Evolutionary Dynamics of Accelerated Antiviral Resistance Development in Hypermutator Herpesvirus.

Antiviral therapy is constantly challenged by the emergence of resistant pathogens. At the same time, experimental approaches to understand and predict resistance are limited by long periods required for evolutionary processes. Here, we present a herpes simplex virus 1 (HSV-1) mutant with impaired proofreading capacity and consequently elevated mutation rates. Comparing this hypermutator to parental wild type virus, we study the evolution of antiviral drug resistance in vitro. We model resistance development and elucidate underlying genetic changes against three antiviral substances. Our analyses reveal no principle difference in the evolutionary behavior of both viruses, adaptive processes are overall similar, however significantly accelerated for the hypermutator. We conclude that hypermutator viruses are useful for modelling adaptation to antiviral therapy. They offer the benefit of expedited adaptation without introducing apparent bias and can therefore serve as an accelerator to predict natural evolution.

Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19.

A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively.

Mucus-Inspired Self-Healing Hydrogels: A Protective Barrier for Cells against Viral Infection.

Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.

Computationally Designed Epitope-Mediated Imprinted Polymers versus Conventional Epitope Imprints for the Detection of Human Adenovirus in Water and Human Serum Samples.

Detection of pathogenic viruses for point-of-care applications has attracted great attention since the COVID-19 pandemic. Current virus diagnostic tools are laborious and expensive, while requiring medically trained staff. Although user-friendly and cost-effective biosensors are utilized for virus detection, many of them rely on recognition elements that suffer major drawbacks. Herein, computationally designed epitope-imprinted polymers (eIPs) are conjugated with a portable piezoelectric sensing platform to establish a sensitive and robust biosensor for the human pathogenic adenovirus (HAdV). The template epitope is selected from the knob part of the HAdV capsid, ensuring surface accessibility. Computational simulations are performed to evaluate the conformational stability of the selected epitope. Further, molecular dynamics simulations are executed to investigate the interactions between the epitope and the different functional monomers for the smart design of eIPs. The HAdV epitope is imprinted via the solid-phase synthesis method to produce eIPs using in silico-selected ingredients. The synthetic receptors show a remarkable detection sensitivity (LOD: 102 pfu mL-1) and affinity (dissociation constant (Kd): 6.48 × 10-12 M) for HAdV. Moreover, the computational eIPs lead to around twofold improved binding behavior than the eIPs synthesized with a well-established conventional recipe. The proposed computational strategy holds enormous potential for the intelligent design of ultrasensitive imprinted polymer binders.

Improving the Antigenicity of SARS-CoV-2 Vaccine Genes by Merging Mutations from Different Variants of Concern.

During the COVID-19 pandemic, the early emergence of viral variants repeatedly undermined the effects of vaccination. Our aim here is to explore strategies for improving spike vaccine gene antigenicity by merging mutations from different variants of concern (VOCs) in a single vaccine gene. To this end, newly developed recombinant vaccine genes were designed, cloned into adenoviral vectors, and applied to C57BL/6 mice; then, serum-neutralizing antibodies against the wildtype SARS-CoV-2 strains were determined in neutralization assays. The merger of mutations from different variants of concern (alpha, beta, gamma, and delta) in a single recombinant spike-based vaccine gene provided a substantial improvement in neutralizing immunity to all variants of concern, including the omicron strains. To date, only unmodified spike genes of the original SARS-CoV-2 Wuhan strain (B.1) or dominant variants (BA.1, BA.5, and XBB.1.5) have been used as vaccine genes. The employment of unmodified vaccine genes is afflicted by limited cross-protection among variant strains. In contrast, recombinant vaccine genes that combine mutations from different strains in a single gene hold the potential to broaden and improve immune protection and might help to reduce the need for frequent vaccine adaptations in the future.

Protective role of the HSP90 inhibitor, STA-9090, in lungs of SARS-CoV-2-infected Syrian golden hamsters.

INTRODUCTION: The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyperinflammatory state generated on infection, reducing lung tissue pathology and inhibiting viral replication. Previous research has pointed to a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. METHODS: In this study, we investigated the effects of the potent HSP90 inhibitor Ganetespib (STA-9090) in vitro on alveolar epithelial cells and alveolar macrophages to characterise its effects on cell activation and viral replication. Additionally, the Syrian hamster animal model was used to evaluate its efficacy in controlling systemic inflammation and viral burden after infection. RESULTS: In vitro, STA-9090 reduced viral replication on alveolar epithelial cells in a dose-dependent manner and lowered significantly the expression of proinflammatory genes, in both alveolar epithelial cells and alveolar macrophages. In vivo, although no reduction in viral load was observed, administration of STA-9090 led to an overall improvement of the clinical condition of infected animals, with reduced oedema formation and lung tissue pathology. CONCLUSION: Altogether, we show that HSP90 inhibition could serve as a potential treatment option for moderate and severe cases of COVID-19.

An intranasal live-attenuated SARS-CoV-2 vaccine limits virus transmission.

The development of effective SARS-CoV-2 vaccines has been essential to control COVID-19, but significant challenges remain. One problem is intramuscular administration, which does not induce robust mucosal immune responses in the upper airways-the primary site of infection and virus shedding. Here we compare the efficacy of a mucosal, replication-competent yet fully attenuated virus vaccine, sCPD9-ΔFCS, and the monovalent mRNA vaccine BNT162b2 in preventing transmission of SARS-CoV-2 variants B.1 and Omicron BA.5 in two scenarios. Firstly, we assessed the protective efficacy of the vaccines by exposing vaccinated male Syrian hamsters to infected counterparts. Secondly, we evaluated transmission of the challenge virus from vaccinated and subsequently challenged male hamsters to naïve contacts. Our findings demonstrate that the live-attenuated vaccine (LAV) sCPD9-ΔFCS significantly outperformed the mRNA vaccine in preventing virus transmission in both scenarios. Our results provide evidence for the advantages of locally administered LAVs over intramuscularly administered mRNA vaccines in preventing infection and reducing virus transmission.

A systematic analysis of anthocyanins inhibiting human, murine, and equine herpesviruses.

BACKGROUND: Herpesviruses are common animal and human pathogens that cause severe health problems in children, immunocompromised patients, and infected animals with a host range from fish to mammals. Anthocyanin-containing plant extracts have been described as potent antivirals, which might cause fewer harmful side effects than direct-acting antivirals. Here, we report that an extract of Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae) (MBE) with a high content of the anthocyanin delphinidin suppresses lytic replication of equine, murine and human herpesviruses of replication in vitro. METHODS: We treated cultured cells with MBE and purified individual anthocyanins present in the extract to determine the most active compound at different concentrations. We subsequently infected the cultures with human herpesviruses 1 (HSV-1) or 8 (HHV-8), murine cytomegalovirus (CMV), or equine herpesviruses 1 (EHV-1) and determined the number of infected cells and viral infectivity. RESULTS: MBE inhibited the HSV-1, murine CMV, and EHV-1 by up to 2 orders of magnitude. In the presence of the stabilizing randomly methylated-beta-cyclodextrin, the inhibitory concentration could be lowered significantly. We identified delphinidin as an active antiviral compound and showed that the non-glycosylated delphinidin solved and stabilized with sulfobutylether-beta-cyclodextrin allowed usage of approximately 50 times lower concentrations. CONCLUSION: Glycosylated delphinidin derivatives were identified as active antiviral compounds of MBE. This suggests that plant extracts rich in delphinidin-anthocyanins have potent antiviral properties that could be used in treatment and prevention.

In vitro and in vivo effects of Pelargonium sidoides DC. root extract EPs® 7630 and selected constituents against SARS-CoV-2 B.1, Delta AY.4/AY.117 and Omicron BA.2.

The occurrence of immune-evasive SARS-CoV-2 strains emphasizes the importance to search for broad-acting antiviral compounds. Our previous in vitro study showed that Pelargonium sidoides DC. root extract EPs® 7630 has combined antiviral and immunomodulatory properties in SARS-CoV-2-infected human lung cells. Here we assessed in vivo effects of EPs® 7630 in SARS-CoV-2-infected hamsters, and investigated properties of EPs® 7630 and its functionally relevant constituents in context of phenotypically distinct SARS-CoV-2 variants. We show that EPs® 7630 reduced viral load early in the course of infection and displayed significant immunomodulatory properties positively modulating disease progression in hamsters. In addition, we find that EPs® 7630 differentially inhibits SARS-CoV-2 variants in nasal and bronchial human airway epithelial cells. Antiviral effects were more pronounced against Omicron BA.2 compared to B.1 and Delta, the latter two preferring TMPRSS2-mediated fusion with the plasma membrane for cell entry instead of receptor-mediated low pH-dependent endocytosis. By using SARS-CoV-2 Spike VSV-based pseudo particles (VSVpp), we confirm higher EPs® 7630 activity against Omicron Spike-VSVpp, which seems independent of the serine protease TMPRSS2, suggesting that EPs® 7630 targets endosomal entry. We identify at least two molecular constituents of EPs® 7630, i.e., (-)-epigallocatechin and taxifolin with antiviral effects on SARS-CoV-2 replication and cell entry. In summary, our study shows that EPs® 7630 ameliorates disease outcome in SARS-CoV-2-infected hamsters and has enhanced activity against Omicron, apparently by limiting late endosomal SARS-CoV-2 entry.

Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease. Materials and methods: We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Results: Using HLA-A*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. Conclusion: PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.

The NSP4 T492I mutation increases SARS-CoV-2 infectivity by altering non-structural protein cleavage.

The historically dominant SARS-CoV-2 Delta variant and the currently dominant Omicron variants carry a T492I substitution within the non-structural protein 4 (NSP4). Based on in silico analyses, we hypothesized that the T492I mutation increases viral transmissibility and adaptability, which we confirmed with competition experiments in hamster and human airway tissue culture models. Furthermore, we showed that the T492I mutation increases the replication capacity and infectiveness of the virus and improves its ability to evade host immune responses. Mechanistically, the T492I mutation increases the cleavage efficiency of the viral main protease NSP5 by enhancing enzyme-substrate binding, which increases production of nearly all non-structural proteins processed by NSP5. Importantly, the T492I mutation suppresses viral-RNA-associated chemokine production in monocytic macrophages, which may contribute to the attenuated pathogenicity of Omicron variants. Our results highlight the importance of NSP4 adaptation in the evolutionary dynamics of SARS-CoV-2.

A non-transmissible live attenuated SARS-CoV-2 vaccine.

Live attenuated vaccines (LAVs) administered via the mucosal route may offer better control of the COVID-19 pandemic than non-replicating vaccines injected intramuscularly. Conceptionally, LAVs have several advantages, including presentation of the entire antigenic repertoire of the virus, and the induction of strong mucosal immunity. Thus, immunity induced by LAV could offer superior protection against future surges of COVID-19 cases caused by emerging SARS-CoV-2 variants. However, LAVs carry the risk of unintentional transmission. To address this issue, we investigated whether transmission of a SARS-CoV-2 LAV candidate can be blocked by removing the furin cleavage site (FCS) from the spike protein. The level of protection and immunity induced by the attenuated virus with the intact FCS was virtually identical to the one induced by the attenuated virus lacking the FCS. Most importantly, removal of the FCS completely abolished horizontal transmission of vaccine virus between cohoused hamsters. Furthermore, the vaccine was safe in immunosuppressed animals and showed no tendency to recombine in vitro or in vivo with a SARS-CoV-2 field strain. These results indicate that removal of the FCS from SARS-CoV-2 LAV is a promising strategy to increase vaccine safety and prevent vaccine transmission without compromising vaccine efficacy.

Mucin-Inspired Single-Chain Polymer (MIP) Fibers as Potent SARS-CoV-2 Inhibitors.

Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450 kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59 nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0 nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.

Live-attenuated vaccine sCPD9 elicits superior mucosal and systemic immunity to SARS-CoV-2 variants in hamsters.

Vaccines play a critical role in combating the COVID-19 pandemic. Future control of the pandemic requires improved vaccines with high efficacy against newly emerging SARS-CoV-2 variants and the ability to reduce virus transmission. Here we compare immune responses and preclinical efficacy of the mRNA vaccine BNT162b2, the adenovirus-vectored spike vaccine Ad2-spike and the live-attenuated virus vaccine candidate sCPD9 in Syrian hamsters, using both homogeneous and heterologous vaccination regimens. Comparative vaccine efficacy was assessed by employing readouts from virus titrations to single-cell RNA sequencing. Our results show that sCPD9 vaccination elicited the most robust immunity, including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue after challenge with heterologous SARS-CoV-2. Overall, our results demonstrate that live-attenuated vaccines offer advantages over currently available COVID-19 vaccines.

Preclinical safety and efficacy of a therapeutic antibody that targets SARS-CoV-2 at the sotrovimab face but is escaped by Omicron.

The recurrent emerging of novel viral variants of concern (VOCs) with evasion of preexisting antibody immunity upholds severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case numbers and maintains a persistent demand for updated therapies. We selected the patient-derived antibody CV38-142 based on its potency and breadth against the VOCs Alpha, Beta, Gamma, and Delta for preclinical development into a therapeutic. CV38-142 showed in vivo efficacy in a Syrian hamster VOC infection model after post-exposure and therapeutic application and revealed a favorable safety profile in a human protein library screen and tissue cross-reactivity study. Although CV38-142 targets the same viral surface as sotrovimab, which maintains activity against Omicron, CV38-142 did not neutralize the Omicron lineages BA.1 and BA.2. These results highlight the contingencies of developing antibody therapeutics in the context of antigenic drift and reinforce the need to develop broadly neutralizing variant-proof antibodies against SARS-CoV-2.

Pet Rats as the Likely Reservoir for Human Seoul Orthohantavirus Infection.

Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient's rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007-2020) as well as 594 wild rats from the Netherlands (2013-2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

Suicidal Phenotype of Proofreading-Deficient Herpes Simplex Virus 1 Polymerase Mutants.

Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants.

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4 nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection.

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.

Protocol to dissociate healthy and infected murine- and hamster-derived lung tissue for single-cell transcriptome analysis.

In infectious disease research, single-cell RNA sequencing allows dissection of host-pathogen interactions. As a prerequisite, we provide a protocol to transform solid and complex organs such as lungs into representative diverse, viable single-cell suspensions. Our protocol describes performance of vascular perfusion, pneumonectomy, enzymatic digestion, and mechanical dissociation of lung tissue, as well as red blood cell lysis and counting of isolated cells. A challenge remains, however, to further increase the proportion of pulmonary endothelial cells without compromising on viability. For complete details on the use and execution of this protocol, please refer to Nouailles et al. (2021),1 Wyler et al. (2022),2 and Ebenig et al. (2022).3.