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.

Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS-CoV-2 detection using a laboratory-developed test.

The objective of this study was to validate the use of spring water gargle (SWG) as an alternative to oral and nasopharyngeal swab (ONPS) for SARS-CoV-2 detection with a laboratory-developed test. Healthcare workers and adults from the general population, presenting to one of two COVID-19 screening clinics in Montréal and Québec City, were prospectively recruited to provide a gargle sample in addition to the standard ONPS. The paired specimens were analyzed using thermal lysis followed by a laboratory-developed nucleic acid amplification test (LD-NAAT) to detect SARS-CoV-2, and comparative performance analysis was performed. An individual was considered infected if a positive result was obtained on either sample. A total of 1297 adult participants were recruited. Invalid results (n = 18) were excluded from the analysis. SARS-CoV-2 was detected in 144/1279 (11.3%) participants: 126 from both samples, 15 only from ONPS, and 3 only from SWG. Overall, the sensitivity was 97.9% (95% CI: 93.7-99.3) for ONPS and 89.6% (95% CI: 83.4-93.6; p = 0.005) for SWG. The mean ONPS cycle threshold (Ct ) value was significantly lower for the concordant paired samples as compared to discordant ones (22.9 vs. 32.1; p < 0.001). In conclusion, using an LD-NAAT with thermal lysis, SWG is a less sensitive sampling method than the ONPS. However, the higher acceptability of SWG might enable a higher rate of detection from a population-based perspective. Nonetheless, in patients with a high clinical suspicion of COVID-19, a repeated analysis with ONPS should be considered. The sensitivity of SWG using NAAT preceded by chemical extraction should be evaluated.

Evaluation of Real-life Use of Point-of-care Rapid Antigen Testing for SARS-CoV-2 in Schools (EPOCRATES): a cohort study.

BACKGROUND: SARS-CoV-2 transmission has an impact on education. In this study, we assessed the performance of rapid antigen detection tests (RADTs) versus polymerase chain reaction (PCR) for the diagnosis of SARS-CoV-2 infection in school settings, and RADT use for monitoring exposed contacts. METHODS: In this real-world, prospective observational cohort study, high-school students and staff were recruited from 2 high schools in Montréal, Canada, and followed from Jan. 25 to June 10, 2021. Twenty-five percent of asymptomatic participants were tested weekly by RADT (nasal) and PCR (gargle). Class contacts of cases were tested. Symptomatic participants were tested by RADT (nasal) and PCR (nasal and gargle). The number of cases and outbreaks were compared with those of other high schools in the same area. RESULTS: Overall, 2099 students and 286 school staff members consented to participate. The overall specificity of RADTs varied from 99.8% to 100%, with a lower sensitivity, varying from 28.6% in asymptomatic to 83.3% in symptomatic participants. Secondary cases were identified in 10 of 35 classes. Returning students to school after a 7-day quarantine, with a negative PCR result on days 6-7 after exposure, did not lead to subsequent outbreaks. Of cases for whom the source was known, 37 of 51 (72.5%) were secondary to household transmission, 13 (25.5%) to intraschool transmission, and 1 to community contacts between students in the same school. INTERPRETATION: Rapid antigen detection tests did not perform well compared with PCR in asymptomatic individuals. Reinforcing policies for symptom screening when entering schools and testing symptomatic individuals with RADTs on the spot may avoid subsequent substantial exposures in class. Preprint: medRxiv - doi.org/10.1101/2021.10.13.21264960.

Uncertainty around the Long-Term Implications of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 231 million people globally, with more than 4.7 million deaths recorded by the World Health Organization as of 26 September 2021. In response to the pandemic, some countries (New Zealand, Vietnam, Taiwan, South Korea and others) have pursued suppression strategies, so-called Zero COVID policies, to drive and maintain infection rates as close to zero as possible and respond aggressively to new cases. In comparison, European countries and North America have adopted mitigation strategies (of varying intensity and effectiveness) that aim primarily to prevent health systems from being overwhelmed. With recent advances in our understanding of SARS-CoV-2 and its biology, and the increasing recognition there is more to COVID-19 beyond the acute infection, we offer a perspective on some of the long-term risks of mutational escape, viral persistence, reinfection, immune dysregulation and neurological and multi-system complications (Long COVID).

Natural spring water gargle and direct RT-PCR for the diagnosis of COVID-19 (COVID-SPRING study).

BACKGROUND: Nasopharyngeal swab has long been considered the specimen of choice for the diagnosis of respiratory viral infections, including SARS-CoV-2 infection, but it suffers from several drawbacks: its discomfort limits screening acceptability, and it is vulnerable to shortages in both specialized materials and trained healthcare workers in the context of a pandemic. METHODS: We prospectively compared natural spring water gargle to combined oro-nasopharyngeal swab (ONPS) for the diagnosis of coronavirus disease 2019 (COVID-19) in paired clinical specimens (1005 ONPS and 1005 gargles) collected from 987 unique early symptomatic as well as asymptomatic individuals from the community. RESULTS: Using a direct RT-PCR method with the Allplex™ 2019-nCoV Assay (Seegene), the clinical sensitivity of the gargle was 95.3% (95% confidence interval [CI], 90.2 - 98.3%), similar to the sensitivity of the ONPS (93.8%; 95% CI, 88.2 - 97.3%), despite significantly lower viral RNA concentration in gargles, as reflected by higher cycle threshold values. No single specimen type detected all COVID-19 cases. SARS-CoV-2 RNA was stable in gargles at room temperature for at least 7 days. CONCLUSION: The simplicity of this sampling method coupled with the accessibility of spring water are clear advantages in a pandemic situation where testing frequency, turnaround time and shortage of consumables and trained staff are critical elements.

Olfactory Bulb and Amygdala Gene Expression Changes in Subjects Dying with COVID-19.

In this study we conducted RNA sequencing on two brain regions (olfactory bulb and amygdala) from subjects who died from COVID-19 or who died of other causes. We found several-fold more transcriptional changes in the olfactory bulb than in the amygdala, consistent with our own work and that of others indicating that the olfactory bulb may be the initial and most common brain region infected. To some extent our results converge with pseudotime analysis towards common processes shared between the brain regions, possibly induced by the systemic immune reaction following SARS-CoV-2 infection. Changes in amygdala emphasized upregulation of interferon-related neuroinflammation genes, as well as downregulation of synaptic and other neuronal genes, and may represent the substrate of reported acute and subacute COVID-19 neurological effects. Additionally, and only in olfactory bulb, we observed an increase in angiogenesis and platelet activation genes, possibly associated with microvascular damages induced by neuroinflammation. Through coexpression analysis we identified two key genes (CAMK2B for the synaptic neuronal network and COL1A2 for the angiogenesis/platelet network) that might be interesting potential targets to reverse the effects induced by SARS-CoV-2 infection. Finally, in olfactory bulb we detected an upregulation of olfactory and taste genes, possibly as a compensatory response to functional deafferentation caused by viral entry into primary olfactory sensory neurons. In conclusion, we were able to identify transcriptional profiles and key genes involved in neuroinflammation, neuronal reaction and olfaction induced by direct CNS infection and/or the systemic immune response to SARS-CoV-2 infection.

Mapping of SARS-CoV-2 Brain Invasion and Histopathology in COVID-19 Disease.

The coronavirus SARS-CoV-2 (SCV2) causes acute respiratory distress, termed COVID-19 disease, with substantial morbidity and mortality. As SCV2 is related to previously-studied coronaviruses that have been shown to have the capability for brain invasion, it seems likely that SCV2 may be able to do so as well. To date, although there have been many clinical and autopsy-based reports that describe a broad range of SCV2-associated neurological conditions, it is unclear what fraction of these have been due to direct CNS invasion versus indirect effects caused by systemic reactions to critical illness. Still critically lacking is a comprehensive tissue-based survey of the CNS presence and specific neuropathology of SCV2 in humans. We conducted an extensive neuroanatomical survey of RT-PCR-detected SCV2 in 16 brain regions from 20 subjects who died of COVID-19 disease. Targeted areas were those with cranial nerve nuclei, including the olfactory bulb, medullary dorsal motor nucleus of the vagus nerve and the pontine trigeminal nerve nuclei, as well as areas possibly exposed to hematogenous entry, including the choroid plexus, leptomeninges, median eminence of the hypothalamus and area postrema of the medulla. Subjects ranged in age from 38 to 97 (mean 77) with 9 females and 11 males. Most subjects had typical age-related neuropathological findings. Two subjects had severe neuropathology, one with a large acute cerebral infarction and one with hemorrhagic encephalitis, that was unequivocally related to their COVID-19 disease while most of the 18 other subjects had non-specific histopathology including focal ß-amyloid precursor protein white matter immunoreactivity and sparse perivascular mononuclear cell cuffing. Four subjects (20%) had SCV2 RNA in one or more brain regions including the olfactory bulb, amygdala, entorhinal area, temporal and frontal neocortex, dorsal medulla and leptomeninges. The subject with encephalitis was SCV2-positive in a histopathologically-affected area, the entorhinal cortex, while the subject with the large acute cerebral infarct was SCV2-negative in all brain regions. Like other human coronaviruses, SCV2 can inflict acute neuropathology in susceptible patients. Much remains to be understood, including what viral and host factors influence SCV2 brain invasion and whether it is cleared from the brain subsequent to the acute illness.

Potential differences in cleavage of the S protein and type-1 interferon together control human coronavirus infection, propagation, and neuropathology within the central nervous system.

Human coronaviruses (HCoV) are respiratory pathogens which have been known since the 1960's. In December 2019, a new betacoronavirus, SARS-CoV-2, was reported and is responsible for one of the biggest pandemics of the last two centuries. Similar to the HCoV-OC43 strain, available evidence suggests SARS-CoV-2 neuroinvasion associated with potential neurological disorders. Coronavirus infection of the central nervous system (CNS) is largely controlled by a viral factor, the spike glycoprotein (S) and a host factor, innate immunity. However, the interaction between these two factors remains elusive. Proteolytic cleavage of the S protein can occur at the interface between receptor binding (S1) and fusion (S2) domains (S1/S2), as well as in a position adjacent to a fusion peptide within S2 (S2'). Herein, using HCoV-OC43 as a surrogate for SARS-CoV-2, we report that both S protein sites are involved in neurovirulence and are required for optimal CNS infection. Whereas efficient cleavage at S1/S2 is associated with decreased virulence, the potentially cleavable putative S2' site is essential for efficient viral infection. Furthermore, type 1 interferon (IFN 1)-related innate immunity also plays an important role in the control of viral spread towards the spinal cord, by preventing infection of ependymal cells. Our results underline the link between the differential S cleavage and IFN 1 in the prevention of viral spread, to control the severity of infection and pathology in both immunocompetent and immunodeficient mice. Taken together, these results point towards two potential therapeutic anti-viral targets: cleavage of the S protein in conjunction with efficient IFN 1-related innate immunity to prevent or at least reduce neuroinvasion, neural spread, and potential associated neurovirulence of human coronaviruses.ImportanceHuman coronaviruses (HCoV) are recognized respiratory pathogens. The emergence of the novel pathogenic member of this family in December 2019 (SARS-CoV-2, which causes COVID-19) poses a global health emergency. As with other coronaviruses reported previously, invasion of the human central nervous system (CNS), associated with diverse neurological disorders, was suggested for SARS-CoV-2. Herein, using the related HCoV-OC43 strain, we show that the viral spike protein constitutes a major neurovirulence factor and that type 1 interferon (IFN 1), in conjunction with cleavage of S protein by host proteases, represent important host factors that participate in the control of CNS infection.To our knowledge, this is the first demonstration of a direct link between cleavage of the S protein, innate immunity and neurovirulence. Understanding mechanisms of viral infection and spread in neuronal cells is essential to better design therapeutic strategies, and to prevent infection by human coronaviruses such as SARS-CoV-2 in human CNS especially in the vulnerable populations such as the elderly and immune-compromised individuals.

Cross-Sectional Evaluation of Humoral Responses against SARS-CoV-2 Spike.

SARS-CoV-2 is responsible for the coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoproteins of SARS-CoV-2 mediate viral entry and are the main targets for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic, and public health interventions. Here, we perform a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against SARS-CoV-2 Spike. Most infected individuals elicit anti-Spike antibodies within 2 weeks of the onset of symptoms. The levels of receptor binding domain (RBD)-specific immunoglobulin G (IgG) persist over time, and the levels of anti-RBD IgM decrease after symptom resolution. Although most individuals develop neutralizing antibodies within 2 weeks of infection, the level of neutralizing activity is significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.

Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike.

The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.

Human Coronaviruses and Other Respiratory Viruses: Underestimated Opportunistic Pathogens of the Central Nervous System?

Respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. However, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). Respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. Furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza A and SARS-CoV, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. For a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (CNS). Viruses infecting human CNS cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. Like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to CNS cells (virus-induced neuropathology). The etiological agent of several neurological disorders remains unidentified. Opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. Herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in CNS infection, with a special emphasis on human coronaviruses.

The human coronavirus HCoV-229E S-protein structure and receptor binding.

The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the cross-species transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion.

Axonal Transport Enables Neuron-to-Neuron Propagation of Human Coronavirus OC43.

Human coronaviruses (HCoVs) are recognized respiratory pathogens for which accumulating evidence indicates that in vulnerable patients the infection can cause more severe pathologies. HCoVs are not always confined to the upper respiratory tract and can invade the central nervous system (CNS) under still unclear circumstances. HCoV-induced neuropathologies in humans are difficult to diagnose early enough to allow therapeutic interventions. Making use of our already described animal model of HCoV neuropathogenesis, we describe the route of neuropropagation from the nasal cavity to the olfactory bulb and piriform cortex and then the brain stem. We identified neuron-to-neuron propagation as one underlying mode of virus spreading in cell culture. Our data demonstrate that both passive diffusion of released viral particles and axonal transport are valid propagation strategies used by the virus. We describe for the first time the presence along axons of viral platforms whose static dynamism is reminiscent of viral assembly sites. We further reveal that HCoV OC43 modes of propagation can be modulated by selected HCoV OC43 proteins and axonal transport. Our work, therefore, identifies processes that may govern the severity and nature of HCoV OC43 neuropathogenesis and will make possible the development of therapeutic strategies to prevent occurrences.IMPORTANCE Coronaviruses may invade the CNS, disseminate, and participate in the induction of neurological diseases. Their neuropathogenicity is being increasingly recognized in humans, and the presence and persistence of human coronaviruses (HCoV) in human brains have been proposed to cause long-term sequelae. Using our mouse model relying on natural susceptibility to HCoV OC43 and neuronal cell cultures, we have defined the most relevant path taken by HCoV OC43 to access and spread to and within the CNS toward the brain stem and spinal cord and studied in cell culture the underlying modes of intercellular propagation to better understand its neuropathogenesis. Our data suggest that axonal transport governs HCoV OC43 egress in the CNS, leading to the exacerbation of neuropathogenesis. Exploiting knowledge on neuroinvasion and dissemination will enhance our ability to control viral infection within the CNS, as it will shed light on underlying mechanisms of neuropathogenesis and uncover potential druggable molecular virus-host interfaces.

The OC43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and CNS pathology.

The OC43 strain of human coronavirus (HCoV-OC43) is an ubiquitous respiratory tract pathogen possessing neurotropic capacities. Coronavirus structural envelope (E) protein possesses specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation, which are known to play various roles including in virion morphology/assembly and in cell response to infection and/or virulence. Making use of recombinant viruses either devoid of the E protein or harboring mutations either in putative transmembrane domain or PDZ-binding motif, we demonstrated that a fully functional HCoV-OC43 E protein is first needed for optimal production of recombinant infectious viruses. Furthermore, HCoV-OC43 infection of human epithelial and neuronal cell lines, of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the E protein is critical for efficient and optimal virus replication and propagation, and thereby for neurovirulence.

Receptor-binding loops in alphacoronavirus adaptation and evolution.

RNA viruses are characterized by a high mutation rate, a buffer against environmental change. Nevertheless, the means by which random mutation improves viral fitness is not well characterized. Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Three extended loops are solely responsible for receptor binding and the evolution of HCoV-229E and its close relatives is accompanied by changing loop-receptor interactions. Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. These RBD classes differ in their affinity for APN and their ability to bind an HCoV-229E neutralizing antibody. Together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding.

Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43.

Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE: Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.

Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.

Human coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express the Renilla luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication in vitro, with a 50% inhibitory concentration (IC50) of 0.33 µM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50 of 10 µM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication.