BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1ß, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression.

How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15-35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19.

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

Injection site vaccinology of a recombinant vaccinia-based vector reveals diverse innate immune signatures.

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1ß, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design.

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives.

BACKGROUND: The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or "cytomorbidity", as distinct from cytotoxicity or loss of viability. METHODS: A simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays. RESULTS: The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity. CONCLUSIONS: We describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.

Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.

HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production.

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.

The protein arginine methyltransferase PRMT6 inhibits HIV-1 Tat nucleolar retention.

The human immunodeficiency virus (HIV)-1 transactivator protein Tat is known to play a key role in HIV infection, integrally related to its role in the host cell nucleus/nucleolus. Here we show for the first time that Tat localisation can be modulated by specific methylation, whereby overexpression of active but not catalytically inactive PRMT6 methyltransferase specifically leads to exclusion of Tat from the nucleolus. An R52/53A mutated Tat derivative does not show this redistribution, implying that R52/53, within Tat's nuclear/nucleolar localisation signal, are the targets of PRMT6 activity. Analysis using fluorescence recovery after photobleaching indicate that Tat nucleolar accumulation is largely through binding to nucleolar components, with methylation of Tat by PRMT6 preventing this. To our knowledge, this is the first report of specific protein methylation inhibiting nucleolar retention.

Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target.

Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3-4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target.

A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes.

BACKGROUND: Nullbasic is a mutant HIV-1 Tat protein that inhibits HIV-1 replication via three independent mechanisms that disrupts 1) reverse transcription of the viral RNA genome into a DNA copy, 2) HIV-1 Rev protein function required for viral mRNA transport from the nucleus to the cytoplasm and 3) HIV-1 mRNA transcription by RNA Polymerase II. The Nullbasic protein is derived from the subtype B strain HIV-1BH10 and has only been tested against other HIV-1 subtype B strains. However, subtype B strains only account for ~10% of HIV-1 infections globally and HIV-1 Tat sequences vary between subtypes especially for subtype C, which is responsible for ~50% HIV-1 infection worldwide. These differences could influence the ability of Tat to interact with RNA and cellular proteins and thus could affect the antiviral activity of Nullbasic. Therefore, Nullbasic was tested against representative HIV-1 strains from subtypes C, D and A/D recombinant to determine if it can inhibit their replication. METHODS: Nullbasic was delivered to human cells using a self-inactivating (SIN) γ-retroviral system. We evaluated Nullbasic-mCherry (NB-mCh) fusion protein activity against the HIV-1 strains in TZM-bl cell lines for inhibition of transactivation and virus replication. We also examined antiviral activity of Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein against the same strains in primary CD4+ T cells. The Nullbasic expression was monitored by western blot and flow cytometry. The effects of Nullbasic on primary CD4+ T cells cytotoxicity, proliferation and apoptosis were also examined. RESULTS: The results show that Nullbasic inhibits Tat-mediated transactivation and virus replication of all the HIV-1 strains tested in TZM-bl cells. Importantly, Nullbasic inhibits replication of the HIV-1 strains in primary CD4+ T cells without affecting cell proliferation, cytotoxicity or level of apoptotic cells. CONCLUSION: A SIN-based γ-retroviral vector used to express Nullbasic fusion proteins improved protein expression particularly in primary CD4+ T cells. Nullbasic has antiviral activity against all strains from the subtypes tested although small differences in viral inhibition were observed. Further improvement of in γ-retroviral vector stable expression of Nullbasic expression may have utility in a future gene therapy approach applicable to genetically diverse HIV-1 strains.

Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains.

A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent.

Comparison of attenuated and virulent West Nile virus strains in human monocyte-derived dendritic cells as a model of initial human infection.

BACKGROUND: The human-pathogenic North American West Nile virus strain (WNVNY99), responsible for the outbreak in New York city in 1999, has caused 41000 infections and 1739 human deaths to date. A new strain of West Nile virus emerged in New South Wales, Australia in 2011 (WNVNSW2011), causing a major encephalitic outbreak in horses with close to 1000 cases and 10-15% mortality. Unexpectedly, no human cases have so far been documented. FINDINGS: We report here, using human monocyte-derived dendritic cells (MoDCs) as a model of initial WNV infection, that the pathogenic New York 99 WNV strain (WNVNY99) replicated better than WNVNSW2011, indicative of increased viral dissemination and pathogenesis in a natural infection. This was attributed to suppressed viral replication and type I interferon (IFN) response in the early phase of WNVNY99 infection, leading to enhanced viral replication at the later phase of infection. In addition, WNVNY99 induced significantly more pro-inflammatory cytokines in MoDCs compared to WNVNSW2011. CONCLUSIONS: Our results suggest that the observed differences in replication and induction of IFN response between WNVNY99 and WNVNSW2011 in MoDCs may be indicative of their difference in virulence for humans.

Microplastics dysregulate innate immunity in the SARS-CoV-2 infected lung.

Introduction: Global microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis, with a growing body of concern surrounding the potential impacts on human health. Methods: Using a mouse model of mild COVID-19, we describe herein the effects of azide-free 1 µm polystyrene MP beads, co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum. The effect of MPs on the host response to SARS-CoV-2 infection was analysed using histopathology and RNA-Seq at 2 and 6 days post-infection (dpi). Results: Although infection reduced clearance of MPs from the lung, virus titres and viral RNA levels were not significantly affected by MPs, and overt MP-associated clinical or histopathological changes were not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 dpi and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the 'cytokine release syndrome' signature observed in some COVID-19 patients. Discussion: The findings are consistent with the recent finding that MPs can inhibit phagocytosis of apoptotic cells via binding of Tim4. They also add to a growing body of literature suggesting that MPs can dysregulate inflammatory processes in specific disease settings.

Generating prophylactic immunity against arboviruses in vertebrates and invertebrates.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Warmer ambient air temperatures reduce nasal turbinate and brain infection, but increase lung inflammation in the K18-hACE2 mouse model of COVID-19.

Warmer climatic conditions have been associated with fewer COVID-19 cases. Herein we infected K18-hACE2 mice housed at the standard animal house temperature of ∼22 °C, or at ∼31 °C, which is considered to be thermoneutral for mice. On day 2 post infection, RNA-Seq analyses showed no significant differential gene expression lung in lungs of mice housed at the two temperatures, with almost identical viral loads and type I interferon responses. There was also no significant difference in viral loads in lungs on day 5, but RNA-Seq and histology analyses showed clearly elevated inflammatory signatures and infiltrates. Thermoneutrality thus promoted lung inflammation. On day 2 post infection mice housed at 31 °C showed reduced viral loads in nasal turbinates, consistent with increased mucociliary clearance at the warmer ambient temperature. These mice also had reduced virus levels in the brain, and an ensuing amelioration of weight loss and a delay in mortality. Warmer air temperatures may thus reduce infection of the upper respiratory track and the olfactory epithelium, resulting in reduced brain infection. Potential relevance for anosmia and neurological sequelae in COVID-19 patients is discussed.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

SARS-CoV-2 omicron BA.5 and XBB variants have increased neurotropic potential over BA.1 in K18-hACE2 mice and human brain organoids.

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential.