MERS-CoV nsp1 regulates autophagic flux via mTOR signalling and dysfunctional lysosomes.

Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signalling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.

From common colds to COVID-19: potential neurological consequences of a coronavirus infection / Des rhumes à la Covid-19 : conséquences neurologiques potentielles d'une infection par les coronavirus

For a large proportion of mankind, the word coronavirus only became a reality in the year 2020, as it was the cause of one of the worst pandemics of the last two centuries. Nevertheless, well before this ominous moment, human coronaviruses (HCoV) were well characterized respiratory pathogens since the 1960s. The most recent discovery of SARS-CoV and MERS-CoV showed that coronaviruses have a pandemic potential with important consequences. With the COVID-19 pandemic caused by SARS-CoV-2, this potential is now certain. Moreover, accumulating evidence support an association between coronaviruses and extra-respiratory pathologies, in particular of the central and peripheral nervous system. Linked or not with a neuro-invasive and neurotropic potential, it is now clear that coronaviruses can be associated with the development of neurological disorders.

Pour une grande partie de l'humanité, le terme coronavirus n'est devenu réalité qu'au début de l'année 2020, associé à une des plus importantes pandémies des deux derniers siècles. Pourtant, bien avant ce moment fatidique, les coronavirus humains (HCoV) étaient bien caractérisés en tant que pathogènes respiratoires depuis la fin des années 1960. Depuis le début du XXIe siècle, deux autres coronavirus pouvant infecter l'humain (SARS-CoV et MERS-CoV), ont montré que ces virus avaient un potentiel pandémique pouvant entraîner des conséquences importantes. Avec la survenue de la pandémie de Covid-19 créée par le SARS-CoV-2, ce potentiel ne fait aujourd'hui plus aucun doute. De plus, un nombre grandissant d'études supporte l'idée d'une association entre les coronavirus et diverses pathologies extra-respiratoires, en particulier au niveau des systèmes nerveux central et périphérique. Liés ou non à un véritable potentiel neuro-invasif et neurotrope, il apparaît maintenant de façon claire que les coronavirus peuvent être associés au développement de divers désordres neurologiques.

Evaluation of alpaca tracheal explants as an ex vivo model for the study of Middle East respiratory syndrome coronavirus (MERS-CoV) infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to public health. Here, we established an ex vivo alpaca tracheal explant (ATE) model using an air-liquid interface culture system to gain insights into MERS-CoV infection in the camelid lower respiratory tract. ATE can be infected by MERS-CoV, being 103 TCID50/mL the minimum viral dosage required to establish a productive infection. IFNs and antiviral ISGs were not induced in ATE cultures in response to MERS-CoV infection, strongly suggesting that ISGs expression observed in vivo is rather a consequence of the IFN induction occurring in the nasal mucosa of camelids.

Thapsigargin: key to new host-directed coronavirus antivirals?

Despite the great success of vaccines that protect against RNA virus infections, and the development and clinical use of a limited number of RNA virus-specific drugs, there is still an urgent need for new classes of antiviral drugs against circulating or emerging RNA viruses. To date, it has proved difficult to efficiently suppress RNA virus replication by targeting host cell functions, and there are no approved drugs of this type. This opinion article discusses the recent discovery of a pronounced and sustained antiviral activity of the plant-derived natural compound thapsigargin against enveloped RNA viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), and influenza A virus. Based on its mechanisms of action, thapsigargin represents a new prototype of compounds with multimodal host-directed antiviral activity.

Global ecological analysis of COVID-19 mortality and comparison between "the East" and "the West".

Although SARS-CoV-2 was first reported in China and neighbouring countries, the pandemic quickly spread around the globe. This paper explores national drivers of the pandemic and the radically different epidemiology and response in the West and in the East. We studied coronavirus disease (COVID-19) mortality until 31st December 2020, using an ecological study design, considering baseline characteristics and responses that might account for the uneven impact of the pandemic. A multivariable regression model was developed to explore key determinants. Key variables in the West were contrasted with those in the East, and speed of response was examined. Worldwide, 2.24 million COVID-19 deaths were documented in 2020. Western countries reported a median mortality 114 times that of the East (684 vs. 6.0 per million). Significant correlates of mortality in countries with at least 1 million population were median age, obesity prevalence, and democracy index; political stability and experience of SARS in 2002-2003 were protective; health system variables and income inequality were not associated. Outputs of the model were consistent when adjusted for stringency index, timeliness of stay-at-home requirements, and geographical autocorrelation. The West experiences a much higher COVID-19 mortality than the East. Despite structural advantages in the West, delays in national responses early on resulted in a loss of control over the spread of SARS-CoV-2. Although the early success of the East was sustained in the second half of 2020, the region remains extremely vulnerable to COVID-19 until enough people are immunized.

The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.

Proteomic landscape of SARS-CoV-2- and MERS-CoV-infected primary human renal epithelial cells.

Acute kidney injury is associated with mortality in COVID-19 patients. However, host cell changes underlying infection of renal cells with SARS-CoV-2 remain unknown and prevent understanding of the molecular mechanisms that may contribute to renal pathology. Here, we carried out quantitative translatome and whole-cell proteomics analyses of primary renal proximal and distal tubular epithelial cells derived from human donors infected with SARS-CoV-2 or MERS-CoV to disseminate virus and cell type-specific changes over time. Our findings revealed shared pathways modified upon infection with both viruses, as well as SARS-CoV-2-specific host cell modulation driving key changes in innate immune activation and cellular protein quality control. Notably, MERS-CoV infection-induced specific changes in mitochondrial biology that were not observed in response to SARS-CoV-2 infection. Furthermore, we identified extensive modulation in pathways associated with kidney failure that changed in a virus- and cell type-specific manner. In summary, we provide an overview of the effects of SARS-CoV-2 or MERS-CoV infection on primary renal epithelial cells revealing key pathways that may be essential for viral replication.

hnRNP C modulates MERS-CoV and SARS-CoV-2 replication by governing the expression of a subset of circRNAs and cognitive mRNAs.

ABSTRACTHost circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA-mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P < 0.05 to P < 0.0001) suppressed MERS-CoV replication in human lung adenocarcinoma (Calu-3) and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 µM) or SARS-CoV-2-infected (IC50: 1.233 µM) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.

A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets.

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity.

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Interspecies Jumping of Bat Coronaviruses.

In the last two decades, several coronavirus (CoV) interspecies jumping events have occurred between bats and other animals/humans, leading to major epidemics/pandemics and high fatalities. The SARS epidemic in 2002/2003 had a ~10% fatality. The discovery of SARS-related CoVs in horseshoe bats and civets and genomic studies have confirmed bat-to-civet-to-human transmission. The MERS epidemic that emerged in 2012 had a ~35% mortality, with dromedaries as the reservoir. Although CoVs with the same genome organization (e.g., Tylonycteris BatCoV HKU4 and Pipistrellus BatCoV HKU5) were also detected in bats, there is still a phylogenetic gap between these bat CoVs and MERS-CoV. In 2016, 10 years after the discovery of Rhinolophus BatCoV HKU2 in Chinese horseshoe bats, fatal swine disease outbreaks caused by this virus were reported in southern China. In late 2019, an outbreak of pneumonia emerged in Wuhan, China, and rapidly spread globally, leading to >4,000,000 fatalities so far. Although the genome of SARS-CoV-2 is highly similar to that of SARS-CoV, patient zero and the original source of the pandemic are still unknown. To protect humans from future public health threats, measures should be taken to monitor and reduce the chance of interspecies jumping events, either occurring naturally or through recombineering experiments.

Promising Antiviral Activities of Natural Flavonoids against SARS-CoV-2 Targets: Systematic Review.

The ongoing COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a globally leading public health concern over the past two years. Despite the development and administration of multiple vaccines, the mutation of newer strains and challenges to universal immunity has shifted the focus to the lack of efficacious drugs for therapeutic intervention for the disease. As with SARS-CoV, MERS-CoV, and other non-respiratory viruses, flavonoids present themselves as a promising therapeutic intervention given their success in silico, in vitro, in vivo, and more recently, in clinical studies. This review focuses on data from in vitro studies analyzing the effects of flavonoids on various key SARS-CoV-2 targets and presents an analysis of the structure-activity relationships for the same. From 27 primary papers, over 69 flavonoids were investigated for their activities against various SARS-CoV-2 targets, ranging from the promising 3C-like protease (3CLpro) to the less explored nucleocapsid (N) protein; the most promising were quercetin and myricetin derivatives, baicalein, baicalin, EGCG, and tannic acid. We further review promising in silico studies featuring activities of flavonoids against SARS-CoV-2 and list ongoing clinical studies involving the therapeutic potential of flavonoid-rich extracts in combination with synthetic drugs or other polyphenols and suggest prospects for the future of flavonoids against SARS-CoV-2.

Multi-level inhibition of coronavirus replication by chemical ER stress.

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

Focus on Middle East respiratory syndrome coronavirus (MERS-CoV).

Since the first case of human infection by the Middle East respiratory syndrome coronavirus (MERS-CoV) in Saudi Arabia in June 2012, more than 2260 cases of confirmed MERS-CoV infection and 803 related deaths have been reported since the 16th of October 2018. The vast majority of these cases (71%) were reported in Saudi Arabia but the epidemic has now spread to 27 countries and has not ceased 6 years later, unlike SARS-CoV that disappeared a little less than 2 years after emerging. Due to the high fatality rate observed in MERS-CoV infected patients (36%), much effort has been put into understanding the origin and pathophysiology of this novel coronavirus to prevent it from becoming endemic in humans. This review focuses in particular on the origin, epidemiology and clinical manifestations of MERS-CoV, as well as the diagnosis and treatment of infected patients. The experience gained over recent years on how to manage the different risks related to this kind of epidemic will be key to being prepared for future outbreaks of communicable disease.

Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV, and MERS-CoV genomes.

During virus infection, host toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and trigger the innate immune response. TLR7/8 can identify the single-stranded RNA (ssRNA) of the virus. This study aimed to search ssRNA sequences recognized by TLR7/8 from the SARS-CoV-2, SARS-CoV, and MERS-CoV whole genomes by a bioinformatic technique. The immunoinformatic approach showed that the SARS-CoV-2 genome has more ssRNA fragments that could be recognized by TLR7/8 than the SARS-CoV genome. These findings suggest innate immune hyperactivation by SARS-CoV-2. This activity is possibly able to provoke a robust proinflammatory response via TLR7/8 recognition and cause acute lung injury.

A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses.

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (-1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro -1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential -1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.

SARS-CoV-2 and other coronaviruses bind to phosphorylated glycans from the human lung.

SARS-CoV, MERS-CoV, and potentially SARS-CoV-2 emerged as novel human coronaviruses following cross-species transmission from animal hosts. Although the receptor binding characteristics of human coronaviruses are well documented, the role of carbohydrate binding in addition to recognition of proteinaceous receptors has not been fully explored. Using natural glycan microarray technology, we identified N-glycans in the human lung that are recognized by various human and animal coronaviruses. All viruses tested, including SARS-CoV-2, bound strongly to a range of phosphorylated, high mannose N-glycans and to a very specific set of sialylated structures. Examination of two linked strains, human CoV OC43 and bovine CoV Mebus, reveals shared binding to the sialic acid form Neu5Gc (not found in humans), supporting the evidence for cross-species transmission of the bovine strain. Our findings, revealing robust recognition of lung glycans, suggest that these receptors could play a role in the initial stages of coronavirus attachment and entry.