Seasonal Human Coronaviruses Research Articles

Impact of COVID-19 Nonpharmaceutical Interventions on Bordetella pertussis, Human Respiratory Syncytial Virus, Influenza Virus, and Seasonal Coronavirus Antibody Levels: A Systematic Review.

During the COVID-19 pandemic, nonpharmaceutical interventions (NPIs) were introduced to reduce the spread of SARS-CoV-2. This also resulted in a reduction of notifications of other acute respiratory infections and an altered seasonality when NPIs were lifted. Without circulation of pathogens, waning of antibodies is expected, which is a first indicator of decreased immunity. Here, by performing a systematic literature review, we investigated whether reduced antibody levels due to waning immunity contributed to the altered seasonality after NPIs were lifted. Thirteen articles met the inclusion criteria and reported antibody levels or seroprevalence of human respiratory syncytial virus, seasonal human coronavirus, Bordetella pertussis, and influenza virus. We show that the COVID-19 pandemic most likely led to waning of pathogen-specific antibodies, with the strongest evidence for human respiratory syncytial virus and seasonal human coronavirus and with a larger decrease in children vs adults. Waning antibodies might have resulted in out-of-season activity for these pathogens.

SARS-CoV-2 natural infection, but not vaccine-induced immunity, elicits cross-reactive immunity to OC43

BackgroundThe recent SARS-CoV-2 pandemic renewed interest toward other non-severe acute respiratory syndrome human coronaviruses. Among these, OC43 is a seasonal human coronavirus widely diffused in the population (90 % seroprevalence in adults) which is responsible for mild respiratory symptoms. As OC43 protective immunity is short lasting, we investigated whether humoral immunity to SARS-CoV-2, induced by vaccination or spontaneous infection, protects against OC43 re-infection at either systemic or mucosal level. MethodsA neutralization assay was conducted against “wild type" SARS-CoV-2 lineage B.1 (EU) and OC43 in VeroE6 cell lines using plasma and saliva samples from 49 subjects who were never infected and received three BNT162b2 RNA vaccine doses (SARS-CoV-2-vaccinated: SV) and from 25 SARS-CoV-2-infected and vaccinated subjects (SIV). The assays were performed right before (T0), fifteen days (T1) and three months (T2) after the third dose administration (SV) or post-infection (SIV). ResultsAfter the third vaccination dose was administered, SARS-CoV-2-specific neutralizing activity (NA) significantly augmented in SV saliva (p < 0.05) and plasma (p < 0.0001); yet, this NA was not protective against OC43. Conversely, in SIV, at T1, natural infection significantly increased NA against both SARS-CoV-2 (p < 0.01) and OC43 (p < 0.05) at systemic as well as mucosal level; still, this cross-reactivity vanished at T2. Of note, NA against SARS-CoV-2 and OC43 was shown to be higher in SIV compared to SV in plasma and saliva, as well; though, statistically significant differences were evident only in the oral mucosa at T1 (p < 0.05). ConclusionsOur findings show that SARS-CoV-2 spontaneous infection triggers a more comprehensive and cross-reactive immunity than vaccine-induced immunity, protecting against OC43 at the systemic and mucosal levels. These results support the development of a pan-coronavirus vaccine able to prompt cross-reactive immunity even against seasonal coronaviruses, which could have enormous economic and health benefits globally.

Indomethacin inhibits human seasonal coronaviruses at late stages of viral replication in lung cells: Impact on virus-induced COX-2 expression

Coronaviruses (CoV), zoonotic viruses periodically emerging worldwide, represent a constant potential threat to humans. To date, seven human coronaviruses (HCoV) have been identified: HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, globally circulating in the human population (seasonal coronaviruses, sHCoV), and three highly-pathogenic coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2. Although sHCoV generally cause only mild respiratory diseases, severe complications may occur in specific populations, highlighting the need for broad-spectrum anti-coronavirus drugs. Herein we show that indomethacin (INDO), a non-steroidal anti-inflammatory drug widely used in the clinic for its potent anti-inflammatory and analgesic properties, effectively inhibits the replication of Alpha-coronavirus HCoV-229E and Beta-coronavirus HCoV-OC43 in human lung-derived cells. Indomethacin does not interfere with HCoV binding or entry into target cells, but acts at late stages of the virus life cycle, inhibiting viral RNA synthesis and infectious viral particles production. Although INDO anti-inflammatory action is mediated by blocking cyclooxygenase-1 and -2 (COX-1/2) enzymatic activity, the antiviral effect appears to be cyclooxygenase-independent and is not mimicked by the potent COX-1/2 inhibitor aspirin. Interestingly we found that both seasonal HCoVs markedly (>100 fold) induce the expression of the pro-inflammatory mediator COX-2 in lung cells; notably, INDO-treatment was found to effectively inhibit virus-induced COX-2 expression at the transcriptional level, revealing an additional mechanism to prevent COX-2-mediated inflammatory reactions in HCoV-infected lung cells, besides COX activity inhibition. Altogether the results indicate that indomethacin, possessing both potent anti-inflammatory properties and a direct antiviral activity against HCoV, could be effective in the treatment of Alpha- and Beta-coronavirus infections.

Do patients infected with human coronavirus before the COVID-19 pandemic have less risk of being infected with COVID-19?

Although seasonal human coronaviruses (HCoVs) have long been recognized as respiratory tract viruses, the newly identified SARS-CoV-2 caused a pandemic associated with severe respiratory failure. We aimed to evaluate the incidence of COVID-19 infection in patients diagnosed in three tertiary teaching hospitals, both with and without prior confirmed HCoV infection, and to compare these cohorts in terms of COVID-19 contraction. In our study, we examined HCoV PCR-positive cases obtained retrospectively between January 2014 and March 2020 from three University Hospital Microbiology Laboratories (Cohort 1), as well as PCR-negative patients detected in the same PCR cycle as the positive cases (Cohort 2). We also evaluated subgroups of HCoV-positive cases. There was no difference in COVID-19 contraction rates between Cohort 1 and Cohort 2 (p = 0.724). When previous HCoV subgroups of COVID-19-positive patients were examined, no significant difference was found between the betacoronavirus and alphacoronavirus subgroups (p = 0.822), among the four groups (NL63, 229E, OC43, HKU-1) (p = 0.207), or between the OC43 subgroup and the other groups (p = 0.295). Being previously infected with HCoV did not provide protection against COVID-19 in our study group. We suggest evaluating the possible effect of previous OC43 infection on COVID-19 contraction in larger cohorts.

A T-Cell-Derived 3-Gene Signature Distinguishes SARS-CoV-2 from Common Respiratory Viruses.

Research on the host responses to respiratory viruses could help develop effective interventions and therapies against the current and future pandemics from the host perspective. To explore the pathogenesis that distinguishes SARS-CoV-2 infections from other respiratory viruses, we performed a multi-cohort analysis with integrated bioinformatics and machine learning. We collected 3730 blood samples from both asymptomatic and symptomatic individuals infected with SARS-CoV-2, seasonal human coronavirus (sHCoVs), influenza virus (IFV), respiratory syncytial virus (RSV), or human rhinovirus (HRV) across 15 cohorts. First, we identified an enhanced cellular immune response but limited interferon activities in SARS-CoV-2 infection, especially in asymptomatic cases. Second, we identified a SARS-CoV-2-specific 3-gene signature (CLSPN, RBBP6, CCDC91) that was predominantly expressed by T cells, could distinguish SARS-CoV-2 infection, including Omicron, from other common respiratory viruses regardless of symptoms, and was predictive of SARS-CoV-2 infection before detectable viral RNA on RT-PCR testing in a longitude follow-up study. Thereafter, a user-friendly online tool, based on datasets collected here, was developed for querying a gene of interest across multiple viral infections. Our results not only identify a unique host response to the viral pathogenesis in SARS-CoV-2 but also provide insights into developing effective tools against viral pandemics from the host perspective.

Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics.

The emergence of the COVID-19 pandemic prompted an increased interest in seasonal human coronaviruses. OC43, 229E, NL63, and HKU1 are endemic seasonal coronaviruses that cause the common cold and are associated with generally mild respiratory symptoms. In this study, we identified cell lines that exhibited cytopathic effects (CPE) upon infection by three of these coronaviruses and characterized their viral replication kinetics and the effect of infection on host surface receptor expression. We found that NL63 produced CPE in LLC-MK2 cells, while OC43 produced CPE in MRC-5, HCT-8, and WI-38 cell lines, while 229E produced CPE in MRC-5 and WI-38 by day 3 post-infection. We observed a sharp increase in nucleocapsid and spike viral RNA (vRNA) from day 3 to day 5 post-infection for all viruses; however, the abundance and the proportion of vRNA copies measured in the supernatants and cell lysates of infected cells varied considerably depending on the virus-host cell pair. Importantly, we observed modulation of coronavirus entry and attachment receptors upon infection. Infection with 229E and OC43 led to a downregulation of CD13 and GD3, respectively. In contrast, infection with NL63 and OC43 leads to an increase in ACE2 expression. Attempts to block entry of NL63 using either soluble ACE2 or anti-ACE2 monoclonal antibodies demonstrated the potential of these strategies to greatly reduce infection. Overall, our results enable a better understanding of seasonal coronaviruses infection kinetics in permissive cell lines and reveal entry receptor modulation that may have implications in facilitating co-infections with multiple coronaviruses in humans.IMPORTANCESeasonal human coronavirus is an important cause of the common cold associated with generally mild upper respiratory tract infections that can result in respiratory complications for some individuals. There are no vaccines available for these viruses, with only limited antiviral therapeutic options to treat the most severe cases. A better understanding of how these viruses interact with host cells is essential to identify new strategies to prevent infection-related complications. By analyzing viral replication kinetics in different permissive cell lines, we find that cell-dependent host factors influence how viral genes are expressed and virus particles released. We also analyzed entry receptor expression on infected cells and found that these can be up- or down-modulated depending on the infecting coronavirus. Our findings raise concerns over the possibility of infection enhancement upon co-infection by some coronaviruses, which may facilitate genetic recombination and the emergence of new variants and strains.

Seasonal human coronavirus humoral responses in AZD1222 (ChaAdOx1 nCoV-19) COVID-19 vaccinated adults reveal limited cross-immunity.

Immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now widespread; however, the degree of cross-immunity between SARS-CoV-2 and endemic, seasonal human coronaviruses (HCoVs) remains unclear. SARS-CoV-2 and HCoV cross-immunity was evaluated in adult participants enrolled in a US sub-study in the phase III, randomized controlled trial (NCT04516746) of AZD1222 (ChAdOx1 nCoV-19) primary-series vaccination for one-year. Anti-HCoV spike-binding antibodies against HCoV-229E, HCoV-HKU1, HCoV-OC43, and HCoV-NL63 were evaluated in participants following study dosing and, in the AZD1222 group, after a non-study third-dose booster. Timing of SARS-CoV-2 seroconversion (assessed via anti-nucleocapsid antibody levels) and incidence of COVID-19 were evaluated in those who received AZD1222 primary-series by baseline anti-HCoV titers. We evaluated 2,020/21,634 participants in the AZD1222 group and 1,007/10,816 in the placebo group. At the one-year data cutoff (March 11, 2022) mean duration of follow up was 230.9 (SD: 106.36, range: 1-325) and 94.3 (74.12, 1-321) days for participants in the AZD1222 (n = 1,940) and placebo (n = 962) groups, respectively. We observed little elevation in anti-HCoV humoral titers post study-dosing or post-boosting, nor evidence of waning over time. The occurrence and timing of SARS-CoV-2 seroconversion and incidence of COVID-19 were not largely impacted by baseline anti-HCoV titers. We found limited evidence for cross-immunity between SARS-CoV-2 and HCoVs following AZD1222 primary series and booster vaccination. Susceptibility to future emergence of novel coronaviruses will likely persist despite a high prevalence of SARS-CoV-2 immunity in global populations.

Exploring Pre-Pandemic SARS-CoV-2-Specific B Cell Responses in the West African Population.

Abstract The emergence of the novel coronavirus, SARS-CoV-2, during the COVID-19 pandemic prompted investigations into pre-existing immunity in human populations. Despite being a new coronavirus, individuals had previous exposure to common seasonal human coronaviruses. In this study, we examined SARS-CoV-2-specific memory B cells in six pre-pandemic Lassa virus-infected patients from West Africa. By incorporating a SARS-CoV-2 Spike Antigen (WA-1 strain) into a broader antigen panel that included Lassa virus GPC and a control, we identified and sorted antigen-positive B cells. Further analysis revealed a distinct population of SARS-CoV-2-specific memory B cells. Native paired monoclonal antibody (mAb) sequences were recovered from 598 SARS-CoV-2 Spike-positive B cells, demonstrating a surprisingly high frequency of pre-existing SARS-CoV-2-specific B cells. Remarkably, Spike-specific mAbs were recovered from all six donors, each contributing to at least one expanded clonal lineage. Notably, most IgM antibodies belonged to the same lineages. These findings suggest a notable prevalence of pre-existing immunity to SARS-CoV-2 in pre-pandemic patients, potentially influencing susceptibility to the virus and contributing to adverse COVID-19 outcomes. This study provides insights for risk management and supports the development of universal coronavirus vaccines by highlighting the complex interplay between pre-existing immunity and susceptibility to SARS-CoV-2.

Seasonal human coronavirus NL63 epidemics in children in Guilin, China, reveal the emergence of a new subgenotype of HCoV-NL63.

Seasonal human coronavirus NL63 (HCoV-NL63) is a frequently encountered virus linked to mild upper respiratory infections. However, its potential to cause more severe or widespread disease remains an area of concern. This study aimed to investigate a rare localized epidemic of HCoV-NL63-induced respiratory infections among pediatric patients in Guilin, China, and to understand the viral subtype distribution and genetic characteristics. In this study, 83 pediatric patients hospitalized with acute respiratory infections and positive for HCoV-NL63 were enrolled. Molecular analysis was conducted to identify the viral subgenotypes and to assess genetic variations in the receptor-binding domain of the spiking protein. Among the 83 HCoV-NL63-positive children, three subgenotypes were identified: C4, C3, and B. Notably, 21 cases exhibited a previously unreported subtype, C4. Analysis of the C4 subtype revealed a unique amino acid mutation (I507L) in the receptor-binding domain of the spiking protein, which was also observed in the previously reported C3 genotype. This mutation may suggest potential increases in viral transmissibility and pathogenicity. The findings of this study highlight the rapid mutation dynamics of HCoV-NL63 and its potential for increased virulence and epidemic transmission. The presence of a unique mutation in the C4 subtype, shared with the C3 genotype, raises concerns about the virus's evolving nature and its potential public health implications. This research contributes valuable insights into the understanding of HCoV-NL63's epidemiology and pathogenesis, which is crucial for effective disease prevention and control strategies. Future studies are needed to further investigate the biological significance of the observed mutation and its potential impact on the virus's transmissibility and pathogenicity.

Faradaic Impedimetric Immunosensor for Label-Free Point-of-Care Detection of COVID-19 Antibodies Using Gold-Interdigitated Electrode Array.

Label-free electrochemical biosensors have many desirable characteristics in terms of miniaturization, scalability, digitization, and other attributes associated with point-of-care (POC) applications. In the era of COVID-19 and pandemic preparedness, further development of such biosensors will be immensely beneficial for rapid testing and disease management. Label-free electrochemical biosensors often employ [Fe(CN)6]-3/4 redox probes to detect low-concentration target analytes as they dramatically enhance sensitivity. However, such Faradaic-based sensors are reported to experience baseline signal drift, which compromises the performance of these devices. Here, we describe the use of a mecaptohexanoic (MHA) self-assembled monolayer (SAM) modified Au-interdigitated electrode arrays (IDA) to investigate the origin of the baseline signal drift, developed a protocol to resolve the issue, and presented insights into the underlying mechanism on the working of label-free electrochemical biosensors. Using this protocol, we demonstrate the application of MHA SAM-modified Au-IDA for POC analysis of human serum samples. We describe the use of a label-free electrochemical biosensor based on covalently conjugated SARS-CoV-2 spike protein for POC detection of COVID-19 antibodies. The test requires a short incubation time (10 min), and has a sensitivity of 35.4/decade (35.4%/10 ng mL-1) and LOD of 21 ng/mL. Negligible cross reactivity to seasonal human coronavirus or other endogenous antibodies was observed. Our studies also show that Faradaic biosensors are ~17 times more sensitive than non-Faradaic biosensors. We believe the work presented here contributes to the fundamental understanding of the underlying mechanisms of baseline signal drift and will be applicable to future development of electrochemical biosensors for POC applications.

1752. Seasonal Human Coronavirus in Pediatric Patients enrolled by the New Vaccine Surveillance Network, 2016-2020

Abstract Background The COVID-19 pandemic highlighted the potential pathogenicity of betacoronaviruses. This elucidates the need to further investigate human coronaviruses (HCoV), which includes both alpha and betacoronaviruses, to better understand their seasonality and severity among children. Methods The New Vaccine Surveillance Network (NVSN) enrolled children at 7 pediatric medical centers in the United States seen at the emergency department (ED) or hospitalized with acute respiratory infection (ARI), during 12/1/2016–9/28/2020. Respiratory specimens were collected and tested using real-time reverse transcription polymerase-chain reaction assays. The frequency of seasonal HCoV (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) detections was measured and symptoms were reported by parent interview. Descriptive statistics were performed to compare HCoV-positive inpatients to those seen in the ED. SARS-CoV-2 infections were excluded. Results Of 34,455 children enrolled, 925 (2.7%) were positive for HCoV, of which 398 (43%) were inpatients and (57%) were ED patients (Table). 346 detections (37.4%) were HCoV-OC43, 298 (32.3%) HCoV-NL63, 186 (20.2%) HCoV-HKU1, and 107 (11.6%) HCoV-229E. More than one coronavirus was detected in 12 children (1.3%). HCoV were primarily detected between October and April of each season; 1 to 2 types dominated each year but not consecutively (Figure). Among HCoV-positive children, 527 (57.0%) received their highest level of care in the ED while 398 (43.0%) were hospitalized. HCoV-229E was the most common HCoV detected among inpatients (57.0%), followed by HCoV-HKU1-(45.2%); HCoV-OC43- (40.8%); and HCoV-NL63 (38.9%). Among those positive for a HCoV age, race, and insurance status were significantly associated with hospitalization. Conclusion HCoV followed a winter-season circulation pattern, but with variation in the predominant type across years. HCoV-OC43 accounted for the largest number of HCoV infections whereas HCoV-229E had the highest proportion hospitalized. Further surveillance is required to evaluate the impact of the emergent circulation of SARS-CoV-2 on seasonal HCoVs. Disclosures Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support Geoffrey A. Weinberg, MD, Merck &amp; Co: Honoraria Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Pedro A. Piedra, MD, Ark Bioscience: Advisor/Consultant|Ark Bioscience: Grant/Research Support|GSK: Grant/Research Support|Icosavax: Advisor/Consultant|Icosavax: Grant/Research Support|Mapp Biologics: Grant/Research Support|Meissa Vaccines: Grant/Research Support|Moderna: Advisor/Consultant|Novavax: Advisor/Consultant|Novavax: Grant/Research Support|Sanofi-Pasteur: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Takeda: Advisor/Consultant Mary A. Staat, MD, MPH, CDC: Grant/Research Support|Cepheid: Grant/Research Support|Merck: Grant/Research Support|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Up-To-Date: Honoraria John V. Williams, MD, Merck: Grant/Research Support|Quidel: Board Member Marian G. Michaels, MD, MPH, Merck: Grant/Research Support|Viracor: Grant/Research Support

Antiviral Potential of Azelastine against Major Respiratory Viruses.

The Coronavirus Disease 2019 (COVID-19) pandemic and the subsequent increase in respiratory viral infections highlight the need for broad-spectrum antivirals to enable a quick and efficient reaction to current and emerging viral outbreaks. We previously demonstrated that the antihistamine azelastine hydrochloride (azelastine-HCl) exhibited in vitro antiviral activity against SARS-CoV-2. Furthermore, in a phase 2 clinical study, a commercial azelastine-containing nasal spray significantly reduced the viral load in SARS-CoV-2-infected individuals. Here, we evaluate the efficacy of azelastine-HCl against additional human coronaviruses, including the SARS-CoV-2 omicron variant and a seasonal human coronavirus, 229E, through in vitro infection assays, with azelastine showing a comparable potency against both. Furthermore, we determined that azelastine-HCl also inhibits the replication of Respiratory syncytial virus A (RSV A) in both prophylactic and therapeutic settings. In a human 3D nasal tissue model (MucilAirTM-Pool, Epithelix), azelastine-HCl protected tissue integrity and function from the effects of infection with influenza A H1N1 and resulted in a reduced viral load soon after infection. Our results suggest that azelastine-HCl has a broad antiviral effect and can be considered a safe option against the most common respiratory viruses to prevent or treat such infections locally in the form of a nasal spray that is commonly available globally.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Pathogens detected from patients with acute respiratory infections negative for SARS-CoV-2, Saitama, Japan, 2020.

During the coronavirus disease pandemic in Japan, all patients with respiratory symptoms were initially tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study describes the respiratory pathogens detected from patients who tested negative for SARS-CoV-2 at the Saitama Institute of Public Health from January to December 2020. We performed pathogen retrieval using multiplex real-time polymerase chain reaction on samples from patients with acute respiratory diseases who tested negative for SARS-CoV-2 in Saitama in 2020 and analysed the results by age and symptoms. There were 1530 patients aged 0-104 years (1727 samples), with 14 pathogens detected from 213 patients (245 samples). Most pathogens were human metapneumovirus (25.4%, 54 cases), rhinovirus (16.4%, 35 cases) and Mycoplasma pneumoniae (13.1%, 23 cases). Human metapneumovirus, human coronavirus (but not NL63) and M. pneumoniae were detected in almost all age groups without any significant bias. Seasonal human coronaviruses, human metapneumovirus, M. pneumoniae and several other pathogens were detected until April 2020. Multiple respiratory pathogens were circulating during 2020 in Saitama, including SARS-CoV-2 and influenza viruses. We suggest introducing a system that can comprehensively monitor the regional prevalence of all viruses that cause acute respiratory infections.

Improved Culture Methods for Human Coronaviruses HCoV-OC43, HCoV-229E, and HCoV-NL63.

HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are four of the seven known human coronaviruses (HCoVs) and, unlike the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, these four so-called seasonal HCoVs generally cause mild upper-respiratory-tract illness. As Biosafety Level 2 (BSL-2) pathogens, the seasonal HCoVs are more accessible and can be used as surrogates for studying the highly pathogenic HCoVs. However, scientists have for many years found these difficult to study because of the lack of a universal culture system and the inability of typical culture methods to yield high-titer infectious stocks. We have developed assays to grow and quantify infectious virus and viral RNA for HCoV-OC43, -229E, and -NL63. We identified which immortalized cell lines should be used to optimize the replication of HCoV-OC43, -229E, and -NL63 in order to generate high titers (Vero E6, Huh-7, and LLC-MK2 cells, respectively). Here we present protocols for improved propagation and quantification of each seasonal HCoV. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Growth of HCoVs Basic Protocol 2: Quantification of HCoV by plaque assay Basic Protocol 3: Quantification of HCoV RNA products of replication Basic Protocol 4: Concentrating HCoVs via ultracentrifugation.

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions.

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, includingthe cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example1. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

Epidemiology and Clinical Characteristics of Seasonal Human Coronaviruses in Children Hospitalized in Hebei Province, China Before and During the COVID-19 Pandemic.

This study aimed to assess the impact of the COVID-19 pandemic on the prevalence and clinical characteristics of seasonal human coronavirus (HCoV) infections among children hospitalized in Hebei, China. We examined nasopharyngeal aspirate (NPA) specimens for seasonal HCoVs from January 2018 to December 2021, at the Children's Hospital of Hebei Province. We used a GeXP-based multiplex reverse transcription PCR assay for the detection of 11 common respiratory viruses (including seasonal HCoVs), chlamydia, and Mycoplasma pneumoniae. The demographic and clinical characteristics of children who tested positive for seasonal HCoVs were recorded and analyzed. A total of 377 (1.96%) of the 19,248 specimens from 2018 to 2019 and 263 (1.96%) of the 13,426 specimens from 2020 to 2021 exhibited seasonal HCoVs. Compared to 2018 and 2019, the positive rate of seasonal HCoVs was lower from January to July of 2020 and increased beginning in August 2020, peaking in the autumn and winter. In 2020-2021, nasal blockage and swollen adenoids were detected more frequently in children who tested positive for seasonal HCoVs. During 2018-2019, however, the duration of fever was significantly longer, and cough and dyspnea were more prominent among children who had fallen ill. In addition, seasonal HCoV-positive patients in 2018-2019 were more likely to experience complications, had a higher risk of severe community-acquired pneumonia (CAP), and had a tendency to require a longer hospital stay than patients in 2020-2021. According to our findings, there were significant changes in the epidemiology of seasonal HCoVs in Hebei, China during the COVID-19 pandemic, and children infected with seasonal HCoVs usually experienced milder clinical symptoms during the pandemic than before it.

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 μg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Pathogen Profiles in Outpatients with Non-COVID-19 during the 7th Prevalent Period of COVID-19 in Gunma, Japan.

The identification of pathogens associated with respiratory symptoms other than the novel coronavirus disease 2019 (COVID-19) can be challenging. However, the diagnosis of pathogens is crucial for assessing the clinical outcome of patients. We comprehensively profiled pathogens causing non-COVID-19 respiratory symptoms during the 7th prevalent period in Gunma, Japan, using deep sequencing combined with a next-generation sequencer (NGS) and advanced bioinformatics technologies. The study included nasopharyngeal swabs from 40 patients who tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) using immuno-chromatography and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Comprehensive pathogen sequencing was conducted through deep sequencing using NGS. Additionally, short reads obtained from NGS were analyzed for comprehensive pathogen estimation using MePIC (Metagenomic Pathogen Identification Pipeline for Clinical Specimens) and/or VirusTap. The results revealed the presence of various pathogens, including respiratory viruses and bacteria, in the present subjects. Notably, human adenovirus (HAdV) was the most frequently detected virus in 16 of the 40 cases (40.0%), followed by coryneforms, which were the most frequently detected bacteria in 21 of the 40 cases (52.5%). Seasonal human coronaviruses (NL63 type, 229E type, HKU1 type, and OC43 type), human bocaviruses, and human herpesviruses (human herpesvirus types 1-7) were not detected. Moreover, multiple pathogens were detected in 50% of the subjects. These results suggest that various respiratory pathogens may be associated with non-COVID-19 patients during the 7th prevalent period in Gunma Prefecture, Japan. Consequently, for an accurate diagnosis of pathogens causing respiratory infections, detailed pathogen analyses may be necessary. Furthermore, it is possible that various pathogens, excluding SARS-CoV-2, may be linked to fever and/or respiratory infections even during the COVID-19 pandemic.

Seroepidemiology of the Seasonal Human Coronaviruses NL63, 229E, OC43 and HKU1 in France.

The seasonal human coronaviruses (HCoV) NL63, 229E, OC43, and HKU1 are globally endemic, yet the majority of HCoV infections remain undiagnosed. In a cross-sectional study, 2389 serum samples were collected from children and adults in France in 2020. In a longitudinal cohort study, 2520 samples were collected from 898 French individuals followed up between 2020 and 2021. Antibodies to HCoVs were measured using a bead-based multiplex assay. The rate of waning of anti-HCoV spike immunoglobulin G antibodies was estimated as 0.22-0.47 year-1 for children, and 0.13-0.27 year-1 for adults. Seroreversion was estimated as 0.31-1.37 year-1 in children and 0.19-0.72 year-1 in adults. The estimated seroconversion rate in children was consistent with 20%-39% of children being infected every year with each HCoV. The high force of infection in children indicates that HCoVs may be responsible for a substantial proportion of fever episodes experienced by children.