Recommendations for Uniform Variant Calling of SARS-CoV-2 Genome Sequence across Bioinformatic Workflows.

Genomic sequencing of clinical samples to identify emerging variants of SARS-CoV-2 has been a key public health tool for curbing the spread of the virus. As a result, an unprecedented number of SARS-CoV-2 genomes were sequenced during the COVID-19 pandemic, which allowed for rapid identification of genetic variants, enabling the timely design and testing of therapies and deployment of new vaccine formulations to combat the new variants. However, despite the technological advances of deep sequencing, the analysis of the raw sequence data generated globally is neither standardized nor consistent, leading to vastly disparate sequences that may impact identification of variants. Here, we show that for both Illumina and Oxford Nanopore sequencing platforms, downstream bioinformatic protocols used by industry, government, and academic groups resulted in different virus sequences from same sample. These bioinformatic workflows produced consensus genomes with differences in single nucleotide polymorphisms, inclusion and exclusion of insertions, and/or deletions, despite using the same raw sequence as input datasets. Here, we compared and characterized such discrepancies and propose a specific suite of parameters and protocols that should be adopted across the field. Consistent results from bioinformatic workflows are fundamental to SARS-CoV-2 and future pathogen surveillance efforts, including pandemic preparation, to allow for a data-driven and timely public health response.

Broad-spectrum activity of bulevirtide against clinical isolates of HDV and recombinant pan-genotypic combinations of HBV/HDV.

Background & Aims: Bulevirtide (BLV) is a small lipopeptide agent that specifically binds to the sodium taurocholate cotransporting polypeptide (NTCP) bile salt transporter and HBV/HDV receptor on the surface of human hepatocytes and inhibits HDV and HBV entry. As a satellite virus of HBV, HDV virions are formed after assembly of HDV RNA with the HBV envelope proteins (HBsAg). Because both viruses exist as eight different genotypes, this creates a potential for high diversity in the HBV/HDV combinations. To investigate the sensitivity of various combinations of HBV/HDV genotypes to BLV, clinical and laboratory strains were assessed. Methods: For the laboratory strains, the different envelopes from HBV genotypes A through H were combined with HDV genotypes 1-8 in cotransfection assays. Clinical plasma isolates were obtained from clinical studies and academic collaborations to maximise the diversity of HBV/HDV genotypes tested. Results: The mean BLV EC50 against HDV laboratory strains ranged from 0.44 to 0.64 nM. Regardless of HBV and HDV genotypes, the clinical isolates showed similar sensitivities to BLV with mean values that ranged from 0.2 to 0.73 nM. Conclusions: These data support the use of BLV in patients infected with any HBV/HDV genotypes. Impact and implications: This study describes the potent activity of BLV against multiple laboratory strains spanning all HBV/HDV A-H/1-8 genotype combinations and the most diverse collection of HDV clinical samples tested to date, including HBV/HDV genotype combinations less frequently observed in the clinic. Overall, all isolates and laboratory strains displayed similar in vitro nanomolar sensitivity to BLV. This broad-spectrum antiviral activity of BLV has direct implications on potential simplified treatment for any patient infected with HDV, regardless of genotype, and supports the new 2023 EASL Clinical Practice Guidelines on HDV that recommend antiviral treatment for all patients with CHD.

No virologic resistance to bulevirtide monotherapy detected in patients through 24 weeks treatment in phase II and III clinical trials for chronic hepatitis delta.

BACKGROUND & AIMS: Bulevirtide (BLV) is a HDV/HBV entry inhibitor that is associated with virologic response (responders, HDV-RNA undetectable or ≥2 log10 IU/ml decrease from baseline) in >50% of patients after a 24-week treatment. However, some patients only achieve a <1 log10 IU/ml decline in HDV-RNA after the 24-week treatment (non-responders). Here, we report a viral resistance analysis in participants receiving BLV monotherapy who were non-responders or experienced virologic breakthrough (VB, i.e., two consecutive increases in HDV-RNA of ≥1 log10 IU/ml from nadir or two consecutive HDV-RNA detectable results if previously undetectable) from the phase II MYR202 and phase III MYR301 study. METHODS: Deep-sequencing of the BLV-corresponding region in HBV PreS1 and of the HDV HDAg gene, as well as in vitro phenotypic testing, were performed for the participant with VB (n = 1) and non-responders (n = 20) at baseline (BL) and Week 24 (WK24). RESULTS: No amino acid exchanges associated with reduced susceptibility to BLV within the BLV-corresponding region or within HDAg were identified in isolates from any of the 21 participants at BL or at WK24. Although variants (HBV n = 1; HDV n = 13) were detected at BL in some non-responders or in the participant with VB, none were associated with reduced sensitivity to BLV in vitro. Furthermore, the same variant was detected in virologic responders. A comprehensive phenotypic analysis demonstrated that the BLV EC50 values from 116 BL samples were similar across non-responders, partial responders (HDV RNA decline ≥1 but <2 log10 IU/ml), and responders regardless of the presence of HBV and/or HDV polymorphisms. CONCLUSIONS: No amino acid substitutions associated with reduced sensitivity to BLV monotherapy were detected at BL or WK24 in non-responders or the participant with VB after 24-week BLV treatment. IMPACT AND IMPLICATIONS: This is the first study investigating the development of resistance in patients treated with BLV. Excluding resistance to BLV as an explanation for an insufficient decrease in HDV-RNA levels during BLV therapy is an important finding for patients, clinicians, and researchers. It demonstrates that BLV has a high barrier to resistance, indicating it is safe and suitable for long-term treatment, although long-term surveillance for resistance should be performed. Our results hint at other still unknown mechanisms as an explanation for the persistence of serum HDV-RNA during inhibition of viral entry. CLINICAL TRIAL NUMBERS: NCT03546621 and NCT03852719.

Sequence characterization of extracellular HBV RNA in patient plasma.

Antiviral nucleos(t)ide analogue therapies inhibit HBV replication and suppress the HBV DNA levels in patients with chronic HBV infection. Since HBV RNAs are expressed from cccDNA or HBV integrated sequences, independently of viral genome replication, levels of HBV RNAs in plasma may remain high following treatment with nucleos(t)ide analogue. Thus, HBV RNAs have been proposed to be used as a viral biomarker for treatment outcome and disease progression. Recent investigations of plasma HBV RNAs described the presence of full length as well as subgenomic forms of RNA. To support the usage of plasma HBV RNAs as a viral biomarker, further understanding of HBV RNA composition in clinical samples is needed. Here, sequence of extracellular HBV RNAs was characterized in plasma samples of patients with chronic HBV infection using two independent RNA amplification methods that do not use HBV-specific primers for amplification: total RNA (NuGEN RNAseq) and mRNA (TruSeq RNAseq). Sequencing coverage was obtained across the full length of HBV genome for both methods, confirming the presence of full-length HBV RNA in plasma. The sequence of HBV RNA was nearly identical to plasma HBV DNA sequence in each sample with only 0-14 (median 4) mismatches over 3 kb. Thus, sequence of HBV RNA plasma reflects the intrahepatic viral reservoir and can be used for monitoring of sequence variants such as resistance in clinical trials. Additionally, RNA splice forms, different polyA tails start positions and presence of HBV-human chimeric transcript were identified.

Towards increased accuracy and reproducibility in SARS-CoV-2 next generation sequence analysis for public health surveillance.

During the COVID-19 pandemic, SARS-CoV-2 surveillance efforts integrated genome sequencing of clinical samples to identify emergent viral variants and to support rapid experimental examination of genome-informed vaccine and therapeutic designs. Given the broad range of methods applied to generate new viral genomes, it is critical that consensus and variant calling tools yield consistent results across disparate pipelines. Here we examine the impact of sequencing technologies (Illumina and Oxford Nanopore) and 7 different downstream bioinformatic protocols on SARS-CoV-2 variant calling as part of the NIH Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Tracking Resistance and Coronavirus Evolution (TRACE) initiative, a public-private partnership established to address the COVID-19 outbreak. Our results indicate that bioinformatic workflows can yield consensus genomes with different single nucleotide polymorphisms, insertions, and/or deletions even when using the same raw sequence input datasets. We introduce the use of a specific suite of parameters and protocols that greatly improves the agreement among pipelines developed by diverse organizations. Such consistency among bioinformatic pipelines is fundamental to SARS-CoV-2 and future pathogen surveillance efforts. The application of analysis standards is necessary to more accurately document phylogenomic trends and support data-driven public health responses.

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants.

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.