One for all-human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses.

In vitro investigations of host-virus interactions are reliant on suitable cell and tissue culture models. Results are only as good as the model they are generated in. However, choosing cell models for in vitro work often depends on availability and previous use alone. Despite the vast increase in coronavirus research over the past few years, scientists are still heavily reliant on: non-human, highly heterogeneous or not fully differentiated, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors. Complex primary or stem cell models are highly representative of human tissues but are expensive and time-consuming to develop and maintain with limited suitability for high-throughput experiments.Using tissue-specific expression patterns, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the well-characterized human kidney cell line Caki-1 for infection with three human coronaviruses (hCoVs): Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus and Alphacoronavirus hCoV 229E. Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. A panel of 26 custom antibodies shows the location of SARS-CoV-2 proteins during replication using immunocytochemistry. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison for a broad range of respiratory viruses. IMPORTANCE Cell lines remain the backbone of virus research, but results are only as good as their originating model. Despite increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models of: non-human origin, incomplete differentiation, or lacking active interferon responses. We identified the human kidney Caki-1 cell line as a potential target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show the localization of 26 SARS-CoV-2 proteins in Caki-1 cells during natural replication and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

In Vitro Models for Studying Entry, Tissue Tropism, and Therapeutic Approaches of Highly Pathogenic Coronaviruses.

Coronaviruses (CoVs) are enveloped nonsegmented positive-sense RNA viruses belonging to the family Coronaviridae that contain the largest genome among RNA viruses. Their genome encodes 4 major structural proteins, and among them, the Spike (S) protein plays a crucial role in determining the viral tropism. It mediates viral attachment to the host cell, fusion to the membranes, and cell entry using cellular proteases as activators. Several in vitro models have been developed to study the CoVs entry, pathogenesis, and possible therapeutic approaches. This article is aimed at summarizing the current knowledge about the use of relevant methodologies and cell lines permissive for CoV life cycle studies. The synthesis of this information can be useful for setting up specific experimental procedures. We also discuss different strategies for inhibiting the binding of the S protein to the cell receptors and the fusion process which may offer opportunities for therapeutic intervention.

A literature review of 2019 novel coronavirus (SARS-CoV2) infection in neonates and children.

At the time of writing, there are already millions of documented infections worldwide by the novel coronavirus 2019 (2019-nCoV or severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), with hundreds of thousands of deaths. The great majority of fatal events have been recorded in adults older than 70 years; of them, a large proportion had comorbidities. Since data regarding the epidemiologic and clinical characteristics in neonates and children developing coronavirus disease 2019 (COVID-19) are scarce and originate mainly from one country (China), we reviewed all the current literature from 1 December 2019 to 7 May 2020 to provide useful information about SARS-CoV2 viral biology, epidemiology, diagnosis, clinical features, treatment, prevention, and hospital organization for clinicians dealing with this selected population. IMPACT: Children usually develop a mild form of COVID-19, rarely requiring high-intensity medical treatment in pediatric intensive care unit. Vertical transmission is unlikely, but not completely excluded. Children with confirmed or suspected COVID-19 must be isolated and healthcare workers should wear appropriate protective equipment. Some clinical features (higher incidence of fever, vomiting and diarrhea, and a longer incubation period) are more common in children than in adults, as well as some radiologic aspects (more patchy shadow opacities on CT scan images than ground-glass opacities). Supportive and symptomatic treatments (oxygen therapy and antibiotics for preventing/treating bacterial coinfections) are recommended in these patients.

Zoonotic evolution and implications of microbiome in viral transmission and infection.

The outbreak and spread of new strains of coronavirus (SARS-CoV-2) remain a global threat with increasing cases in affected countries. The evolutionary tree of SARS-CoV-2 revealed that Porcine Reproductive and Respiratory Syndrome virus 2, which belongs to the Beta arterivirus genus from the Arteriviridae family is possibly the most ancient ancestral origin of SARS-CoV-2 and other Coronaviridae. This review focuses on phylogenomic distribution and evolutionary lineage of zoonotic viral cross-species transmission of the Coronaviridae family and the implications of bat microbiome in zoonotic viral transmission and infection. The review also casts light on the role of the human microbiome in predicting and controlling viral infections. The significance of microbiome-mediated interventions in the treatment of viral infections is also discussed. Finally, the importance of synthetic viruses in the study of viral evolution and transmission is highlighted.

Roles of flavonoids against coronavirus infection.

In terms of public health, the 21st century has been characterized by coronavirus pandemics: in 2002-03 the virus SARS-CoV caused SARS; in 2012 MERS-CoV emerged and in 2019 a new human betacoronavirus strain, called SARS-CoV-2, caused the unprecedented COVID-19 outbreak. During the course of the current epidemic, medical challenges to save lives and scientific research aimed to reveal the genetic evolution and the biochemistry of the vital cycle of the new pathogen could lead to new preventive and therapeutic strategies against SARS-CoV-2. Up to now, there is no cure for COVID-19 and waiting for an efficacious vaccine, the development of "savage" protocols, based on "old" anti-inflammatory and anti-viral drugs represents a valid and alternative therapeutic approach. As an alternative or additional therapeutic/preventive option, different in silico and in vitro studies demonstrated that small natural molecules, belonging to polyphenol family, can interfere with various stages of coronavirus entry and replication cycle. Here, we reviewed the capacity of well-known (e.g. quercetin, baicalin, luteolin, hesperetin, gallocatechin gallate, epigallocatechin gallate) and uncommon (e.g. scutellarein, amentoflavone, papyriflavonol A) flavonoids, secondary metabolites widely present in plant tissues with antioxidant and anti-microbial functions, to inhibit key proteins involved in coronavirus infective cycle, such as PLpro, 3CLpro, NTPase/helicase. Due to their pleiotropic activities and lack of systemic toxicity, flavonoids and their derivative may represent target compounds to be tested in future clinical trials to enrich the drug arsenal against coronavirus infections.

[Neurological damage linked to coronaviruses : SARS-CoV-2 and other human coronaviruses]. / Les atteintes neurologiques liées au SARS-CoV-2 et autres coronavirus humains.

The recent emergence of a new coronavirus, SARS-CoV-2, responsible for COVID-19, is a new warning of the risk to public health represented by viral zoonoses and in particular by coronaviruses. Mainly described as being able to infect the upper and lower respiratory tract, coronaviruses can also infect the central and peripheral nervous systems as many other respiratory viruses, such as influenza or respiratory syncytial virus. Viral infections of the nervous system are a major public health concern as they can cause devastating illnesses up to death, especially when they occur in the elderly, who are more susceptible to these infections. Knowledge concerning the pathophysiology of recently emerging coronaviruses (MERS-CoV, SARS-CoV and SARS-CoV-2) and how they reach the central nervous system are very sketchy and the work in progress aims in particular to better understand their biology and the mechanisms associated with neurological damage. In this review we will discuss the current state of knowledge on the neurotropism of human coronaviruses and the associated mechanisms by developing in particular the latest data concerning SARS-CoV-2.

TITLE: Les atteintes neurologiques liées au SARS-CoV-2 et autres coronavirus humains. ABSTRACT: L'émergence récente d'un nouveau coronavirus, le SARS-CoV-2, responsable de la maladie appelée COVID-19, est un nouvel avertissement du risque pour la santé publique représenté par les zoonoses virales et notamment par les coronavirus. Principalement connus pour leur capacité à infecter les voies respiratoires supérieures et inférieures, les coronavirus peuvent également affecter le système nerveux central et périphérique, comme c'est le cas pour de nombreux virus respiratoires, tels que les virus influenza ou le virus respiratoire syncytial. Les infections du système nerveux sont un problème important de santé publique car elles peuvent provoquer des atteintes dévastatrices allant jusqu'au décès du patient, en particulier lorsqu'elles surviennent chez les personnes fragilisées ou âgées plus sensibles à ce type d'infection. Les connaissances de la physiopathologie des infections par les coronavirus émergents (MERS-CoV, SARS-CoV et SARS-CoV-2) et leurs moyens d'accéder au système nerveux central sont, pour l'heure, très sommaires. Les travaux en cours visent notamment à mieux appréhender les mécanismes associés aux atteintes neurologiques observées. Dans cette revue nous aborderons l'état des connaissances actuelles sur le neurotropisme des coronavirus humains et les mécanismes associés en développant tout particulièrement les dernières données concernant le SARS-CoV-2.

The neurology of COVID-19 revisited: A proposal from the Environmental Neurology Specialty Group of the World Federation of Neurology to implement international neurological registries.

A comprehensive review of the neurological disorders reported during the current COVID-19 pandemic demonstrates that infection with SARS-CoV-2 affects the central nervous system (CNS), the peripheral nervous system (PNS) and the muscle. CNS manifestations include: headache and decreased responsiveness considered initial indicators of potential neurological involvement; anosmia, hyposmia, hypogeusia, and dysgeusia are frequent early symptoms of coronavirus infection. Respiratory failure, the lethal manifestation of COVID-19, responsible for 264,679 deaths worldwide, is probably neurogenic in origin and may result from the viral invasion of cranial nerve I, progressing into rhinencephalon and brainstem respiratory centers. Cerebrovascular disease, in particular large-vessel ischemic strokes, and less frequently cerebral venous thrombosis, intracerebral hemorrhage and subarachnoid hemorrhage, usually occur as part of a thrombotic state induced by viral attachment to ACE2 receptors in endothelium causing widespread endotheliitis, coagulopathy, arterial and venous thromboses. Acute hemorrhagic necrotizing encephalopathy is associated to the cytokine storm. A frontal hypoperfusion syndrome has been identified. There are isolated reports of seizures, encephalopathy, meningitis, encephalitis, and myelitis. The neurological diseases affecting the PNS and muscle in COVID-19 are less frequent and include Guillain-Barré syndrome; Miller Fisher syndrome; polyneuritis cranialis; and rare instances of viral myopathy with rhabdomyolysis. The main conclusion of this review is the pressing need to define the neurology of COVID-19, its frequency, manifestations, neuropathology and pathogenesis. On behalf of the World Federation of Neurology we invite national and regional neurological associations to create local databases to report cases with neurological manifestations observed during the on-going pandemic. International neuroepidemiological collaboration may help define the natural history of this worldwide problem.

Environmental pollution and COVID-19: the molecular terms and predominant disease outcomes of their sweetheart agreement.

As the Coronavirus situation (COVID-19) continues to evolve, many questions concerning the factors relating to the diffusion and severity of the disease remain unanswered.Whilst opinions regarding the weight of evidence for these risk factors, and the studies published so far are often inconclusive or offer contrasting results, the role of comorbidities in the risk of serious adverse outcomes in patients affected with COVID-19 appears to be evident since the outset. Hypertension, diabetes, and obesity are under discussion as important factors affecting the severity of disease. Air pollution has been considered to play a role in the diffusion of the virus, in the propagation of the contagion, in the severity of symptoms, and in the poor prognosis. Accumulating evidence supports the hypothesis that environmental particulate matter (PM) can trigger inflammatory responses at molecular, cellular, and organ levels, sustaining respiratory, cardiovascular, and dysmetabolic diseases.To better understand the intricate relationships among pre-existing conditions, PM, and viral infection, we examined the response at the molecular level of T47D human breast adenocarcinoma cells exposed to different fractions of PM. T47D cells express several receptors, including the aryl hydrocarbon receptor (AhR), and ACE2, the main - but not the only - receptor for SARS-CoV-2 entry.PM samples were collected in an urban background site located in the Northern area of the City of Bologna (Emilia-Romagna Region, Northern Italy) during winter 2013. T47D cells were exposed to organic or aqueous (inorganic) extracts at the final concentration of 8 m3 for a 4-hour duration. Both the concentration and the exposure time were chosen to resemble an average outdoor exposure. RNA was extracted from cells, purified and hybridised on 66k microarray slides from Agilent.The lists of differentially expressed genes in PM organic extracts were evaluated by using Metacore, and an enrichment analysis was performed to identify pathways maps, process networks, and disease by biomarkers altered after T47D treatment.The analysis of the modulated genes gave evidence for the involvement of PM in dysmetabolic diseases, including diabetes and obesity, and hypertension through the activation of the aryl hydrocarbon receptor (AhR) canonical pathway.On the basis of current knowledge, existing data, and exploratory experimental evidence, we tease out the likely molecular interplay that can ultimately tip the disease outcome into severity. Looking beyond ACE2, several additional key markers are identified. Disruption of these targets worsens pre-existing conditions and/or exacerbates the adverse effects induced by SARS-CoV-2 infection. Whilst appropriately designed, epidemiological studies are very much needed to investigate these associations based on our hypothesis of investigation, by reviewing recent experimental and epidemiological evidence, here we speculate and provide new insights on the possible role of environmental pollution in the exacerbation of effects by SARS-CoV-2 and other respiratory viruses. This work is intended to assist in the development of appropriate investigative approaches to protect public health.

Functional Characterization and Proteomic Analysis of Porcine Deltacoronavirus Accessory Protein NS7.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in neonatal piglets. Like other coronaviruses, PDCoV encodes at least three accessory or species-specific proteins; however, the biological roles of these proteins in PDCoV replication remain undetermined. As a first step toward understanding the biology of the PDCoV accessory proteins, we established a stable porcine cell line constitutively expressing the PDCoV NS7 protein in order to investigate the functional characteristics of NS7 for viral replication. Confocal microscopy and subcellular fractionation revealed that the NS7 protein was extensively distributed in the mitochondria. Proteomic analysis was then conducted to assess the expression dynamics of the host proteins in the PDCoV NS7-expressing cells. Highresolution two-dimensional gel electrophoresis initially identified 48 protein spots which were differentially expressed in the presence of NS7. Seven of these spots, including two upregulated and five down-regulated protein spots, showed statistically significant alterations, and were selected for subsequent protein identification. The affected cellular proteins identified in this study were classified into functional groups involved in various cellular processes such as cytoskeleton networks and cell communication, metabolism, and protein biosynthesis. A substantial down-regulation of α-actinin-4 was confirmed in NS7-expressing and PDCoV-infected cells. These proteomic data will provide insights into the understanding of specific cellular responses to the accessory protein during PDCoV infection.

Glycan Shield and Fusion Activation of a Deltacoronavirus Spike Glycoprotein Fine-Tuned for Enteric Infections.

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5-Å-resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified Deltacoronavirus genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved compared with the human respiratory coronavirus NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates in rendering the spike largely protease resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.IMPORTANCE Coronaviruses use transmembrane S glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic-resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic PDCoV, which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease resistant, which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.

Tubulins interact with porcine and human S proteins of the genus Alphacoronavirus and support successful assembly and release of infectious viral particles.

Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell ß-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles.

SARS-like WIV1-CoV poised for human emergence.

Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.

Hajj-associated viral respiratory infections: A systematic review.

Respiratory tract infections (RTI) are the most common infections transmitted between Hajj pilgrims. The aim of this systematic review was to determine the prevalence of virus carriage potentially responsible for RTI among pilgrims before and after participating in the Hajj. A systematic search for relevant literature was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. 31 studies were identified. Severe Acute Respiratory Syndrome coronavirus and Middle East Respiratory Syndrome coronavirus (MERS) were never isolated in Hajj pilgrims. The viruses most commonly isolated from symptomatic patients during the Hajj by PCR were rhinovirus (5.9-48.8% prevalence), followed by influenza virus (4.5-13.9%) and non-MERS coronaviruses (2.7-13.2%) with most infections due to coronavirus 229E; other viruses were less frequently isolated. Several viruses including influenza A, rhinovirus, and non-MERS coronaviruses had low carriage rates among arriving pilgrims and a statistically significant increase in their carriage rate was observed, following participation in the Hajj. Further research is needed to assess the role of viruses in the pathogenesis of respiratory symptoms and their potential role in the severity of the symptoms.

New insights on the role of paired membrane structures in coronavirus replication.

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication.

Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis.

Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity.

Membranous replication factories induced by plus-strand RNA viruses.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two "morphotypes" of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.