Laying the foundation for single-cell studies in bats.

Bats serve as hosts of viruses that can cause disease in humans. In this issue of Immunity, Gamage et al. characterize the immune cell repertoire in Eonycteris spelaea bat lung tissue using single-cell transcriptomics, providing insight into the in vivo immune response to infection with a Pteropine orthoreovirus (PRV3M) and establishing a paradigm for future comparative immunology studies.

The translational potential of studying bat immunity.

Molecular studies in bats have led to the discovery of antiviral adaptations that may explain how some bat species have evolved enhanced immune tolerance towards viruses. Accumulating data suggest that some bat species have also evolved remarkable features of longevity and low rates of cancer. Furthermore, recent research strongly suggests that discovering immune adaptations in bat models can be translated to develop immune modulators and recognize alternate therapeutic strategies for diseases affecting humans. We posit that research in bat immunology will lead to discoveries that can potentially be translated to improve health outcomes in humans.

A Significant Contribution of the Classical Pathway of Complement in SARS-CoV-2 Neutralization of Convalescent and Vaccinee Sera.

Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Human coronaviruses disassemble processing bodies.

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this submission, no information was known about how human coronaviruses (CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that transcripts encoding TNF and IL-6 localized to PBs in control cells. PB loss correlated with the increased cytoplasmic localization of these transcripts in SARS-CoV-2 N protein-expressing cells. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to the dysregulation of proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

Clash of the titans: interferons and SARS-CoV-2.

Interferons are our first line of defense against invading viruses. However, viruses encode effector proteins that can modulate human interferon responses. In this forum article, we highlight important discoveries and discuss outstanding questions that will enable us to better understand the nuances of this evolutionary battle between interferons and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada.

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/ß) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

Evolutionary trajectory of SARS-CoV-2 and emerging variants.

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and more recently, the independent evolution of multiple SARS-CoV-2 variants has generated renewed interest in virus evolution and cross-species transmission. While all known human coronaviruses (HCoVs) are speculated to have originated in animals, very little is known about their evolutionary history and factors that enable some CoVs to co-exist with humans as low pathogenic and endemic infections (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1), while others, such as SARS-CoV, MERS-CoV and SARS-CoV-2 have evolved to cause severe disease. In this review, we highlight the origins of all known HCoVs and map positively selected for mutations within HCoV proteins to discuss the evolutionary trajectory of SARS-CoV-2. Furthermore, we discuss emerging mutations within SARS-CoV-2 and variants of concern (VOC), along with highlighting the demonstrated or speculated impact of these mutations on virus transmission, pathogenicity, and neutralization by natural or vaccine-mediated immunity.

Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2.

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.

Predicting the recombination potential of severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged to cause widespread infections in humans. SARS-CoV-2 infections have been reported in the Kingdom of Saudi Arabia, where Middle East respiratory syndrome coronavirus (MERS-CoV) causes seasonal outbreaks with a case fatality rate of ~37 %. Here we show that there exists a theoretical possibility of future recombination events between SARS-CoV-2 and MERS-CoV RNA. Through computational analyses, we have identified homologous genomic regions within the ORF1ab and S genes that could facilitate recombination, and have analysed co-expression patterns of the cellular receptors for SARS-CoV-2 and MERS-CoV, ACE2 and DPP4, respectively, to identify human anatomical sites that could facilitate co-infection. Furthermore, we have investigated the likely susceptibility of various animal species to MERS-CoV and SARS-CoV-2 infection by comparing known virus spike protein-receptor interacting residues. In conclusion, we suggest that a recombination between SARS-CoV-2 and MERS-CoV RNA is possible and urge public health laboratories in high-risk areas to develop diagnostic capability for the detection of recombined coronaviruses in patient samples.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Median Tissue Culture Infectious Dose 50 (TCID50) Assay to Determine Infectivity of Cytopathic Viruses.

The emergence of zoonotic viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have significantly impacted global health and economy. The discovery of other viruses in wildlife reservoir species present a threat for future emergence in humans and animals. Therefore, assays that are less reliant on virus-specific information, such as neutralization assays, are crucial to rapidly develop diagnostics, understand virus replication and pathogenicity, and assess the efficacy of therapeutics against newly emerging viruses. Here, we describe the discontinuous median tissue culture infectious dose 50 (TCID50) assay to quantitatively determine the titer of any virus that can produce a visible cytopathic effect in infected cells.

Applications of Quantitative PCR (qPCR) in Studies of Virus-Host Interactions.

Emerging viruses pose significant threats to human health and the global economy. In the past two decades, three different coronaviruses have emerged to cause worldwide public health concerns. The advent of high throughput genomic and transcriptomic technologies facilitated the study of virus-host interactions, accelerating the development of diagnostics, vaccines, and therapeutics. Here, we describe quantitative PCR (qPCR) in studies of virus-host interactions to dissect host responses and viral kinetics and how these relate to one another.

Establishing Air-Liquid Interface (ALI) Airway Culture Models for Infectious Disease Research.

Air-liquid interface (ALI) airway culture models serve as a powerful tool to emulate the characteristic features of the respiratory tract in vitro. These models are particularly valuable for studying emerging respiratory viral and bacterial infections. Here, we describe an optimized protocol to obtain the ALI airway culture models using normal human bronchial epithelial cells (NHBECs). The protocol outlined below enables the generation of differentiated mucociliary airway epithelial cultures by day 28 following exposure to air.

Metatranscriptomic RNA-Seq Data Analysis of Virus-Infected Host Cells.

RNA sequencing (RNA-seq) analysis of virus-infected host cells enables researchers to study a wide range of phenomena involving host-virus interactions. This includes genomic analysis of the viral population itself, as well as analysis of the transcriptional dynamics of the virus and host during infection. In this chapter, we provide a guide for researchers interested in performing RNA-seq data analysis of virus-infected host cells or cell lines. We outline several bioinformatic protocols for quantifying viral abundance, assembling viral genomes from mixed samples, and performing differential expression analysis, among other common workflows. These workflows can be used as starting points for researchers aiming to analyze RNA-seq datasets of mixed samples containing both host and viral RNA, such as virus-infected cell lines or clinical samples.

Efficacy of a stable broadly protective subunit vaccine platform against SARS-CoV-2 variants of concern.

The emergence and ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid vaccine development platforms that can be updated to counteract emerging variants of currently circulating and future emerging coronaviruses. Here we report the development of a "train model" subunit vaccine platform that contains a SARS-CoV-2 Wuhan S1 protein (the "engine") linked to a series of flexible receptor binding domains (RBDs; the "cars") derived from SARS-CoV-2 variants of concern (VOCs). We demonstrate that these linked subunit vaccines when combined with Sepivac SWE™, a squalene in water emulsion (SWE) adjuvant, are immunogenic in Syrian hamsters and subsequently provide protection from infection with SARS-CoV-2 VOCs Omicron (BA.1), Delta, and Beta. Importantly, the bivalent and trivalent vaccine candidates offered protection against some heterologous SARS-CoV-2 VOCs that were not included in the vaccine design, demonstrating the potential for broad protection against a range of different VOCs. Furthermore, these formulated vaccine candidates were stable at 2-8 °C for up to 13 months post-formulation, highlighting their utility in low-resource settings. Indeed, our vaccine platform will enable the development of safe and broadly protective vaccines against emerging betacoronaviruses that pose a significant health risk for humans and agricultural animals.

Versatile use of bat ACE2 for cellular entry by MERS-CoV-like viruses.

Cellular entry receptors for bat MERS-CoV-like viruses NeoCoV and PDF-2180 were unknown, leaving their zoonotic potential ambiguous. A recent study by Xiong et al. published in Nature identified bat ACE2 as the cellular entry receptor for both viruses, highlighting the ability of coronaviruses to utilize a range of entry receptors.

Mycobacterium tuberculosis and SARS-CoV-2 co-infections: The knowns and unknowns.

Health impacts of Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 co-infections are not fully understood. Both pathogens modulate host responses and induce immunopathology with extensive lung damage. With a quarter of the world's population harboring latent TB, exploring the relationship between SARS-CoV-2 infection and its effect on the transition of Mtb from latent to active form is paramount to control this pathogen. The effects of active Mtb infection on establishment and severity of COVID-19 are also unknown, despite the ability of TB to orchestrate profound long-lasting immunopathologies in the lungs. Absence of mechanistic studies and co-infection models hinder the development of effective interventions to reduce the health impacts of SARS-CoV-2 and Mtb co-infection. Here, we highlight dysregulated immune responses induced by SARS-CoV-2 and Mtb, their potential interplay, and implications for co-infection in the lungs. As both pathogens master immunomodulation, we discuss relevant converging and diverging immune-related pathways underlying SARS-CoV-2 and Mtb co-infections.