Impact Of Low-Frequency HIV-1 Drug Resistance Mutations On Antiretroviral Therapy Outcomes.

BACKGROUND: The association between low-frequency HIV-1 drug resistance mutations (DRMs) and treatment failure (TF) is controversial. We explore this association using NGS methods that accurately sample low-frequency DRMs. METHODS: We enrolled women with HIV-1 in Malawi who were either ART naïve (A), had ART failure (B), or had discontinued ART (C). At entry, A and C began an NNRTI-based regimen and B started a PI-based regimen. We used Primer ID MiSeq to identify regimen-relevant DRMs in entry and TF plasma samples, and a Cox proportional hazards model to calculate hazard ratios (HRs) for entry DRMs. Low-frequency DRMs were defined as ≤ 20%. RESULTS: We sequenced 360 participants. Cohort B and C participants were more likely to have TF than Cohort A participants. The presence of K103N at entry significantly increased TF risk among A and C participants at both high and low frequency, with HR of 3.12 [1.58-6.18, 95% CI] and 2.38 [1.00-5.67, 95% CI] respectively. At TF, 45% of participants showed selection of DRMs while in the remaining participants there was an apparent lack of selective pressure from ART. CONCLUSIONS: Using accurate NGS for DRM detection may benefit an additional 10% of the patients by identifying low-frequency K103N mutations.

Impact of Low-Frequency Human Immunodeficiency Virus Type 1 Drug Resistance Mutations on Antiretroviral Therapy Outcomes.

BACKGROUND: The association between low-frequency human immunodeficiency virus type 1 (HIV-1) drug resistance mutations (DRMs) and treatment failure (TF) is controversial. We explore this association using next-generation sequencing (NGS) methods that accurately sample low-frequency DRMs. METHODS: We enrolled women with HIV-1 in Malawi who were either antiretroviral therapy (ART) naive (cohort A), had ART failure (cohort B), or had discontinued ART (cohort C). At entry, cohorts A and C began a nonnucleoside reverse transcriptase inhibitor-based regimen and cohort B started a protease inhibitor-based regimen. We used Primer ID MiSeq to identify regimen-relevant DRMs in entry and TF plasma samples, and a Cox proportional hazards model to calculate hazard ratios (HRs) for entry DRMs. Low-frequency DRMs were defined as ≤20%. RESULTS: We sequenced 360 participants. Cohort B and C participants were more likely to have TF than cohort A participants. The presence of K103N at entry significantly increased TF risk among A and C participants at both high and low frequency, with HRs of 3.12 (95% confidence interval [CI], 1.58-6.18) and 2.38 (95% CI, 1.00-5.67), respectively. At TF, 45% of participants showed selection of DRMs while in the remaining participants there was an apparent lack of selective pressure from ART. CONCLUSIONS: Using accurate NGS for DRM detection may benefit an additional 10% of patients by identifying low-frequency K103N mutations.

Use of Next-Generation Sequencing in a State-Wide Strategy of HIV-1 Surveillance: Impact of the SARS-COV-2 Pandemic on HIV-1 Diagnosis and Transmission.

BACKGROUND: The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic posed an unpreceded threat to the management of other pandemics such as human immunodeficiency virus-1 (HIV-1) in the United States. The full impact of the SARS-CoV-2 pandemic on the HIV-1 pandemic needs to be evaluated. METHODS: All individuals with newly reported HIV-1 diagnoses from NC State Laboratory of Public Health were enrolled in this prospective observational study, 2018-2021. We used a sequencing-based recency assay to identify recent HIV-1 infections and to determine the days postinfection (DPI) for each person at the time of diagnosis. RESULTS: Sequencing used diagnostic serum samples from 814 individuals with new HIV-1 diagnoses spanning this 4-year period. Characteristics of individuals diagnosed in 2020 differed from those in other years. People of color diagnosed in 2021 were on average 6 months delayed in their diagnosis compared to those diagnosed in 2020. There was a trend that genetic networks were more known for individuals diagnosed in 2021. We observed no major integrase resistance mutations over the course of the study. CONCLUSIONS: SARS-CoV-2 pandemic may contribute to the spread of HIV-1. Public health resources need to focus on restoring HIV-1 testing and interrupting active, ongoing, transmission.

ß-d-N4-hydroxycytidine Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells.

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in genomes of RNA viruses during viral replication. ß-d-N4-hydroxycytidine (NHC, initial metabolite of molnupiravir) is >100-fold more active than ribavirin or favipiravir against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate highly active mutagenic ribonucleosides may hold risk for the host.

N4 -Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness.

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N 4 -hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5'-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.

Unique Molecular Identifiers and Multiplexing Amplicons Maximize the Utility of Deep Sequencing To Critically Assess Population Diversity in RNA Viruses.

Next generation sequencing (NGS)/deep sequencing has become an important tool in the study of viruses. The use of unique molecular identifiers (UMI) can overcome the limitations of PCR errors and PCR-mediated recombination and reveal the true sampling depth of a viral population being sequenced in an NGS experiment. This approach of enhanced sequence data represents an ideal tool to study both high and low abundance drug resistance mutations and more generally to explore the genetic structure of viral populations. Central to the use of the UMI/Primer ID approach is the creation of a template consensus sequence (TCS) for each genome sequenced. Here we describe a series of experiments to validate several aspects of the Multiplexed Primer ID (MPID) sequencing approach using the MiSeq platform. We have evaluated how multiplexing of cDNA synthesis and amplicons affects the sampling depth of the viral population for each individual cDNA and amplicon to understand the relationship between broader genome coverage versus maximal sequencing depth. We have validated reproducibility of the MPID assay in the detection of minority mutations in viral genomes. We have also examined the determinants that allow sequencing reads of PCR recombinants to contaminate the final TCS data set and show how such contamination can be limited. Finally, we provide several examples where we have applied MPID to analyze features of minority variants and describe limits on their detection in viral populations of HIV-1 and SARS-CoV-2 to demonstrate the generalizable utility of this approach with any RNA virus.

Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome.

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that ß-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.

Correction: Mechanical rigidity of a shape-memory metal-organic framework increases by crystal downsizing.

Correction for 'Mechanical rigidity of a shape-memory metal-organic framework increases by crystal downsizing' by Al A. Tiba et al., Chem. Commun., 2021, 57, 89-92, DOI: 10.1039/D0CC05684G.

Mechanical rigidity of a shape-memory metal-organic framework increases by crystal downsizing.

Soft porous nanocrystals with a pronounced shape-memory effect exhibit two- to three-fold increase in elastic modulus compared to the microcrystalline counterpart as determined by atomic force microscopy nanoindentation. The increase in rigidity is consistent with the known shape-memory effect displayed by the framework solid at the nanoscale. Crystal downsizing can offer new avenues for tailoring the mechanical properties of metal-organic frameworks.

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.