Middle East Respiratory Syndrome Coronavirus Research Articles

Structural basis for human DPP4 receptor recognition by a pangolin MERS-like coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) and the pangolin MERS-like coronavirus MjHKU4r-CoV-1 employ dipeptidyl peptidase 4 (DPP4) as an entry receptor. MjHKU4r-CoV-1 could infect transgenic mice expressing human DPP4. To understand the mechanism of MjHKU4r-CoV-1 entry into cells, we determined the crystal structures of the receptor binding domain (RBD) of MjHKU4r-CoV-1 spike protein bound to human DPP4 (hDPP4) and Malayan pangolin DPP4 (MjDPP4), respectively. The overall hDPP4-binding mode of MjHKU4r-CoV-1 RBD is similar to that of MERS-CoV RBD. MjHKU4r-CoV-1 RBD shows higher binding affinity to hDPP4 compared to the bat MERS-like coronavirus Ty-BatCoV-HKU4. Via swapping residues between MjHKU4r-CoV-1 RBD and Ty-BatCoV-HKU4 RBD, we identified critical determinants on MjHKU4r-CoV-1 that are responsible for virus usage of hDPP4. Our study suggests that MjHKU4r-CoV-1 is more adapted to the human receptor compared to the bat HKU4 coronavirus and highlights the potential of virus emergence into the human population.

An Immunoinformatic Approach for Identifying and Designing Conserved Multi-Epitope Vaccines for Coronaviruses

Background/Objectives: The COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has exposed the vulnerabilities and unpreparedness of the global healthcare system in dealing with emerging zoonoses. In the past two decades, coronaviruses (CoV) have been responsible for three major viral outbreaks, and the likelihood of future outbreaks caused by these viruses is high and nearly inevitable. Therefore, effective prophylactic universal vaccines targeting multiple circulating and emerging coronavirus strains are warranted. Methods: This study utilized an immunoinformatic approach to identify evolutionarily conserved CD4+ (HTL) and CD8+ (CTL) T cells, and B-cell epitopes in the coronaviral spike (S) glycoprotein. Results: A total of 132 epitopes were identified, with the majority of them found to be conserved across the bat CoVs, pangolin CoVs, endemic coronaviruses, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Their peptide sequences were then aligned and assembled to identify the overlapping regions. Eventually, two major peptide assemblies were derived based on their promising immune-stimulating properties. Conclusions: In this light, they can serve as lead candidates for universal coronavirus vaccine development, particularly in the search for pan-coronavirus multi-epitope universal vaccines that can confer protection against current and novel coronaviruses.

Comparative profile of the human coronaviruses

The coronavirus disease (COVID-19), a respiratory illness is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is a strain of human corona virus (HCoV) and is infectious in humans. Seven types of human coronaviruses, namely, human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus SARS-CoV-1, human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), middle east respiratory syndrome-related coronavirus (MERS-CoV) and SARS-CoV-2 have been identified so far. Almost all these viruses spread between people through respiratory droplets produced from coughs or sneezes; the target cells being the epithelial cells of the respiratory tract. In this review, a brief comparative study of these pathogenic CoVs in humans is done which enables an understanding of the evolution in these diseases over a span of almost a century. This review will provide an insight into all HCoVs identified till date.

Rapid-response RNA-fluorescence in situ hybridization (FISH) assay platform for coronavirus antiviral high-throughput screening

Over the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of a novel alphacoronavirus canine CoV (CCoV-HuPn2018) in human patients in Malaysia underscores the potential for crossover infections to humans. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools that can be quickly implemented to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence in situ hybridization (FISH) imaging assay as a 384-well, high-throughput rapid response platform for antiviral drug discovery. RNA-FISH is a powerful tool to visualize specific mRNA in cultured cells using a high-content imaging platform. The flexibility of RNA-FISH probe sets allows for the rapid design of viral genome-specific probes, enabling in vitro assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors. Screening of 170 antiviral compounds in concentration-response demonstrates a strong correlation between the RNA-FISH assay and an immunofluorescence assay (IFA) for both human coronaviruses HCoV-OC43 and HCoV-229E. Additionally, we successfully applied this methodology in the context of CCoV strain 1–71, proving rapid development and deployment, opening new avenues for the evaluation of antiviral drugs to potential future emerging threats.

Acquisition of a multibasic cleavage site does not increase MERS-CoV entry into Calu-3 human lung cells.

Human-to-human transmission of the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) is currently inefficient. However, there is concern that the virus might mutate and thereby increase its transmissibility and thus pandemic potential. The pandemic SARS-CoV-2 depends on a highly cleavable furin motif at the S1/S2 site of the viral spike (S) protein for efficient lung cell entry, transmission, and pathogenicity. Here, by employing pseudotyped particles, we investigated whether augmented cleavage at the S1/S2 site also increases MERS-CoV entry into Calu-3 human lung cells. We report that polymorphism T746K at the S1/S2 cleavage site or optimization of the furin motif increases S protein cleavage but not lung cell entry. These findings suggest that, unlike what has been reported for SARS-CoV-2, a highly cleavable S1/S2 site might not augment MERS-CoV infectivity for human lung cells.IMPORTANCEThe highly cleavable furin motif in the spike protein is required for robust lung cell entry, transmission, and pathogenicity of SARS-CoV-2. In contrast, it is unknown whether optimization of the furin motif in the spike protein of the pre-pandemic MERS-CoV increases lung cell entry and allows for robust human-human transmission. The present study indicates that this might not be the case. Thus, neither a naturally occurring polymorphism that increased MERS-CoV spike protein cleavage nor artificial optimization of the cleavage site allowed for increased spike-protein-driven entry into Calu-3 human lung cells.

Broad cross neutralizing antibodies against sarbecoviruses generated by SARS-CoV-2 infection and vaccination in humans

The outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 highlight the need for countermeasures to prevent future coronavirus pandemics. Given the unpredictable nature of spillover events, preparing antibodies with broad coronavirus-neutralizing activity is an ideal proactive strategy. Here, we investigated whether SARS-CoV-2 infection and vaccination could provide cross-neutralizing antibodies (nAbs) against zoonotic sarbecoviruses. We evaluated the cross-neutralizing profiles of plasma and monoclonal antibodies constructed from B cells from coronavirus disease 2019 (COVID-19) convalescents and vaccine recipients; against sarbecoviruses originating from bats, civets, and pangolins; and against SARS-CoV-1 and SARS-CoV-2. We found that the majority of individuals with natural infection and vaccination elicited broad nAb responses to most tested sarbecoviruses, particularly to clade 1b viruses, but exhibited very low cross-neutralization to SARS-CoV-1 in both natural infection and vaccination, and vaccination boosters significantly augmented the magnitude and breadth of nAbs to sarbecoviruses. Of the nAbs, several exhibited neutralization activity against multiple sarbecoviruses by targeting the spike receptor-binding domain (RBD) and competing with angiotensin-converting enzyme 2 (ACE2) binding. SCM12-61 demonstrated exceptional potency, with half-maximal inhibitory concentration (IC50) values of 0.001–0.091 μg/mL against tested sarbecoviruses; while VSM9-12 exhibited remarkable cross-neutralizing breadth against sarbecoviruses and SARS-CoV-2 Omicron subvariants, highlighting the potential of these two nAbs in combating sarbecoviruses and SARS-CoV-2 Omicron subvariants. Collectively, our findings suggest that vaccination with an ancestral SARS-CoV-2 vaccine, in combination with broad nAbs against sarbecoviruses, may provide a countermeasure for preventing further sarbecovirus outbreaks in humans.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host’s antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host’s innate antiviral immunity and thus provides insights into viral pathogenesis.

Mutation of a highly conserved isoleucine residue in loop 2 of several β-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental for replication.

The conserved coronavirus (CoV) macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases and is critical for CoV replication and pathogenesis. As such, Mac1 is a potential therapeutic target for CoV-induced disease. However, we lack a basic knowledge of how several residues in its ADP-ribose binding pocket contribute to its biochemical and virological functions. We engineered mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and viruses for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Interestingly, a Mac1 isoleucine-to-alanine mutant protein had enhanced ADP-ribose binding which proved to be detrimental for virus replication, indicating that this isoleucine controls ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

Implementation and validation of MICaFVi: A highly efficient nanotechnology-based method for coronaviruses detection

Coronaviruses have emerged as a significant public health concern due to the global impact of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which cause COVID-19. The development of sensitive and accurate detection methods is critical for early diagnosis, disease management, and outbreak control. In a previous study, we developed and optimized a nanobased detection methodology called Magnetic Immuno-Capture Followed by Flow Virometry (MICaFVi) using virus-mimicking silica nanoparticles and MERS-CoV/SARS-CoV-2 pseudoviral particles. In the present study, we have extended this methodology to evaluate its specificity and sensitivity for detecting wild-type MERS-CoV and SARS-CoV-2 in human and camel samples. Our results demonstrated that MICaFVi successfully detected MERS-CoV and SARS-CoV-2 viruses with high sensitivity and specificity, although it showed reduced performance for samples with Ct values of 30 or lower compared to qPCR. Despite some limitations in detection speed and sensitivity, MICaFVi represents a significant advancement in diagnostic methodologies by combining nanotechnology with flow cytometry. Additionally, we adapted the MICaFVi methodology to simultaneously detect MERS-CoV and SARS-CoV-2 in a single multiplex assay. The successful implementation of this advanced detection approach has important implications for improving early detection, surveillance, and control of both current and future viral or bacterial pandemics. Our results underscore the potential of MICaFVi as a valuable tool for monitoring the spread of these viruses and highlight its role in advancing diagnostic technologies. This extension of our earlier work offers new insights into the application of nanotechnology and flow cytometry for viral detection.

MERS-CoV Infection and Its Impact on the Expression of TSLP Cytokine and IgG Antibodies: An In Vivo and In Vitro Study.

Thymic stromal lymphopoietin (TSLP) is a proinflammatory cytokine produced by epithelial cells that is involved in the activation of allergic disorders. To date, no study has examined TSLP induction during Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Herein, we aimed to study the effects of the recombinant spike protein of MERS-CoV on TSLP production. Additionally, the effects of recombinant human TSLP (rhTSLP) on B cell survival and antibody production were investigated. B cells were separated using the Human B Cell Enrichment Kit, and B cell survival was measured using the WST-1 Assay Kit. Enzyme-linked immunosorbent assay (ELISA) was used to measure TSLP levels in the sera of both MERS-CoV-infected (n=4; median age, 53 years) and healthy individuals (n=5; median age, 35 years). We showed that the group of infected patients had significantly higher levels of TSLP than healthy controls (37.6 pg/mL vs 19.8 pg/mL, *p<0.05). The levels of TSLP in A549 cells were remarkably increased after 48h of stimulation with recombinant full-length spike protein (rSP) (32.2 pg/mL, p=0.01). B cell survival was greatly enhanced by rhTSLP alone or in combination with rSP (0.02 vs 0.046, and 0.045; **p<0.01, respectively). Our data also showed a significant synergistic effect of rhTSLP and rSP on the augmented response of IgG antibodies against the spike protein of MERS-CoV compared with unstimulated cells (0.156 vs 0.22; *p<0.05). TSLP production is induced in vivo after MERS-CoV infection and in vitro after treatment with the rSP of MERS-CoV, which has a significant effect on the survival of B cells. Our data suggest that TSLP can be used as a strong mucosal adjuvant for vaccine development against MERS-CoV infection. However, further investigation is required to study the functional role of TSLP in MERS-CoV infection.

The CoV-Y domain of SARS-CoV-2 Nsp3 interacts with BRAP to stimulate NF-κB signaling and induce host inflammatory responses

Non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome. It consists of multiple domains that perform critical functions during the viral life cycle. CoV-Y is the most C-terminal domain of Nsp3, and it exhibits evolutionary conservation across diverse CoVs; however, the exact biological function of CoV-Y remains unclear. Here, we determined the crystal structure of CoV-Y of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp3 using the single-wavelength anomalous diffraction method. We revealed the interaction between CoV-Y and the host BRCA1-associated protein (BRAP) using immunoprecipitation-mass spectrometry experiments. This interaction was subsequently confirmed in cellular assays, and the precise binding-regions between these two proteins were clarified. We found that this interaction is conserved in SARS-CoV and Middle East respiratory syndrome coronavirus. Next, we demonstrated that CoV-Y enhances IκBα and IκBβ phosphorylation and promotes the nuclear translocation of the downstream NF-κB members p50 and p65 through binding to BRAP. The CoV-Y–BRAP interaction can upregulate the transcript levels of the host inflammatory cytokines. Overall, our findings illustrate the biological function of CoV-Y for the first time and provide novel insights into coronavirus regulation of host inflammatory responses, as well as a possible target for antiviral drug development.

Whole genome characteristics of hedgehog coronaviruses from Poland and analysis of the evolution of the Spike protein for its interspecies transmission potential

BackgroundThe hedgehogs have been recently identified as possible reservoir of Middle East respiratory syndrome coronavirus like (MERS-CoV-like). These viruses were classified as a distinct Betacoronavirus erinacei (BCoV-Eri) species within the MerBCoV-Eriirus subgenus. As coronaviruses are known for their ability to jump between different hosts, including humans, this can pose a particular threat to people in direct contact with hedgehogs, such as those working at animal asylums. Our previous studies have shown the presence of BCoV-Eri strains in animals collected in the wildlife rehabilitation centre. This study aimed to investigate the presence of CoV in subsequent hedgehogs collected from the urban area of Poland and their molecular characteristics.ResultsMonitoring for the presence of coronavirus infection in hedgehogs revealed five positive individuals. The presence of BCoV-Eri was found in a total of 20% of animals tested. Our analyses revealed no correlation between CoVs positivity and animal health conditions but a higher probability of such infection in juveniles and females. The whole genome of two Polish Hedgehog coronavirus 1 strains were sequenced and compared with available counterparts from European and Asian countries. Phylogenetic analysis showed that both CoV strains formed common cluster with other similar MerBCoV-Eriirus, but they were also found to be genetically variable and most changes in the S protein were identified. Our analysis revealed that some S protein sites of the Hedgehog coronavirus 1 strains evolved under positive selection pressure and of five such sites, three are in the S1 region while the other two in the S2 region of the Spike.ConclusionsBCoV-Eri is to some extent prevalent in wildlife asylums in Poland. Given that the S protein of BCoVs-Eri is highly variable and that some sites of this protein evolve under positive selection pressure, these strains could potentially acquire a favourable feature for cross-species transmission. Consequently, the threat to humans working in such asylums is particularly high. Adequate biosecurity safeguards, but also human awareness of such risks, are therefore essential.

COVID-19 mutations: An overview.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the genus Beta coronavirus and the family of Coronaviridae. It is a positive-sense, non-segmented single-strand RNA virus. Four common types of human coronaviruses circulate globally, particularly in the fall and winter seasons. They are responsible for 10%-30% of all mild upper respiratory tract infections in adults. These are 229E, NL63 of the Alfacoronaviridae family, OC43, and HKU1 of the Betacoronaviridae family. However, there are three highly pathogenic human coronaviruses: SARS-CoV-2, Middle East respiratory syndrome coronavirus, and the latest pandemic caused by the SARS-CoV-2 infection. All viruses, including SARS-CoV-2, have the inherent tendency to evolve. SARS-CoV-2 is still evolving in humans. Additionally, due to the development of herd immunity, prior infection, use of medication, vaccination, and antibodies, the viruses are facing immune pressure. During the replication process and due to immune pressure, the virus may undergo mutations. Several SARS-CoV-2 variants, including the variants of concern (VOCs), such as B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617/B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron) have been reported from various parts of the world. These VOCs contain several important mutations; some of them are on the spike proteins. These mutations may lead to enhanced infectivity, transmissibility, and decreased neutralization efficacy by monoclonal antibodies, convalescent sera, or vaccines. Mutations may also lead to a failure of detection by molecular diagnostic tests, leading to a delayed diagnosis, increased community spread, and delayed treatment. We searched PubMed, EMBASE, Covariant, the Stanford variant Database, and the CINAHL from December 2019 to February 2023 using the following search terms: VOC, SARS-CoV-2, Omicron, mutations in SARS-CoV-2, etc. This review discusses the various mutations and their impact on infectivity, transmissibility, and neutralization efficacy.

Prevalence of COVID-19 Incidence Rates in Hypertension Patients at Ibnu Sina Hospital Makassar in 2020

The COVID-19 virus, which is caused by SARS-CoV-2, is a global health problem that affects many nations. Similar to the H7N9, SARS, and MERS viruses, COVID-19 can be more severe in hypertensive individuals. With a prevalence of 50.1% among COVID-19 patients, hypertension is a significant comorbidity that increases the risk of death by 1.37 times due to the increased number of ACE2 receptors that facilitate the virus's spread. This study uses a descriptive cross-sectional design with secondary data from medical records of COVID-19 patients with hypertension at Rumah Sakit Ibnu Sina Makassar. Data were collected through total sampling after obtaining permission from the hospital and then processed and analyzed descriptively. The study shows that the majority of COVID-19 patients with hypertension are aged 65 and over (33.4%). Male patients are slightly more prevalent (53%) compared to females (47%), possibly due to genetic factors affecting the immune system. Seventy-three percent of patients adhered to hypertension treatment, while 27% did not. The function of the ACE-2 enzyme, which is also affected by antihypertensive treatment, may have an impact on the relationship between COVID-19 and hypertension. These findings emphasize the significance of treating the elderly and remaining on treatment in order to reduce the likelihood of contracting COVID-19. The survey derives that the vast majority of Covid patients with hypertension are developed 65 and over (33.4%), with an insignificantly higher inescapability of folks (53.3%) diverged from females. Adherence to hypertension treatment is high, with 73.3% of patients following their prescribed regimen.

Resistance to SARS-CoV-2 infection in camelid nasal organoids is associated with lack of ACE2 expression

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects a variety of animal species. Susceptibility to SARS-CoV-2 is primarily determined by the utilization of the viral receptor, ACE2. SARS-CoV-2 can utilize a broad range of animal ACE2 isoforms in vitro, including the ACE2 from various camelid species. However, experimental infection of these animals does not lead to productive infection or seroconversion. In this study, we investigate the susceptibility of camelids to SARS-CoV-2 using novel well-differentiated camelid nasal organoids. We show that camelid nasal organoids are highly susceptible to Middle East respiratory syndrome coronavirus (MERS-CoV) infection, but not to infection with different SARS-CoV-2 variants (614G, BA.1 or EG.5.1.1). All viruses efficiently infected human airway organoids. Immunohistochemistry analysis revealed the absence of ACE2 on camelid nasal organoids and dromedary camel upper respiratory tract. In contrast, DPP4 was expressed in both camelid nasal organoids and the camel upper respiratory tract, which correlates with MERS-CoV infection. This study indicates that the camelid upper respiratory tract lacks expression of ACE2, which is associated with resistance to SARS-CoV-2 infection.

Construction of pseudotyped human coronaviruses and detection of pre-existing antibodies in the human population

In order to clarify the pre-exist immunity background of different human coronaviruses (HCoV), this study investigated the positive rate of spike (S) protein antibodies of HCoV, including HCoV- severe acute respiratory syndrome (SARS) −associated coronavirus (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43, before and after the Coronavirus Disease 2019 (COVID-19) outbreak. We utilized pseudotyped virus-based neutralization assays (PBNA) or enzyme-linked immunosorbent assays (ELISA) to detect antibody levels against HCoV in serum samples collected in 2009–2010 and 2023. The PBNA results showed that neutralizing antibodies against SARS-CoV-1 and the MERS-CoV were negative. In the serum samples from 2009 to 2010, neutralizing antibodies against SARS-CoV-2 (D614G) were negative, whereas in the serum samples from 2023, 73 samples (73 %) showed neutralizing reactions with the SARS-CoV-2 D614G strain, 96 samples (96 %) with the BA.5 strain, and 91 samples (91 %) with the BF.7 strain. Among pre-COVID-19 samples, 33 % (33/100) showed neutralizing reactions with HCoV-229E and 63 % (63/100) with HCoV-NL63. Among post-COVID-19 samples, 50 % (50/100) showed neutralizing reactions with HCoV-229E and 49 % (49/100) with HCoV-NL63. Due to the different receptors of alpha coronavirus genus compared to other beta coronavirus genus, neutralizing antibodies against HCoV-OC43 and HCoV-HKU1 virus cannot be detected by constructing corresponding pseudotyped virus. Binding antibodies against HCoV-OC43 and HCoV-HKU1 virus were detected using ELISA. The results revealed that among pre-COVID-19 samples, 83 % (83/100) and 45 % (45/100) had binding activity with HCoV-OC43 and HCoV-HKU1, respectively. Among post-COVID-19 samples, 100 % (100/100) and 81 % (81/100) had binding activity with HCoV-OC43 and HCoV-HKU1, respectively.

Differential regulation of viral entry-associated genes modulated by inflammatory cytokines in the nasal epithelium.

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections.

The Role of Biosensors in Detection of SARS-CoV-2: State-of-the-Art and Future Prospects

Abstract: The world is fighting a pandemic so grave that perhaps it has never been witnessed before; COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2). As of August 31st, 2022, the WHO declared the total number of confirmed cases was 599,825,400, with 6,469,458 confirmed deaths from 223 countries under the scourge of this deadly virus. The SARS-CoV-2 is a β-coronavirus, which is an enveloped non-segmented positive- sense RNA virus. It is a close relative of the SARS and MERS viruses and has probably entered humans through bats. Human-to-human transmission is very rapid. People in contact with the patient or even the carriers became infected, leading to a widespread chain of contamination. We are presenting a mini-review on the role of biosensors in detecting SARS-CoV-2. Biosensors have been used for a very long time for viral detection and can be utilized for the prompt detection of the novel coronavirus. This article aims to provide a mini-review on the application of biosensors for the detection of the novel coronavirus with a focus on costeffective paper-based sensors, nanobiosensors, Field effect transistors (FETs), and lab-on-chip integrated platforms. Background: Biosensors have played a crucial role in viral detection for a long time. Objective: To present a comprehensive review of the biosensor application in SARS-Cov-2. Method: We have presented state-of-the-art work in the biosensors field for SARS-Cov-2 detection. Results: The biosensors presented here provide an innovative approach to detecting SARS-Cov- 2 infections early. Conclusion: Biosensors have tremendous potential in accurately detecting viral infections in pandemics requiring rapid screening.

Regulation viral RNA transcription and replication by higher-order RNA structures within the nsp1 coding region of MERS coronavirus

Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3′ untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA.

Transmission dynamics of MERS-CoV in a transgenic human DPP4 mouse model

Since 2002, three novel coronavirus outbreaks have occurred: severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. A better understanding of the transmission potential of coronaviruses will result in adequate infection control precautions and an early halt of transmission within the human population. Experiments on the stability of coronaviruses in the environment, as well as transmission models, are thus pertinent.Here, we show that transgenic mice expressing human DPP4 can be infected with MERS-CoV via the aerosol route. Exposure to 5 × 106 TCID50 and 5 × 104 TCID50 MERS-CoV per cage via fomites resulted in transmission in 15 out of 20 and 11 out of 18 animals, respectively. Exposure of sentinel mice to donor mice one day post inoculation with 105 TCID50 MERS-CoV resulted in transmission in 1 out of 38 mice via direct contact and 4 out of 54 mice via airborne contact. Exposure to donor mice inoculated with 104 TCID50 MERS-CoV resulted in transmission in 0 out of 20 pairs via direct contact and 0 out of 5 pairs via the airborne route. Our model shows limited transmission of MERS-CoV via the fomite, direct contact, and airborne routes. The hDPP4 mouse model will allow assessment of the ongoing evolution of MERS-CoV in the context of acquiring enhanced human-to-human transmission kinetics and will inform the development of other transmission models.