Current methods for diagnosis of human coronaviruses: pros and cons.

The current global fight against coronavirus disease (COVID-19) to flatten the transmission curve is put forth by the World Health Organization (WHO) as there is no immediate diagnosis or cure for COVID-19 so far. In order to stop the spread, researchers worldwide are working around the clock aiming to develop reliable tools for early diagnosis of severe acute respiratory syndrome (SARS-CoV-2) understanding the infection path and mechanisms. Currently, nucleic acid-based molecular diagnosis (real-time reverse transcription polymerase chain reaction (RT-PCR) test) is considered the gold standard for early diagnosis of SARS-CoV-2. Antibody-based serology detection is ineffective for the purpose of early diagnosis, but a potential tool for serosurveys, providing people with immune certificates for clearance from COVID-19 infection. Meanwhile, there are various blooming methods developed these days. In this review, we summarise different types of coronavirus discovered which can be transmitted between human beings. Methods used for diagnosis of the discovered human coronavirus (SARS, MERS, COVID-19) including nucleic acid detection, gene sequencing, antibody detection, antigen detection, and clinical diagnosis are presented. Their merits, demerits and prospects are discussed which can help the researchers to develop new generation of advanced diagnostic tools for accurate and effective control of human coronavirus transmission in the communities and hospitals.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors.

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.

Focus on Middle East respiratory syndrome coronavirus (MERS-CoV).

Since the first case of human infection by the Middle East respiratory syndrome coronavirus (MERS-CoV) in Saudi Arabia in June 2012, more than 2260 cases of confirmed MERS-CoV infection and 803 related deaths have been reported since the 16th of October 2018. The vast majority of these cases (71%) were reported in Saudi Arabia but the epidemic has now spread to 27 countries and has not ceased 6 years later, unlike SARS-CoV that disappeared a little less than 2 years after emerging. Due to the high fatality rate observed in MERS-CoV infected patients (36%), much effort has been put into understanding the origin and pathophysiology of this novel coronavirus to prevent it from becoming endemic in humans. This review focuses in particular on the origin, epidemiology and clinical manifestations of MERS-CoV, as well as the diagnosis and treatment of infected patients. The experience gained over recent years on how to manage the different risks related to this kind of epidemic will be key to being prepared for future outbreaks of communicable disease.

Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.

Antigen Capture Enzyme-Linked Immunosorbent Assay for Detecting Middle East Respiratory Syndrome Coronavirus in Humans.

The Middle East respiratory syndrome (MERS) is the second novel zoonotic disease infecting humans caused by coronavirus (CoV) in this century. To date, more than 2200 laboratory-confirmed human cases have been identified in 27 countries, and more than 800 MERS-CoV associated deaths have been reported since its outbreak in 2012. Rapid laboratory diagnosis of MERS-CoV is the key to successful containment and prevention of the spread of infection. Though the gold standard for diagnosing MERS-CoV infection in humans is still nucleic acid amplification test (NAAT) of the up-E region, an antigen capture enzyme-linked immunosorbent assay (ELISA) could also be of use for early diagnosis in less developed locations. In the present method, a step-by-step guide to perform a MERS-CoV nucleocapsid protein (NP) capture ELISA using two NP-specific monoclonal antibodies is provided for readers to develop their in-house workflow or diagnostic kit for clinical use and for mass-screening project of animals (e.g., dromedaries and bats) to better understand the spread and evolution of the virus.

Evaluating MERS-CoV Entry Pathways.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic pathogen with a broad host range. The extent of MERS-CoV in nature can be traced to its adaptable cell entry steps. The virus can bind host-cell carbohydrates as well as proteinaceous receptors. Following receptor interaction, the virus can utilize diverse host proteases for cleavage activation of virus-host cell membrane fusion and subsequent genome delivery. The fusion and genome delivery steps can be completed at variable times and places, either at or near cell surfaces or deep within endosomes. Investigators focusing on the CoVs have developed several methodologies that effectively distinguish these different cell entry pathways. Here we describe these methods, highlighting virus-cell entry factors, entry inhibitors, and viral determinants that specify the cell entry routes. While the specific methods described herein were utilized to reveal MERS-CoV entry pathways, they are equally suited for other CoVs, as well as other protease-dependent viral species.

Quantification of the Middle East Respiratory Syndrome-Coronavirus RNA in Tissues by Quantitative Real-Time RT-PCR.

Since the emergence of the Middle East respiratory syndrome-coronavirus (MERS-CoV) in 2012, more than 2280 confirmed human infections and 800 associated deaths had been reported to the World Health Organization. MERS-CoV is a single-stranded RNA virus that belongs to the Coronaviridae family. MERS-CoV infection leads to a variety of clinical outcomes in humans ranging from asymptomatic and mild infection to severe acute lung injury and multi-organ failure and death. To study the pathogenesis of MERS-CoV infection and development of medical countermeasures (MCMs) for MERS, a number of genetically modified mouse models have been developed, including various versions of transgenic mice expressing the human DPP4 viral receptor. Tracking and quantifying viral infection, among others, in permissive hosts is a key endpoint for studying MERS pathogenesis and evaluating the efficacy of selected MCMs developed for MERS. In addition to quantifying infectious progeny virus which requires high-containment biosafety level (BSL)-3 laboratory, here we outlined an established real-time quantitative RT-PCR (RT-qPCR)-based procedure to unequivocally quantify MERS-CoV-specific RNAs within the lungs of infected human DPP4 (hDPP4, transgenic (hDPP4 Tg) mice under a standard BSL-2 laboratory.

Molecular detections of coronavirus: current and emerging methodologies.

INTRODUCTION: Seven coronavirus species have been identified that can infect humans. While human coronavirus infections had been historically associated with only mild respiratory symptoms similar to the common cold, three coronaviruses identified since 2003, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, cause life-threatening severe respiratory syndromes. The coronavirus disease 2019 (COVID-19) caused by the highly transmissible SARS-CoV-2 has triggered a worldwide health emergency. Due to the lack of effective drugs and vaccination, rapid and reliable detection is of vital importance to control coronavirus epidemics/pandemics. AREA COVERED: A literature search was performed in Pubmed covering the detections and diagnostics of SARS, MERS and SARS-CoV-2. This review summarized the current knowledge of established and emerging methods for coronavirus detection. The characteristics of different diagnostic approaches were described, and the strengths and weaknesses of each method were analyzed and compared. In addition, future trends in the field of coronavirus detection were also discussed. EXPERT OPINION: Nucleic acid-based RT-PCR is the current golden-standard of coronavirus detection, while immunoassays provide history of coronavirus infection besides diagnostic information. Integrated high-throughput system holds the great potential and is the trend of future detection and diagnosis of virus infection.

Coronaviruses in Bats: A Review for the Americas.

The SARS-CoV-2 coronavirus is the focus of attention as it has caused more than three million human deaths globally. This and other coronaviruses, such as MERS-CoV, have been suggested to be related to coronaviruses that are hosted in bats. This work shows, through a bibliographic review, the frequency of detection of coronavirus in bats species of the Americas. The presence of coronavirus in bats has been examined in 25 investigations in 11 countries of the Americas between 2007 and 2020. Coronaviruses have been explored in 9371 individuals from 160 species of bats, and 187 coronavirus sequences have been deposited in GenBank distributed in 43 species of bats. While 91% of the coronaviruses sequences identified infect a single species of bat, the remainder show a change of host, dominating the intragenera change. So far, only Mex-CoV-6 is related to MERS-CoV, a coronavirus pathogenic for humans, so further coronavirus research effort in yet unexplored bat species is warranted.

Innate and Adaptive Immune Genes Associated with MERS-CoV Infection in Dromedaries.

The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.

Epidemiology and evolution of Middle East respiratory syndrome coronavirus, 2012-2020.

BACKGROUND: The ongoing transmission of the Middle East respiratory syndrome coronavirus (MERS-CoV) in the Middle East and its expansion to other regions are raising concerns of a potential pandemic. An in-depth analysis about both population and molecular epidemiology of this pathogen is needed. METHODS: MERS cases reported globally as of June 2020 were collected mainly from World Health Organization official reports, supplemented by other reliable sources. Determinants for case fatality and spatial diffusion of MERS were assessed with Logistic regressions and Cox proportional hazard models, respectively. Phylogenetic and phylogeographic analyses were performed to examine the evolution and migration history of MERS-CoV. RESULTS: A total of 2562 confirmed MERS cases with 150 case clusters were reported with a case fatality rate of 32.7% (95% CI: 30.9‒34.6%). Saudi Arabia accounted for 83.6% of the cases. Age of ≥ 65 years old, underlying conditions and ≥ 5 days delay in diagnosis were independent risk factors for death. However, a history of animal contact was associated with a higher risk (adjusted OR = 2.97, 95% CI: 1.10-7.98) among female cases < 65 years but with a lower risk (adjusted OR = 0.31, 95% CI: 0.18-0.51) among male cases ≥ 65 years old. Diffusion of the disease was fastest from its origin in Saudi Arabia to the east, and was primarily driven by the transportation network. The most recent sub-clade C5.1 (since 2013) was associated with non-synonymous mutations and a higher mortality rate. Phylogeographic analyses pointed to Riyadh of Saudi Arabia and Abu Dhabi of the United Arab Emirates as the hubs for both local and international spread of MERS-CoV. CONCLUSIONS: MERS-CoV remains primarily locally transmitted in the Middle East, with opportunistic exportation to other continents and a potential of causing transmission clusters of human cases. Animal contact is associated with a higher risk of death, but the association differs by age and sex. Transportation network is the leading driver for the spatial diffusion of the disease. These findings how this pathogen spread are helpful for targeting public health surveillance and interventions to control endemics and to prevent a potential pandemic.

The prevalence of MERS-CoV among military personnel and their families: A single-center study.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a Betacoronavirus that results in a severe fatal respiratory disease; however, it is also associated with mild inapparent infections. The western part of the Kingdom of Saudi Arabia (KSA) contains the holy places where millions of Muslims gathered from all over the world, all year round, with a high probability of mass disease transmission. The aim of this study was to estimate the prevalence of MERS-CoV among military personnel and their families during the period 2014-2019, in the western part of the KSA. A total of 35,203 sputum samples collected from patients with respiratory distress were screened for the presence of MERS-CoV using real-time reverse-transcription polymerase chain reaction in the examined patients. MERS-CoV infections were detected at a very low percentage in the examined patients. Only 42 of the examined subjects (0.12%) were found positive for MERS-CoV. Most infected cases (32/42) cases were detected in 2014, and the rest of the cases were reported in 2015-2019. The cases with fatal consequences (n = 20) were only detected in 2014. It was concluded that there is a very low prevalence of MERS-CoV infections among the military personnel and their families.

Animal Reservoirs and Hosts for Emerging Alphacoronaviruses and Betacoronaviruses.

The ongoing global pandemic caused by coronavirus disease has once again demonstrated the role of the family Coronaviridae in causing human disease outbreaks. Because severe acute respiratory syndrome coronavirus 2 was first detected in December 2019, information on its tropism, host range, and clinical manifestations in animals is limited. Given the limited information, data from other coronaviruses might be useful for informing scientific inquiry, risk assessment, and decision-making. We reviewed endemic and emerging infections of alphacoronaviruses and betacoronaviruses in wildlife, livestock, and companion animals and provide information on the receptor use, known hosts, and clinical signs associated with each host for 15 coronaviruses detected in humans and animals. This information can be used to guide implementation of a One Health approach that involves human health, animal health, environmental, and other relevant partners in developing strategies for preparedness, response, and control to current and future coronavirus disease threats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) coinfection: A unique case series.

INTRODUCTION: The emergence of the Severe Acute Respiratory Syndrome Coroanvirus 2 (SARS-CoV-2) had raised possibilities of coinfection with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in countries were these two viruses were reported. In this study, we describe the clinical presentation and demographics of eight patients who were coinfected with SARS-CoV-2 and MERS-CoV. MATERIALS AND METHODS: This is a case series of hospitalized patients admitted to intensive care units (ICUs). We collected demographics, underlying conditions, presenting symptoms and clinical outcome from the patients' medical records. RESULTS: During the study period from March 14, 2020 to October 19, 2020, there was a total of 67 SARS-CoV-2 ICU admitted patients who underwent simultaneous SARS-CoV-2 and MERS-CoV testing by PCR. Of those patients, 8 (12%) tested positive for both SARS-CoV-2 and MERS-CoV. There were 6 (75%) males, the mean age ± SD was 44.4 ± 11.8 years, and 7 (87.5%) were obese. Of the patients, 7 (87.5%) were non-smokers, 1 (12.5%) had diabetes mellitus, 1 (12.5%) had heart failure, and 1 (12.5%) had been on anti-platelet therapy. The mean hospital length of stay (LOS) was 21.1 ± 11.6 days and the average ICU LOS was 10.9 ± 6.03 days. All patients received supportive therapy and all were treated with corticosteroid. Of all the patients, 4 (50%) were discharged home and 3 (37.5%) died. CONCLUSION: This case series is an important addition to the medical knowledge as it showed the interaction of the coinfection of SARS-CoV-2 and MERS-CoV.

Design, synthesis and biological evaluation of 2-aminoquinazolin-4(3H)-one derivatives as potential SARS-CoV-2 and MERS-CoV treatments.

Despite the rising threat of fatal coronaviruses, there are no general proven effective antivirals to treat them. 2-Aminoquinazolin-4(3H)-one derivatives were newly designed, synthesized, and investigated to show the inhibitory effects on SARS-CoV-2 and MERS-CoV. Among the synthesized derivatives, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (9g) and 2-((3,5-dichlorophenyl)amino)-5-hydroxyquinazolin-4 (3H)-one (11e) showed the most potent anti-SARS-CoV-2 activities (IC50 < 0.25 µM) and anti-MERS-CoV activities (IC50 < 1.1 µM) with no cytotoxicity (CC50 > 25 µM). In addition, both compounds showed acceptable results in metabolic stabilities, hERG binding affinities, CYP inhibitions, and preliminary PK studies.

Testing early warning and response systems through a full-scale exercise in Vietnam.

BACKGROUND: Simulation exercises can functionally validate World Health Organization (WHO) International Health Regulations (IHR 2005) core capacities. In 2018, the Vietnam Ministry of Health (MOH) conducted a full-scale exercise (FSX) in response to cases of severe viral pneumonia with subsequent laboratory confirmation for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to evaluate the country's early warning and response capabilities for high-risk events. METHODS: An exercise planning team designed a complex fictitious scenario beginning with one case of severe viral pneumonia presenting at the hospital level and developed all the materials required for the exercise. Actors, controllers and evaluators were trained. In August 2018, a 3-day exercise was conducted in Quang Ninh province and Hanoi city, with participation of public health partners at the community, district, province, regional and national levels. Immediate debriefings and an after-action review were conducted after all exercise activities. Participants assessed overall exercise design, conduction and usefulness. RESULTS: FSX findings demonstrated that the event-based surveillance component of the MOH surveillance system worked optimally at different administrative levels. Detection and reporting of signals at the community and health facility levels were appropriate. Triage, verification and risk assessment were successfully implemented to identify a high-risk event and trigger timely response. The FSX identified infection control, coordination with internal and external response partners and process documentation as response challenges. Participants positively evaluated the exercise training and design. CONCLUSIONS: This exercise documents the value of exercising surveillance capabilities as part of a real-time operational scenario before facing a true emergency. The timing of this exercise and choice of disease scenario was particularly fortuitous given the subsequent appearance of COVID-19. As a result of this exercise and subsequent improvements made by the MOH, the country may have been better able to deal with the emergence of SARS-CoV-2 and contain it.

Clinical and radiological findings of adult hospitalized patients with community-acquired pneumonia from SARS-CoV-2 and endemic human coronaviruses.

Endemic human coronaviruses (HCoVs) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are members of the family Coronaviridae. Comparing the findings of the infections caused by these viruses would help reveal the novel characteristics of SARS-CoV-2 and provide insight into the unique pathogenesis of SARS-CoV-2 infection. This study aimed to compare the clinical and radiological characteristics of SARS-CoV-2 and endemic HCoVs infection in adult hospitalized patients with community-acquired pneumonia (CAP). This study was performed at a university-affiliated tertiary hospital in the Republic of Korea, between January 1, 2015, and July 31, 2020. A total of 109 consecutive patients who were over 18 years of age with confirmed SARS-CoV-2 and endemic HCoVs were enrolled. Finally, 19 patients with SARS-CoV-2 CAP were compared to 40 patients with endemic HCoV CAP. Flu-like symptoms such as cough, sore throat, headache, myalgia, and prolonged fever were more common in SARS-CoV-2 CAP, whereas clinical findings suggestive of bacterial pneumonia such as dyspnea, leukocytosis with left shift, and increased C-reactive protein were more common in endemic HCoV CAP. Bilateral peripherally distributed ground-glass opacities (GGOs) were typical radiologic findings in SARS-CoV-2 CAP, whereas mixed patterns of GGOs, consolidations, micronodules, and pleural effusion were observed in endemic HCoV CAP. Coinfection was not observed in patients with SARS-CoV-2 CAP, but was observed in more than half of the patients with endemic HCoV CAP. There were distinctive differences in the clinical and radiologic findings between SARS-CoV-2 and endemic HCoV CAP. Further investigations are required to elucidate the mechanism underlying this difference. Follow-up observations are needed to determine if the presentation of SARS-CoV-2 CAP changes with repeated infection.

MERS-CoV: epidemiology, molecular dynamics, therapeutics, and future challenges.

The Severe Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has gained research attention worldwide, given the current pandemic. Nevertheless, a previous zoonotic and highly pathogenic coronavirus, the Middle East Respiratory Syndrome coronavirus (MERS-CoV), is still causing concern, especially in Saudi Arabia and neighbour countries. The MERS-CoV has been reported from respiratory samples in more than 27 countries, and around 2500 cases have been reported with an approximate fatality rate of 35%. After its emergence in 2012 intermittent, sporadic cases, nosocomial infections and many community clusters of MERS continued to occur in many countries. Human-to-human transmission resulted in the large outbreaks in Saudi Arabia. The inherent genetic variability among various clads of the MERS-CoV might have probably paved the events of cross-species transmission along with changes in the inter-species and intra-species tropism. The current review is drafted using an extensive review of literature on various databases, selecting of publications irrespective of favouring or opposing, assessing the merit of study, the abstraction of data and analysing data. The genome of MERS-CoV contains around thirty thousand nucleotides having seven predicted open reading frames. Spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins are the four main structural proteins. The surface located spike protein (S) of betacoronaviruses has been established to be one of the significant factors in their zoonotic transmission through virus-receptor recognition mediation and subsequent initiation of viral infection. Three regions in Saudi Arabia (KSA), Eastern Province, Riyadh and Makkah were affected severely. The epidemic progression had been the highest in 2014 in Makkah and Riyadh and Eastern Province in 2013. With a lurking epidemic scare, there is a crucial need for effective therapeutic and immunological remedies constructed on sound molecular investigations.