Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development. © 2022 by the authors.

The original strain of SARS-CoV-2, the Delta variant, and the Omicron variant infect microglia efficiently, in contrast to their inability to infect neurons: Analysis using 2D and 3D cultures.

COVID-19 causes neurological damage, systemic inflammation, and immune cell abnormalities. COVID-19-induced neurological impairment may be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which directly infects cells of the central nervous system (CNS) and exerts toxic effects. Furthermore, SARS-CoV-2 mutations occur constantly, and it is not well understood how the infectivity of the virus to cells of the CNS changes as the virus mutates. Few studies have examined whether the infectivity of cells of CNS - neural stem/progenitor cells (NS/PCs), neurons, astrocytes, and microglia - varies among SARS-CoV-2 mutant strains. In this study, therefore, we investigated whether SARS-CoV-2 mutations increase infectivity to CNS cells, including microglia. Since it was essential to demonstrate the infectivity of the virus to CNS cells in vitro using human cells, we generated cortical neurons, astrocytes, and microglia from human induced pluripotent stem cells (hiPSCs). We added pseudotyped lentiviruses of SARS-CoV-2 to each type of cells, and then we examined their infectivity. We prepared three pseudotyped lentiviruses expressing the S protein of the original strain (the first SARS-CoV-2 discovered in the world), the Delta variant, and the Omicron variant on their envelopes and analyzed differences of their ability to infect CNS cells. We also generated brain organoids and investigated the infectivity of each virus. The viruses did not infect cortical neurons, astrocytes, or NS/PCs, but microglia were infected by the original, Delta, and Omicron pseudotyped viruses. In addition, DPP4 and CD147, potential core receptors of SARS-CoV-2, were highly expressed in the infected microglia, while DPP4 expression was deficient in cortical neurons, astrocytes, and NS/PCs. Our results suggest that DPP4, which is also a receptor for Middle East respiratory syndrome-coronavirus (MERS-CoV), may play an essential role in the CNS. Our study is applicable to the validation of the infectivity of viruses that cause various infectious diseases in CNS cells, which are difficult to sample from humans.

Cortical-blood vessel assembloids exhibit Alzheimer's disease pathologies by activating glia after SARS-CoV-2 infection

A correlation between COVID-19 and Alzheimer's disease (AD) has been proposed recently. Although the number of case reports on neuroinflammation in COVID-19 patients has increased, studies of SARSCoV- 2 neurotrophic pathology using brain organoids have restricted recapitulation of those phenotypes due to insufficiency of immune cells and absence of vasculature. To overcome these limitations, we developed fused cortical-blood vessel organoids to provide blood vessels to brain organoids and obtained the characteristics of increased expression of glial cells and blood-brain barrier-like structures in brain organoids. Furthermore, we observed AD pathologies, including Abeta plaques, which were affected by the inflammatory response from SARS-CoV-2 infection. These findings provide an advanced platform to investigate human neurotrophic diseases, including COVID-19, and suggest that neuroinflammation caused by viral infection facilitates AD pathology.

Infection of 3D Brain Organoids with Human Pathogenic Viruses Under Biosafety Level-3 Conditions with Subsequent Inactivation to Study Viral Replication, Pathomechanisms, and Other Viral Infection-Mediated Effects

Viral infectious diseases may cause neurological symptoms primarily in two nonexclusive ways. Infection may lead to an excessive inflammatory response that damages the neuronal system, which is referred to as immunopathology, or the pathogen is able to infect brain cells, such as neurons or nonneuronal glial cells, like oligodendrocytes, microglia, and astrocytes. Viruses that target and infect cells of the CNS, in general, are called neurotropic. 3D brain organoids provide favorable conditions to study target cells and induced pathomechanisms associated with such viral neurotropism. In the context of highly pathogenic viruses, strict safety precautions (safety level-3 laboratory) must be taken if infectious laboratory strains or strains from clinical samples are to be used for infection experiments. Likewise, safe inactivation protocols must be used for subsequent analysis. This chapter will discuss appropriate protocols, focusing on methodological aspects for each of these steps, and discuss advantages and disadvantages when working with 3D brain organoids while handling biosafety level-3 pathogens based on our work with severe acute respiratory virus type 2 (SARS-CoV-2). Copyright © 2023, Springer Science+Business Media, LLC, part of Springer Nature.

Brain Organoids as a Model to Study Zika Virus and SARS-CoV-2 Infections

In recent years, we are living through different viral pandemics that result in neurological impairments. Given the human-specific nature of brain development, physiology, and pathology, it is imperative to use human models to investigate the neurological impact of viral infections, such as Zika virus and SARS-CoV-2. Brain organoids are powerful in vitro platforms for the analysis of the effects of viral infections on brain development and function, with prospective application to new emerging viral threats. Using brain organoids, it was possible to show that Zika virus infects neural stem cells, disrupting the cell cycle and neurogenesis, leading to microcephaly, a severe reduction of the brain. On the other hand, while it is still under investigation how SARS-CoV-2 might enter and alter the brain, organoid studies are helping to characterize its neurotropism and potential mechanisms of neurovirulence. Here, we describe a method for the infection of human brain organoid cultures with Zika and Sars-CoV-2 viruses that can be used to study neurodevelopmental phenotypes, alteration in neuronal functionality, host-pathogen interactions, as well as for drug testing. Copyright © 2023, Springer Science+Business Media, LLC, part of Springer Nature.

NEUROPILIN-1 MEDIATES SARS-CoV-2 INFECTION of ASTROCYTES PROMOTING NEURON DYSFUNCTION

Background: SARS-CoV-2 primarily infects the lung but may also damage other organs including the brain, heart, kidney, and intestine. Central nervous system (CNS) disorders include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Methods: We used iPSC-derived brain organoids and primary human astrocytes from cerebral cortex to study direct SARS-CoV-2 infection, as confirmed by Spike and Nucleocapsid immunostaining and RT-qPCR. siRNAs, blocking antibodies, and small molecule inhibitors were used to assess SARS-CoV-2 receptor candidates. Bulk RNA-seq, DNA methylation seq, and Nanostring GeoMx digital spatial profiling were utilized to identify virus-induced changes in host gene expression. Results: Astrocytes were robustly infected by SARS-CoV-2 in brain organoids while neurons and neuroprogenitor cells supported only low-level infection. Based on siRNA knockdowns, Neuropilin-1, not ACE2, functioned as the primary receptor for SARS-CoV-2 in astrocytes. The endolysosomal two-pore channel protein, TPC, also facilitated infection likely through its regulatory effects on endocytosis. Other alternative receptors, including the AXL tyrosine kinase, CD147, and dipeptidyl protease 4 (DPP4), did not function as SARS-CoV-2 receptors in astrocytes. SARS-CoV-2 infection dynamically induced type I, II, and III interferons, and genes involved in Toll-like receptor signaling, MDA5 and RIG-I sensing of double-stranded RNA, and production of inflammatory cytokines. Genes activating apoptosis were also increased. Down-regulated genes included those involved in water, ion and lipid transport, synaptic transmission, and formation of cell junctions. Epigenetic analyses revealed transcriptional changes related to DNA methylation states, particularly decreased DNA methylation in interferon-related genes. Long-term viral infection of brain organoids resulted in progressive neuronal degeneration and death. Conclusion: Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. Deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

CROI 2022

The proceedings contain 936 papers. The topics discussed include: vaccine strategies for HIV-1 and COVID-19;we?re still here: HIV, aging, and the invisible generation;HIV prevention in vulnerable populations: generating evidence to reduce inequalities;liver steatosis in persons living with HIV;deep mutational scanning to interpret viral evolution;the original delta virus or D like the devil;assessing vaccine effectiveness in the real world;studying covid treatment outcomes: why disparate results?;analysis approaches to correlates of vaccine efficacy;HIV nonhuman primate models for studies of virus pathogenesis, persistence, and cure;children exposed to HIV, but uninfected: evidence for action;macrophages are the primary source of virus in semen in acutely infected macaques;HIV infection of brain organoid microglia induces inflammation and neuronal death;and Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.

Brain surgical tissue for advanced tumour models in precision medicine: Developing the brain-stat pathway

AIMS: There are approximately four thousand neuro-oncology procedures in the UK per annum. Many of these result in tissue and biofluid specimens that are surplus to diagnostic requirement and can be collected as standard of clinical care. However, developing technologies and treatments for precision medicine require access to a range of individualised biospecimens paired with deep clinical phenotyping data. Here, we present Brain Surgical Tissue for Advanced Tumour Models (BRAINSTAT) programme, an infrastructure that has been established between Queen Elizabeth Hospital, Birmingham and the University of Birmingham, to collect, structure and store these resources and also maximise their value for research over the long-term. Using this approach our aim is to provide high-quality, annotated resources to help develop novel treatments for patients with brain tumours. METHOD: BRAINSTAT infrastructure allows: Prospective consent Biospecimens, including tumour tissue (brain and other primary in the case of metastasis), cyst fluid, dura, skin, CSF, blood (matched "germ-line" and for circulating cell free tumour DNA analysis), urine and saliva can be collected. Consent for long term follow-up, is either via clinic or NHS digital. More limited consent for non-oncological neurosurgical cohorts (e.g. epilepsy or vascular) and healthy volunteers allow healthy access-tissue and biofluids to be collected. B. Rapid transfer of fresh surgical tissue samples: Strong collaborative links and close physical proximity between operating theatre and laboratory allows rapid transfer of biospecimens minimising transit time. C. Standardised annotation across disciplines The RedCAP database system allows granular control over data-access, and each specialist research team is provided access only to the sub-sections relevant to them. All users must have Good Clinical Practice certification and GDPR training, prior to access of the BRAINSTAT database. RESULTS: Between 25/11/2019-16/03/2020 and 27/07/2020-16/11/2020, 65 patients were consented for BRAINSTAT at the weekly neurosurgical oncology clinic. (Recruitment gaps due to the SARS-COVID 19 pandemic). Pathological diagnosis of surplus tissue collected included: 37 high grade glioma, 3 low grade glioma and 16 brain metastasis including: (6 lung, 6 breast, 2 colorectal, 1 oesophageal, 1 endometrial). Meningioma (5 WHO I;1 WHO III) 1 patient undergoing anterior temporal lobectomy for hippocampal sclerosis contributed access tissue from the lateral neocortex. 1 patient had a non-neoplastic, non-diagnostic sample. All patients had matched "germline" blood samples. Median time from resection to arrival in the laboratory was 10 minutes (range 4-31). Standardised operating protocols to optimise this have been developed. Glioblastoma and breast-brain metastasis tumourspheres and cerebral organoids are currently being validated. CONCLUSION: Despite the challenges of the pandemic we have established a viable tissue pipeline from neurosurgical operating theatre to our university laboratories. We are developing clinically annotated human brain tumour cell lines, stem cells and 3D organoid models, principally for commonly encountered brain tumours such as glioma and metastasis. The research sets the foundation for a multitude of downstream applications including:-Building more complex organoid cultures e.g. by including other cell types such as healthy brain cells and endothelial cells allowing future experiments to more accurately model tumour growth.-Developing high-throughput, patient-specific drug screens of novel drugs and drug combinations using these 3D tumour models aiming to more effectively treat tumour proliferation and spread. These patient avatars will help inform and test more "stratified" personal medical treatments and will provide opportunities to allow earlier intervention with the aim of improving survival, coupled with a better quality of life.