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 SARS-CoV-2 pandemic posed an unpreceded threat to the management of other pandemics such as 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 from 2018 to 2021. We used a sequencing-based recency assay to identify recent HIV-1 infections and to determine the days post infection (DPI) for each person at the time of diagnosis. RESULTS: Sequencing was done using 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 from other years. DPI analysis showed that 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.

Promising Best Practices Implemented in Long-Term Care Facilities During the COVID-19 Pandemic to Address Social Isolation and Loneliness: A Scoping Review

Context: Throughout the current COVID-19 pandemic, tremendous effort has been made to implement innovative practices to address social isolation and loneliness (SIL) in long-term care facilities (LTCFs), disproportionally affected by COVID-19. These interventions have not yet been synthesized. This review intended to gather the current promising best practices (PBPs) implemented in LTCFs to alleviate SIL in older persons during the COVID-19 pandemic as well as during the SARS and H1N1 pandemics, using an intersectional lens. Methods: An extensive search was done in nine electronic databases. Arksey and O'Malley's framework was used to format the scoping review. Two independent reviewers screened citations for inclusion, blindly. The selection of articles was conducted blindly by two coauthors. Finally, 16 studies were analyzed out of 9,077 records. Results: Two main themes of findings arose from this review. They comprised proximal PBPs directly addressing SIL in LTCF residents such as pseudo-contact interventions (e.g., chat from balcony or behind transparent barriers/glasses), remote communication tools (e.g., phone or video chat, voice mail/text messaging), and humanoid robots. Distal PBPs included measures implemented to prevent or mitigate the development of COVID-19, including COVID-19 screening approaches, outbreak preparedness, quarantining approaches for both residents and staff. Conclusion: This scoping review found varied PBP implemented during the multiple waves of the COVID-19 pandemic as well as evidence supporting their effectiveness. The contribution of this study is significant as most of the PBP investigated should be prioritized by public policymakers or institutions to provide more satisfactory services to the elderly and their families. © 2022 The Author(s).

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.

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.

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.

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.

Sinus computed tomography findings in patients with COVID-19

OBJECTIVE: To analyze computed tomography scans of paranasal sinuses of a series of patients with coronavirus disease 2019, and correlate the findings with the disease. METHODS: Computed tomography scans of 95 adult patients who underwent a polymerase chain reaction test for severe acute respiratory syndrome coronavirus 2 were analyzed. Clinical data were obtained from patients' records and telephone calls. Paranasal sinus opacification was graded and compared according to severe acute respiratory syndrome coronavirus 2 positivity. RESULTS: Of the patients 28 (29.5%) tested positive for severe acute respiratory syndrome coronavirus 2 (median age 52 [range 26-95] years) and 67 were negative (median age 50 [range 18-95] years). Mucosal thickening was present in 97.4% of maxillary sinuses, 80% of anterior ethmoid air cells, 75.3% of posterior ethmoid air cells, 74.7% of frontal sinuses, and 66.3% of sphenoid sinuses. Minimal or mild mucosal thickening (score 1)and normally aerated sinuses (score 0) corresponded to 71.4% and 21.3% of all paranasal sinuses, respectively. The mean score of each paranasal sinus among severe acute respiratory syndrome coronavirus 2 positive and negative patients was 0.85+/-0.27 and 0.87+/-0.38, respectively (p=0.74). Median paranasal sinus opacification score among severe acute respiratory syndrome coronavirus 2 positive patients was 9 (interquartile range 8-10) compared to 9 (interquartile range 5-10) in negative patients (p=0.89). There was no difference in mean score adjusted for age and sex. Nasal congestion was more frequent in severe acute respiratory syndrome coronavirus 2 positive than negative patients (p=0.05). CONCLUSION: Severe acute respiratory syndrome coronavirus 2 infection was associated with patient recall of nasal congestion, but showed no correlation with opacification of paranasal sinuses.