Virus Evolution最新文献

Public databases of protein sequences, such as the National Center for Biotechnology Information (NCBI) Protein repository and UniProt, contain millions of proteins identified in samples from specific species but named as uncharacterized or hypothetical due to a lack of information about their function. Many such sequences are actually derived from RNA viruses, either due to viral infection of the original sample, contamination, or endogenous viral elements (EVEs) integrated into the genome of the sample species. Many proteins from RNA virus discovery research are also deposited into these repositories but are labelled as uncharacterized and only classified taxonomically at a superkingdom or realm level. Sequences from protein repositories not labelled specifically as being derived from the RNA-viral RNA-dependent RNA polymerase (RdRp) protein are often used as negative controls when looking to identify viral RdRp sequences, so the presence of unlabelled viruses amongst these datasets is problematic. These sequences also represent a source of information about novel viruses and EVEs. In this study, we screened uncharacterized proteins from two large public protein repositories-NCBI Protein and UniProt-to identify sequences likely to be derived from RNA viral RdRp and to perform detailed characterization of sequences of interest. We identified 3560 such sequences, many derived from EVEs. Many are previously unknown EVEs, which led to characterization of additional, related sequences. For example, a group of orbi-like viruses infecting nematodes was uncovered that appears to have both ancient endogenous and circulating exogenous members. Many integrations of mito-like viruses into plant genomes were also found. In several host taxonomic groups, the first example of an EVE, and in some cases the first example of any RNA virus, was uncovered. The large number of EVEs uncovered by this relatively small-scale search suggests that only a fraction of the true diversity of EVEs is currently known. We also provide provisional taxonomic annotations for RdRps, currently only listed as members of the Riboviria realm. A number of sequences are identified that are indistinguishable from viruses but are labelled as bacteria, seemingly as a result of mislabelling or contamination. Non-RdRp sequences that share near-significant similarity with RdRp are also characterized. Finally, recommendations are made for generating useful negative controls and sets of negative control sequences are provided.

Glycoproteins cover the surface of enveloped viruses such as herpes simplex virus 1 (HSV-1). Whilst essential for cellular attachment and entry, they also are excellent targets for host immune responses. This dichotomy culminates in an evolutionary struggle in which receptor recognition and immune escape are intricately balanced. Herpesviruses feature a variety of different glycoproteins with diverse molecular functions. Here, we describe the rapid evolution of HSV-1 towards syncytial plaque phenotypes in Vero cell culture, as well as anti-gD antibody resistance in human foreskin fibroblast cells. Using a mild hypermutator virus to accelerate experimental evolution, we identified multiple genetic variants leading to syncytial plaques. Strikingly, these variants differentially affect interactions within viral populations. Whilst gK mutants engage in collective syncytia formation upon entry, accelerate superinfection exclusion and maintain fitness advantages at high multiplicities of infection, gB and gD mutants do not. Furthermore, we find gE mutants which lead to mouse anti-gD antibody resistance and cross protect wt virus in mixed populations. Our findings suggest complex social interactions within herpesvirus populations and illustrate the evolutionary plasticity and diverse function of their glycoproteins.

Anelloviridae members are ubiquitous viruses with a small, negative sense, single-stranded DNA genome which is replicated by host cell DNA polymerases. Anelloviruses are postulated to interact with the host cell nuclear transport machinery, however, the lack of reliable cell culture models strongly limits our knowledge regarding Anelloviridae-host interactions. In particular, capsid nuclear import is a largely uncharacterized process. We addressed this by investigating the relationship between host cell nuclear transport receptors (NTRs) and ORF1, the putative capsid protein from torque teno douroucouli virus (TTDoV). We identified the subcellular targeting signals and NTRs responsible for its nucleolar and nuclear localization, and characterized their relative contribution to ORF1 subcellular localization. In the absence of other viral proteins, ORF1 accumulated in the nucleoli. Bioinformatics analysis revealed a putative classical nuclear localization signal (cNLS) within the highly conserved N-terminal arginine rich motif (ARM) ('NLSn', 27-RRWRRRPRRRRRPYRRRPYRRYGRRRKVRRR-57), and an additional C-terminal cNLS ('NLSc', 632-LPPPEKRARWGF-643), which has been specifically acquired by Anelloviridae capsids with larger projection domains. Such NLSs play distinct roles in ORF1 subcellular localization by interacting with specific NTRs. NLSn, a non-classical NLS, features broad importin (IMP) binding affinity yet plays a minor role in nuclear import, being responsible for nucleolar targeting likely through interaction with nucleolar components. NLSc, a bona fide cNLS, specifically interacts with IMPα and is the main driver of active nuclear transport in an IMPα/β1-dependent fashion. These findings suggest an evolutionary correlation between the acquisition of progressively larger projection domains and the presence of additional cNLSs in Anelloviridae capsids, aimed at maximizing IMPα/β1-mediated nuclear import.

Swine vesicular disease virus (SVDV) was considered endemic in Italy until 2015, when eradication was achieved. From 1992, when the most recent antigenic-genomic variant of SVDV was identified, to 2015, ~685 SVD outbreaks occurred. In order to understand the evolutionary drivers of SVDV in Italy, 152 viral strains were selected and whole-genome sequencing was performed. Comparison of the polyprotein coding region revealed a minimum pairwise identity of 85% and 98% at the nucleotide and amino acid levels, respectively, consistent with the antigenic homogeneity of the analysed strains. Phylogenetic analysis confirmed the presence of two sublineages: sublineage 1, which evolved and circulated specifically in Italy since 1995, and sublineage 2, which also includes strains circulating in both Spain and Portugal in 1993 and in Portugal during 2003. Recombination analysis revealed a breakpoint site within the 2B coding region, resulting in a recombination strain originating from viruses belonging to the two sublineages, which was dated to the beginning of 2008. This single recombination event gave rise to at least 20 recombinant strains, which circulated together with their parental strains until 2010 and from then on circulated on their own until eradication in 2015. Our data showed that, apart from the recombination event, SVDV, which has been present on Italian territory for >20 years, has not been subject to positive selective pressures that would have conferred a possible evolutionary advantage.

Ribgrass mosaic virus (RMV) and related viruses of the genus Tobamovirus (family Virgaviridae) are cruciferous plant pathogens that represent a threat to global horticultural systems. In Australia, they are considered exotic biosecurity threats, and an incursion of these viruses would require rapid and strict control efforts. However, current surveillance methods for these viruses are limited. We examined whether RMV and related tobamoviruses could be detected through the analysis of mammalian gut metatranscriptomes. Accordingly, we identified five different tobamoviruses in one or more gut metatranscriptomes of the Eastern grey kangaroo, fallow deer, domestic dog, spotted-tailed quoll, feral cat, and the Tasmanian devil. One of the tobamoviruses was also detected in a tick metatranscriptome. The five tobamoviruses detected were: (i) RMV, (ii) a novel relative of RMV, (iii and iv) two highly diverse novel tobamoviruses, and (v) the plant pathogen tobacco mild green mosaic virus (TMGMV) already known to be present in Australia. Subsequent phylogenetic analysis provided information on their origin and spread through Australia. RMV was detected at multiple sites in both the Australian Capital Territory (ACT) and Tasmania, two regions separated by ~700 km of land and 200 km of water. The novel relative of RMV was detected in the ACT and New South Wales (NSW), the highly divergent novel tobamoviruses were each detected in a single state (NSW and Queensland, QLD), while TMGMV was detected in QLD. This work highlights the potential utility of metatranscriptomic sequencing of wild animal gut samples for the surveillance of biosecurity threats to native and agricultural plant species and for studying their evolution in new environments.

Time-stamped genomic sequences from rapidly evolving pathogens can be used to estimate the rates of evolution through molecular tip-dating. The validity of this approach, however, depends on whether detectable levels of genetic variation have accumulated over the given sampling interval, generating a temporal signal. Moreover, molecular dating methods have demonstrated varying degrees of systematic biases under different biologically realistic scenarios, such as the presence of phylo-temporal clustering. Persistent SARS-CoV-2 infections in immunocompromised individuals have been linked to accelerated intrahost molecular rates compared to those of global lineages, facilitating the emergence of novel viral lineages. Yet, studies reporting elevated rates lack assessment of data properties, such as evaluation of temporal signal and comparison of multiple methods of inference, both crucial for robust rate estimation. In this study, we applied a range of molecular dating approaches to reassess the rate of SARS-CoV-2 intrahost evolution in immunocompromised individuals using publicly available datasets. Our findings suggest that even during long-term infections, the limited number of genetic changes accumulating may pose a challenge for robust inference of within-host evolutionary rates, particularly when relying on consensus sequences and when datasets are small or unevenly sampled. Moreover, our results highlight that when certain methodological limitations are overlooked, evolutionary rates can be significantly overestimated. In general, our findings demonstrate that estimating within-host evolutionary rates is a challenging question necessitating thorough assessment of data quality, careful selection of appropriate methods, and cautious interpretation of the resulting estimates. Whereas our phylogenetic analyses of viral consensus sequences provide no evidence of elevated evolutionary rates across the complete genome during chronic SARS-CoV-2 infection, prolonged viral shedding may nevertheless promote the emergence of new viral variants in immunocompromised individuals.

Heartland virus (HRTV) is an emerging tick-borne virus associated with severe illness in the USA. There are large gaps in knowledge of HRTV diversity, evolution, and transmission due to a paucity of HRTV-positive samples and genome sequences. We identified a focal site of HRTV-positive Amblyomma americanum ticks in central Georgia and developed a novel multiplex-amplicon sequencing assay to generate full HRTV genome sequences. By screening over 21 000 field-collected ticks from 2021 to 2023, we identified six positive pools. Five were collected from the site in central Georgia where our group first detected HRTV-positive ticks in 2019 and one from a site in western Georgia ~175 km away. The HRTV genome sequences from Georgia were highly related, even across this distance and over five years. Reference HRTV genome sequences from across the USA were also geographically clustered. Time-scaled phylogenetic analysis suggested a recent spread of HRTV in the USA, with all available sequences sharing a common ancestor within the last 75 years, since the mid-1900s, and sequences from Georgia sharing a common ancestor within the last 15 years, since 2010. Our observed spatial clustering of HRTV and the high degree of genetic conservation in our persistent focus suggest the importance of small spatial dynamics in HRTV transmission ecology.

Amplicon-based next-generation sequencing (aNGS) is a powerful tool in diagnostics and genetic studies. We developed an aNGS approach to study the population structure of the Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), a specific pathogen of the spongy moth Lymantria dispar, a devastating lepidopteran pest in European, Asian, and American deciduous forests. Naturally occurring pathogens, such as LdMNPV, are frequently reported to cause epizootics and a rapid decline of insect pest populations. DNA samples of pooled LdMNPV-infected larvae from forest regions in Northern Bavaria (Germany) were subjected to whole genome sequencing (WGS) and aNGS optimization. Then, five marker regions were identified in the genome of LdMNPV for PCR amplification, covering 21 highly specific single-nucleotide polymorphism (SNP) positions that enabled comprehensive analysis at the intra- and intersample levels. These markers were used in aNGS analyses of 70 single larvae collected in 12 forest sites, followed by SNP-based hierarchical clustering on principal components (HCPC). This approach identified three LdMNPV population clusters consisting of homogenous (pure) and heterogeneous (mixed) LdMNPV samples. To explain the genetic variability within each sample, a model based on linear optimization was developed and validated by comparing the predictions from aNGS and WGS data. The analyses showed that LdMNPV from Bavarian forests carried genetic variants highly similar to those present in the commercial product Gypchek®, developed for biocontrol. The distribution of genetic characteristics showed some trends of geographic and temporal prevalence, which are indicative of short-distance and long-distance transmission. The aNGS approach offers a fast, cost-effective, and comprehensive insight into the natural population structure of LdMNPV.

Betacoronaviruses (β-CoVs) display divergent mechanisms to evade host antiviral responses, yet the evolutionary origin and functional relevance of their strategies remain unclear. Stress granules (SGs), central to host defenses, are disrupted by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein via its interaction with G3BP1 mediated by an ¹⁵ITFG¹⁸ motif. This interaction inhibits SG assembly, enhancing viral replication and immune evasion. Here, we analyzed 179 N protein sequences across β-CoVs subgenera and identified the ITFG motif in sarbecoviruses but not in MERS-CoV or embecoviruses. Among tested CoVs, only SARS-CoV-2 N showed robust G3BP1 binding in vitro and in infected cells. Phylogenetic analyses revealed that the ΦxFG motif emerged independently in sarbecoviruses and a bat-infecting merbecovirus clade. The VGTF motif in these merbecoviruses also binds to G3BP1, suggesting convergent evolution in viral evasion strategies. The emergence of this motif was unrelated to 4a protein inactivation, another viral protein that inhibits SG formation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans and animals and is therefore a pathogen of grave concern within a One Health framework. Identifying animal-adaptive mutations is critical to preserving One Health, as these mutations could also lead to the persistence of SARS-CoV-2 in animal reservoirs with continual spillover to humans. Therefore, we sought to pair experimental evolution and epidemiological data to identify putative human- and animal-adaptive viral residues and determine their impact on replication-competent SARS-CoV-2 in both human and animal cells. We passaged SARS-CoV-2 in cells expressing human, dog, cat, mink, and white-tailed deer ACE2 and sequenced the passaged populations. In addition, we searched SARS-CoV-2 sequences for mutations following patterns of convergent evolution that were common to both human- and animal-derived SARS-CoV-2 sequences. We identified the epidemiologically relevant Spike A222V mutation from our passaging experiment in cells expressing cat ACE2, a mutation that has also arisen independently across eight lineages of SARS-CoV-2 from human- and animal-derived sequences. To assess its impact on replication in human and animal cells, we constructed SARS-CoV-2 Spike A222V in the Wuhan-Hu-1 backbone with Spike D614G; this virus replicated similarly to the WT SARS-CoV-2 in human lung epithelial cells. In contrast, SARS-CoV-2 Spike A222V demonstrated an advantage in replication in primary deer lung cells, which was not mediated by the deer ACE2 receptor. Infection via the human, dog, cat, and mink ACE2 receptor resulted in reduced replication of SARS-CoV-2 Spike A222V. Our experiments identified Spike A222V as a putatively deer-adaptive mutation. Future studies should assess Spike A222V's relevance to transmission within deer and to other animal species in contact with deer.