Whole genome sequence analysis of shallot virus X from India reveals it to be a natural recombinant with positive selection pressure.

BACKGROUND: Shallots are infected by various viruses like Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Shallot virus X (ShVX). In India, they have been found to be persistently infected by ShVX. ShVX also infects onion and garlic in combination with other carlaviruses and potyviruses. ShVX is a member of genus Allexivirus of family Alphaflexiviridae. ShVX has a monopartite genome, which is represented by positive sense single-stranded RNA. Globally, only six complete and 3 nearly complete genome sequences of ShV X are reported to date. This number is insufficient to measure a taxon's true molecular diversity. Moreover, the complete genome sequence of ShVX from Asia has not been reported as yet. Therefore, this study was undertaken to generate a complete genome sequence of ShVX from India. RESULTS: Shallot virus X (ShVX) is one of the significant threats to Allium crop production. In this study, we report the first complete genome sequence of the ShVX from India through Next-generation sequencing (NGS). The complete genome of the ShVX (Accession No. OK104171), from this study comprised 8911 nucleotides. In-silico analysis of the sequence revealed variability between this isolate and isolates from other countries. The dissimilarities are spread all over the genome specifically some non-coding intergenic regions. Statistical analysis of individual genes for site-specific selection indicates a positive selection in NABP region. The presence of a recombination event was detected in coat protein region. The sequence similarity percentage and phylogenetic analysis indicate ShVX Indian isolate is a distinctly different isolate. Recombination and site-specific selection may have a function in the evolution of this isolate. This is the first detailed study of the ShVX complete genome sequence from Southeast Asia. CONCLUSION: This study presents the first report of the entire genome sequence of an Indian isolate of ShVX along with an in-depth exploration of its evolutionary traits. The findings highlight the Indian variant as a naturally occurring recombinant, emphasizing the substantial role of recombination in the evolution of this viral species. This insight into the molecular diversity of strains within a specific geographical region holds immense significance for comprehending and forecasting potential epidemics. Consequently, the insights garnered from this research hold practical value for shaping ShVX management strategies and providing a foundation for forthcoming studies delving into its evolutionary trajectory.

A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

Molecular detection and genetic characteristics of porcine circovirus 3 and porcine circovirus 4 in central China.

Porcine circoviruses (PCVs) are a significant cause of concern for swine health, with four genotypes currently recognized. Two of these, PCV3 and PCV4, have been detected in pigs across all age groups, in both healthy and diseased animals. These viruses have been associated with various clinical manifestations, including porcine dermatitis and nephropathy syndrome (PDNS) and respiratory and enteric signs. In this study, we detected PCV3 and PCV4 in central China between January 2022 and February 2023. We tested fecal swabs and tissue samples from growing-finishing and suckling pigs with or without respiratory and systemic manifestations and found the prevalence of PCV3 to be 15.15% (15/99) and that of PCV3/PCV4 coinfection to be 4.04% (4/99). This relatively low prevalence might be attributed to the fact that most of the clinical samples were collected from pigs exhibiting respiratory signs, with only a few samples having been obtained from pigs with diarrhea. In some cases, PCV2 was also detected, and the coinfection rates of PCV2/3, PCV2/4, and PCV2/3/4 were 6.06% (6/99), 5.05% (5/99), and 3.03% (3/99), respectively. The complete genomic sequences of four PCV3 and two PCV4 isolates were determined. All four of the PCV3 isolates were of subtype PCV3b, and the two PCV4 isolates were of subtype PCV4b. Two mutations (A24V and R27K) were found in antibody recognition domains of PCV3, suggesting that they might be associated with immune escape. This study provides valuable insights into the molecular epidemiology and evolution of PCV3 and PCV4 that will be useful in future investigations of genotyping, immunogenicity, and immune evasion strategies.

Complete genome sequence of a novel totivirus isolated from the leaves of Myrica rubra.

Chinese bayberry is a fruit that is appreciated for its taste. A novel totivirus associated with rolling, disfiguring, chlorotic and vein-clearing symptoms on the leaf apices of Chinese bayberry was identified by transcriptome sequencing and reverse transcription PCR (RT-PCR). The complete genome of the virus was determined to be 4959 nucleotides long, and it contains two open reading frames (ORFs). Its genomic organization is similar to that of previously reported totiviruses. ORF1 encodes a putative coat protein (CP) of 765 aa, and ORF2 encodes an RNA-dependent RNA polymerase (RdRp) of 815 aa. These two putative proteins share 55.1% and 62.6%, amino acid sequence identity, respectively, with the corresponding proteins of Panax notoginseng virus A, respectively. According to the demarcation criteria for totivirus species established by the International Committee on Taxonomy of Viruses (ICTV), the new virus should be considered a member of a new species in the genus totivirus, family Orthototiviridae, which we have tentatively named ''Myrica rubra-associated totivirus'' (MRaTV).

Genome analysis of gyroviruses identified in waterfowl in Arizona (USA).

Gyroviruses are small single-stranded DNA (ssDNA) viruses that are largely associated with birds. Chicken anemia virus is the most extensively studied gyrovirus due to its disease impact on the poultry industry. However, we know much less about gyroviruses infecting other avian species. To investigate gyroviruses infecting waterfowl, we determined six complete genome sequences that fall into three gyrovirus groups, referred to as waterfowl gyrovirus 1 (n = 3), 2 (n = 2), and 3 (n = 1), in organs from hunter-harvested waterfowl from Arizona (USA). The waterfowl gyrovirus 1 variants were identified in multiple organs of a single American wigeon and represent a tentative new species. The waterfowl gyrovirus 2 variants were identified in the livers of two American wigeons and share >70% VP1 nucleotide sequence identity with gyrovirus 9, previously identified in the spleen of a Brazilian Pekin duck (MT318123) and a human fecal sample (KP742975). Waterfowl gyrovirus 3 was identified in a northern pintail spleen sample, and it shares >73% VP1 nucleotide sequence identity with two gyrovirus 13 sequences previously identified in Brazilian Pekin duck spleens (MT318125 and MT318127). These gyroviruses are the first to be identified in waterfowl in North America, as well as in American wigeons and northern pintails.

Genomic characterization of Influenza A (H1N1)pdm09 and SARS-CoV-2 from Influenza Like Illness (ILI) and Severe Acute Respiratory Illness (SARI) cases reported between July-December, 2022.

Influenza Like Illness (ILI) and Severe Acute Respiratory Infection (SARI) cases are more prone to Influenza and SARS-CoV-2 infection. Accordingly, we genetically characterized Influenza and SARS-CoV-2 in 633 ILI and SARI cases by rRT-PCR and WGS. ILI and SARI cases showed H1N1pdm09 prevalence of 20.9% and 23.2% respectively. 135 (21.3%) H1N1pdm09 and 23 (3.6%) H3N2 and 5 coinfection (0.78%) of H1N1pdm09 and SARS-CoV-2 were detected. Phylogenetic analysis revealed H1N1pdm09 resemblance to clade 6B.1A.5a.2 and their genetic relatedness to InfA/Perth/34/2020, InfA/Victoria/88/2020 and InfA/Victoria/2570/2019. Pan 24 HA and 26 NA nonsynonymous mutations and novel HA (G6D, Y7F, Y78H, P212L, G339R, T508K and S523T) and NA (S229A) mutations were observed. S74R, N129D, N156K, S162N, K163Q and S164T alter HA Cb and Sa antibody recognizing site. Similarly, M19T, V13T substitution and multiple mutations in transmembrane and NA head domain drive antigenic drift. SARS-CoV-2 strains genetically characterized to Omicron BA.2.75 lineage containing thirty nonsynonymous spike mutations exhibited enhanced virulence and transmission rates. Coinfection although detected very minimal, the mutational changes in H1N1pdm09 and SARS-CoV-2 virus infected individuals could alter antibody receptor binding sites, allowing the viruses to escape immune response resulting in better adaptability and transmission. Thus continuous genomic surveillance is required to tackle any future outbreak.

Exploring the diversity and evolutionary strategies of prophages in Hyphomicrobiales, comparing animal-associated with non-animal-associated bacteria.

The Hyphomicrobiales bacterial order (previously Rhizobiales) exhibits a wide range of lifestyle characteristics, including free-living, plant-association, nitrogen-fixing, and association with animals (Bartonella and Brucella). This study explores the diversity and evolutionary strategies of bacteriophages within the Hyphomicrobiales order, comparing animal-associated (AAB) with non-animal-associated bacteria (NAAB). We curated 560 high-quality complete genomes of 58 genera from this order and used the PHASTER server for prophage annotation and classification. For 19 genera with representative genomes, we curated 96 genomes and used the Defense-Finder server to summarize the type of anti-phage systems (APS) found in this order. We analyzed the genetic repertoire and length distributions of prophages, estimating evolutionary rates and comparing intact, questionable, and incomplete prophages in both groups. Analyses of best-fit parameters and bootstrap sensitivity were used to understand the evolutionary processes driving prophage gene content. A total of 1860 prophages distributed in Hyphomicrobiales were found, 695 in AAB and 1165 in the NAAB genera. The results revealed a similar number of prophages per genome in AAB and NAAB and a similar length distribution, suggesting shared mechanisms of genetic acquisition of prophage genes. Changes in the frequency of specific gene classes were observed between incomplete and intact prophages, indicating preferential loss or enrichment in both groups. The analysis of best-fit parameters and bootstrap sensitivity tests indicated a higher selection coefficient, induction rate, and turnover in NAAB genomes. We found 68 types of APS in Hyphomicrobiales; restriction modification (RM) and abortive infection (Abi) were the most frequent APS found for all Hyphomicrobiales, and within the AAB group. This classification of APS showed that NAAB genomes have a greater diversity of defense systems compared to AAB, which could be related to the higher rates of prophage induction and turnover in the latter group. Our study provides insights into the distributions of both prophages and APS in Hyphomicrobiales genomes, demonstrating that NAAB carry more defense systems against phages, while AAB show increased prophage stability and an increased number of incomplete prophages. These results suggest a greater role for domesticated prophages within animal-associated bacteria in Hyphomicrobiales.

Long-read powered viral metagenomics in the oligotrophic Sargasso Sea.

Dominant microorganisms of the Sargasso Sea are key drivers of the global carbon cycle. However, associated viruses that shape microbial community structure and function are not well characterised. Here, we combined short and long read sequencing to survey Sargasso Sea phage communities in virus- and cellular fractions at viral maximum (80 m) and mesopelagic (200 m) depths. We identified 2,301 Sargasso Sea phage populations from 186 genera. Over half of the phage populations identified here lacked representation in global ocean viral metagenomes, whilst 177 of the 186 identified genera lacked representation in genomic databases of phage isolates. Viral fraction and cell-associated viral communities were decoupled, indicating viral turnover occurred across periods longer than the sampling period of three days. Inclusion of long-read data was critical for capturing the breadth of viral diversity. Phage isolates that infect the dominant bacterial taxa Prochlorococcus and Pelagibacter, usually regarded as cosmopolitan and abundant, were poorly represented.

Identification and molecular characterization of two novel viruses from Allamanda cathartica in China.

Allamanda cathartica is an ornamental medicinal plant that grows widely in the tropics. In the present study, two novel viruses, Allamanda chlorotic virus A (AlCVA) and Allamanda chlorotic virus B (AlCVB), were identified in an A. cathartica plant with interveinal chlorosis by ribosomal RNA-depleted total-RNA sequencing. Phylogenetic analysis and sequence comparisons confirmed that AlCVA and AlCVB belong to the families Closteroviridae and Betaflexiviridae, respectively. Long, flexuous, filamentous virus particles approximately 12 nm in diameter and 784-2291 nm in length were observed using transmission electron microscopy. A specific RT-PCR assay was used to demonstrate a consistent association of viral infection with symptoms.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Molecular characterization of emerging Echovirus 11 (E11) shed light on the recombinant origin of a variant associated with severe hepatitis in neonates.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3Cpro and 3Dpol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections.

Prevalence and diversity of imported severe acute respiratory syndrome coronavirus 2 variants in China, 2021-2022.

The causative agent of coronavirus disease 2019 (COVID-19), known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread accumulatively to 240 countries and continues to evolve. To gain a comprehensive understanding of the epidemiological characteristics of imported variants in China and their correlation with global circulating variants, genomic surveillance data from 11 139 imported COVID-19 cases submitted by Chinese provincial CDC laboratories between 2021 and 2022 were analyzed. Consensus sequences underwent rigorous quality checks, followed by amino acid mutations analysis using Nextclade. Sequences with satisfactory quality control status were classified according to the Pango nomenclature. The results showed that the dominant variants in imported cases reflected the global epidemic trend. An increase in the number of imported SARS-CoV-2 lineages monitored in China in the second half of 2022, and the circulating Omicron subvariants changed from the ancestral lineages of BA.5 and BA.2 into the lineages containing key amino acid mutations of spike protein. There was significant variation in the detection of Omicron subvariants among continents (χ2 = 321.968, p < 0.001) in the second half of 2022, with four lineages (BA.2.3.7, BA.2.2, BA.5.2.7, and XBB.1.2) identified through imported surveillance mainly prevalent respectively in Taiwan, China, Hong Kong SAR, China, Russian Federation, and Singapore. These findings revealed the alterations in circulating imported variants from 2021 to 2022 in China, reflecting the higher diversity of lineages in the second half of 2022, and revealed the predominant lineages of countries or regions that are in close contacts to China, providing new insights into the global prevalence of SARS-CoV-2.

Unveiling hidden structural patterns in the SARS-CoV-2 genome: Computational insights and comparative analysis.

SARS-CoV-2, the causative agent of COVID-19, is known to exhibit secondary structures in its 5' and 3' untranslated regions, along with the frameshifting stimulatory element situated between ORF1a and 1b. To identify additional regions containing conserved structures, we utilized a multiple sequence alignment with related coronaviruses as a starting point. We applied a computational pipeline developed for identifying non-coding RNA elements. Our pipeline employed three different RNA structural prediction approaches. We identified forty genomic regions likely to harbor structures, with ten of them showing three-way consensus substructure predictions among our predictive utilities. We conducted intracomparisons of the predictive utilities within the pipeline and intercomparisons with four previously published SARS-CoV-2 structural datasets. While there was limited agreement on the precise structure, different approaches seemed to converge on regions likely to contain structures in the viral genome. By comparing and combining various computational approaches, we can predict regions most likely to form structures, as well as a probable structure or ensemble of structures. These predictions can be used to guide surveillance, prophylactic measures, or therapeutic efforts. Data and scripts employed in this study may be found at https://doi.org/10.5281/zenodo.8298680.

Early detection of emerging viral variants through analysis of community structure of coordinated substitution networks.

The emergence of viral variants with altered phenotypes is a public health challenge underscoring the need for advanced evolutionary forecasting methods. Given extensive epistatic interactions within viral genomes and known viral evolutionary history, efficient genomic surveillance necessitates early detection of emerging viral haplotypes rather than commonly targeted single mutations. Haplotype inference, however, is a significantly more challenging problem precluding the use of traditional approaches. Here, using SARS-CoV-2 evolutionary dynamics as a case study, we show that emerging haplotypes with altered transmissibility can be linked to dense communities in coordinated substitution networks, which become discernible significantly earlier than the haplotypes become prevalent. From these insights, we develop a computational framework for inference of viral variants and validate it by successful early detection of known SARS-CoV-2 strains. Our methodology offers greater scalability than phylogenetic lineage tracing and can be applied to any rapidly evolving pathogen with adequate genomic surveillance data.

Genomic characterization of human respiratory syncytial virus circulating in Islamabad, Pakistan, during an outbreak in 2022-2023.

In this study, conducted at the National Institute of Health, Islamabad, during an outbreak of human respiratory syncytial virus (hRSV) from December 2022 to January 2023, the first whole-genome sequences of hRSV isolates from Islamabad, Pakistan, were determined. Out of 10 positive samples, five were sequenced, revealing the presence of two genotypes: RSV-A (GA2.3.5, ON1 strain) and RSV-B (GB5.0.5.a, BA-10 strain). A rare non-synonymous substitution (E232G) in G the protein and N276S in the F protein were found in RSV-A. In RSV-B, the unique mutations K191R, Q209R, and I206M were found in the F protein. These mutations could potentially influence vaccine efficacy and viral pathogenicity. This research underscores the importance of genomic surveillance for understanding RSV diversity and guiding public health responses in Pakistan.

Substantial viral diversity in bats and rodents from East Africa: insights into evolution, recombination, and cocirculation.

BACKGROUND: Zoonotic viruses cause substantial public health and socioeconomic problems worldwide. Understanding how viruses evolve and spread within and among wildlife species is a critical step when aiming for proactive identification of viral threats to prevent future pandemics. Despite the many proposed factors influencing viral diversity, the genomic diversity and structure of viral communities in East Africa are largely unknown. RESULTS: Using 38.3 Tb of metatranscriptomic data obtained via ultradeep sequencing, we screened vertebrate-associated viromes from 844 bats and 250 rodents from Kenya and Uganda collected from the wild. The 251 vertebrate-associated viral genomes of bats (212) and rodents (39) revealed the vast diversity, host-related variability, and high geographic specificity of viruses in East Africa. Among the surveyed viral families, Coronaviridae and Circoviridae showed low host specificity, high conservation of replication-associated proteins, high divergence among viral entry proteins, and frequent recombination. Despite major dispersal limitations, recurrent mutations, cocirculation, and occasional gene flow contribute to the high local diversity of viral genomes. CONCLUSIONS: The present study not only shows the landscape of bat and rodent viromes in this zoonotic hotspot but also reveals genomic signatures driven by the evolution and dispersal of the viral community, laying solid groundwork for future proactive surveillance of emerging zoonotic pathogens in wildlife. Video Abstract.

Genomic analyses of Symbiomonas scintillans show no evidence for endosymbiotic bacteria but does reveal the presence of giant viruses.

Symbiomonas scintillans Guillou et Chrétiennot-Dinet, 1999 is a tiny (1.4 µm) heterotrophic microbial eukaryote. The genus was named based on the presence of endosymbiotic bacteria in its endoplasmic reticulum, however, like most such endosymbionts neither the identity nor functional association with its host were known. We generated both amplification-free shotgun metagenomics and whole genome amplification sequencing data from S. scintillans strains RCC257 and RCC24, but were unable to detect any sequences from known lineages of endosymbiotic bacteria. The absence of endobacteria was further verified with FISH analyses. Instead, numerous contigs in assemblies from both RCC24 and RCC257 were closely related to prasinoviruses infecting the green algae Ostreococcus lucimarinus, Bathycoccus prasinos, and Micromonas pusilla (OlV, BpV, and MpV, respectively). Using the BpV genome as a reference, we assembled a near-complete 190 kbp draft genome encoding all hallmark prasinovirus genes, as well as two additional incomplete assemblies of closely related but distinct viruses from RCC257, and three similar draft viral genomes from RCC24, which we collectively call SsVs. A multi-gene tree showed the three SsV genome types branched within highly supported clades with each of BpV2, OlVs, and MpVs, respectively. Interestingly, transmission electron microscopy also revealed a 190 nm virus-like particle similar the morphology and size of the endosymbiont originally reported in S. scintillans. Overall, we conclude that S. scintillans currently does not harbour an endosymbiotic bacterium, but is associated with giant viruses.

A giant virus infecting the amoeboflagellate Naegleria.

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.