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Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection., Remdesivir is an approved antiviral treatment for COVID-19, but it needs to be administered intravenously. Here, Cox et al. show that GS-621763, a prodrug of remdesivir parent nucleoside GS-441524 has good oral bioavailability and inhibits SARS-CoV-2 and variants of concerns in ferrets.

Real‐time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real‐time. However, sequencing can take time and may not be accessible in all laboratories. Genotyping by RT‐ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS‐CoV‐2 detected in Washington State by using a combination of reverse‐transcription polymerase chain reaction (RT‐PCR), RT‐ddPCR, and next‐generation sequencing. We initially screened 1035 samples positive for SARS‐CoV‐2 by our CDC‐based laboratory‐developed assay using ThermoFisher's multiplex RT‐PCR COVID‐19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT‐ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69‐70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow‐up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT‐ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT‐ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS‐CoV‐2., Highlights ‐B.1.1.7 (alpha) lineage of SARS‐CoV‐2 was detected in Washington State using RT‐ddPCR.‐Specific allelic discrimination can be used to distinguish SARS‐CoV‐2 variants of concern.‐Allelic discrimination can identify variants of concern more rapidly than conventional sequencing techniques.‐S‐gene target failures can be used to screen for certain SARS‐CoV‐2 variants.

Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309—a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development. In this Article, the authors report the discovery of a small molecule (GHP-88309) that is a new class of allosteric viral polymerase inhibitor that works against two different genera of paramyxoviruses.

Rapid dissemination of SARS-CoV-2 sequencing data to public repositories has enabled widespread study of viral genomes, but studies of longitudinal specimens from infected persons are relatively limited. Analysis of longitudinal specimens enables understanding of how host immune pressures drive viral evolution in vivo. Here we performed sequencing of 49 longitudinal SARS-CoV-2-positive samples from 20 patients in Washington State collected between March and September of 2020. Viral loads declined over time with an average increase in RT-QPCR cycle threshold of 0.87 per day. We found that there was negligible change in SARS-CoV-2 consensus sequences over time, but identified a number of nonsynonymous variants at low frequencies across the genome. We observed enrichment for a relatively small number of these variants, all of which are now seen in consensus genomes across the globe at low prevalence. In one patient, we saw rapid emergence of various low-level deletion variants at the N-terminal domain of the spike glycoprotein, some of which have previously been shown to be associated with reduced neutralization potency from sera. In a subset of samples that were sequenced using metagenomic methods, differential gene expression analysis showed a downregulation of cytoskeletal genes that was consistent with a loss of ciliated epithelium during infection and recovery. We also identified co-occurrence of bacterial species in samples from multiple hospitalized individuals. These results demonstrate that the intrahost genetic composition of SARS-CoV-2 is dynamic during the course of COVID-19, and highlight the need for continued surveillance and deep sequencing of minor variants.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited. Methods We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples. Results We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January. Conclusions We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ~6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ∼6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

Amplicon-based sequencing methods have been central in characterizing the diversity, transmission and evolution of SARS-CoV-2, but need to be rigorously assessed for clinical utility. Here, we validated the Swift Biosciences’ SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens with a 95% limit of detection of ≥ 40.08 SARS-CoV-2 copies/PCR reaction. Breadth of genome recovery was evaluated across a range of Ct values (11.3 – 36.7, median 21.6). Out of 428 positive samples, 406 (94.9%) generated genomes with < 10% Ns, with a mean genome coverage of 13,545X ± SD 8,382X. No genomes were recovered from PCR-negative specimens (n = 30), or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared to whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of C

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [β]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Sequencing of plasma cell-free DNA (cfDNA) is a promising milieu for broad-based cancer and infectious disease diagnostics. The performance of cfDNA sequencing for infectious disease diagnostics is chiefly limited by inadequate analytical sensitivity. The current study investigated whether the analytical sensitivity of cfDNA sequencing for viral diagnostics could be improved by selective sequencing of short cfDNA fragments, given prior observations of shorter fragment size distribution in microbial and cytomegalovirus-derived cfDNA compared with human-derived cfDNA. It shows that the shorter plasma cfDNA fragment size distribution is a general feature of multiple DNA viruses, including adenovirus [interquartile range (IQR), 87 to 165 bp], herpes simplex virus 2 (IQR, 114 to 195 bp), human herpesvirus 6 (IQR, 145 to 176 bp), and varicella zoster virus (IQR, 98 to 182 bp), compared with human (IQR, 148 to 178 bp). It was used to further optimize a size selection–based cfDNA sequencing method, demonstrating an enrichment of viral sequences up to 16.6-fold, with a median fold enrichment of 6.7×, 4.6×, 2.2×, and 10.3× for adenovirus, herpes simplex virus 2, human herpesvirus 6, and varicella zoster virus, respectively. These findings demonstrate a simple yet scalable method for enhanced detection of DNA viremia that maintains the unbiased nature of cfDNA sequencing.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host., Author summary Syphilis continues to be a disease of global and public health concern, even though the infection can be easily diagnosed and effectively treated with penicillin. Although infected individuals often develop a strong immunity to the pathogen, repeated infection with the syphilis agent, Treponema pallidum subspecies pallidum (T. pallidum), is possible. Several studies point at antigenic variation of the T. pallidum TprK protein as the mechanism responsible for evasion of the immunity that develops during infection, pathogen persistence, and re-infection. Past studies have highlighted the importance of immune clearance of dominant variants that, in turn, allows less represented variants to emerge. The contribution of an immunity-independent baseline generation of variability in the tprK gene is less clear. Here, we used deep sequencing to profile tprK variants using a laboratory-isolated T. pallidum strain nearly isogenic for tprK that was propagated over time in vitro, where no immune pressure is exerted on the pathogen, as well as in samples obtained from immunosuppressed and immunocompetent rabbits infected with the same strain. We confirmed that tprK accumulates significantly more diversity under immune pressure, and demonstrated a low but discernible basal rate of gene conversion in complete absence of immune pressure.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains – including 191 T. pallidum subsp. pallidum – sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.Author SummaryEach year, millions of new cases of venereal and congenital syphilis, caused by the bacterium Treponema pallidum (T. pallidum) subsp. pallidum, are diagnosed worldwide, resulting in significant morbidity and mortality. Alongside endemic circulation of syphilis in low-income countries, disease resurgence in high-income nations has underscored the need for a vaccine. Due to prior technological limitations in culturing and sequencing the organism, the extent of the genetic diversity within modern strains of T. pallidum subsp. pallidum remains poorly understood, hampering development of a broadly protective vaccine. In this study, we obtained 196 near-complete T. pallidum genomes directly from clinical swabs from eight countries across six continents. Of these, 191 were identified as T. pallidum subsp. pallidum, including 90 Nichols clade genomes. Bayesian analysis revealed a high degree of variance in mutation rate among subclades. Interestingly, a Nichols subclade with a particularly high mutation rate harbors a non-synonymous mutation in a putative DNA repair helicase. Coupling sequencing data with protein structure prediction, we identified multiple novel amino acid variants in several proteins previously identified as potential vaccine candidates. Our data help inform current efforts to develop a broadly protective syphilis vaccine.

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.Author SummarySyphilis continues to be a disease of global and public health concern, even though the infection can be easily diagnosed and effectively treated with penicillin. Although infected individuals often develop a strong immunity to the pathogen, repeated infection with the syphilis agent, Treponema pallidum subspecies pallidum (T. pallidum), is possible. Several studies point at antigenic variation of the T. pallidum TprK protein as the mechanism responsible for evasion of the immunity that develops during infection, pathogen persistence, and re-infection. Past studies have highlighted the importance of immune clearance of dominant variants that, in turn, allows less represented variants to emerge. The contribution of an immunity-independent baseline generation of variability in the tprK gene is less clear. Here, we used deep sequencing to profile tprK variants using a laboratory-isolated T. pallidum strain nearly isogenic for tprK that was propagated over time in vitro, where no immune pressure is exerted on the pathogen, as well as in samples obtained from immunosuppressed and immunocompetent rabbits infected with the same strain. We confirmed that tprK accumulates significantly more diversity under immune pressure, and demonstrated a low but discernible basal rate of gene conversion in complete absence of immune pressure.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory ddPCR of urine samples taken from immunosuppressed individuals, we show clinical BKPyV and JCPyV strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were universally derived from type I subtype BKPyV. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCEDefective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens -- no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes with the majority affecting genes required for viral replication. Longitudinal analysis showed these DVGs can persist during an infection, but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests these parasitic sequences exist across the many classes of viruses capable of causing human disease.

BackgroundRapid dissemination of SARS-CoV-2 sequencing data to public repositories has enabled widespread study of viral genomes, but studies of longitudinal specimens from infected persons are relatively limited. Analysis of longitudinal specimens enables understanding of how host immune pressures drive viral evolutionin vivo.Methods and findingsHere we performed sequencing of 49 longitudinal SARS-CoV-2-positive samples from 20 patients in Washington State collected between March and September of 2020. Viral loads declined over time with an average increase in RT-PCR cycle threshold (Ct) of 0.87 per day. We found that there was negligible change in SARS-CoV-2 consensus sequences over time, but identified a number of nonsynonymous variants at low frequencies across the genome. We observed enrichment for a relatively small number of these variants, all of which are now seen in consensus genomes across the globe at low prevalence. In one patient, we saw rapid emergence of various low-level deletion variants at the N-terminal domain of the spike glycoprotein, some of which have previously been shown to be associated with reduced neutralization potency from sera. In a subset of samples that were sequenced using metagenomic methods, differential gene expression analysis showed a downregulation of cytoskeletal genes that was consistent with a loss of ciliated epithelium during infection and recovery. We also identified co-occurrence of bacterial species in samples from multiple hospitalized individuals.ConclusionsThese results demonstrate that the intrahost genetic composition of SARS-CoV-2 is dynamic during the course of COVID-19, and highlight the need for continued surveillance and deep sequencing of minor variants.

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN’s receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

Whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a better understanding of the virus’s origin, transmission, and evolution ([1][1][–][2][6][3]). Multiplex amplicon sequencing for viral WGS is a preferred approach to library preparation

Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host., Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein’s seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection.

Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein’s seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection. IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

Developing a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times., Author summary The causative agent of syphilis, Treponema pallidum subspecies pallidum, is capable of repeat infections in people, suggesting that the human immune response does not develop sufficiently broad or long-lasting immunity to cover treponemal diversity. Here, we examined the genomes from two blood-derived isolates of T. pallidum derived 6 years apart from a patient who had syphilis four times during the same period to understand the genetic basis of reinfection. We found a paucity of coding changes across the genome outside of the highly variable tprK gene. Using deep profiling of the full-length tprK gene, we found surprisingly that the two isolates did not share any full-length TprK sequences.

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

With their small genomes, fast evolutionary rates, and clinical significance, viruses have long been fodder for studies of whole genome evolution. One common need in these studies is the analysis of viral evolution over time through longitudinal sampling. However, there exists no simple tool to automate such analyses. We created a simple command-line visualization tool called LAVA (Longitudinal Analysis of Viral Alleles). LAVA allows dynamic and interactive visualization of viral evolution across the genome and over time. Results are easily shared via a single HTML file that also allows interactive analysis based on read depth and allele frequency. LAVA requires minimal input and runs in minutes for most use cases. LAVA is programmed mainly in Python 3 and is compatible with Mac and Linux machines. LAVA is a user-friendly command-line tool for generating, visualizing, and sharing the results of longitudinal viral genome evolution analysis. Instructions for downloading, installing, and using LAVA can be found at https://github.com/michellejlin/lava.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

BackgroundWith sequencing technologies becoming cheaper and easier to use, more groups are able to obtain whole genome sequences of viruses of public health and scientific importance. Submission of genomic data to NCBI GenBank is a requirement prior to publication and plays a critical role in making scientific data publicly available.GenBank currently has automatic prokaryotic and eukaryotic genome annotation pipelines but has no viral annotation pipeline beyond influenza virus. Annotation and submission of viral genome sequence is a non-trivial task, especially for groups that do not routinely interact with GenBank for data submissions.ResultsWe present Viral Annotation Pipeline and iDentification (VAPiD), a portable and lightweight command-line tool for annotation and GenBank deposition of viral genomes. VAPiD supports annotation of nearly all unsegmented viral genomes. The pipeline has been validated on human immunodeficiency virus, human parainfluenza virus 1-4, human metapneumovirus, human coronaviruses (229E/OC43/NL63/HKU1/SARS/MERS), human enteroviruses/rhinoviruses, measles virus, mumps virus, Hepatitis A-E Virus, Chikungunya virus, dengue virus, and West Nile virus, as well the human polyomaviruses BK/JC/MCV, human adenoviruses, and human papillomaviruses. The program can handle individual or batch submissions of different viruses to GenBank and correctly annotates multiple viruses, including those that contain ribosomal slippage or RNA editing without prior knowledge of the virus to be annotated. VAPiD is programmed in Python and is compatible with Windows, Linux, and Mac OS systems.ConclusionsWe have created a portable, lightweight, user-friendly, internet-enabled, open-source, command-line genome annotation and submission package to facilitate virus genome submissions to NCBI GenBank. Instructions for downloading and installing VAPiD can be found athttps://github.com/rcs333/VAPiD.

With sequencing technologies becoming cheaper and easier to use, more groups are able to obtain whole genome sequences of viruses of public health and scientific importance. Submission of genomic data to NCBI GenBank is a requirement prior to publication and plays a critical role in making scientific data publicly available. GenBank currently has automatic prokaryotic and eukaryotic genome annotation pipelines but has no viral annotation pipeline beyond influenza virus. Annotation and submission of viral genome sequence is a non-trivial task, especially for groups that do not routinely interact with GenBank for data submissions. We present Viral Annotation Pipeline and iDentification (VAPiD), a portable and lightweight command-line tool for annotation and GenBank deposition of viral genomes. VAPiD supports annotation of nearly all unsegmented viral genomes. The pipeline has been validated on human immunodeficiency virus, human parainfluenza virus 1–4, human metapneumovirus, human coronaviruses (229E/OC43/NL63/HKU1/SARS/MERS), human enteroviruses/rhinoviruses, measles virus, mumps virus, Hepatitis A-E Virus, Chikungunya virus, dengue virus, and West Nile virus, as well the human polyomaviruses BK/JC/MCV, human adenoviruses, and human papillomaviruses. The program can handle individual or batch submissions of different viruses to GenBank and correctly annotates multiple viruses, including those that contain ribosomal slippage or RNA editing without prior knowledge of the virus to be annotated. VAPiD is programmed in Python and is compatible with Windows, Linux, and Mac OS systems. We have created a portable, lightweight, user-friendly, internet-enabled, open-source, command-line genome annotation and submission package to facilitate virus genome submissions to NCBI GenBank. Instructions for downloading and installing VAPiD can be found at https://github.com/rcs333/VAPiD .