health (U.S.) (Grant R01 NS088538), National Institutes of Health (U.S.) (Grant U19 AI131135), National Institutes of Health (U.S.) (Grant R33 AI100190), Simons Foundation (Grant SFARI 204106)

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. A global reduction in ribosome levels in Diamond-Blackfan anemia profoundly alters translation of a select subset of transcripts, thereby impeding erythroid lineage commitment. Keyword: hematopoiesis; lineage commitment; GATA1; genetics; ribosome; translation; Diamond-Blackman anemia; erythropoiesis, National Institutes of Health (U.S.) (Grants R01 DK103794), National Institutes of Health (U.S.) (Grant R33 HL120791)

Maternal Zika virus (ZIKV) infection during pregnancy is recognized as the cause of an epidemic of microcephaly and other neurological anomalies in human fetuses. It remains unclear how ZIKV accesses the highly vulnerable population of neural progenitors of the fetal central nervous system (CNS), and which cell types of the CNS may be viral reservoirs. In contrast, the related dengue virus (DENV) does not elicit teratogenicity. To model viral interaction with cells of the fetal CNS in vitro, we investigated the tropism of ZIKV and DENV for different induced pluripotent stem cell-derived human cells, with a particular focus on microglia-like cells. We show that ZIKV infected isogenic neural progenitors, astrocytes, and microglia-like cells (pMGLs), but was only cytotoxic to neural progenitors. Infected glial cells propagated ZIKV and maintained ZIKV load over time, leading to viral spread to susceptible cells. DENV triggered stronger immune responses and could be cleared by neural and glial cells more efficiently. pMGLs, when cocultured with neural spheroids, invaded the tissue and, when infected with ZIKV, initiated neural infection. Since microglia derive from primitive macrophages originating in proximity to the maternal vasculature, they may act as a viral reservoir for ZIKV and establish infection of the fetal brain. Infection of immature neural stem cells by invading microglia may occur in the early stages of pregnancy, before angiogenesis in the brain rudiments. Our data are also consistent with ZIKV and DENV affecting the integrity of the blood–brain barrier, thus allowing infection of the brain later in life. Keywords: Zika; microglia; organoids; interferon; iPS, National Institutes of Health (U.S.) (Grant AI100190)