D614G mutation in the SARS-CoV-2 spike protein enhances viral fitness by desensitizing it to temperature-dependent denaturation

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.     

Making sense of spike D614G in SARS-CoV-2 transmission

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), the etiologic agent of the current coronavirus disease 2019(COVID-19) pandemic, has evolved to adapt to human host and transmission over the past 12 months. One prominent adaptive mutation is the asparagine-to-glycine substitution at amino acid position 614 in the viral spike protein(D614G), which has become dominant in the currently circulating virus strains. Since spike protein determines host ranges, tissue tropism, and pathogenesis through binding to the cellular receptor of angiotensin converting enzyme 2(ACE2), the D614G mutation is hypothesized to enhance viral fitness in human host, leading to increased transmission during the global pandemic. Here we summarize the recent progress on the role of the D614G mutation in viral replication, pathogenesis, transmission, and vaccine and therapeutic antibody development. These findings underscore the importance in closely monitoring viral evolution and defining their functions to ensure countermeasure efficacy against newly emerging variants.     

Modeling the onset of symptoms of COVID-19: Effects of SARS-CoV-2 variant

Identifying order of symptom onset of infectious diseases might aid in differentiating symptomatic infections earlier in a population thereby enabling non-pharmaceutical interventions and reducing disease spread. Previously, we developed a mathematical model predicting the order of symptoms based on data from the initial outbreak of SARS-CoV-2 in China using symptom occurrence at diagnosis and found that the order of COVID-19 symptoms differed from that of other infectious diseases including influenza. Whether this order of COVID-19 symptoms holds in the USA under changing conditions is unclear. Here, we use modeling to predict the order of symptoms using data from both the initial outbreaks in China and in the USA. Whereas patients in China were more likely to have fever before cough and then nausea/vomiting before diarrhea, patients in the USA were more likely to have cough before fever and then diarrhea before nausea/vomiting. Given that the D614G SARS-CoV-2 variant that rapidly spread from Europe to predominate in the USA during the first wave of the outbreak was not present in the initial China outbreak, we hypothesized that this mutation might affect symptom order. Supporting this notion, we found that as SARS-CoV-2 in Japan shifted from the original Wuhan reference strain to the D614G variant, symptom order shifted to the USA pattern. Google Trends analyses supported these findings, while weather, age, and comorbidities did not affect our model’s predictions of symptom order. These findings indicate that symptom order can change with mutation in viral disease and raise the possibility that D614G variant is more transmissible because infected people are more likely to cough in public before being incapacitated with fever.     

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.     

D614G substitution at the hinge region enhances the stability of trimeric SARS-CoV-2 spike protein

Mutations in the spike protein of SARS-CoV-2 are the major causes for the modulation of ongoing COVID-19 infection. Currently, the D614G substitution in the spike protein has become dominant worldwide. It is associated with higher infectivity than the ancestral (D614)variant. We demonstrate using Gaussian network model-based normal mode analysis that the D614G substitution occurs at the hinge region that facilitates domain-domain motions between receptor binding domain and S2 region of the spike protein. Computer-aided mutagenesis and inter-residue energy calculations reveal that contacts involving D614 are energetically frustrated. However, contacts involving G614 are energetically favourable, implying the substitution strengthens residue contacts that are formed within as well as between protomers. We also find that the free energy difference (ΔΔG) between two variants is -2.6 kcal/mol for closed and -2.0 kcal/mol for 1-RBD up conformation. Thus, the thermodynamic stability has increased upon D614G substitution. Whereas the reverse mutation in spike protein structures having G614 substitution has resulted in the free energy differences of 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations, respectively, indicating that the overall thermodynamic stability has decreased. These results suggest that the D614G substitution modulates the flexibility of spike protein and confers enhanced thermodynamic stability irrespective of conformational states. This data concurs with the known information demonstrating increased availability of the functional form of spikeprotein trimer upon D614G substitution.     

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).     

Mutational sensitivity of D614G in spike protein of SARS-CoV-2 in Jordan

BackgroundSpike protein is the surface glycoprotein of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) necessary for the entry of the virus via the transmembrane receptors of the human respiratory cells causing COVID-19 disease.AimHere, we aimed to predict the three-dimensional monomer structure of spike protein of SARS-CoV-2 from 20 Jordanian nasopharyngeal samples and to determine the percentage of single amino acid variants (SAV) in the spike protein of SARS-CoV-2.MethodsThe output of the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) found four single amino acid variants in the spike gene.ResultsThe first variant represented by 5% of samples that showed tyrosine deletion at Y144 located in the N terminal domain. The second and the dominant variant, represented by 62%, showed aspartate a coil amino acid substitution to glycine an extracellular amino acid at D614G located in the spike recognition binding site. The third variant, represented by 5%, showed aspartate substitution to tyrosine at D1139Y, and the fourth variant, represented by 5% glycine substitution to serine at G1167S.ConclusionOur results have shown low mutational sensitivity in all variants except to D614G the one with the most likely neutral mutational sensitivity that all variants might not explicitly affect the function of spike glycoprotein. However, D614G might change the viral conformational plasticity and hence a potential viral fitness gain but one must be cautious about drawing any concrete conclusions about the severity of symptoms and viral transmission from genomic data only.General significanceStudying mutations such as D614G in deep is essential to control the pandemic in terms of immune systems, antibodies, or even vaccines.     

Non-synonymous mutations of SARS-CoV-2 leads epitope loss and segregates its variants

The non-synonymous mutations of SARS-CoV-2 isolated from across the world have been identified during the last few months. The surface glycoprotein spike of SARS-CoV-2 forms the most important hotspot for amino acid alterations followed by the ORF1a/ORF1ab poly-proteins. It is evident that the D614G mutation in spike glycoprotein and P4715L in RdRp is the important determinant of SARS-CoV-2 evolution since its emergence. P4715L in RdRp, G251V in ORF3a and S1498F of Nsp3 is associated with the epitope loss that may influence pathogenesis caused by antibody escape variants. The phylogenomics distinguished the ancestral viral samples from China and most part of Asia, isolated since the initial outbreak and the later evolved variants isolated from Europe and Americas. The evolved variants have been found to predominant globally with the loss of epitopes from its proteins. These have implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.     

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.     

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.     

马铃薯Y病毒RPA-CRISPR/Cas12a检测技术体系的建立与应用

植物病毒病是制约农作物安全生产的重要因素, 病毒检测能够发现病毒并确定病毒的种类, 是病害监测预警和防控的关键。该研究以马铃薯Y病毒(PVY)为检测对象, 建立了基于RPA-CRISPR/Cas12a的检测体系。结果表明, (1) CRISPR/ Cas12a检测体系内Cas12a及各组分为检测顺利进行所必要; (2) crRNA的靶点位置对Cas12a蛋白活性有较大影响, 当crRNA的靶点包含部分PAM位点序列时, 反应效率最高; (3) RPA-CRISPR/Cas12a检测模板的最低限度为3×10² copies∙μL^-1, 灵敏度高于PCR及qPCR检测法; (4) RPA-CRISPR/Cas12a检测体系与核酸粗提及逆转录反应联合, 可在非实验室环境下进行PVY检测, 整个过程耗时约60分钟。该研究建立了基于RPA-CRISPR/Cas12a的PVY检测技术体系, 为在非实验室条件下实时快速检测植物病毒提供了一种有效方法。     

输入病毒导致的十起本土疫情首例病例新冠病毒基因特征分析

2020年4月中国阻断湖北省武汉市新冠肺炎疫情传播后,中国国内报道了多起由境外输入导致的本土聚集性新冠肺炎疫情。为分析引起聚集性疫情的输入性新型冠状病毒(SARS-CoV-2)的基因组特征,本研究对2020年4-11月份十起输入相关本土疫情首例病例的SARS-CoV-2全基因组基因特征进行分析,系统阐述了相关SARS-CoV-2的全基因组和氨基酸变异特征。结果显示,与武汉参考株相比,十起本土聚集性疫情首例病例的SARS-CoV-2核苷酸突变中位数为10个(8个-26个),氨基酸突变的中位数为6个(4个-16个),且刺突(spike,S)蛋白只有D614G一个氨基酸发生突变。除分支位点外,10条SARS-CoV-2全基因组序列的65个核苷酸突变位点以及35个氨基酸突变仅出现1-2次,呈现随机性。全基因组分析表明,这十起本土疫情的首例病例基因组按照中国分型法可划分为4个型,按照Pangolin分型法可划分为7个型,与我国2020年1-3月份武汉流行的毒株属于不同基因型,不是本土SARS-CoV-2的持续传播。与2020年9-12月英国和南非变异株属于不同基因型,无相关性。本文系统分析了2020年由输入病毒导致的十起本土疫情首例病例的SARS-CoV-2核苷酸与氨基酸变异特征,为我国新冠防控策略的制定以及后续新冠疫情的溯源提供了参考依据。     

Modelling conformational state dynamics and its role on infection for SARS-CoV-2 Spike protein variants

The SARS-CoV-2 Spike protein needs to be in an open-state conformation to interact with ACE2 to initiate viral entry. We utilise coarse-grained normal mode analysis to model the dynamics of Spike and calculate transition probabilities between states for 17081 variants including experimentally observed variants. Our results correctly model an increase in open-state occupancy for the more infectious D614G via an increase in flexibility of the closed-state and decrease of flexibility of the open-state. We predict the same effect for several mutations on glycine residues (404, 416, 504, 252) as well as residues K417, D467 and N501, including the N501Y mutation recently observed within the B.1.1.7, 501.V2 and P1 strains. This is, to our knowledge, the first use of normal mode analysis to model conformational state transitions and the effect of mutations on such transitions. The specific mutations of Spike identified here may guide future studies to increase our understanding of SARS-CoV-2 infection mechanisms and guide public health in their surveillance efforts.     

The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters

As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel SARS-CoV-2 variant designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and it was shown to have enhanced infectivity in vitro and decreased antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both variants exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most marked body weight loss among the 3 variants. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three variants. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the oropharynx but not in the lungs. In multi-virus in-vivo competition experiments, we found that B.1. (614G), epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the nasal cavity, B.1. (614G), gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) and WA-1 variants in hamsters. These results demonstrate enhanced virulence and high relative oropharyngeal replication of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) variant.     

Genomic and epidemiological characteristics of SARS-CoV-2 in Africa

Since late 2019, the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly evolved to become a global pandemic. Each country was affected but with a varying number of infected cases and mortality rates. Africa was hit late by the pandemic but the number of cases rose sharply. In this study, we investigated 224 SARS-CoV-2 genome sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) in the early part of the outbreak, of which 69 were from Africa. We analyzed a total of 550 mutations by comparing them with the reference SARS-CoV-2 sequence from Wuhan. We classified the mutations observed based on country and region, and afterwards analyzed common and unique mutations on the African continent as a whole. Correlation analyses showed that the duo variants ORF1ab/RdRp 4715L and S protein 614G variants, which are strongly linked to fatality rate, were not significantly and positively correlated with fatality rates (r = -0.03757, P = 0.5331 and r = -0.2876, P = 0.6389, respectively), although increased number of cases correlated with number of deaths (r = 0.997, P = 0.0002). Furthermore, most cases in Africa were mainly imported from American and European countries, except one isolate with no mutation and was similar to the original isolate from Wuhan. Moreover, unique mutations specific to countries were identified in the early phase of the outbreak but these mutations were not regional-specific. There were common mutations in all isolates across the continent as well as similar isolate-specific mutations in different regions. Our findings suggest that mutation is rapid in SARS-CoV-2 in Africa and although these mutations spread across the continent, the duo variants could not possibly be the sole cause of COVID-19 deaths in Africa in the early phase of the outbreak.     

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12–April 1 2020) and 31 from later time-points ( 25–27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5–99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.     

In-silico genomic landscape characterization and evolution of SARS-CoV-2 variants isolated in India shows significant drift with high frequency of mutations

In-silico studies on SARS-CoV-2 genome are considered important to identify the significant pattern of variations and its possible effects on the structural and functional characteristics of the virus. The current study determined such genetic variations and their possible impact among SARS-CoV-2 variants isolated in India. A total of 546 SARS-CoV-2 genomic sequences (India) were retrieved from the gene bank (NCBI) and subjected to alignment against the Wuhan variant (NC_045512.2), the corresponding amino acid changes were analyzed using NCBI Protein-BLAST. These 546 variants revealed 841 mutations; most of these were non-synonymous 464/841 (55.1%), there was no identical variant compared to the original strain. All genes; coding and non-coding showed nucleotide changes, most of the structural genes showed frequent nonsynonymous mutations. The most affected genes were ORF1a/b followed by the S gene which showed 515/841 (61.2%) and 120/841 (14.3%) mutations, respectively. The most frequent non-synonymous mutation 486/546 (89.01%) occurred in the S gene (structural gene) at position 23,403 where A changed to G leading to the replacement of aspartic acid by glycine in position (D614G). Interestingly, four variants also showed deletion. The variants MT800923 and MT800925 showed 12 consecutive nucleotide deletion in position 21982–21993 resulting in 4 consecutive amino acid deletions that were leucine, glycine, valine, and tyrosine in positions 141, 142, 143, and 144 respectively. The present study exhibited a higher mutations rate per variant compared to other studies carried out in India.     

Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment

Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR–based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.     

SARS-CoV-2 variants reveal features critical for replication in primary human cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

SARS-CoV-2 variants have appeared throughout the emergence of the current COVID-19 pandemic. In this study, characterization of a panel of infectious SARS-CoV-2 isolates in primary human respiratory cells allows the identification of important sequence features that contribute to virus replication.     

新型冠状病毒(SARS-CoV-2)SNV分型检测技术

新型冠状病毒肺炎(COVID-19)的全球大流行对整个人类社会造成了重大影响,人类面临着财政刺激、金融压力、债务重整等挑战。在特效治疗药物与方法出现之前,大规模的人群筛查隔离成为现在疫情治理的最有效方法。然而,这一次的新冠病毒(SARS-CoV-2)展示出了极高的遗传变异性,截至2022年3月31日统计突变率超过了2.3‰,迄今为止高传染性的新病毒株不断出现,被世界卫生组织正式警告的变异株就达到了7个。因此,在接下来的病毒防控与研究中,不但需要检测SARS-CoV-2,更需要精准、实用的单核苷酸变异(single nucleotide variation, SNV)基因分型技术,特别针对大规模人群筛查中,不仅需要获得SRAS-CoV-2的信息,还需要精准快速区分具有更高传染性与毒性的变异株感染。对病毒的感染和突变机制进行了简要介绍,并着重对现有主要的SARS-CoV-2 SNV分型技术进行了分类综述,希望为新型检测技术的开发提供参考。