Neutralizing immunity against coronaviruses in Tanzanian health care workers.

The ongoing vaccination efforts and exposure to endemic and emerging coronaviruses can shape the population's immunity against this group of viruses. In this study, we investigated neutralizing immunity against endemic and emerging coronaviruses in 200 Tanzanian frontline healthcare workers (HCWs). Despite low vaccination rates (19.5%), we found a high SARS-CoV-2 seroprevalence (94.0%), indicating high exposure in these HCWs. Next, we determined the neutralization capacity of antisera against human coronavirus NL63, and 229E, SARS-CoV-1, MERS-CoV and SARS-CoV-2 (including Omicron subvariants: BA.1, BQ.1.1 and XBB.1.5) using pseudovirus neutralization assay. We observed a broad range of neutralizing activity in HCWs, but no neutralization activity detected against MERS-CoV. We also observed a strong correlation between neutralizing antibody titers for SARS-CoV-2 and SARS-CoV-1, but not between other coronaviruses. Cross-neutralization titers against the newer Omicron subvariants, BQ.1.1 and XBB.1.5, was significantly reduced compared to BA.1 and BA.2 subvariants. On the other hand, the exposed vaccinated HCWs showed relatively higher median cross-neutralization titers against both the newer Omicron subvariants and SARS-CoV-1, but did not reach statistical significance. In summary, our findings suggest a broad range of neutralizing potency against coronaviruses in Tanzanian HCWs with detectable neutralizing immunity against SARS-CoV-1 resulting from SARS-CoV-2 exposure.

Influenza-like illness symptoms due to endemic human coronavirus reinfections are not influenced by the length of the interval separating reinfections.

After 3 years of its introduction to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared as endemic. Little is known about the severity of the disease manifestation that future infections may cause, especially when reinfections occur after humoral immunity from a previous infection or vaccination has waned. Such knowledge could inform policymakers regarding the frequency of vaccination. Reinfections by endemic human coronaviruses (HCoVs) can serve as a model system for SARS-CoV-2 endemicity. We monitored 44 immunocompetent male adults with blood sampling every 6 months (for 17 years), for the frequency of HCoV (re-)infections, using rises in N-antibodies of HCoV-NL63, HCoV-29E, HCoV-OC43, and HCoV-HKU1 as markers of infection. Disease associations during (re-)infections were examined by comparison of self-reporting records of influenza-like illness (ILI) symptoms, every 6 months, by all participants. During 8,549 follow-up months, we found 364 infections by any HCoV with a median of eight infections per person. Symptoms more frequently reported during HCoV infection were cough, sore throat, and myalgia. Two hundred fifty-one of the 364 infections were species-specific HCoV-reinfections, with a median interval of 3.58 (interquartile range 1.92-5.67) years. The length of the interval between reinfections-being either short or long-had no influence on the frequency of reporting ILI symptoms. All HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 (re-)infections are associated with the reporting of ILIs. Importantly, in immunocompetent males, these symptoms are not influenced by the length of the interval between reinfections. IMPORTANCE: Little is known about the disease following human coronavirus (HCoV) reinfection occurring years after the previous infection, once humoral immunity has waned. We monitored endemic HCoV reinfection in immunocompetent male adults for up to 17 years. We found no influence of reinfection interval length in the disease manifestation, suggesting that immunocompetent male adults are adequately protected against future HCoV infections.

Remdesivir Derivative VV116 Is a Potential Broad-Spectrum Inhibitor of Both Human and Animal Coronaviruses.

Coronaviruses represent a significant threat to both human and animal health, encompassing a range of pathogenic strains responsible for illnesses, from the common cold to more severe diseases. VV116 is a deuterated derivative of Remdesivir with oral bioavailability that was found to potently inhibit SARS-CoV-2. In this work, we investigated the broad-spectrum antiviral activity of VV116 against a variety of human and animal coronaviruses. We examined the inhibitory effects of VV116 on the replication of the human coronaviruses HCoV-NL63, HCoV-229E, and HCoV-OC43, as well as the animal coronaviruses MHV, FIPV, FECV, and CCoV. The findings reveal that VV116 effectively inhibits viral replication across these strains without exhibiting cytotoxicity, indicating its potential for safe therapeutic use. Based on the results of a time-of-addition assay and an rNTP competitive inhibition assay, it is speculated that the inhibitory mechanism of VV116 against HCoV-NL63 is consistent with its inhibition of SARS-CoV-2. Our work presents VV116 as a promising candidate for broad-spectrum anti-coronavirus therapy, with implications for both human and animal health, and supports the expansion of its therapeutic applications as backed by detailed experimental data.

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.

Multicenter analysis of epidemiological and clinical features of pediatric acute lower respiratory tract infections associated with common human coronaviruses in China, 2014-2019.

The common human coronaviruses (HCoVs) HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 which are members of the coronavirus family are long co-existed with humans and widely distributed globally. Common HCoVs usually cause mild, self-limited upper respiratory tract infections (URTI), and also associated with lower respiratory tract infections (LRTI), especially in children. However, there are little multicentre studies have been conducted in children of several different areas in China, and the epidemic potential of common HCoVs remains unclear. Understanding of the common HCoVs is valuable for clinical and public health. Herein, we retrospectively analysed the medical records of children with acute lower respiratory tract infection admitted to 9 hospitals from different regions in China from 2014 to 2019. Of the 124 patients who tested positive for coronaviruses, OC43 was the predominant type, accounting for 36.3% (45/124) of the detections. Children aged ≤ 6 months and 12-23 months had the highest detection rate of common HCoVs, and the detection rate gradually declined after 2 years old. These four HCoVs could be detected all year round. Among the areas of our study, the overall positive rate was higher in southern China, especially in Guangzhou (29/124, 23.4%). Moreover, common HCoV-positive patients were codetected with 9 other common respiratory pathogens. 229E (11/13, 84.6%) was the most frequently associated with codetection, with EV/RhV was the most frequently codetected virus. Cough (113/124, 91.1%) and fever (73/124, 58.9%) were the most common symptoms of common HCoVs infection.

Previous infection with seasonal coronaviruses does not protect male Syrian hamsters from challenge with SARS-CoV-2.

SARS-CoV-2 variants and seasonal coronaviruses continue to cause disease and coronaviruses in the animal reservoir pose a constant spillover threat. Importantly, understanding of how previous infection may influence future exposures, especially in the context of seasonal coronaviruses and SARS-CoV-2 variants, is still limited. Here we adopted a step-wise experimental approach to examine the primary immune response and subsequent immune recall toward antigenically distinct coronaviruses using male Syrian hamsters. Hamsters were initially inoculated with seasonal coronaviruses (HCoV-NL63, HCoV-229E, or HCoV-OC43), or SARS-CoV-2 pango B lineage virus, then challenged with SARS-CoV-2 pango B lineage virus, or SARS-CoV-2 variants Beta or Omicron. Although infection with seasonal coronaviruses offered little protection against SARS-CoV-2 challenge, HCoV-NL63-infected animals had an increase of the previously elicited HCoV-NL63-specific neutralizing antibodies during challenge with SARS-CoV-2. On the other hand, primary infection with HCoV-OC43 induced distinct T cell gene signatures. Gene expression profiling indicated interferon responses and germinal center reactions to be induced during more similar primary infection-challenge combinations while signatures of increased inflammation as well as suppression of the antiviral response were observed following antigenically distant viral challenges. This work characterizes and analyzes seasonal coronaviruses effect on SARS-CoV-2 secondary infection and the findings are important for pan-coronavirus vaccine design.

Continuous evolution and emerging lineage of seasonal human coronaviruses: A multicenter surveillance study.

The seasonal human coronaviruses (HCoVs) have zoonotic origins, repeated infections, and global transmission. The objectives of this study are to elaborate the epidemiological and evolutionary characteristics of HCoVs from patients with acute respiratory illness. We conducted a multicenter surveillance at 36 sentinel hospitals of Beijing Metropolis, China, during 2016-2019. Patients with influenza-like illness (ILI) and severe acute respiratory infection (SARI) were included, and submitted respiratory samples for screening HCoVs by multiplex real-time reverse transcription-polymerase chain reaction assays. All the positive samples were used for metatranscriptomic sequencing to get whole genomes of HCoVs for genetical and evolutionary analyses. Totally, 321 of 15 677 patients with ILI or SARI were found to be positive for HCoVs, with an infection rate of 2.0% (95% confidence interval, 1.8%-2.3%). HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 infections accounted for 18.7%, 38.3%, 40.5%, and 2.5%, respectively. In comparison to ILI cases, SARI cases were significantly older, more likely caused by HCoV-229E and HCoV-OC43, and more often co-infected with other respiratory pathogens. A total of 179 full genome sequences of HCoVs were obtained from 321 positive patients. The phylogenetical analyses revealed that HCoV-229E, HCoV-NL63 and HCoV-OC43 continuously yielded novel lineages, respectively. The nonsynonymous to synonymous ratio of all key genes in each HCoV was less than one, indicating that all four HCoVs were under negative selection pressure. Multiple substitution modes were observed in spike glycoprotein among the four HCoVs. Our findings highlight the importance of enhancing surveillance on HCoVs, and imply that more variants might occur in the future.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Seroprevalence of four endemic human coronaviruses and, reactivity and neutralization capability against SARS-CoV-2 among children in the Philippines.

Four endemic human coronaviruses (HCoV), HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are closely related to SARS-CoV-2. These coronaviruses are known to infect humans living in temperate areas, including children under 5 years old; however, the seroprevalence of four HCoVs among children in tropical areas, including the Philippines, remains unclear. This study aimed to assess the prevalence of antibodies against four HCoVs and to determine the reactivity and neutralization of these antibodies against SARS-CoV-2 among children in the Philippines. A total of 315 serum samples collected from 2015 to 2018, before the emergence of SARS-CoV-2, in Biliran island, Philippines, were tested for the presence of antibodies against four HCoVs and SARS-CoV-2 using recombinant spike ectodomain proteins by IgG-enzyme-linked immunosorbent assay (ELISA). Reactivity to and neutralization of SARS-CoV-2 were also investigated. The seroprevalence of the four HCoVs was 63.8% for HCoV-229E, 71.4% for HCoV-NL63, 76.5% for HCoV-HKU1, and 83.5% for HCoV-OC43 by ELISA. Age group analysis indicated that seropositivity to all HCoVs reached 80% by 2-3 years of age. While 69/315 (21.9%) of the samples showed reactive to SARS-CoV-2, almost no neutralization against SARS-CoV-2 was detected using neutralization assay. Reactivity of antibodies against SARS-CoV-2 spike protein obtained by ELISA may not correlate with neutralization capability.

Molecular mechanisms of human coronavirus NL63 infection and replication.

Human coronavirus NL63 (HCoV-NL63) is spread globally, causing upper and lower respiratory tract infections mainly in young children. HCoV-NL63 shares a host receptor (ACE2) with severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 but, unlike them, HCoV-NL63 primarily develops into self-limiting mild to moderate respiratory disease. Although with different efficiency, both HCoV-NL63 and SARS-like CoVs infect ciliated respiratory cells using ACE2 as receptor for binding and cell entry. Working with SARS-like CoVs require access to BSL-3 facilities, while HCoV-NL63 research can be performed at BSL-2 laboratories. Thus, HCoV-NL63 could be used as a safer surrogate for comparative studies on receptor dynamics, infectivity and virus replication, disease mechanism, and potential therapeutic interventions against SARS-like CoVs. This prompted us to review the current knowledge on the infection mechanism and replication of HCoV-NL63. Specifically, after a brief overview on the taxonomy, genomic organization and virus structure, this review compiles the current HCoV-NL63-related research in virus entry and replication mechanism, including virus attachment, endocytosis, genome translation, and replication and transcription. Furthermore, we reviewed cumulative knowledge on the susceptibility of different cells to HCoV-NL63 infection in vitro, which is essential for successful virus isolation and propagation, and contribute to address different scientific questions from basic science to the development and assessment of diagnostic tools, and antiviral therapies. Finally, we discussed different antiviral strategies that have been explored to suppress replication of HCoV-NL63, and other related human coronaviruses, by either targeting the virus or enhancing host antiviral mechanisms.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

Household Transmission Dynamics of Seasonal Human Coronaviruses.

BACKGROUND: Household transmission studies inform how viruses spread among close contacts, but few characterize household transmission of endemic coronaviruses. METHODS: We used data collected from 223 households with school-age children participating in weekly disease surveillance over 2 respiratory virus seasons (December 2015 to May 2017), to describe clinical characteristics of endemic human coronaviruses (HCoV-229E, HcoV-HKU1, HcoV-NL63, HcoV-OC43) infections, and community and household transmission probabilities using a chain-binomial model correcting for missing data from untested households. RESULTS: Among 947 participants in 223 households, we observed 121 infections during the study, most commonly subtype HCoV-OC43. Higher proportions of infected children (<19 years) displayed influenza-like illness symptoms than infected adults (relative risk, 3.0; 95% credible interval [CrI], 1.5-6.9). The estimated weekly household transmission probability was 9% (95% CrI, 6-13) and weekly community acquisition probability was 7% (95% CrI, 5-10). We found no evidence for differences in community or household transmission probabilities by age or symptom status. Simulations suggest that our study was underpowered to detect such differences. CONCLUSIONS: Our study highlights the need for large household studies to inform household transmission, the challenges in estimating household transmission probabilities from asymptomatic individuals, and implications for controlling endemic CoVs.

Low pre-existing endemic human coronavirus (HCoV-NL63)-specific T cell frequencies are associated with impaired SARS-CoV-2-specific T cell responses in people living with HIV.

Background: Understanding how HIV affects SARS-CoV-2 immunity is crucial for managing COVID-19 in sub-Saharan populations due to frequent coinfections. Our previous research showed that unsuppressed HIV is associated with weaker immune responses to SARS-CoV-2, but the underlying mechanisms are unclear. We investigated how pre-existing T cell immunity against an endemic human coronavirus HCoV-NL63 impacts SARS-CoV-2 T cell responses in people living with HIV (PLWH) compared to uninfected individuals, and how HIV-related T cell dysfunction influences responses to SARS-CoV-2 variants. Methods: We used flow cytometry to measure T cell responses following PBMC stimulation with peptide pools representing beta, delta, wild-type, and HCoV-NL63 spike proteins. Luminex bead assay was used to measure circulating plasma chemokine and cytokine levels. ELISA and MSD V-PLEX COVID-19 Serology and ACE2 Neutralization assays were used to measure humoral responses. Results: Regardless of HIV status, we found a strong positive correlation between responses to HCoV-NL63 and SARS-CoV-2. However, PLWH exhibited weaker CD4+ T cell responses to both HCoV-NL63 and SARS-CoV-2 than HIV-uninfected individuals. PLWH also had higher proportions of functionally exhausted (PD-1high) CD4+ T cells producing fewer proinflammatory cytokines (IFNγ and TNFα) and had elevated plasma IL-2 and IL-12(p70) levels compared to HIV-uninfected individuals. HIV status didn't significantly affect IgG antibody levels against SARS-CoV-2 antigens or ACE2 binding inhibition activity. Conclusion: Our results indicate that the decrease in SARS-CoV-2 specific T cell responses in PLWH may be attributable to reduced frequencies of pre-existing cross-reactive responses. However, HIV infection minimally affected the quality and magnitude of humoral responses, and this could explain why the risk of severe COVID-19 in PLWH is highly heterogeneous.

Hold your horses: The receptor-binding domains of SARS-CoV-2, SARS-CoV, and hCoV-NL63 bind equine ACE2.

In this issue of Structure, Lan and colleagues seek to identify regions on the ACE2 receptor and coronavirus spikes that are essential for the viral attachment. They achieve it through a detailed comparative analysis of the binding of coronaviruses NL63, SARS-CoV, and several SARS-CoV-2 variants with human and horse ACE2.

More tools for our toolkit: The application of HEL-299 cells and dsRNA-nanoparticles to study human coronaviruses in vitro.

Human coronaviruses (HCoVs) are important human pathogens, as exemplified by the current SARS-CoV-2 pandemic. While the ability of type I interferons (IFNs) to limit coronavirus replication has been established, the ability of double-stranded (ds)RNA, a potent IFN inducer, to inhibit coronavirus replication when conjugated to a nanoparticle is largely unexplored. Additionally, the number of IFN competent cell lines that can be used to study coronaviruses in vitro are limited. In the present study, we show that poly inosinic: poly cytidylic acid (pIC), when conjugated to a phytoglycogen nanoparticle (pIC+NDX) is able to protect IFN-competent human lung fibroblasts (HEL-299 cells) from infection with different HCoV species. HEL-299 was found to be permissive to HCoV-229E, -OC43 and MERS-CoV-GFP but not to HCoV-NL63 or SARS-CoV-2. Further investigation revealed that HEL-299 does not contain the required ACE2 receptor to enable propagation of both HCoV-NL63 and SARS-CoV-2. Following 24h exposure, pIC+NDX was observed to stimulate a significant, prolonged increase in antiviral gene expression (IFNß, CXCL10 and ISG15) when compared to both NDX alone and pIC alone. This antiviral response translated into complete protection against virus production, for 4 days or 7 days post treatment with HCoV-229E or -OC43 when either pre-treated for 6h or 24h respectively. Moreover, the pIC+NDX combination also provided complete protection for 2d post infection when HEL-299 cells were infected with MERS-CoV-GFP following a 24h pretreatment with pIC+NDX. The significance of this study is two-fold. Firstly, it was revealed that HEL-299 cells can effectively be used as an IFN-competent model system for in vitro analysis of MERS-CoV. Secondly, pIC+NDX acts as a powerful inducer of type I IFNs in HEL-299, to levels that provide complete protection against coronavirus replication. This suggests an exciting and novel area of investigation for antiviral therapies that utilize innate immune stimulants. The results of this study will help to expand the range of available tools scientists have to investigate, and thus further understand, human coronaviruses.

Are higher antibody levels against seasonal human coronaviruses associated with a more robust humoral immune response after SARS-CoV-2 vaccination?

The SARS-CoV-2 belongs to the coronavirus family, which also includes common endemic coronaviruses (HCoVs). We hypothesized that immunity to HCoVs would be associated with stronger immunogenicity from SARS-CoV-2 vaccines. The study included samples from the COSRIP observational cohort study of adult paramedics in Canada. Participants provided blood samples, questionnaire data, and results of COVID-19 testing. Samples were tested for anti-spike IgG against SARS-CoV-2, HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43 antigens. We first compared samples from vaccinated and unvaccinated participants, to determine which HCoV antibodies were affected by vaccination. We created scatter plots and performed correlation analysis to estimate the extent of the linear relationship between HCoVs and SARS-CoV-2 anti-spike antibodies. Further, using adjusted log-log multiple regression, we modeled the association between each strain of HCoV and SARS-CoV-2 antibodies. Of 1510 participants (mean age of 39 years), 94 (6.2%) had a history of COVID-19. There were significant differences between vaccinated and unvaccinated participant in anti-spike antibodies to HCoV-HKU1, and HCoV-OC43; however, levels for HCoV-229E and HCoV-NL63 were similar (suggesting that vaccination did not affect these baseline values). Among vaccinated individuals without prior COVID-19 infection, SARS-COV-2 anti-spike IgG demonstrated a weak positive relationship between both HCoV-229E (r = 0.11) and HCoV-NL63 (r = 0.12). From the adjusted log-log multiple regression model, higher HCoV-229E and HCoV-NL63 anti-spike IgG antibodies were associated with increased SARS-COV-2 anti-spike IgG antibodies. Vaccination appears to result in measurable increases in HCoV-HKU1, and HCoV-OC43 IgG levels. Anti-HCoV-229E and HCoV-NL63 antibodies were unaffected by vaccination, and higher levels were associated with significantly higher COVID-19 vaccine-induced SARS-COV-2 antibodies.

Structural insights into the binding of SARS-CoV-2, SARS-CoV, and hCoV-NL63 spike receptor-binding domain to horse ACE2.

Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus (hCoV)-NL63 utilize ACE2 as the functional receptor for cell entry, which leads to zoonotic infection. Horses (Equus caballus) attracted our attention because the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV-2-related coronaviruses bind equine ACE2 (eACE2) with high affinity. Here we show that eACE2 binds the RBDs of these three coronaviruses and also SARS-CoV-2 variants but with lower affinities compared with human ACE2 (hACE2). Structural analysis and mutation assays indicated that eACE2-H41 accounts for the lower binding affinity of eACE2 to the RBDs of SARS-CoV-2 variants (Alpha, Beta, and Gamma), SARS-CoV, and hCoV-NL63. Pseudovirus infection assays showed that the SARS-CoV-2 Delta strain (B.1.617.2) displayed a significantly increased infection efficiency in eACE2-expressing HeLa cells. Our results reveal the molecular basis of eACE2 binding to the RBDs of SARS-CoV, SARS-CoV-2, and hCoV-NL63, which provides insights into the potential animal transmission of these ACE2-dependent coronaviruses.

The Flexible, Extended Coil of the PDZ-Binding Motif of the Three Deadly Human Coronavirus E Proteins Plays a Role in Pathogenicity.

The less virulent human (h) coronaviruses (CoVs) 229E, NL63, OC43, and HKU1 cause mild, self-limiting respiratory tract infections, while the more virulent SARS-CoV-1, MERS-CoV, and SARS-CoV-2 have caused severe outbreaks. The CoV envelope (E) protein, an important contributor to the pathogenesis of severe hCoV infections, may provide insight into this disparate severity of the disease. We, therefore, generated full-length E protein models for SARS-CoV-1 and -2, MERS-CoV, HCoV-229E, and HCoV-NL63 and docked C-terminal peptides of each model to the PDZ domain of the human PALS1 protein. The PDZ-binding motif (PBM) of the SARS-CoV-1 and -2 and MERS-CoV models adopted a more flexible, extended coil, while the HCoV-229E and HCoV-NL63 models adopted a less flexible alpha helix. All the E peptides docked to PALS1 occupied the same binding site and the more virulent hCoV E peptides generally interacted more stably with PALS1 than the less virulent ones. We hypothesize that the increased flexibility of the PBM in the more virulent hCoVs facilitates more stable binding to various host proteins, thereby contributing to more severe disease. This is the first paper to model full-length 3D structures for both the more virulent and less virulent hCoV E proteins, providing novel insights for possible drug and/or vaccine development.

Seasonality of Common Human Coronaviruses, United States, 2014-20211.

The 4 common types of human coronaviruses (HCoVs)-2 alpha (HCoV-NL63 and HCoV-229E) and 2 beta (HCoV-HKU1 and HCoV-OC43)-generally cause mild upper respiratory illness. Seasonal patterns and annual variation in predominant types of HCoVs are known, but parameters of expected seasonality have not been defined. We defined seasonality of HCoVs during July 2014-November 2021 in the United States by using a retrospective method applied to National Respiratory and Enteric Virus Surveillance System data. In the 6 HCoV seasons before 2020-21, season onsets occurred October 21-November 12, peaks January 6-February 13, and offsets April 18-June 27; most (>93%) HCoV detection was within the defined seasonal onsets and offsets. The 2020-21 HCoV season onset was 11 weeks later than in prior seasons, probably associated with COVID-19 mitigation efforts. Better definitions of HCoV seasonality can be used for clinical preparedness and for determining expected patterns of emerging coronaviruses.

Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells.

Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.