Viral Genome Research Articles

Exploration of the genetic landscape of bacterial dsDNA viruses reveals an ANI gap amid extensive mosaicism.

Average nucleotide identity (ANI) is a widely used metric to estimate genetic relatedness, especially in microbial species delineation. While ANI calculation has been well optimized for bacteria and closely related viral genomes, accurate estimation of ANI below 80%, particularly in large reference data sets, has been challenging due to a lack of accurate and scalable methods. To bridge this gap, we introduce MANIAC, an efficient computational pipeline optimized for estimating ANI and alignment fraction (AF) in viral genomes with divergence around ANI of 70%. Using a rigorous simulation framework, we demonstrate MANIAC's accuracy and scalability compared to existing approaches, even to data sets of hundreds of thousands of viral genomes. Applying MANIAC to a curated data set of complete bacterial dsDNA viruses revealed a multimodal ANI distribution, with a distinct gap around 80%, akin to the bacterial ANI gap (~90%) but shifted, likely due to viral-specific evolutionary processes such as recombination dynamics and mosaicism. We then evaluated ANI and AF as predictors of genus-level taxonomy using a logistic regression model. We found that this model has strong predictive power (PR-AUC = 0.981), but that it works much better for virulent (PR-AUC = 0.997) than temperate (PR-AUC = 0.847) bacterial viruses. This highlights the complexity of taxonomic classification in temperate phages, known for their extensive mosaicism, and cautions against over-reliance on ANI in such cases. MANIAC can be accessed at https://github.com/bioinf-mcb/MANIAC.IMPORTANCEWe introduce a novel computational pipeline called MANIAC, designed to accurately assess average nucleotide identity (ANI) and alignment fraction (AF) between diverse viral genomes, scalable to data sets of over 100k genomes. Using computer simulations and real data analyses, we show that MANIAC could accurately estimate genetic relatedness between pairs of viral genomes of around 60%-70% ANI. We applied MANIAC to investigate the question of ANI discontinuity in bacterial dsDNA viruses, finding evidence for an ANI gap, akin to the one seen in bacteria but around ANI of 80%. We then assessed the ability of ANI and AF to predict taxonomic genus boundaries, finding its strong predictive power in virulent, but not in temperate phages. Our results suggest that bacterial dsDNA viruses may exhibit an ANI threshold (on average around 80%) above which recombination helps maintain population cohesiveness, as previously argued in bacteria.

Tetraspanins 10 and 15 support Venezuelan equine encephalitis virus replication in astrocytoma cells.

Tetraspanins (Tspans) are transmembrane proteins that coordinate life cycle steps of viruses from distinct families. Here, we identify the human Tspan10 and Tspan15, both members of the TspanC8 subfamily, as replication factors for alphavirus Venezuelan equine encephalitis virus (VEEV) in astrocytoma cells. Pharmacological inhibition and siRNA-mediated silencing of TspanC8 interactor a disintegrin and metalloproteinase 10 (ADAM10) reduced VEEV infection. Silencing of Tspan10, Tspan15 and ADAM10 did not affect VEEV entry but diminished viral genome replication. We report that Tspan10 is important for VEEV infection of several cell lines, while silencing of Tspan15 diminishes infection with several alphaviruses, but not flaviviruses, in astrocytoma cells. Conversely, we demonstrate that siRNA-mediated silencing of Tspan14, another member of the TspanC8 family, enhances infection with lentiviral pseudoparticles harbouring the envelope proteins of VEEV, identifying it as a restriction factor for VEEV entry. Silencing of ADAM10/Tspan15 substrate neuronal (N)-cadherin reduced VEEV infectivity, suggesting potential roles of ADAM10 substrates in VEEV infection. In sum, our study identifies three TspanC8s and ADAM10 as important modulators of VEEV infectivity.

Identification of patient demographic, clinical, and SARS-CoV-2 genomic factors associated with severe COVID-19 using supervised machine learning: a retrospective multicenter study

BackgroundDrivers of COVID-19 severity are multifactorial and include multidimensional and potentially interacting factors encompassing viral determinants and host-related factors (i.e., demographics, pre-existing conditions and/or genetics), thus complicating the prediction of clinical outcomes for different severe acute respiratory syndrome coronavirus (SARS-CoV-2) variants. Although millions of SARS-CoV-2 genomes have been publicly shared in global databases, linkages with detailed clinical data are scarce. Therefore, we aimed to establish a COVID-19 patient dataset with linked clinical and viral genomic data to then examine associations between SARS-CoV-2 genomic signatures and clinical disease phenotypes.MethodsA cohort of adult patients with laboratory confirmed SARS-CoV-2 from 11 participating healthcare institutions in the Greater Toronto Area (GTA) were recruited from March 2020 to April 2022. Supervised machine learning (ML) models were developed to predict hospitalization using SARS-CoV-2 lineage-specific genomic signatures, patient demographics, symptoms, and pre-existing comorbidities. The relative importance of these features was then evaluated.ResultsComplete clinical data and viral whole genome level information were obtained from 617 patients, 50.4% of whom were hospitalized. Notably, inpatients were older with a mean age of 66.67 years (SD ± 17.64 years), whereas outpatients had a mean age of 44.89 years (SD ± 16.00 years). SHapley Additive exPlanations (SHAP) analyses revealed that underlying vascular disease, underlying pulmonary disease, and fever were the most significant clinical features associated with hospitalization. In models built on the amino acid sequences of functional regions including spike, nucleocapsid, ORF3a, and ORF8 proteins, variants preceding the emergence of variants of concern (VOCs) or pre-VOC variants, were associated with hospitalization.ConclusionsViral genomic features have limited utility in predicting hospitalization across SARS-CoV-2 diversity. Combining clinical and viral genomic datasets provides perspective on patient specific and virus-related factors that impact COVID-19 disease severity. Overall, clinical features had greater discriminatory power than viral genomic features in predicting hospitalization.

Characterizations of cytokines and viral genomes in serum of patients with Dabie bandavirus infection

Characterizations of cytokines and viral genomes in serum of patients with Dabie bandavirus infection

Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS immune sensing.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

Web Visualization for Spatiotemporal Genomic Epidemiology, Annotation, and Mutation Dynamics of Orthohantavirus hantanense Using Nextstrain.

The phylogeographic inference approach aims to connect genomic data with epidemiology to understand the spread and evolution of pathogens using visualization of spatiotemporal reconstructions. Orthohantavirus hantanense (HTNV), the causative agent of hemorrhagic fever with renal syndrome (HFRS), represents a significant global public health concern. Here, we introduce a localized Nextstrain platform for HTNV, offering a comprehensive resource for facilitating spatiotemporal genomic surveillance and the study of evolutionary dynamics of viral genomes. Nextstrain enables web-based visualization and simple sharing of graphic and numeric data through unique web addresses. The Nextstrain build for HTNV stands out for its user-friendly interface and is readily accessible online at https://nextstrain.org/community/KU-MV/Hantavirus. This study provides valuable insights into genomic surveillance, viral phylodynamics, and the evolutionary history of orthohantaviruses for the development of public health policies against endemic HFRS outbreaks.

Sequence Notes: Characterization of Two Novel HIV-1 Unique Recombinant Forms (CRF01_AE/B) in Hebei Province.

Many new circulating recombinant forms (CRFs) and unique recombinant forms (URFs) of human immunodeficiency virus type-1 (HIV-1) have been discovered in populations with multiple circulating HIV-1 genotypes. In this study, we report two novel URFs derived from two HIV-1-positive individuals in Hebei, China, who were infected through homosexual (BDD142) and heterosexual (BDD154) contact. Phylogenetic and recombinant analyses of the two NFLG revealed that they are second-generation recombinant strains originating from the CRF01_AE cluster 4/B and CRF01_AE cluster 5/B strains. The BDD142 viral genome consists of a subtype B fragment inserted into a CRF01_AE backbone, whereas the BDD154 virus genome consists of two subtype B fragments inserted into a CRF01_AE backbone. Prompt monitoring of molecular epidemiological shifts of HIV-1 within sexually transmitted populations and enhanced behavioral interventions targeting this group are imperative to mitigate the spread of HIV-1 effectively.

Machine learning models reveal microbial signatures in healthy human tissues, challenging the sterility of human organs

BackgroundThe presence of microbes within healthy human internal organs still remains under question. Our study endeavors to discern microbial signatures within normal human internal tissues using data from the Genotype-Tissue Expression (GTEx) consortium. Machine learning (ML) models were developed to classify each tissue type based solely on microbial profiles, with the identification of tissue-specific microbial signatures suggesting the presence of distinct microbial communities inside tissues.MethodsWe analyzed 13,871 normal RNA-seq samples from 28 tissues obtained from the GTEx consortium. Unaligned sequencing reads with the human genome were processed using AGAMEMNON, an algorithm for metagenomic microbial quantification, with a reference database comprising bacterial, archaeal, and viral genomes, alongside fungal transcriptomes. Gradient-boosting ML models were trained to classify each tissue against all others based on its microbial profile. To validate the findings, we analyzed 38 healthy living tissue samples (samples from healthy tissues obtained from living individuals, not deceased) from an independent study, as the GTEx samples were derived from post-mortem biopsies.ResultsTissue-specific microbial signatures were identified in 11 out of the 28 tissues while the signatures for 8 tissues (Muscle, Heart, Stomach, Colon tissue, Testis, Blood, Liver, and Bladder tissue) demonstrated resilience to in silico contamination. The models for Heart, Colon tissue, and Liver displayed high discriminatory performance also in the living dataset, suggesting the presence of a tissue-specific microbiome for these tissues even in a living state. Notably, the most crucial features were the fungus Sporisorium graminicola for the heart, the gram-positive bacterium Flavonifractor plautii for the colon tissue, and the gram-negative bacterium Bartonella machadoae for the liver.ConclusionThe presence of tissue-specific microbial signatures in certain tissues suggests that these organs are not devoid of microorganisms even in healthy conditions and probably they harbor low-biomass microbial communities unique to each tissue. The discoveries presented here confront the enduring dogma positing the sterility of internal tissues, yet further validation through controlled laboratory experiments is imperative to substantiate this hypothesis. Exploring the microbiome of internal tissues holds promise for elucidating the pathophysiology underlying both health and a spectrum of diseases, including sepsis, inflammation, and cancer.

Evaluation of Genomic Surveillance of SARS-CoV-2 Virus Isolates and Comparison of Mutational Spectrum of Variants in Bangladesh

The SARS-CoV-2-induced disease, COVID-19, remains a worldwide public health concern due to its high rate of transmission, even in vaccinated and previously infected people. In the endemic state, it continues to cause significant pathology. To elu- cidate the viral mutational changes and screen the emergence of new variants of concern, we conducted this study in Bangladesh. The viral RNA genomes extracted from 25 ran- domly collected samples of COVID-19-positive patients from March 2021 to February 2022 were sequenced using Illumina COVID Seq protocol and genomic data processing, as well as evaluations performed in DRAGEN COVID Lineage software. In this study, the percentage of Delta, Omicron, and Mauritius variants identified were 88%, 8%, and 4%, respectively. All of the 25 samples had 23,403 A>G (D614G, S gene), 3037 C>T (nsp3), and 14,408 C>T (nsp12) mutations, where 23,403 A>G was responsible for increased transmis- sion. Omicron had the highest number of unique mutations in the spike protein (i.e., sub- stitutions, deletions, and insertions), which may explain its higher transmissibility and immune-evading ability than Delta. A total of 779 mutations were identified, where 691 substitutions, 85 deletions, and 3 insertion mutations were observed. To sum up, our study will enrich the genomic database of SARS-CoV-2, aiding in treatment strategies along with understanding the virus’s preferences in both mutation type and mutation site for predicting newly emerged viruses’ survival strategies and thus for preparing to coun- teract them.

Unraveling the Kaposi Sarcoma-Associated Herpesvirus (KSHV) Lifecycle: An Overview of Latency, Lytic Replication, and KSHV-Associated Diseases

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of several diseases. These include the malignancies Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), as well as the inflammatory disorder KSHV inflammatory cytokine syndrome (KICS). The KSHV lifecycle is characterized by two phases: a default latent phase and a lytic replication cycle. During latency, the virus persists as an episome within host cells, expressing a limited subset of viral genes to evade immune surveillance while promoting cellular transformation. The lytic phase, triggered by various stimuli, results in the expression of the full viral genome, production of infectious virions, and modulation of the tumor microenvironment. Both phases of the KSHV lifecycle play crucial roles in driving viral pathogenesis, influencing oncogenesis and immune evasion. This review dives into the intricate world of the KSHV lifecycle, focusing on the molecular mechanisms that drive its latent and lytic phases, their roles in disease progression, and current therapeutic strategies.

Abstract B030: Novel strategy to improve response of high risk HPV+ HNSCC to radiation therapy

Abstract Background: Improving understanding of head and neck squamous cell carcinoma (HNSCC) has led to the discovery of novel molecular markers to stratify risk and guide treatment decisions. Recently, we have identified two subtypes of HPV+ OPSCC – one with good prognosis and one with poor prognosis. The subtypes are distinct in many ways – somatic genes mutated, HPV viral oncogenes expression, the physical state of the viral genome (integrated vs episomal), as well as global mutation and methylation profiles. Intriguingly, all differences converge on intrinsic tumor NF-κB activity with constitutively active NF-κB (usually arising from genetic defects in NF-κB regulators, including TRAF3 and CYLD) driving significantly elevated radiation sensitivity and improved patient survival. NF-κB-based groups define an important clinically actionable prognostic biomarker in HPV+ HNSCC: one important goal is to de-escalate therapy for patients with molecularly low-risk tumors to improve quality of life amongst survivors, while another goal is to improve the cure for of patients with a high risk of treatment failure. Material and methods: We utilized HPV+ head and neck cancer cells, HPV+ HNSCC xenografted mouse model, patient-derived xenografts (PDX), as well as our novel fully immunocompetent mouse model of high risk HPV+ HNSCC; we applied targeted fractionated radiation. To activate NF-κB, we used two clinically applicable drugs with different mechanisms of action: Toll-like receptor 5 (TLR5) agonist and mitochondria-derived activator of caspases (SMAC) mimetic. Custom NF-κB NanoString panel contains our recently developed NF-κB gene signature along with HPV genes and housekeeping genes that are used to normalize the gene expression data. Results: Both drugs activated NF-κB in HPV+ HNSCC and significantly improved response of HPV+ head and neck cancer cells and tumors to radiation. Interestingly, TLR5 agonist alone had a strong tumor suppressing effect in low NF-κB HPV+ HNSCC; this effect was significantly better than 32 Gy focused radiation given in fractions of 4 Gy. Custom NF-κB NanoString panel is robustly prognostic with NF-κB active tumors having improved outcomes after chemoradiation. Conclusions: Because of the average relatively favorable prognosis of HPV+ HNSCC, urgent need for improved therapy for patients with aggressive high risk tumors is greatly undervalued. Here, we present a clinical assay to risk stratify patients with HPV+ HNSCC and begin to explore pharmacological activation of NF-κB as a strategy to sensitize high risk HPV+ HNSCC to radiation therapy. TLR5 agonist is well known for the remarkable normal tissue protective effects against radiation and other damaging conditions; the dual effect of entolimod – sensitization of HPV+ HNSCC to radiation, while simultaneously protecting normal tissues from severe side effects - may be useful for patients with high-risk HPV+ head and neck cancer in combination with radiotherapy. Citation Format: Sri Vemulamanda, Natalia Issaeva1, Aditi Kothari, Travis Schrank, Wendell G. Yarbrough. Novel strategy to improve response of high risk HPV+ HNSCC to radiation therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B030.

An efficient assembly method for a viral genome based on T7 endonuclease Ⅰ-mediated error correction

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.

Structural insights into HIV-2 CA lattice formation and FG-pocket binding revealed by single-particle cryo-EM.

One of the striking features of human immunodeficiency virus (HIV) is the capsid, a fullerene cone comprised of pleomorphic capsid protein (CA) that shields the viral genome and recruits cofactors. Despite significant advances in understanding the mechanisms of HIV-1 CA assembly and host factor interactions, HIV-2 CA assembly remains poorly understood. By templating the assembly of HIV-2 CA on functionalized liposomes, we report high-resolution structures of the HIV-2 CA lattice, including both CA hexamers and pentamers, alone and with peptides of host phenylalanine-glycine (FG)-motif proteins Nup153 and CPSF6. While the overall fold and mode of FG-peptide binding is conserved with HIV-1, this study reveals distinctive features of the HIV-2 CA lattice, including differing structural character at regions of host factor interactions and divergence in the mechanism of formation of CA hexamers and pentamers. This study extends our understanding of HIV capsids and highlights an approach facilitating the study of lentiviral capsid biology.

Lake salinization on the Qinghai–Tibetan Plateau alters viral community composition and lifestyles

Global warming has accelerated lake salinization, driving changes in microbial community structure and function. However, the dynamics of viral communities in response to salinity remain unclear. Here, we apply metagenomic sequencing to a lake on the Qinghai–Tibet Plateau, spanning a broad salinity gradient, to investigate viral community dynamics. Our findings reveal that salinity strongly influences viral composition and modulates viral lifestyles. Temperate viruses increase in relative abundance along the salinity gradient, whereas virulent viruses show a corresponding decline. These shifts are mirrored in the prokaryotic communities, with Alphaproteobacteria and their infecting temperate viruses, notably Casadabanvirus, becoming more prevalent in higher salinity zones. Viral genomes encode genes associated with osmotic stress adaptation, DNA recombination, and nutrient transport, which may facilitate host adaptation to saline stress. This study provides valuable insights into the interplay between viral and prokaryotic communities in response to lake salinization.

Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.

The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge. Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR3pot to be one-pot assay, CasTDR1pot can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30min without pre-amplification. The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR1pot has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA. Our study provides a rapid and accurate molecular diagnostic tool for point-of-care testing, epidemiological screening, and the detection of diseases associated with other RNA biomarkers with excellent single nucleotide differentiation, high sensitivity, and simplicity. National Key Research and Development Program of China (No. 2023YFB3208302), National Natural Science Foundation of China (No. 22377110, 22034004, 82402749, 82073787, 22122409), National Key Research and Development Program of China (No. 2021YFA1200104), Henan Province Fund for Cultivating Advantageous Disciplines (No. 222301420019).

Evolution of SARS-CoV-2 in white-tailed deer in Pennsylvania 2021-2024.

SARS-CoV-2 continues to transmit and evolve in humans and animals. White-tailed deer (Odocoileus virginianus) have been previously identified as a zoonotic reservoir for SARS-CoV-2 with high rates of infection and probable spillback into humans. Here we report sampling 1,127 white-tailed deer (WTD) in Pennsylvania, and a genomic analysis of viral dynamics spanning 1,017 days between April 2021 and January 2024. To assess viral load and genotypes, RNA was isolated from retropharyngeal lymph nodes and analyzed using RT-qPCR and viral whole genome sequencing. Samples showed a 14.64% positivity rate by RT-qPCR. Analysis showed no association of SARS-CoV-2 prevalence with age, sex, or diagnosis with Chronic Wasting Disease. From the 165 SARS-CoV-2 positive WTD, we recovered 25 whole genome sequences and an additional 17 spike-targeted amplicon sequences. The viral variants identified included 17 Alpha, 11 Delta, and 14 Omicron. Alpha largely stopped circulating in humans around September 2021, but persisted in WTD as recently as March of 2023. Phylodynamic analysis of pooled genomic data from Pennsylvania documents at least 12 SARS-CoV-2 spillovers from humans into WTD, including a recent series of Omicron spillovers. Prevalence was higher in WTD in regions with crop coverage rather than forest, suggesting an association with proximity to humans. Analysis of seasonality showed increased prevalence in winter and spring. Multiple examples of recurrent mutations were identified associated with transmissions, suggesting WTD-specific evolutionary pressures. These data document ongoing infections in white-tailed deer, probable onward transmission in deer, and a remarkable rate of new spillovers from humans.

Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch.

The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza A virus catalyzes viral RNA transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) by adopting different conformations. A switch from transcription to replication occurs at a relatively late stage of infection. We recently reported that the viral NS2 protein, expressed at later stages from a spliced transcript of the NS segment messenger RNA (mRNA), inhibits transcription, promotes replication and plays a key role in the transcription-to-replication switch. In this study, we performed comprehensive functional analyses to elucidate how NS2 promotes viral genome replication. Using a cell-based single-step RNP reconstitution assay, we found that NS2 specifically promotes the first-step vRNA-to-cRNA synthesis. Further investigation revealed that this promotion is tightly associated with the intrinsic properties of the 3'-vRNA promoter. Employing a highly sensitive complementation reporter assay, we demonstrated that NS2 associates more strongly with the vRNA-resident RdRp than the cRNA-resident RdRp. These findings were further validated through in vitro replication analyses. We, therefore, propose that influenza A virus NS2 protein targets vRNA-resident RdRp to drive the transcription-to-replication switch during infection.

Evaluation of long-read sequencing for Ostreid herpesvirus type 1 genome characterization from Magallana gigas infected tissues.

Since the 1990s, the Pacific oyster Magallana gigas has faced significant mortality, which has been associated with the detection of the Ostreid Herpesvirus type 1 (OsHV-1). Due to the complex genomic architecture and the presence of multiple genomic isomers, short-read sequencing using Illumina method struggles to accurately assemble tandem and repeat regions and to identify and characterize large structural variations in the OsHV-1 genome. Third-generation sequencing technologies, as long-read real-time nanopore sequencing from Oxford Nanopore Technologies (ONT), offer new possibilities for OsHV-1 whole-genome analysis. Identification of the best method for extraction of high molecular weight (HMW) DNA and development of accurate bioinformatic pipelines for its characterization are now required. To this end, we evaluated and compared six HMW methods and one conventional DNA extraction kit for their ability to extract OsHV-1 DNA from M. gigas-infected tissues. We then evaluated the ability of ONT sequencing to produce an accurate OsHV-1 genome from both whole-genome and "adaptive sampling" (AS) sequencing approaches. Finally, we evaluated the efficiency of bioinformatics tools for de novo assembly and consensus calling to generate accurate OsHV-1 genomes. The HMW DNA extraction kit coupled with ONT sequencing and dedicated bioinformatics tools allowed us to produce accurate OsHV-1 genomes compared to those assembled using Illumina technology. The AS approach allowed up to 60% enrichment for viral data, and the long reads generated by ONT allowed the characterization of OsHV-1 isomers. Together with its portability, this sequencing shows great promise as a diagnostic tool for the characterization of unculturable aquatic viruses directly from host tissues.IMPORTANCEMany aquatic viruses threaten commercially valuable species and cause significant economic losses during outbreaks. To improve our understanding of the origin, transmission patterns and spread of these viruses, additional genomic data are essential. However, genomic characterization of unculturable large DNA viruses is a major challenge. In the present study, we have successfully evaluated the ability of ONT sequencing and adaptive sequencing (AS) to sequence and assemble the complete OsHV-1 genome. Our results show that it is now possible to sequence the whole genome of large DNA viruses directly from infected host tissue, without the need for prior in vitro propagation or prior laboratory steps for virus enrichment.

Passive infusion of an S2-Stem broadly neutralizing antibody protects against SARS-CoV-2 infection and lower airway inflammation in rhesus macaques.

The continued evolution of SARS-CoV-2 variants capable of subverting vaccine and infection-induced immunity suggests the advantage of a broadly protective vaccine against betacoronaviruses (β-CoVs). Recent studies have isolated monoclonal antibodies (mAbs) from SARS-CoV-2 recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other β-CoVs. Many of these mAbs target the conserved S2 stem region of the SARS-CoV-2 spike protein, rather than the receptor binding domain contained within S1 primarily targeted by current SARS-CoV-2 vaccines. One of these S2-directed mAbs, CC40.8, has demonstrated protective efficacy in small animal models against SARS-CoV-2 challenge. As the next step in the pre-clinical testing of S2-directed antibodies as a strategy to protect from SARS-CoV-2 infection, we evaluated the in vivo efficacy of CC40.8 in a clinically relevant non-human primate model by conducting passive antibody transfer to rhesus macaques (RM) followed by SARS-CoV-2 challenge. CC40.8 mAb was intravenously infused at 10mg/kg, 1mg/kg, or 0.1 mg/kg into groups (n = 6) of RM, alongside one group that received a control antibody (PGT121). Viral loads in the lower airway were significantly reduced in animals receiving higher doses of CC40.8. We observed a significant reduction in inflammatory cytokines and macrophages within the lower airway of animals infused with 10mg/kg and 1mg/kg doses of CC40.8. Viral genome sequencing demonstrated a lack of escape mutations in the CC40.8 epitope. Collectively, these data demonstrate the protective efficiency of broadly neutralizing S2-targeting antibodies against SARS-CoV-2 infection within the lower airway while providing critical preclinical work necessary for the development of pan-β-CoV vaccines.

MA104 cell line is permissive for human bocavirus 1 infection.

Human bocavirus 1 (HBoV1) has appeared as an emerging pathogen, causing mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children and immunocompromised individuals. The lack of cell lines suitable for culturing replicative viruses hinders research on HBoV1. Here, we characterized the susceptibility to HBoV1 of 29 human and 7 animal cell lines, and identified a permissive cell line, MA104. The complete HBoV1 life cycle was achieved in MA104 cells, including viral entry, complete replication, and infectious progeny virion production. Additionally, the suppression of the interferon pathway facilitated the viral genome replication in MA104 cells. RNA-sequencing showed that innate immunity, inflammation, the PI3K-Akt and MAPK signaling pathways, and the cellular membrane system were mobilized in response to HBoV1 infection. Overall, our study is the first to identify a cell line, MA104, that supports the complete HBoV1 life cycle, which will promote research on HBoV1 virology and pathogenesis and benefit drug and vaccine development.IMPORTANCEHBoV1 is an emerging pathogen that mainly causes respiratory tract infections, while the lack of cell lines suitable for culture replicative viruses hindered research on HBoV1. Here, we identify a permissive cell line for HBoV1 infection, MA104, and reveal that the complete life cycle of HBoV1 was supported in MA104 cells. Our findings provide a suitable cell model for the study of HBoV1 and explore its application for antiviral drug evaluation, which is vital for research on HBoV1 virology and pathogenesis, as well as for drug and vaccine development.