Global Prevalence of Adaptive and Prolonged Infections’ Mutations in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

From MDPI via Jisc Publications RouterHistory: accepted 2021-09-27, pub-electronic 2021-09-30Publication status: PublishedSeveral vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called ‘adaptive mutations’ and ‘mutations identified in the prolonged infections’, in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections’ mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus

SARS-CoV-2 variants of concern and spike protein mutational dynamics in a Swedish cohort during 2021, studied by Nanopore sequencing

From Springer Nature via Jisc Publications RouterHistory: received 2022-04-21, rev-recd 2022-09-08, accepted 2022-10-05, registration 2022-10-10, pub-electronic 2022-10-18, online 2022-10-18, collection 2022-12Acknowledgements: Acknowledgements: We are immensely grateful to all our co-workers at the Section for Clinical Microbiology and Hospital Hygiene at Uppsala University Hospital, who PCR tested all the COVID-19 samples and consequently extracted the viral RNA for us from the positive samples. Secondly, we are deeply thankful to Tor-Elesh Albrigtsen for the remarkable assistance with data science, analysis and programming in Python.Publication status: PublishedFunder: Science for Life Laboratory; doi: http://dx.doi.org/10.13039/501100009252; Grant(s): ZSC – National core facility for pandemic preparednessFunder: Scandinavian Society for Antimicrobial Chemotherapy Foundation; doi: http://dx.doi.org/10.13039/501100011777; Grant(s): SLS-961049Funder: Erik, Karin and Gösta Selander Foundation; Grant(s): 2022Funder: Regionala Forskningsrådet Uppsala/Örebro; doi: http://dx.doi.org/10.13039/100019032; Grant(s): RFR-930984Funder: Uppsala UniversityAbstract: Background: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. Methods: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. Results: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant’s signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. Conclusion: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Lastly, we report multiple systematic errors that occurred when following the ARTIC Network SARS-CoV-2 tiled amplicon approach using the V3 primers and Nanopore sequencing, which led to the masking of some of the important signature mutations in the Delta sequences

Prevalence of SARS-CoV-2 Omicron Sublineages and Spike Protein Mutations Conferring Resistance against Monoclonal Antibodies in a Swedish Cohort during 2022–2023

Monoclonal antibodies (mAbs) are an important treatment option for COVID-19 caused by SARS-CoV-2, especially in immunosuppressed patients. However, this treatment option can become ineffective due to mutations in the SARS-CoV-2 genome, mainly in the receptor binding domain (RBD) of the spike (S) protein. In the present study, 7950 SARS-CoV-2 positive samples from the Uppsala and Örebro regions of central Sweden, collected between March 2022 and May 2023, were whole-genome sequenced using amplicon-based sequencing methods on Oxford Nanopore GridION, Illumina MiSeq, Illumina HiSeq, or MGI DNBSEQ-G400 instruments. Pango lineages were determined and all single nucleotide polymorphism (SNP) mutations that occurred in these samples were identified. We found that the dominant sublineages changed over time, and mutations conferring resistance to currently available mAbs became common. Notable ones are R346T and K444T mutations in the RBD that confer significant resistance against tixagevimab and cilgavimab mAbs. Further, mutations conferring a high-fold resistance to bebtelovimab, such as the K444T and V445P mutations, were also observed in the samples. This study highlights that resistance mutations have over time rendered currently available mAbs ineffective against SARS-CoV-2 in most patients. Therefore, there is a need for continued surveillance of resistance mutations and the development of new mAbs that target more conserved regions of the RBD.Acknowledgements: We would like to thank the personnel at Clinical Microbiology and Hospital Hygiene, Uppsala University Hospital for their help with handling samples, RNA extraction, PCR, and whole-genome sequencing.Funder: Regional Research Council Mid Sweden; Grant(s): RFR-98011

Environmental Levels of the Antiviral Oseltamivir Induce Development of Resistance Mutation H274Y in Influenza A/H1N1 Virus in Mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals

Detection of Resistance Mutations to Antivirals Oseltamivir and Zanamivir in Avian Influenza A Viruses Isolated from Wild Birds

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are the first-line of defense against potentially fatal variants of influenza A pandemic strains. However, if resistant virus strains start to arise easily or at a high frequency, a new anti-influenza strategy will be necessary. This study aimed to investigate if and to what extent NA inhibitor–resistant mutants exist in the wild population of influenza A viruses that inhabit wild birds. NA sequences of all NA subtypes available from 5490 avian, 379 swine and 122 environmental isolates were extracted from NCBI databases. In addition, a dataset containing 230 virus isolates from mallard collected at Ottenby Bird Observatory (Öland, Sweden) was analyzed. Isolated NA RNA fragments from Ottenby were transformed to cDNA by RT-PCR, which was followed by sequencing. The analysis of genotypic profiles for NAs from both data sets in regard to antiviral resistance mutations was performed using bioinformatics tools. All 6221 sequences were scanned for oseltamivir- (I117V, E119V, D198N, I222V, H274Y, R292K, N294S and I314V) and zanamivir-related mutations (V116A, R118K, E119G/A/D, Q136K, D151E, R152K, R224K, E276D, R292K and R371K). Of the sequences from the avian NCBI dataset, 132 (2.4%) carried at least one, or in two cases even two and three, NA inhibitor resistance mutations. Swine and environmental isolates from the same data set had 18 (4.75%) and one (0.82%) mutant, respectively, with at least one mutation. The Ottenby sequences carried at least one mutation in 15 cases (6.52%). Therefore, resistant strains were more frequently found in Ottenby samples than in NCBI data sets. However, it is still uncertain if these mutations are the result of natural variations in the viruses or if they are induced by the selective pressure of xenobiotics (e.g., oseltamivir, zanamivir)

Global Prevalence of Adaptive and Prolonged Infections' Mutations in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

From PubMed via Jisc Publications RouterHistory: received 2021-08-06, revised 2021-09-23, accepted 2021-09-27Publication status: epublishSeveral vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called 'adaptive mutations' and 'mutations identified in the prolonged infections', in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections' mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus

Determination of Hepatitis C (HCV) Genotypes and Drug Resistances By a Efficient and Cost-effective Sequence Analysis Method : One cDNA synthesis, two assays

A more efficient, high specific and cost-effective RT-PCR sequencing method has developed for a correct HCV genotype and study the natural genetic variability and drug resistance within HCV non-structure region. Infection with hepatitis C virus (HCV) frequently leads to chronic hepatitis with an increased risk for the development of liver cirrhosis and liver cancer. HCV is classified into eleven major (designated 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains based on the sequence heterogeneity. In Sweden, the genotype distribution was different from that in studies from other parts of the world, with a lower frequency of genotype 1b and a higher frequency of genotype 1a and 3a. HCVgenotype differences affect responses to antiviral therapy, for exemple, patient infected with genotype 1 responds only 50% to PEG-IFN-á and ribavirin treatment in 48 weeks and approximately 80% of patients infected with HCV genotypes 2 and 3 treated with PEG-IFN-á plus ribavirin in 24 weeks achieve a sustained virological response. It has been suggest that the therapeutic strategy should be different for genotype 1 (and 4-6) and genotypes 2 and 3, respectively. Therefore, determination of HCV genotype and antiviral resistance are important and must be performed after the diagnosis of chronic hepatitis C, in order to provide the most effective treatment for HCV infected patients. To identify a correct genotypes and mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a "One cDNA synthesis and two assays" RT-nestPCR method where we sequenced the HCV NS5b region for the genotyping and protease gene for determination of resistance mutations. cDNA was synthesis using random primer and PCR primers were designed from the NS5b region for genotyping and NS3 regions for determining mutations which covers known 10 protease resistance in NS3, including R155K and V36M. Sequences were then analyzed and phylogenetic tree was made for genotypes according to alignment, and identification of resistance substitutions in the NS3 protease was performed by Seqscape software. RT-nestPCR assay was successful in samples containing >100IU/mL HCV RNA. The accuracy of this method has been validated by QCMD (Quality Control for Molecular Diagnosis, UK). Our method represents a more efficient in identifying mixture of genotypes (2k/1b, 2a/2c/2i), specific and reliable method for differentiation between all genotypes and subtypes, economic and is useful in study natural genetic, mutations and polymorphism within HCV NS3 protease region. This simple, more efficient, specific, low-cost and reproducible method can be used as a routine diagnostic and should also be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden

Determination of Hepatitis C (HCV) Genotypes and Drug Resistances By a Efficient and Cost-effective Sequence Analysis Method : One cDNA synthesis, two assays

A more efficient, high specific and cost-effective RT-PCR sequencing method has developed for a correct HCV genotype and study the natural genetic variability and drug resistance within HCV non-structure region. Infection with hepatitis C virus (HCV) frequently leads to chronic hepatitis with an increased risk for the development of liver cirrhosis and liver cancer. HCV is classified into eleven major (designated 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains based on the sequence heterogeneity. In Sweden, the genotype distribution was different from that in studies from other parts of the world, with a lower frequency of genotype 1b and a higher frequency of genotype 1a and 3a. HCVgenotype differences affect responses to antiviral therapy, for exemple, patient infected with genotype 1 responds only 50% to PEG-IFN-á and ribavirin treatment in 48 weeks and approximately 80% of patients infected with HCV genotypes 2 and 3 treated with PEG-IFN-á plus ribavirin in 24 weeks achieve a sustained virological response. It has been suggest that the therapeutic strategy should be different for genotype 1 (and 4-6) and genotypes 2 and 3, respectively. Therefore, determination of HCV genotype and antiviral resistance are important and must be performed after the diagnosis of chronic hepatitis C, in order to provide the most effective treatment for HCV infected patients. To identify a correct genotypes and mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a "One cDNA synthesis and two assays" RT-nestPCR method where we sequenced the HCV NS5b region for the genotyping and protease gene for determination of resistance mutations. cDNA was synthesis using random primer and PCR primers were designed from the NS5b region for genotyping and NS3 regions for determining mutations which covers known 10 protease resistance in NS3, including R155K and V36M. Sequences were then analyzed and phylogenetic tree was made for genotypes according to alignment, and identification of resistance substitutions in the NS3 protease was performed by Seqscape software. RT-nestPCR assay was successful in samples containing >100IU/mL HCV RNA. The accuracy of this method has been validated by QCMD (Quality Control for Molecular Diagnosis, UK). Our method represents a more efficient in identifying mixture of genotypes (2k/1b, 2a/2c/2i), specific and reliable method for differentiation between all genotypes and subtypes, economic and is useful in study natural genetic, mutations and polymorphism within HCV NS3 protease region. This simple, more efficient, specific, low-cost and reproducible method can be used as a routine diagnostic and should also be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden

Determination of Hepatitis C (HCV) Genotypes and Drug Resistances By a Efficient and Cost-effective Sequence Analysis Method : One cDNA synthesis, two assays

A more efficient, high specific and cost-effective RT-PCR sequencing method has developed for a correct HCV genotype and study the natural genetic variability and drug resistance within HCV non-structure region. Infection with hepatitis C virus (HCV) frequently leads to chronic hepatitis with an increased risk for the development of liver cirrhosis and liver cancer. HCV is classified into eleven major (designated 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains based on the sequence heterogeneity. In Sweden, the genotype distribution was different from that in studies from other parts of the world, with a lower frequency of genotype 1b and a higher frequency of genotype 1a and 3a. HCVgenotype differences affect responses to antiviral therapy, for exemple, patient infected with genotype 1 responds only 50% to PEG-IFN-á and ribavirin treatment in 48 weeks and approximately 80% of patients infected with HCV genotypes 2 and 3 treated with PEG-IFN-á plus ribavirin in 24 weeks achieve a sustained virological response. It has been suggest that the therapeutic strategy should be different for genotype 1 (and 4-6) and genotypes 2 and 3, respectively. Therefore, determination of HCV genotype and antiviral resistance are important and must be performed after the diagnosis of chronic hepatitis C, in order to provide the most effective treatment for HCV infected patients. To identify a correct genotypes and mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a "One cDNA synthesis and two assays" RT-nestPCR method where we sequenced the HCV NS5b region for the genotyping and protease gene for determination of resistance mutations. cDNA was synthesis using random primer and PCR primers were designed from the NS5b region for genotyping and NS3 regions for determining mutations which covers known 10 protease resistance in NS3, including R155K and V36M. Sequences were then analyzed and phylogenetic tree was made for genotypes according to alignment, and identification of resistance substitutions in the NS3 protease was performed by Seqscape software. RT-nestPCR assay was successful in samples containing >100IU/mL HCV RNA. The accuracy of this method has been validated by QCMD (Quality Control for Molecular Diagnosis, UK). Our method represents a more efficient in identifying mixture of genotypes (2k/1b, 2a/2c/2i), specific and reliable method for differentiation between all genotypes and subtypes, economic and is useful in study natural genetic, mutations and polymorphism within HCV NS3 protease region. This simple, more efficient, specific, low-cost and reproducible method can be used as a routine diagnostic and should also be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden