Viral Genome Sequences Research Articles

Marked neurotropism and potential adaptation of H5N1 clade 2.3.4.4.b virus in naturally infected domestic cats

ABSTRACT In April 2024, ten cats died in a rural South Dakota (SD) residence, showing respiratory and neurological symptoms. Necropsy and laboratory testing of two cats confirmed H5N1 clade 2.3.4.4b infection. The viral genome sequences are closely related to recent SD cattle H5N1 sequences. Cat H5N1 genomes had unique mutations, including T143A in haemagglutinin, known to affect infectivity and immune evasion, and two novel mutations in PA protein (F314L, L342Q) that may affect polymerase activity and virulence, suggesting potential virus adaptation. Dead cats showed systemic infection with lesions and viral antigens in multiple organs. Higher viral RNA and antigen in the brain indicated pronounced neurotropism. Lectin-histochemistry revealed widespread co-expression of sialic acid α-2,6 and α-2,3 receptors, suggesting cats could serve as mixing vessels for reassortment of avian and mammalian influenza viruses. No differences in clade 2.2 or 2.3.4.4b H5 pseudoviruses binding to cat lung/brain tissues indicated the neurotropism is unlikely mediated by receptor binding affinity.

Polyphonia: detecting inter-sample contamination in viral genomic sequencing data.

In viral genomic research and surveillance, inter-sample contamination can affect variant detection, analysis of within-host evolution, outbreak reconstruction, and detection of superinfections and recombination events. While sample barcoding methods exist to track inter-sample contamination, they are not always used and can only detect contamination in the experimental pipeline from the point they are added. The underlying genomic information in a sample, however, carries information about inter-sample contamination occurring at any stage. Here, we present Polyphonia, a tool for detecting inter-sample contamination directly from deep sequencing data without the need for additional controls, using intrahost variant frequencies. We apply Polyphonia to 1 102 SARS-CoV-2 samples sequenced at the Broad Institute and already tracked using molecular barcoding for comparison. Polyphonia is available as a standalone Docker image and is also included as part of viral-ngs, available in Dockstore. Full documentation, source code, and instructions for use are available at https://github.com/broadinstitute/polyphonia. Data for reproducing results are available at Bioinformatics online.

The Arrival of Highly Pathogenic Avian Influenza Viruses in North America, Ensuing Epizootics in Poultry and Dairy Farms and Difficulties in Scientific Naming.

The highly pathogenic avian influenza virus (HPAIV) H5N1, first isolated in 1996 in China, spread rapidly across Eurasia and caused major epizootics in wild and domesticated birds, as well as spillover infections in humans characterised by high mortality. Avian influenza viruses are therefore candidate viruses for a human pandemic. Surprisingly, HPAIV was not isolated in North America until 2014. With the help of intensive biological sampling and viral genome sequencing, the intrusion of HPAIV into North America could be retraced to two separate events. First, migratory birds carried HPAIV from East Siberia via Beringia and dispersed the virus along the Pacific flyway. After reassortment with genes of local low pathogenic avian influenza viruses, HPAIV H5 caused 2015 a major epizootic on poultry farms in the US Mid-West. After costly containment, HPAIV dropped below the detection limit. In 2021, Eurasian HPAIV H5 viruses arrived a second time in North America, carried by migratory birds to Canada via the Atlantic flyway, using Iceland as a stop. The H5 virus then spread with water birds along the East Coast of the United States and dispersed across the United States. In contrast to the 2015 poultry outbreak, spillover infections into diverse species of mammals were now observed. The events culminated in the 2024 HPAIV H5 epizootic in dairy cows affecting 300 dairy herds in 14 US states. The cattle epizootic was spread mainly by milking machinery and animal transport. On affected farms infected cats developed fatal neurological diseases. Retail milk across the United States frequently contains viral RNA, but so far only a few milk farm workers have developed mild symptoms. The tracing of HPAIV with viral genome sequencing complicated the taxonomical naming of influenza viruses raising fundamental problems in how to mirror biological complexity in written plain language, rendering communication with the lay public difficult.

Long-term co-circulation of multiple arboviruses insoutheast Australia revealed by xeno-monitoring andviral whole-genome sequencing.

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. Polymerase chain reaction (PCR)-based screening coupled with RNA sequencing is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of ≤200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterized using RNA sequencing generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like, and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, demonstrating that different epidemiological, immunological, and evolutionary processes may actively shape the evolution of Australian arboviruses. These results underscore the need for more genomic surveillance data to explore the complex evolutionary pressures acting on arboviruses. Overall, our findings highlight the effectiveness of our methodology, which can be applied broadly to enhance arbovirus surveillance in various ecological contexts and improve understanding of transmission dynamics.

The Growing Phenomenon of ‘Frozen’ Virus Genome Sequences and Their Likely Origin in Research Facility Escapes

‘Frozen’ virus genome sequences are sampled from outbreaks and have unusually low sequence divergence when compared to genome sequences from historical strains. A growing number of ‘frozen’ virus genome sequences are being reported as virus genome sequencing becomes more common. Examples of ‘frozen’ sequences include the 1977 H1N1 ‘Russian’ flu; Venezuelan Equine Encephalitis Virus from Venezuela and Colombia in 1995; E71 sequences from a Hand, Foot and Mouth outbreak in 2007–2009 in China; and a polio strain isolated in 2014 from Anhui, China. The origin of these ‘frozen’ sequences has been attributed to escapes from research facilities and often appears to be associated with vaccine work. Consequently, a new paradigm for pathogen emergence appears in operation, that involves laboratory research or vaccine production which utilizes ‘live’ virus isolates of historical strains. The accidental release and re-emergence of such strains are straightforward to detect from their genome sequences and should spur the routine sequencing and publication of all known pathogenic viral strains undergoing experimentation, or being used for vaccine manufacture, in order to facilitate tracing. However, it is noted that novel pathogenic viruses accidentally released into the population from research facilities are harder to detect if their sequence has first not been made public, which should prompt the routine sequencing and reporting of all novel pathogenic viruses before experimentation.

Dimensionality reduction distills complex evolutionary relationships in seasonal influenza and SARS-CoV-2.

Public health researchers and practitioners commonly infer phylogenies from viral genome sequences to understand transmission dynamics and identify clusters of genetically-related samples. However, viruses that reassort or recombine violate phylogenetic assumptions and require more sophisticated methods. Even when phylogenies are appropriate, they can be unnecessary or difficult to interpret without specialty knowledge. For example, pairwise distances between sequences can be enough to identify clusters of related samples or assign new samples to existing phylogenetic clusters. In this work, we tested whether dimensionality reduction methods could capture known genetic groups within two human pathogenic viruses that cause substantial human morbidity and mortality and frequently reassort or recombine, respectively: seasonal influenza A/H3N2 and SARS-CoV-2. We applied principal component analysis, multidimensional scaling (MDS), t-distributed stochastic neighbor embedding (t-SNE), and uniform manifold approximation and projection to sequences with well-defined phylogenetic clades and either reassortment (H3N2) or recombination (SARS-CoV-2). For each low-dimensional embedding of sequences, we calculated the correlation between pairwise genetic and Euclidean distances in the embedding and applied a hierarchical clustering method to identify clusters in the embedding. We measured the accuracy of clusters compared to previously defined phylogenetic clades, reassortment clusters, or recombinant lineages. We found that MDS embeddings accurately represented pairwise genetic distances including the intermediate placement of recombinant SARS-CoV-2 lineages between parental lineages. Clusters from t-SNE embeddings accurately recapitulated known phylogenetic clades, H3N2 reassortment groups, and SARS-CoV-2 recombinant lineages. We show that simple statistical methods without a biological model can accurately represent known genetic relationships for relevant human pathogenic viruses. Our open source implementation of these methods for analysis of viral genome sequences can be easily applied when phylogenetic methods are either unnecessary or inappropriate.

VISTA: A Tool for Fast Taxonomic Assignment of Viral Genome Sequences.

The rapid expansion of the number of viral genome sequences in public databases necessitates a scalable, universal, and automated preliminary taxonomic framework for comprehensive virus studies. Here, we introduce VISTA (Virus Sequence-based Taxonomy Assignment), a computational tool that employs a novel pairwise sequence comparison system and an automatic demarcation threshold identification framework for virus taxonomy. Leveraging physio-chemical property sequences, k-mer profiles, and machine learning techniques, VISTA constructs a robust distance-based framework for taxonomic assignment. Functionally similar to PASC (Pairwise Sequence Comparison), a widely used virus assignment tool based on pairwise sequence comparison, VISTA demonstrates superior performance by providing significantly improved separation for taxonomic groups, more objective taxonomic demarcation thresholds, greatly enhanced speed, and a wider application scope. We successfully applied VISTA to 38 virus families, as well as to the class Caudoviricetes. This demonstrates VISTA's scalability, robustness, and ability to automatically and accurately assign taxonomy to both prokaryotic and eukaryotic viruses. Furthermore, the application of VISTA to 679 unclassified prokaryotic virus genomes recovered from metagenomic data identified 46 novel virus families. VISTA is available as both a command line tool and a user-friendly web portal at https://ngdc.cncb.ac.cn/vista.

Assessing the de novo assemblers: a metaviromic study of apple and first report of citrus concave gum-associated virus, apple rubbery wood virus 1 and 2 infecting apple in India

BackgroundThe choice of de novo assembler for high-throughput sequencing (HTS) data remains a pivotal factor in the HTS-based discovery of viral pathogens. This study assessed de novo assemblers, namely Trinity, SPAdes, and MEGAHIT for HTS datasets generated on the Illumina platform from 23 apple samples, representing 15 exotic and indigenous apple varieties and a rootstock. The assemblers were compared based on assembly quality metrics, including the largest contig, total assembly length, genome coverage, and N50.ResultsMEGAHIT was most efficient assembler according to the metrics evaluated in this study. By using multiple assemblers, near-complete genome sequences of citrus concave gum-associated virus (CCGaV), apple rubbery wood virus 1 (ARWV-1), ARWV-2, apple necrotic mosaic virus (ApNMV), apple mosaic virus, apple stem pitting virus, apple stem grooving virus, apple chlorotic leaf spot virus, apple hammerhead viroid and apple scar skin viroid were reconstructed. These viruses were further confirmed through Sanger sequencing in different apple cultivars. Among them, CCGaV, ARWV-1 and ARWV-2 were recorded from apples in India for the first time. The analysis of virus richness revealed that ApNMV was dominant, followed by ARWV-1 and CCGaV. Moreover, MEGAHIT identified novel single-nucleotide variants.ConclusionsOur analyses highlight the crucial role of assembly methods in reconstructing near-complete apple virus genomes from the Illumina reads. This study emphasizes the significance of employing multiple assemblers for de novo virus genome assembly in vegetatively propagated perennial fruit crops.

Clinical and Genomic Epidemiology of Coxsackievirus A21 and Enterovirus D68 in Homeless Shelters, King County, Washington, USA, 2019-2021.

Congregate homeless shelters are disproportionately affected by infectious disease outbreaks. We describe enterovirus epidemiology across 23 adult and family shelters in King County, Washington, USA, during October 2019-May 2021, by using repeated cross-sectional respiratory illness and environmental surveillance and viral genome sequencing. Among 3,281 participants >3 months of age, we identified coxsackievirus A21 (CVA21) in 39 adult residents (3.0% [95% CI 1.9%-4.8%] detection) across 7 shelters during October 2019-February 2020. We identified enterovirus D68 (EV-D68) in 5 adult residents in 2 shelters during October-November 2019. Of 812 environmental samples, 1 was EV-D68-positive and 5 were CVA21-positive. Other enteroviruses detected among residents, but not in environmental samples, included coxsackievirus A6/A4 in 3 children. No enteroviruses were detected during April 2020-May 2021. Phylogenetically clustered CVA21 and EV-D68 cases occurred in some shelters. Some shelters also hosted multiple CVA21 lineages.

Rapid generation and characterization of recombinant HCoV-OC43-VR1558 infectious clones expressing reporter Renilla luciferase

Viral infectious clones (ICs) serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics. However, the molecular profiles and complex limitations of human coronaviruses (HCoVs) pose a challenge to ICs development. In this study, we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination (TAR) technology. Recombinant HCoV-OC43-VR1558, named as rOC43(1558)-WT, was rapidly generated by TAR. In addition, recombinant HCoV-OC43-VR1558-expressing reporter genes, named as rOC43(1558)-ns2FusionRluc, was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase (Rluc). We further characterized their replication through virus titration using 50% tissue culture infective dose (TCID50) and indirect immunofluorescence assay (IFA), luciferase reporter assay, and western blotting (WB) assay. The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells. These reporter viruses were validated as screening tools for inhibitors in vitro by evaluating the antiviral activities of remdesivir and chloroquine. The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were compared in vitro and in vivo. The TAR-based one-step assembly of IC was successfully applied, facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms, development of vaccines and diagnostic tests, and screening inhibitors of HCoVs.

How Trustworthy Are the Genomic Sequences of SARS-CoV-2 in GenBank?

Well-annotated gene and genomic sequences serve as a foundation for making inferences in molecular biology and evolution and can directly impact public health. The first SARS-CoV-2 genome was submitted to the GenBank database hosted by the U.S. National Center for Biotechnology Information and used to develop the two successful vaccines. Conserved protein domains are often chosen as targets for developing antiviral medicines or vaccines. Mutation and substitution patterns provide crucial information not only on functional motifs and genome/protein interactions but also for characterizing phylogenetic relationships among viral strains. These patterns, together with the collection time of viral samples, serve as the basis for addressing the question of when and where the host-switching event occurred. Unfortunately, viral genomic sequences submitted to GenBank undergo little quality control, and critical information in the annotation is frequently changed without being recorded. Researchers often have no choice but to hold blind faith in the authenticity of the sequences. There have been reports of incorrect genome annotation but no report that casts doubt on the genomic sequences themselves because it seems theoretically impossible to identify genomic sequences that may not be authentic. This paper takes an innovative approach to show that some SARS-CoV-2 genomes submitted to GenBank cannot possibly be authentic. Specifically, some SARS-CoV-2 genomic sequences deposited in GenBank with collection times in 2023 and 2024, isolated from saliva, nasopharyngeal, sewage, and stool, are identical to the reference genome of SARS-CoV-2 (NC_045512). The probability of such occurrence is effectively 0. I also compile SARS-CoV-2 genomes with changed sample collection times. One may be led astray in bioinformatic analysis without being aware of errors in sequences and sequence annotation.

Sheeppox virus genome sequences from the European outbreaks in Spain, Bulgaria, and Greece in 2022–2024

In 2022-2024, three outbreaks of sheeppox (SPP) were reported in the European Union. These occurred in Spain, Bulgaria, and Greece and had serious economic consequences due to animal losses and trade restrictions. Five sheeppox virus (SPPV) whole-genome sequences (WGSs) were determined from samples collected during these outbreaks and analyzed in the context of all other published WGSs. Sheeppox virus strains can be divided in two, or possibly three, main groups. The isolates from the recent outbreaks belong to clade A2, which includes strains historically circulating in the Middle East and Northern Africa. Sequence divergence was low among the isolates that caused the recent European outbreaks. These results highlight the need for more regular and dense surveillance in under-sampled areas and the use of WGS to increase the chance of pinpointing the origin of an introduction, identifying potential introduction routes, and providing insights into SPPV evolution.

Genome sequences of dengue virus serotypes 2, 3, and 4 isolated from adult patients in Thailand.

Here, we present the genome sequences of dengue viruses (DENV) isolated from adult patients in Thailand during 2016-2017: DENV2 (412749), DENV3 (416384), and DENV4 (416709). These sequences provide valuable genetic and evolutionary information for dengue research and antiviral development.

A Novel and Rapid Selective Viral Genome Amplification and Sequencing Method for African Swine Fever Virus.

African swine fever virus (ASFV) is the etiological agent of African swine fever, a highly contagious hemorrhagic disease affecting both wild boars and domestic pigs with lethality rates up to 100%. Until now, the most effective measure to prevent an outbreak of ASFV was early detection. In this situation, whole genome sequencing (WGS) allows the gathering of detailed information about the identity and epidemiology of the virus. However, due to the large genome size and complex genome ends, WGS is challenging. Current WGS workflows require either elaborate enrichment methods or are based on tiled PCR approaches, which are susceptible to genetic differences between ASFV strains. To overcome this, we developed a novel approach for WGS of ASFV, using the Phi29 DNA polymerase-based multiple displacement amplification in combination with only seven primers. Furthermore, we applied an alkaline-based DNA denaturation step to significantly increase the number of viral reads, which resolves the near-full genome of ASFV. This novel isothermal WGS approach can be used in authorized laboratories for the genomic epidemiological analysis of ASFV outbreaks caused by different genotypes.

Analysis of Powassan Virus Genome Sequences from Human Cases Reveals Substantial Genetic Diversity with Implications for Molecular Assay Development.

Powassan virus (POWV) is an emerging tick-borne virus that causes severe meningoencephalitis in the United States, Canada, and Russia. Serology is generally the preferred diagnostic modality, but PCR on cerebrospinal fluid, blood, or urine has an important role, particularly in immunocompromised patients who are unable to mount a serologic response. Although the perceived poor sensitivity of PCR in the general population may be due to the biology of infection and health-seeking behavior (with short viremic periods that end before hospital presentation), limitations in assay design may also contribute. Genome sequences from clinical POWV cases are extremely scarce; PCR assay design has been informed by those available, but the numbers are limited. Larger numbers of genome sequences from tick-derived POWV are available, but it is not known if POWV genomes from human infections broadly mirror genomes from tick hosts, or if human infections are caused by a subset of more virulent strains. We obtained viral genomic data from 10 previously unpublished POWV human infections and showed that they broadly mirror the diversity of genome sequences seen in ticks, including all three major clades (lineage I, lineage II Northeast, and lineage II Midwest). These newly published clinical POWV genome sequences include the first confirmed lineage I infection in the United States, highlighting the relevance of all clades in human disease. An in silico analysis of published POWV PCR assays shows that many assays were optimized against a single clade and have mismatches that may affect their sensitivity when applied across clades. This analysis serves as a launching point for improved PCR design for clinical diagnostics and environmental surveillance.

Next-Generation Sequencing Methods for Near-Real-Time Molecular Epidemiology of HIV and HCV.

The World Health Organisation has set targets of reducing the transmission of new hepatitis C (HCV) infections by 90%, and ending human immunodeficiency virus-1 (HIV) as a public health threat, by 2030. To achieve this, efficient and timely viral surveillance, and effective public health interventions, are required. Traditional epidemiological methods are largely dependent on the recognition of incident cases with symptomatic illness; acute HIV and HCV infections are commonly asymptomatic, which may lead to delays in the recognition of such new infections. Instead, for these viruses, molecular epidemiology may improve the detection of, and response to, clusters of viral transmission. Molecular epidemiology using historical datasets has highlighted key populations that may have benefitted from a timely intervention. Similar analyses performed on contemporary samples are needed to underpin the 2030 targets, but this requires the generation of a cohesive dataset of viral genome sequences in near-real-time. To generate such data, methodologies harnessing next-generation sequencing (NGS) should be utilised. Here we discuss the opportunity presented by NGS for public health surveillance of HIV and HCV, and discuss three methods that can generate sequences for such analysis. These include full-length genome amplification, utilised for analysis of HCV in the research space; tiling PCR, which was the method of choice for many diagnostic laboratories in the SARS-CoV-2 pandemic; and bait-capture hybridisation, which has been utilised in local HIV outbreaks. These techniques could be applied for near-real-time HIV and HCV surveillance, informing public health strategies that will be key to achieving 2030 targets.

Alterations of the gut microbiome in HIV infection highlight human anelloviruses as potential predictors of immune recovery

BackgroundHIV-1 infection is characterized by a massive depletion of mucosal CD4 T cells that triggers a cascade of events ultimately linking gut microbial dysbiosis to HIV-1 disease progression and pathogenesis. The association between HIV infection and the enteric virome composition is less characterized, although viruses are an essential component of the gut ecosystem. Here, we performed a cross-sectional analysis of the fecal viral (eukaryotic viruses and bacteriophages) and bacterial microbiome in people with HIV (PWH) and in HIV-negative individuals. To gain further insight into the association between the gut microbiome composition, HIV-associated immunodeficiency, and immune recovery, we carried out a longitudinal study including 14 PWH who initiated antiretroviral therapy (ART) and were followed for 24 months with samplings performed at baseline (before ART) and at 2, 6, 12, and 24 months post-ART initiation.ResultsOur data revealed a striking expansion in the abundance and prevalence of several human virus genomic sequences (Anelloviridae, Adenoviridae, and Papillomaviridae) in stool samples of PWH with severe immunodeficiency (CD4 < 200). We also noted a decreased abundance of sequences belonging to two plant viruses from the Tobamovirus genus, a reduction in bacterial alpha diversity, and a decrease in Inoviridae bacteriophage sequences. Short-term ART (24 months) was linked to a significant decrease in human Anelloviridae sequences. Remarkably, the detection of Anellovirus sequences at baseline independently predicted poor immune recovery, as did low CD4 T cell counts. The bacterial and bacteriophage populations were unique to each PWH with individualized trajectories; we found no discernable pattern of clustering after 24 months on ART.ConclusionAdvanced HIV-1 infection was associated with marked alterations in the virome composition, in particular a remarkable expansion of human anelloviruses, with a gradual restoration after ART initiation. In addition to CD4 T cell counts, anellovirus sequence detection might be useful to predict and monitor immune recovery. This study confirms data on the bacteriome and expands our knowledge on the viral component of the gut microbiome in HIV-1 infection.3oR8qDfXoWY9s_P5ES648YVideo

Direct genome sequencing of respiratory viruses from low viral load clinical specimens using the target capture sequencing technology.

The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging-especially in the case of respiratory viruses-due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2,000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/µL of SARS-CoV-2 or 625.1 copies/µL of influenza A virus. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/µL, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.IMPORTANCETarget capture sequencing has been developed and applied for direct genome sequencing of viruses in clinical specimens to overcome the low detection sensitivity of metagenomic next-generation sequencing. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A, focusing on clinical specimens containing low copy numbers of viral genomes. Our results showed that the target capture sequencing yielded dramatically higher read counts than metagenomic sequencing for both viruses. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with other viruses, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.

Integrated analyses of the transmission history of SARS-CoV-2 and its association with molecular evolution of the virus underlining the pandemic outbreaks in Italy, 2019-2023

BackgroundItaly was significantly affected by the COVID-19 pandemic, experiencing multiple waves of infection following the sequential emergence of new variants. Understanding the transmission patterns and evolution of SARS-CoV-2 is vital for future preparedness. MethodsWe conducted an analysis of viral genome sequences, integrating epidemiological and phylodynamic approaches, to characterize how SARS-CoV-2 variants have spread within the country. ResultsOur findings indicate bidirectional international transmission, with Italy transitioning between importing and exporting the virus. Italy experienced four distinct epidemic waves, each associated with a significant reduction in fatalities from 2021 to 2023. These waves were primarily driven by the emergence of VOCs such as Alpha, Delta, and Omicron, which were reflected in observed transmission dynamics and effectiveness of public health measures. ConclusionsThe changing patterns of viral spread and variant prevalence throughout Italy's pandemic response underscore the continued importance of flexible public health strategies and genomic surveillance, both of which are crucial for tracking the evolution of variants and adapting control measures effectively to ensure preparedness for future outbreaks.

Differences in HIV-1 reservoir size, landscape characteristics and decay dynamics in acute and chronic treated HIV-1 Clade C infection.

Persisting HIV reservoir viruses in resting CD4 T cells and other cellular subsets are the main barrier to cure efforts. Antiretroviral therapy (ART) intensification by early initiation has been shown to enable post-treatment viral control in some cases but the underlying mechanisms are not fully understood. We hypothesized that ART initiated during the hyperacute phase of infection before peak will affect the size, decay dynamics and landscape characteristics of HIV-1 subtype C viral reservoirs. We studied 35 women at high risk of infection from Durban, South Africa identified with hyperacute HIV infection by twice weekly testing for plasma HIV-1 RNA. Study participants included 11 who started ART at a median of 456 (297-1203) days post onset of viremia (DPOV), and 24 who started ART at a median of 1 (1-3) DPOV. We used peripheral blood mononuclear cells (PBMC) to measure total HIV-1 DNA by ddPCR and to sequence reservoir viral genomes by full length individual proviral sequencing (FLIP-seq) from onset of detection of HIV up to 1 year post treatment initiation. Whereas ART in hyperacute infection blunted peak viremia compared to untreated individuals (p<0.0001), there was no difference in total HIV-1 DNA measured contemporaneously (p=0.104). There was a steady decline of total HIV DNA in early treated persons over 1 year of ART (p=0.0004), with no significant change observed in the late treated group. Total HIV-1 DNA after one year of treatment was lower in the early treated compared to the late treated group (p=0.02). Generation of 697 single viral genome sequences revealed a difference in the longitudinal proviral genetic landscape over one year between untreated, late treated, and early treated infection: the relative contribution of intact genomes to the total pool of HIV-1 DNA after 1 year was higher in untreated infection (31%) compared to late treated (14%) and early treated infection (0%). Treatment initiated in both late and early infection resulted in a more rapid decay of intact (13% and 51% per month) versus defective (2% and 35% per month) viral genomes. However, intact genomes were still observed one year post chronic treatment initiation in contrast to early treatment where intact genomes were no longer detectable. Moreover, early ART reduced phylogenetic diversity of intact genomes and limited the seeding and persistence of cytotoxic T lymphocyte immune escape variants in the reservoir. Overall, our results show that whereas ART initiated in hyperacute HIV-1 subtype C infection did not impact reservoir seeding, it was nevertheless associated with more rapid decay of intact viral genomes, decreased genetic complexity and immune escape in reservoirs, which could accelerate reservoir clearance when combined with other interventional strategies.