Desorption Separation Ionization Mass Spectrometry (DSI-MS) for Rapid Analysis of COVID-19.

During the coronavirus disease 2019 (COVID-19) pandemic, which has witnessed over 772 million confirmed cases and over 6 million deaths globally, the outbreak of COVID-19 has emerged as a significant medical challenge affecting both affluent and impoverished nations. Therefore, there is an urgent need to explore the disease mechanism and to implement rapid detection methods. To address this, we employed the desorption separation ionization (DSI) device in conjunction with a mass spectrometer for the efficient detection and screening of COVID-19 urine samples. The study encompassed patients with COVID-19, healthy controls (HC), and patients with other types of pneumonia (OP) to evaluate their urine metabolomic profiles. Subsequently, we identified the differentially expressed metabolites in the COVID-19 patients and recognized amino acid metabolism as the predominant metabolic pathway involved. Furthermore, multiple established machine learning algorithms validated the exceptional performance of the metabolites in discriminating the COVID-19 group from healthy subjects, with an area under the curve of 0.932 in the blind test set. This study collectively suggests that the small-molecule metabolites detected from urine using the DSI device allow for rapid screening of COVID-19, taking just three minutes per sample. This approach has the potential to expand our understanding of the pathophysiological mechanisms of COVID-19 and offers a way to rapidly screen patients with COVID-19 through the utilization of machine learning algorithms.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

First detection of Hedgehog coronavirus 1 in Poland.

Hedgehogs are common in the majority of European countries and are known to host various pathogens, including viruses. The recent discovery of MERS-related coronaviruses (CoVs) in hedgehogs from Germany, France, the UK, China, and Italy suggests that hedgehogs may represent a wild reservoir of betacoronaviruses. This study reports the first detection and characterization of novel betacoronovirus, subgenus Merbecovirus in wild hedgehogs in Poland. The CoV RNA was detected in 10 out of 40 hedgehogs' rectal swabs and in 1 out of 18 samples of the lung. No viral RNA was identified in the duodenum and kidney. There was no significant relationship between clinical status, gender, hedgehogs' age, and coronaviral RNA detection. Phylogenetic analysis showed that CoVs detected in our study grouped together with other representatives of Hedgehog coronavirus 1 species identified in Western Europe. Our findings provide further evidence that hedgehogs are a natural reservoir of Merbecovirus. Considering the high mutation rate of CoVs and their potential for crossing interspecies barriers, the proper management of hedgehogs admitted to wildlife rehabilitation centres is needed. It cannot be excluded that merbecovirus strains detected in hedgehogs may recombine with other CoVs leading to new viruses with potential for interspecies transmission.

The unresolved question on COVID-19 virus origin: The three cards game?

The ongoing discussion about the real origin of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) feeds acrimonious debates. Where did SARS-CoV-2 come from? Was SARS-CoV-2 transmitted in the wild from an animal to a person before exploding in Wuhan or was it an engineered virus that escaped from research or a laboratory in Wuhan? Right now, we still don't know enough whether SARS-CoV-2 is human-made or not, and lab-leak theories remain essentially speculative. Many recent studies have pointed out several plausible scenarios. Anyhow, currently, even if suspicions by some about the possibility of lab-leak hypothesis still remain, the consensus view is that the pandemic probably started from a natural source and, to determine the real origin of the SARS-CoV-2 virus, further research is needed.

Utilizing the VirIdAl Pipeline to Search for Viruses in the Metagenomic Data of Bat Samples.

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.

Metagenomic identification of a new sarbecovirus from horseshoe bats in Europe.

The source of the COVID-19 pandemic is unknown, but the natural host of the progenitor sarbecovirus is thought to be Asian horseshoe (rhinolophid) bats. We identified and sequenced a novel sarbecovirus (RhGB01) from a British horseshoe bat, at the western extreme of the rhinolophid range. Our results extend both the geographic and species ranges of sarbecoviruses and suggest their presence throughout the horseshoe bat distribution. Within the spike protein receptor binding domain, but excluding the receptor binding motif, RhGB01 has a 77% (SARS-CoV-2) and 81% (SARS-CoV) amino acid homology. While apparently lacking hACE2 binding ability, and hence unlikely to be zoonotic without mutation, RhGB01 presents opportunity for SARS-CoV-2 and other sarbecovirus homologous recombination. Our findings highlight that the natural distribution of sarbecoviruses and opportunities for recombination through intermediate host co-infection are underestimated. Preventing transmission of SARS-CoV-2 to bats is critical with the current global mass vaccination campaign against this virus.

Etiology and clinical characteristics of SARS-CoV-2 and other human coronaviruses among children in Zhejiang Province, China 2017-2019.

BACKGROUND: A novel coronavirus (SARS-CoV-2) emerging has put global public health institutes on high alert. Little is known about the epidemiology and clinical characteristics of human coronaviruses infections in relation to infections with other respiratory viruses. METHODS: From February 2017 to December 2019, 3660 respiratory samples submitted to Zhejiang Children Hospital with acute respiratory symptoms were tested for four human coronaviruses RNA by a novel two-tube multiplex reverse transcription polymerase chain reaction assays. Samples were also screened for the occurrence of SARS-CoV-2 by reverse transcription-PCR analysis. RESULTS: Coronavirus RNAs were detected in 144 (3.93%) specimens: HCoV-HKU1 in 38 specimens, HCoV-NL63 in 62 specimens, HCoV-OC43 in 38 specimens and HCoV-229E in 8 specimens. Genomes for SARS-CoV-2 were absent in all specimens by RT-PCR analysis during the study period. The majority of HCoV infections occurred during fall months. No significant differences in gender, sample type, year were seen across species. 37.5 to 52.6% of coronaviruses detected were in specimens testing positive for other respiratory viruses. Phylogenic analysis identified that Zhejiang coronaviruses belong to multiple lineages of the coronaviruses circulating in other countries and areas. CONCLUSION: Common HCoVs may have annual peaks of circulation in fall months in the Zhejiang province, China. Genetic relatedness to the coronaviruses in other regions suggests further surveillance on human coronaviruses in clinical samples are clearly needed to understand their patterns of activity and role in the emergence of novel coronaviruses.

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.

Detection and differentiation of respiratory syncytial virus subgroups A and B with colorimetric toehold switch sensors in a paper-based cell-free system.

Respiratory syncytial virus (RSV) infection is the most common clinical infectious disease threatening the safety of human life. Herein, we provided a sensitive and specific method for detection and differentiation of RSV subgroups A (RSVA) and B (RSVB) with colorimetric toehold switch sensors in a paper-based cell-free system. In this method, we applied the toehold switch, an RNA-based riboswitch, to regulate the translation level of ß-galactosidase (lacZ) gene. In the presence of target trigger RNA, the toehold switch sensor was activated and the expressed LacZ hydrolyzed chromogenic substrates to produce a colorimetric result that can be observed directly with the naked eye in a cell-free system. In addition, nucleic acid sequence-based amplification (NASBA) was used to improve the sensitivity by amplifying target trigger RNAs. Under optimal conditions, our method produced a visible result for the detection of RSVA and RSVB with the detection limit of 52 aM and 91 aM, respectively. The cross-reaction of this method was validated with other closely related respiratory viruses, including human coronavirus HKU1 (HCoV-HKU1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Furthermore, we used the paper-based carrier material that allows stable storage of our detection elements and rapid detection outside laboratory. In conclusion, this method can sensitively and specifically differentiate RSVA and RSVB and generate a visible colorimetric result without specialized operators and sophisticated equipment. Based on these advantages above, this method serves as a simple and portable detector in resource-poor areas and point-of-care testing (POCT) scenarios.

Orientaciones para la selección de muestras de SARS-CoV-2 para caracterización y vigilancia genómica / Guidance for the selection of SARS-CoV-2 samples for genomic characterization and surveillance

La secuenciación genómica ha sido una herramienta esencial para generar datos virológicos, impulsar la respuesta del laboratorio y comprender mejor los patrones evolutivos y de dispersión del SARS-CoV-2. Además de la caracterización de los patrones de circulación global, la detección temprana de las variantes del SARS-CoV-2 dentro de cada país es fundamental para complementar la vigilancia epidemiológica y virológica.

Ultra-sensitive viral glycoprotein detection NanoSystem toward accurate tracing SARS-CoV-2 in biological/non-biological media.

Rapid person-to-person transfer of viruses such as SARS-CoV-2 and their occasional mutations owing to the human activity and climate/ecological changes by the mankind led to creation of wrecking worldwide challenges. Such fast transferable pathogens requiring practical diagnostic setups to control their transfer chain and stop sever outbreaks in early stages of their appearance. Herein, we have addressed this urgent demand by designing a rapid electrochemical diagnostic kit composed of fixed/screen printed electrodes that can detect pathogenic viruses such as SARS-CoV-2 and/or animal viruses through the differentiable fingerprint of their viral glycoproteins at different voltage positions. The working electrode of developed sensor is activated upon coating a layer of coupled graphene oxide (GO) with sensitive chemical compounds along with gold nanostars (Au NS) that can detect the trace of viruses in any aquatic biological media (e.g., blood, saliva and oropharyngeal/nasopharyngeal swab) through interaction with active functional groups of their glycoproteins. The method do not require any extraction and/or biomarkers for detection of target viruses and can identify trace of different pathogenic viruses in about 1 min. The nanosensor also demonstrated superior limit of detection (LOD) and sensitivity of 1.68 × 10-22 µg mL-1 and 0.0048 µAµg.mL-1. cm-2, respectively, toward detection of SARS-CoV-2 in biological media, while blind clinical evaluations of 100 suspected samples furtherly confirmed the superior sensitivity/specificity of developed nanosystem toward rapid identification of ill people even at incubation and prodromal periods of illness.

Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva.

The diffusion of novel SARS-CoV-2 coronavirus over the world generated COVID-19 pandemic event as reported by World Health Organization on March 2020. The huge issue is the high infectivity and the absence of vaccine and customised drugs allowing for hard management of this outbreak, thus a rapid and on site analysis is a need to contain the spread of COVID-19. Herein, we developed an electrochemical immunoassay for rapid and smart detection of SARS-CoV-2 coronavirus in saliva. The electrochemical assay was conceived for Spike (S) protein or Nucleocapsid (N) protein detection using magnetic beads as support of immunological chain and secondary antibody with alkaline phosphatase as immunological label. The enzymatic by-product 1-naphtol was detected using screen-printed electrodes modified with carbon black nanomaterial. The analytical features of the electrochemical immunoassay were evaluated using the standard solution of S and N protein in buffer solution and untreated saliva with a detection limit equal to 19 ng/mL and 8 ng/mL in untreated saliva, respectively for S and N protein. Its effectiveness was assessed using cultured virus in biosafety level 3 and in saliva clinical samples comparing the data using the nasopharyngeal swab specimens tested with Real-Time PCR. The agreement of the data, the low detection limit achieved, the rapid analysis (30 min), the miniaturization, and portability of the instrument combined with the easiness to use and no-invasive sampling, confer to this analytical tool high potentiality for market entry as the first highly sensitive electrochemical immunoassay for SARS-CoV-2 detection in untreated saliva.

A computational simulation platform for designing real-time monitoring systems with application to COVID-19.

With the aim of contributing to the fight against the coronavirus disease 2019 (COVID-19), numerous strategies have been proposed. While developing an effective vaccine can take months up to years, detection of infected patients seems like one of the best ideas for controlling the situation. The role of biosensors in containing highly pathogenic viruses, saving lives and economy is evident. A new competitive numerical platform specifically for designing microfluidic-integrated biosensors is developed and presented in this work. Properties of the biosensor, sample, buffer fluid and even the microfluidic channel can be modified in this model. This feature provides the scientific community with the ability to design a specific biosensor for requested point-of-care (POC) applications. First, the validation of the presented numerical platform against experimental data and then results and discussion, highlighting the important role of the design parameters on the performance of the biosensor is presented. For the latter, the baseline case has been set on the previous studies on the biosensors suitable for SARS-CoV, which has the highest similarity to the 2019 nCoV. Subsequently, the effects of concentration of the targeted molecules in the sample, installation position and properties of the biosensor on its performance were investigated in 11 case studies. The presented numerical framework provides an insight into understanding of the virus reaction in the design process of the biosensor and enhances our preparation for any future outbreaks. Furthermore, the integration of biosensors with different devices for accelerating the process of defeating the pandemic is proposed.

Comparative evaluation of nasopharyngeal swab and saliva specimens for the molecular detection of SARS-CoV-2 RNA in Japanese patients with COVID-19.

Considering the issues of shortage of medical resources and the invasiveness and infection risk involved in the collection of nasopharyngeal swab specimens, there is a need for an effective alternative test specimen for SARS-CoV-2 RNA detection. Here, we investigated suitability of saliva as a non-invasively obtained specimen for molecular detection of SARS-CoV-2 RNA in Japanese patients with COVID-19. In total, 28 paired clinical specimens of saliva and nasopharyngeal swabs were collected from 12 patients at various time points after symptom onset. Each specimen was assayed using reverse transcription real-time polymerase chain reaction (rRT-PCR) on the BD MAX open system using primers and probes targeting the N-gene. The saliva and nasopharyngeal swab specimens showed 19 and 15 positive results, respectively. No invalid (PCR inhibition) result was observed for any specimen. The qualitative results of each specimen obtained in the period immediately after symptom onset were similar. Three convalescent patients presented saliva-positive results, whereas their nasopharyngeal swabs were negative at four different time points, suggesting that saliva may be superior to nasopharyngeal swabs in terms of obtaining stable assay result of SARS-CoV-2. In conclusion, our results suggest that saliva can potentially serve as an alternative to nasopharyngeal swabs as a specimen for SARS-CoV-2 rRT-PCR. As saliva can be collected by patients themselves, it may be an effective way to overcome the shortage of personal protective equipment and specimen sampling tools.