scholarly journals 511. Treatment with Molnupiravir in the MOVe-In and MOVe-Out Clinical Trials Results in an Increase in Transition Mutations Across the SARS-CoV-2 Genome

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S357-S358
Author(s):  
Julie Strizki ◽  
Jay Grobler ◽  
Ying Zhang ◽  
Jiejun Du ◽  
Shunbing Zhao ◽  
...  
Keyword(s):  
Nasal Swab ◽  
Mutation Rates ◽  
Ion Torrent ◽  
Rt Pcr ◽  
Entire Genome ◽  
Error Catastrophe ◽  
Viral Mutation ◽  
Frequent Mutations ◽  
Ion Torrent Sequencing ◽  
Combined Data

Abstract Background Molnupiravir (MOV), (MK-4482, EIDD-2801) is being clinically developed for the treatment of COVID-19 disease caused by SARS-CoV-2. MOV is the orally administered 5′-isobutyrate prodrug of the active, antiviral ribonucleoside analogue, N-hydroxycytidine (NHC, EIDD-1931) which inhibits viral replication by induction of mutations in the viral genome, leading to viral error catastrophe. In 2 clinical studies, hospitalized (MOVe-In) and non-hospitalized (MOVe-Out) participants were treated for 5 days with MOV and followed up to Day 29. Viral RNA isolated from nasal swab samples were sequenced to determine the rate, distribution and type of viral mutations observed after MOV treatment. Methods RNA isolated from nasopharangeal swab samples collected during study conduct was quantified by RT-PCR. Samples containing >22,000 copies/mL of RNA underwent complete genome NGS using the Ion AmpliSeq SARS-CoV-2 research panel and Ion Torrent sequencing. Mutation rates were calculated by determining the number of nucleotide changes observed across the entire genome at Day 3 and/or Day 5 compared to baseline. Results Combined data from both studies showed an increase of ~2-4 fold in the viral mutation rate post-baseline in MOV treated compared with placebo. Mutations were distributed across the entire genome with only a minority being observed in more than one sample. The most frequent mutations were transitions of C to U observed in the highest MOV dose group (800 mg/BID). Conclusion Consistent with the proposed mechanism of action of MOV, an increase in the rate of transition mutations in the virus was observed in post-baseline nasal swab samples from participants treated with MOV compared with placebo. Disclosures Julie Strizki, PhD, Merck & Co., Inc. (Employee, Shareholder) Jay Grobler, PhD, Merck & Co., Inc. (Employee, Shareholder) Ying Zhang, PhD, Merck & Co., Inc. (Employee, Shareholder) Jiejun Du, PhD, Merck & Co., Inc. (Employee, Shareholder) Shunbing Zhao, PhD, Merck & Co., Inc. (Employee, Shareholder) Diane Levitan, PhD, Merck & Co., Inc. (Employee, Shareholder) Alex Therien, PhD, Merck & Co., Inc. (Employee, Shareholder) Joan R. Butterton, MD, Merck Sharp & Dohme Corp. (Employee, Shareholder) Nicholas Murgolo, PhD, Merck & Co., Inc. (Employee, Shareholder)

scholarly journals Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity

2015 ◽  
Vol 112 (11) ◽  
pp. 3229-3234 ◽  
Author(s):  
Chunte Sam Peng ◽  
Bogdan I. Fedeles ◽  
Vipender Singh ◽  
Deyu Li ◽  
Tiffany Amariuta ◽  
...  
Keyword(s):  
Ir Spectroscopy ◽  
Mutation Rates ◽  
Viral Population ◽  
Lethal Mutagenesis ◽  
2D Ir ◽  
Viral Mutation ◽  
Unique Shape ◽  
Ph Dependent ◽  
Mutagenic Property ◽  
Anti Hiv

Antiviral drugs designed to accelerate viral mutation rates can drive a viral population to extinction in a process called lethal mutagenesis. One such molecule is 5,6-dihydro-5-aza-2′-deoxycytidine (KP1212), a selective mutagen that induces A-to-G and G-to-A mutations in the genome of replicating HIV. The mutagenic property of KP1212 was hypothesized to originate from its amino–imino tautomerism, which would explain its ability to base pair with either G or A. To test the multiple tautomer hypothesis, we used 2D IR spectroscopy, which offers subpicosecond time resolution and structural sensitivity to distinguish among rapidly interconverting tautomers. We identified several KP1212 tautomers and found that >60% of neutral KP1212 is present in the enol–imino form. The abundant proportion of this traditionally rare tautomer offers a compelling structure-based mechanism for pairing with adenine. Additionally, the pKa of KP1212 was measured to be 7.0, meaning a substantial population of KP1212 is protonated at physiological pH. Furthermore, the mutagenicity of KP1212 was found to increase dramatically at pH <7, suggesting a significant biological role for the protonated KP1212 molecules. Overall, our data reveal that the bimodal mutagenic properties of KP1212 result from its unique shape shifting ability that utilizes both tautomerization and protonation.

scholarly journals Prolonged Detection of SARS CoV2 RNA in Extracellular Vesicles in Nasal Swab RT-PCR Negative Patients

2021 ◽  
Author(s):  
P Debishree Subudhi ◽  
Sheetalnath Rooge ◽  
Swati Thangriyal ◽  
Reshu Aggarwal ◽  
Ekta Gupta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Viral Load ◽  
Liver Disease ◽  
Chronic Liver Disease ◽  
Extracellular Vesicles ◽  
Nasal Swab ◽  
High Viral Load ◽  
Chronic Liver ◽  
Rt Pcr ◽  
Route Of Transmission

Background: There is a prolonged RT PCR positivity seen in COVID-19 infected patients up to 2 to 3 months. It is assumed that this virus is usually non-infective but there are hardly any study on the reactivation of this virus within the respiratory tract. We aim to investigate the presence of viral particles inside Extracellular vesicles (EV) and its role in underlying liver disease patients. Methods: SARS CoV2 nasal and throat swab RT-PCR positive n=78 {n=24(66.6%) chronic liver disease (CLD); n=52 (81.3%) non liver disease} n=5 RT PCR negative subjects (HC) were studied. SARS CoV2 patients were also followed up for day (d) 7, 14 and 28. Nasal swab [collected in viral transport media (VTM)] and plasma samples were investigated at each time point. Extracellular vesicles were isolated using differential ultracentrifugation. SARS CoV2 RNA was measured using qRT-PCR by Altona Real Star kit. Cellular origin of EV was confirmed using epithelial cells (Epcam+ CK19+ CDh1+), endothelial cells (CD31+CD45-), and hepatocytes (ASGPR+) surface markers by Flow cytometry. Results: The COVID19 patients {Mean age 54±23 years; 41 males} were having severity between moderate to severe. In patients with cirrhosis, the most common aetiology of liver disease was alcohol (MELD 22±8). In baseline RT-PCR positive patients, SARS-CoV2 RNA inside the EV was present in 64/74 (82%) patients with comparable viral load between VTM and EV (mean 1/CT 0.033±0.005 vs. 1/CT 0.029±0.014, p=ns). On follow-up at day 7, of the 24 patients negative for COVID19, 10 (41%) had persistence of virus in the EV (1/CT 0.028±0.004) and on day 14, 14 of 40 (35%) negative RT-PCR had EVs with SARS CoV2 RNA (1/CT 0.028±0.06). The mean viral load decreased at day7 and day14 in nasal swab from baseline (p=0.001) but not in EV. SARS-CoV2 RNA otherwise undetectable in plasma, was found to be positive in EV in 12.5% of COVID19 positive patients. Interestingly, significantly prolonged and high viral load was found in EV at day 14 in CLD COVID19 patients compared to COVID19 alone (p=0.002). The high cellular injury was seen in CLD COVID19 infected patients with significant high levels of EV associated with endothelial cells and hepatocytes than COVID19 alone (p=0.004; 0.001). Conclusion: Identification of SARS-CoV2 RNA in EV, in RT-PCR negative patients indicates persistence of infection for and likely recurrence of the infection. It is suggestive of another route of transmission as EV harbour SARS CoV2 RNA. EV associated RNA may determine the ongoing inflammation and clinical course of subjects with undetectable SARS-CoV2 virus and this may also have relevance in management of chronic liver disease patients.

scholarly journals Viral Mutation Rates

2010 ◽  
Vol 84 (19) ◽  
pp. 9733-9748 ◽  
Author(s):  
Rafael Sanjuán ◽  
Miguel R. Nebot ◽  
Nicola Chirico ◽  
Louis M. Mansky ◽  
Robert Belshaw
Keyword(s):  
Mutation Rate ◽  
Rna Viruses ◽  
Experimental Testing ◽  
Mutation Rates ◽  
Cell Infection ◽  
Nucleotide Substitutions ◽  
Data Set ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Viral Mutation

ABSTRACT Accurate estimates of virus mutation rates are important to understand the evolution of the viruses and to combat them. However, methods of estimation are varied and often complex. Here, we critically review over 40 original studies and establish criteria to facilitate comparative analyses. The mutation rates of 23 viruses are presented as substitutions per nucleotide per cell infection (s/n/c) and corrected for selection bias where necessary, using a new statistical method. The resulting rates range from 10−8 to10−6 s/n/c for DNA viruses and from 10−6 to 10−4 s/n/c for RNA viruses. Similar to what has been shown previously for DNA viruses, there appears to be a negative correlation between mutation rate and genome size among RNA viruses, but this result requires further experimental testing. Contrary to some suggestions, the mutation rate of retroviruses is not lower than that of other RNA viruses. We also show that nucleotide substitutions are on average four times more common than insertions/deletions (indels). Finally, we provide estimates of the mutation rate per nucleotide per strand copying, which tends to be lower than that per cell infection because some viruses undergo several rounds of copying per cell, particularly double-stranded DNA viruses. A regularly updated virus mutation rate data set will be available at www.uv.es/rsanjuan/virmut .

High Throughput Semi-Automated SARS-CoV-2 Library Preparation Protocol for Ion Torrent Sequencing using Opentrons, New England Biolabs Kit, and ARTIC Primers v2

protocols.io ◽  
2021 ◽  
Author(s):  
Elias Dahdouh ◽  
Fernando Lázaro Perona ◽  
María Rodríguez Tejedor ◽  
Rubén Cáceres Sánchez ◽  
Iván Bloise Sánchez ◽  
...  
Keyword(s):  
New England ◽  
High Throughput ◽  
Ion Torrent ◽  
Library Preparation ◽  
England Biolabs ◽  
Ion Torrent Sequencing ◽  
Library Preparation Protocol

Error Thresholds in Genetic Algorithms

2006 ◽  
Vol 14 (2) ◽  
pp. 157-182 ◽  
Author(s):  
Gabriela Ochoa
Keyword(s):  
Genetic Algorithms ◽  
Selection Pressure ◽  
Evolutionary Process ◽  
Critical Value ◽  
Mutation Rates ◽  
Genomic Information ◽  
Quasispecies Model ◽  
Error Catastrophe ◽  
Important Notion ◽  
Quasispecies Evolution

The error threshold of replication is an important notion in the quasispecies evolution model; it is a critical mutation rate (error rate) beyond which structures obtained by an evolutionary process are destroyed more frequently than selection can reproduce them. With mutation rates above this critical value, an error catastrophe occurs and the genomic information is irretrievably lost. Therefore, studying the factors that alter this magnitude has important implications in the study of evolution. Here we use a genetic algorithm, instead of the quasispecies model, as the underlying model of evolution, and explore whether the phenomenon of error thresholds is found on finite populations of bit strings evolving on complex landscapes. Our empirical results verify the occurrence of error thresholds in genetic algorithms. In this way, this notion is brought from molecular evolution to evolutionary computation. We also study the effect of modifying the most prominent evolutionary parameters on the magnitude of this critical value, and found that error thresholds depend mainly on the selection pressure and genotype length.

Quality control of Ion Torrent sequencing library

2014 ◽  
Vol 14 (2-3) ◽  
pp. 93-101 ◽  
Author(s):  
Laura-Ancuţa Pop ◽  
Emil Puscas ◽  
Valentina Pileczki ◽  
Roxana Cojocneanu-Petric ◽  
Cornelia Braicu ◽  
...  
Keyword(s):  
Quality Control ◽  
Ion Torrent ◽  
Sequencing Library ◽  
Ion Torrent Sequencing

scholarly journals Novel viruses detected in bats in the Republic of Korea

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sook-Young Lee ◽  
Chul-Un Chung ◽  
Jun Soo Park ◽  
Jae-Ku Oem
Keyword(s):  
Genetic Relationships ◽  
Rt Pcr ◽  
Entire Genome ◽  
Rotavirus A ◽  
Zoonotic Viruses ◽  
Feces Sample ◽  
The Family ◽  
The Republic ◽  
Viral Sequences ◽  
Generation Sequencing

AbstractBats are natural reservoirs for potential zoonotic viruses. In this study, next-generation sequencing was performed to obtain entire genome sequences of picornavirus from a picornavirus-positive bat feces sample (16BF77) and to explore novel viruses in a pooled bat sample (16BP) from samples collected in South Korea, 2016. Fourteen mammalian viral sequences were identified from 16BF77 and 29 from 16BP, and verified by RT-PCR. The most abundant virus in 16BF77 was picornavirus. Highly variable picornavirus sequences encoding 3Dpol were classified into genera Kobuvirus, Shanbavirus, and an unassigned group within the family Picornaviridae. Amino acid differences between these partial 3Dpol sequences were ≥ 65.7%. Results showed that one bat was co-infected by picornaviruses of more than two genera. Retrovirus, coronavirus, and rotavirus A sequences also were found in the BP sample. The retrovirus and coronavirus genomes were identified in nine and eight bats, respectively. Korean bat retroviruses and coronavirus demonstrated strong genetic relationships with a Chinese bat retrovirus (RfRV) and coronavirus (HKU5-1), respectively. A co-infection was identified in one bat with a retrovirus and a coronavirus. Our results indicate that Korean bats were multiply infected by several mammal viruses.

scholarly journals A Fatal Case of COVID-19 in an Infant with Severe Acute Malnutrition Admitted to a Paediatric Ward in Niger

2020 ◽  
Vol 2020 ◽  
pp. 1-3
Author(s):  
Alido Soumana ◽  
Aboubacar Samaila ◽  
Lamine Mahaman Moustapha ◽  
Moumouni Kamaye ◽  
Balkissa Daouda ◽  
...  
Keyword(s):  
Nasal Swab ◽  
Severe Acute Malnutrition ◽  
Fatal Case ◽  
Acute Malnutrition ◽  
Rt Pcr ◽  
X Ray ◽  
Laboratory Confirmation ◽  
History Of ◽  
Chest X Ray ◽  
The Right

While there have been very few fatal cases, SARS-CoV-2 has been reported in paediatric patients. This study aims to describe a fatal case of COVID-19 in a child with severe acute malnutrition. The eight-month-old child presented with fever, diarrhoea, and difficulty in breathing. The mother of the child had fever and shortness of breath four weeks before she died. Physical examination revealed lethargy, dehydration, and severe weight loss with a weight of 5 kg at a height of 78 cm tall. The weight-for-height index was less than three Z-scores, which corresponds to severe acute malnutrition. The pulmonary examination revealed moderate respiratory distress, and the chest X-ray presented features suggestive of pneumonia in the right lung area. In the context of the COVID-19 outbreak in Niger and the circumstances of the mother’s death, a nasal swab was taken for laboratory confirmation. Treatment provided to the child included intranasal oxygen, antibiotics, and a dietary program with therapeutic milk. The child died 48 hours after his admission. The history of contact with a SARS-CoV-2 suspect or positive patient should lead to screening for infection by using RT-PCR. It is important to investigate malnutrition as a potential risk factor for severe SARS-CoV-2 infection and resultant mortality.

scholarly journals Identification of Genetic Mutations in Human Lung Cancer by Targeted Sequencing

2015 ◽  
Vol 14 ◽  
pp. CIN.S22941 ◽  
Author(s):  
Hongxiang Feng ◽  
Xiaowei Wang ◽  
Zhenrong Zhang ◽  
Chuanning Tang ◽  
Hua Ye ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Human Lung ◽  
Chinese Patients ◽  
Human Lung Cancer ◽  
Genetic Mutations ◽  
Ion Torrent ◽  
Cancer Subtypes ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Frequent Mutations

Lung cancer remains the most prevalent malignancy and the primary cause of cancer-related deaths worldwide. Unique mutations patterns can be found in lung cancer subtypes, in individual cancers, or within a single tumor, and drugs that target these genetic mutations and signal transduction pathways are often beneficial to patients. In this study, we used the Ion Torrent AmpliSeq Cancer Panel to sequence 737 loci from 45 cancer-related genes and oncogenes to identify genetic mutations in 48 formalin-fixed, paraffin-embedded (FFPE) human lung cancer samples from Chinese patients. We found frequent mutations in EGFR, KRAS, PIK3CA, and TP53 genes. Moreover, we observed that a portion of the lung cancer samples harbored two or more mutations in these key genes. This study demonstrates the feasibility of using the Ion Torrent sequencing to efficiently identify genetic mutations in individual tumors for targeted lung cancer therapy.

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