High temporal resolution Nanopore sequencing dataset of SARS-CoV-2 and host cell RNAs.

BACKGROUND: Recent studies have disclosed the genome, transcriptome, and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for noncanonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (multiplicity of infection [MOI] = 0.1) applied for the infection. It has never been tested whether the alteration in the host gene expressions is caused by aging of the cells or by the viral infection. FINDINGS: In this study, we used Oxford Nanopore's direct cDNA and direct RNA sequencing methods for the generation of a high-coverage, high temporal resolution transcriptomic dataset of SARS-CoV-2 and of the primate host cells, using a high infection titer (MOI = 5). Sixteen sampling time points ranging from 1 to 96 hours with a varying time resolution and 3 biological replicates were used in the experiment. In addition, for each infected sample, corresponding noninfected samples were employed. The raw reads were mapped to the viral and to the host reference genomes, resulting in 49,661,499 mapped reads (54,62 Gbs). The genome of the viral isolate was also sequenced and phylogenetically classified. CONCLUSIONS: This dataset can serve as a valuable resource for profiling the SARS-CoV-2 transcriptome dynamics, the virus-host interactions, and the RNA base modifications. Comparison of expression profiles of the host gene in the virally infected and in noninfected cells at different time points allows making a distinction between the effect of the aging of cells in culture and the viral infection. These data can provide useful information for potential novel gene annotations and can also be used for studying the currently available bioinformatics pipelines.

Middle East Respiratory Syndrome Coronavirus Gene 5 Modulates Pathogenesis in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia that emerged in 2012. There is limited information on MERS-CoV pathogenesis, as data from patients are scarce and the generation of animal models reproducing MERS clinical manifestations has been challenging. Human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice and a mouse-adapted MERS-CoV strain (MERSMA-6-1-2) were recently described. hDPP4-KI mice infected with MERSMA-6-1-2 show pathological signs of respiratory disease, high viral titers in the lung, and death. In this work, a mouse-adapted MERS-CoV infectious cDNA was engineered by introducing nonsynonymous mutations contained in the MERSMA-6-1-2 genome into a MERS-CoV infectious cDNA, leading to a recombinant mouse-adapted virus (rMERS-MA) that was virulent in hDDP4-KI mice. MERS-CoV adaptation to cell culture or mouse lungs led to mutations and deletions in genus-specific gene 5 that prevented full-length protein expression. In contrast, analysis of 476 MERS-CoV field isolates showed that gene 5 is highly stable in vivo in both humans and camels. To study the role of protein 5, two additional viruses were engineered expressing a full-length gene 5 (rMERS-MA-5FL) or containing a complete gene 5 deletion (rMERS-MA-Δ5). rMERS-MA-5FL virus was unstable, as deletions appeared during passage in different tissue culture cells, highlighting MERS-CoV instability. The virulence of rMERS-MA-Δ5 was analyzed in a sublethal hDPP4-KI mouse model. Unexpectedly, all mice died after infection with rMERS-MA-Δ5, in contrast to those infected with the parental virus, which contains a 17-nucleotide (nt) deletion and a stop codon in protein 5 at position 108. Expression of interferon and proinflammatory cytokines was delayed and dysregulated in the lungs of rMERS-MA-Δ5-infected mice. Overall, these data indicated that the rMERS-MA-Δ5 virus was more virulent than the parental one and suggest that the residual gene 5 sequence present in the mouse-adapted parental virus had a function in ameliorating severe MERS-CoV pathogenesis.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus causing human infections with high mortality rate (∼35%). Animal models together with reverse-genetics systems are essential to understand MERS-CoV pathogenesis. We developed a reverse-genetics system for a mouse-adapted MERS-CoV that reproduces the virus behavior observed in humans. This system is highly useful to investigate the role of specific viral genes in pathogenesis. In addition, we described a virus lacking gene 5 expression that is more virulent than the parental one. The data provide novel functions in IFN modulation for gene 5 in the context of viral infection and will help to develop novel antiviral strategies.

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.

In-Yeast Assembly of Coronavirus Infectious cDNA Clones Using a Synthetic Genomics Pipeline.

The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).

A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture.

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.

An Infectious cDNA Clone of SARS-CoV-2.

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 106 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.

Epidemiological and clinical characteristics of coronavirus disease (COVID-19) cases at a screening clinic during the early outbreak period: a single-centre study.

Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by Severe Acute Respiratory Corona Virus-2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-2020 corona virus pandemic. SARS-CoV-2 is closely related to the original SARS-CoV. It is thought to have a zoonotic origin. The virus is primarily spread between people during close contact, often via small droplets produced by coughing, sneezing or talking. People may also become infected by touching a contaminated surface and then touching their face. COVID-19 patients currently remain the primary source of infection. An epidemiological survey indicated that the general population is susceptible to SARS-CoV-2. The spectrum of this disease ranges from mild to life-threatening. Fever is the most common symptom, although older people and those with comorbidities may experience fever later in the disease. Other common symptoms include cough, loss of appetite, fatigue, shortness of breath, sputum production, and muscle and joint pains. Symptoms such as nausea, vomiting and diarrhea have been observed in varying percentages. Some cases might progress promptly to acute respiratory distress syndrome (ARDS) and/or multiple organ function failure. Asymptomatic carriers and those in the incubation period may also be infectious.Aim. To determine the epidemiological and clinical characteristics of patients presenting with COVID-19 at the screening clinic of a tertiary care hospital in Peshawar, Pakistan.Methodology. In this descriptive study, we analysed data of patients presenting to a newly established Covid-19 screening clinic in Rehman Medical Institute. Anyone who reported with new onset fever and/or cough was tested for SARS-CoV-2 in the screening clinic. We documented and analysed demographic, epidemiological and clinical characteristics, which included age, sex, travel history, clinical features, comorbidities and laboratory data of patients confirmed by real-time reverse-transcription (RT)-PCR at Rehman Medical Institute, Peshawar, Pakistan from 15 March till 21 April 2020. Paired specimens of throat swabs and nasal swabs were obtained from 845 patients, ribonucleic acid (RNA) was extracted and tested for SARS-CoV-2 by the RT-PCR assay.Results. A total of 845 specimens were taken as described above. The positive rate for SARS-CoV-2 was about 14.3%. Male and older population had a significantly higher positive rate. Of the 121 patients infected with SARS-CoV-2, the mean age was 43.19 years (sd, 17.57) and the infections were more frequent among male gender accounting for 85 (70.25 %) patients. Common symptoms included fever (88 patients, 72 %), cough (72 patients, 59.5 %) and shortness of breath (69 patients, 57 %). Twenty-two (18 %) patients had recent travel history outside Pakistan in the previous 14 days, the majority of whom had returned back from Saudi Arabia.Conclusion. In this single-centre, prospective, descriptive study, fever, cough and shortness of breath were the most common symptoms. Old age (>50 years), chronic underlying comorbidities and travel history may be risk factors. Therefore, we concluded that viral nucleic acid amplification tests (NAAT) played an important role in identifying SARS-CoV-2 infection in a screening clinic, which helped with isolation and cohorting of these patients.

[A race against the clock: creation of SARS-Cov-2 in the laboratory, a month after its emergence!] / Une course contre la montre - Création du SARS-CoV-2 en laboratoire, un mois après son émergence !

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2, which emerged in China at the end of 2019, is responsible for a global health crisis resulting in the confinement of more than 3 billion people worldwide and the sharp decline of the world economy. In this context, a race against the clock is launched in order to develop a treatment to stop the pandemic as soon as possible. A study published in Nature by the Volker Thiel team reports the development of reverse genetics for SARS-CoV-2 allowing them to recreate the virus in just a few weeks. The perspectives of this work are very interesting since it will allow the genetic manipulation of the virus and thus the development of precious tools which will be useful to fight the infection. Even though this approach represents a technological leap that will improve our knowledge of the virus, it also carries the germ of possible misuse and the creation of the virus for malicious purposes. The advantages and disadvantages of recreating SARS-CoV-2 in this pandemic period are discussed in this mini-synthesis.

TITLE: Une course contre la montre - Création du SARS-CoV-2 en laboratoire, un mois après son émergence ! ABSTRACT: Le SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2), qui a émergé à la fin de l'année 2019 en République populaire de Chine, est responsable d'une crise sanitaire mondiale qui a entraîné le confinement de plus de 3 milliards d'individus et l'arrêt brutal de l'économie planétaire. Dans ce contexte, une course contre la montre est lancée afin de développer, dans les plus brefs délais, un traitement permettant d'enrayer la pandémie. Une étude de l'équipe de Volker Thiel, parue dans le journal Nature, rapporte la mise au point d'une technique de génétique inverse pour le SARS-CoV-2, leur ayant permis de recréer le virus en seulement quelques semaines. Les perspectives de ces travaux sont très intéressantes puisqu'elles permettent d'envisager la manipulation génétique du virus et ainsi le développement d'outils précieux qui seront utiles pour combattre l'infection. Si la technique représente également un saut technologique qui permettra d'améliorer nos connaissances sur le virus, elle porte aussi en elle le germe d'un possible mésusage et la création d'un virus à des fins malveillantes. Les avantages et inconvénients de recréer le SARS-CoV-2 dans cette période de pandémie sont discutés dans cet article.

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.

SARS-CoV-2 orf1b Gene Sequence in the NTNG1 Gene on Human Chromosome 1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.