The H9N2 subtype of avian influenza viruses (AIVs) is widely prevalent in poultry and wild birds globally, with occasional transmission to humans. In comparison to other H9N2 lineages, the BJ/94 lineage has raised more public health concerns; however, its evolutionary dynamics and transmission patterns remain poorly understood. In this study, we demonstrate that over three decades (1994-2023), BJ/94 lineage has undergone substantial expansion in its geographical distribution, interspecies transmission, and viral reassortment with other AIV subtypes, increasing associated public health risks. These changes were primarily driven by the emergence of a dominant genotype G57. In the first decade, G57 emerged in East China and rapidly adapted to chickens and spread across China. Since 2013, the G57 genotype has expanded beyond China into eight other countries and reassorted with various AIV subtypes to form new zoonotic reassortants. Chickens have played a key role in the generation and circulation of the G57 viruses, with ducks and other poultry species likely assuming an increasingly importantly role. Over the past decade, G57 has been more frequently detected in wild birds, mammals, and humans. Additionally, Vietnam has emerged as a new hotspot for the international spread of G57. Our results suggest that the BJ/94 lineage H9N2 virus may continue to overcome geographical and species barriers, with potentially more severe consequences.

Reverse-transcribing viruses (RTVs) characterized by reverse transcription required for their replication infect nearly all the eukaryotes. After decades of extensive analyses and discoveries, the understanding of the diversity of RTVs has largely stagnated. Herein, we discover a previously neglected lineage of RTVs, designated Kuafuorterviruses, in animals. Through screening over 8000 eukaryote genomes, we identify the presence of endogenous Kuafuorterviruses in the genomes of 169 eumetazoans dispersed across 11 animal phyla. Phylogenetic analyses and sequence similarity networks indicate that Kuafuorterviruses constitute a novel major lineage of RTVs. The discovery of Kuafuorterviruses refines our understanding of the diversity, evolution, and classification of RTVs and has implications in annotating animal genomes.

The ability of viruses to emerge in new species is influenced by aspects of host biology and ecology, with some taxa harbouring a high diversity and abundance of viruses. However, how these factors shape virus diversity at the ecosystem scale is often unclear. To better understand the pattern and determinants of viral diversity within an ecosystem, and to describe the novel avian viruses infecting an individual avian community, we performed a metagenomic snapshot of the virome from the entire avian community on remote Pukenui/Anchor Island in Aotearoa New Zealand. Through total RNA sequencing of 18 bird species, we identified 50 avian viruses from 9 viral families, of which 96% were novel. Of note, passerines (perching birds) exhibited high viral abundance and diversity, with viruses found across all nine viral families identified. We also identified numerous viruses infecting seabirds on the Island, including megriviruses, hepaciviruses, and hepatoviruses, while parrots exhibited an extremely low diversity of avian viruses. Within passerines, closely related astroviruses and hepatoviruses, and multiple identical hepe-like viruses, were shared among host species. Phylogenetic reconciliation analysis of these viral groups revealed a mixture of co-divergence and cross-species transmission, with virus host-jumping relatively frequent among passerines. In contrast, there was no evidence for recent cross-species virus transmission in parrots or seabirds. The novel pegiviruses and a flavivirus identified here also pose intriguing questions regarding their origins, pathogenicity, and potential impact on vertebrate hosts. Overall, these results highlight the importance of understudied remote island ecosystems as refugia for novel viruses, as well as the intricate interplay between host ecology and behaviour in shaping viral communities.

Geese, both wild and domestic, are generally considered part of the natural reservoir for influenza A viruses. The highly pathogenic H5 Goose/Guangdong avian influenza virus lineage that is still causing outbreaks worldwide was first detected in domestic geese in 1996. However, while wild geese might have a somewhat restricted role in the influenza ecosystem, the role of domestic geese is little studied. Here, 109 H6 viruses isolated from domestic geese during 2001-2018 in southern China had their phylogeny, evolutionary dynamics, and molecular signatures characterized to examine the role of domestic geese. Our findings demonstrated that all geese H6 viruses were derived from H6 viruses established in ducks and that they subsequently formed three distinct hemagglutinin lineages. Rapid evolution of the hemagglutinin genes was not detected after the duck-to-goose transmissions of H6 viruses that then circulated in geese. Despite long-term persistence in geese, H6 viruses were rarely observed to transmit back to ducks or terrestrial poultry and never exchanged genes with viruses from other subtypes. Most geese H6 viruses maintained the primary molecular signatures of their duck precursors. This study raises the possibility that, rather than being part of the natural reservoir, domestic geese might be more like an aberrant host species for influenza A viruses, and perhaps a "dead-end" host.

Different theoretical frameworks have been invoked to guide the study of virus evolution. Three of the more prominent ones are (i) the evolution of virulence, (ii) life history theory, and (iii) the generalism-specialism dichotomy. All involve purported tradeoffs between traits that define the evolvability and constraint of virus-associated phenotypes. However, as popular as these frameworks are, there is a surprising paucity of direct laboratory tests of the frameworks that support their utility as broadly applicable theoretical pillars that can guide our understanding of disease evolution. In this study, we conduct a meta-analysis of direct experimental evidence for these three frameworks across several widely studied virus-host systems: plant viruses, fungal viruses, animal viruses, and bacteriophages. We extracted 60 datasets from 28 studies and found a range of relationships between traits in different analysis categories (e.g., frameworks, virus-host systems). Our work demonstrates that direct evidence for relationships between traits is highly idiosyncratic and specific to the host-virus system and theoretical framework. Consequently, scientists researching viral pathogens from different taxonomic groups might reconsider their allegiance to these canons as the basis for expectation, explanation, or prediction. Future efforts could benefit from consistent definitions, and from developing frameworks that are compatible with the evidence and apply to particular biological and ecological contexts.

Swine influenza A viruses (swIAVs) are a major cause of respiratory disease in pigs worldwide, presenting significant economic and health risks. These viruses can reassort, creating new strains with varying pathogenicity and cross-species transmissibility. This study aimed to monitor the genetic and antigenic evolution of swIAV in France from 2019 to 2022. Molecular subtyping revealed a marked increase in H1N2 cases from 2020 onwards, altering the previously stable subtypes' distribution. Whole-genome sequencing and phylogenetic analyses of H1 (1C) strains identified 10 circulating genotypes, including 5 new genotypes. The most predominant genotype from 2020 onwards, denominated H1N2#E, was characterized by an HA-1C.2.4, an N2-Gent/84, and internal protein-encoding genes belonging to a newly defined subclade within the Eurasian avian-like (EA) lineage termed EA-DK. H1N2#E emerged in Brittany, the country's most pig-dense region, and rapidly became the most frequently detected swIAV genotype across France. This drastic change in the swIAV lineage proportions at a national scale was unprecedented, making H1N2#E a unique case for understanding swIAV evolution and spreading patterns. Phylogenetic analyses suggested an introduction of the H1N2#E genotype from a restricted source, likely originating from Denmark. It spread rapidly with low genetic diversity at the start of the epizootic in 2020, showing increasing diversification in 2021 and 2022 as the inferred population size grew and stabilized, and exhibited reassortments with other enzootic genotypes. Amino acid sequence alignments of H1N2#E antigenic sites revealed major mutations and deletions compared to commercial vaccine 1C strain (HA-1C.2.2) and previously predominant H1N1 strains (HA-1C.2.1). Antigenic cartography confirmed significant antigenic distances between H1N2#E and other 1C strains, suggesting that the new genotype has escaped the pre-existing immunity of the swine population. Epidemiologically, the H1N2#E virus exhibited epizootic hallmarks with more severe clinical outcomes compared to H1N1 viruses. These factors likely contributed to the spread of H1N2#E within the pig population. The rapid rise of H1N2#E highlighted the dynamic nature of swIAV genetic and antigenic diversity, underscoring the importance of tailored surveillance programs to support risk assessment during potential new outbreaks. It also demonstrates the need to strengthen biosecurity measures when introducing pigs into a herd, including swIAV positivity assessment followed by quarantine, and restrict the trade of swIAV-excreting live swine between European countries.

Infectious disease transmission to different host species makes eradication very challenging and expands the diversity of evolutionary trajectories taken by the pathogen. Since the beginning of the ongoing COVID-19 pandemic, SARS-CoV-2 has been transmitted from humans to many different animal species, in which viral variants of concern could potentially evolve. Previously, using available whole genome consensus sequences of SARS-CoV-2 from four commonly sampled animals (mink, deer, cat, and dog), we inferred similar numbers of transmission events from humans to each animal species. Using a genome-wide association study, we identified 26 single nucleotide variants (SNVs) that tend to occur in deer-more than any other animal-suggesting a high rate of viral adaptation to deer. The reasons for this rapid adaptive evolution remain unclear, but within-host evolution-the ultimate source of the viral diversity that transmits globally-could provide clues. Here, we quantify intra-host SARS-CoV-2 genetic diversity across animal species and show that deer harbor more intra-host SNVs (iSNVs) than other animals, providing a larger pool of genetic diversity for natural selection to act upon. Mixed infections involving more than one viral lineage are unlikely to explain the higher diversity within deer. Rather, a combination of higher mutation rates, longer infections, and species-specific selective pressures are likely explanations. Combined with extensive deer-to-deer transmission, the high levels of within-deer viral diversity help explain the apparent rapid adaptation of SARS-CoV-2 to deer.

The enormous diversity of bacteriophages and their bacterial hosts presents a significant challenge to predict which phages infect a focal set of bacteria. Infection is largely determined by complementary-and largely uncharacterized-genetics of adsorption, injection, cell take-over, and lysis. Here we present a machine learning approach to predict phage-bacteria interactions trained on genome sequences of and phenotypic interactions among 51 strains and 45 phage λ strains that coevolved in laboratory conditions for 37 days. Leveraging multiple inference strategies and without knowledge of driver mutations, this framework predicts both who infects whom and the quantitative levels of infections across a suite of 2,295 potential interactions. We found that the most effective approach inferred interaction phenotypes from independent contributions from phage and bacteria mutations, accurately predicting 86% of interactions while reducing the relative error in the estimated strength of the infection phenotype by 40%. Feature selection revealed key phage λ and mutations that have a significant influence on the outcome of phage-bacteria interactions, corroborating sites previously known to affect phage λ infections, as well as identifying mutations in genes of unknown function not previously shown to influence bacterial resistance. The method's success in recapitulating strain-level infection outcomes arising during coevolutionary dynamics may also help inform generalized approaches for imputing genetic drivers of interaction phenotypes in complex communities of phage and bacteria.

Advancements in high-throughput sequencing and associated bioinformatics methods have significantly expanded the RNA virus repertoire, including novel viruses with highly divergent genomes encoding "orphan" proteins that apparently lack homologous sequences. This absence of homologs in routine sequence similarity search complicates their taxonomic classification and raises a fundamental question: Do these orphan viral genomes represent viruses? In 2022, an orphan viral genome encoding a large polyprotein was identified in alfalfa () and thrips (), and named Snake River alfalfa virus (SRAV). SRAV was initially proposed as an uncommon flavi-like virus identified in a plant host distantly related to family . Subsequently, another research group showed its common occurrence in alfalfa but challenged its taxonomic position, suggesting it belongs to the family . In this study, a large-scale analysis of 77 publicly available small RNA datasets indicates that SRAV could be detected across various tissues and cultivars of alfalfa, and has a broad geographical distribution. Moreover, profiles of the SRAV-derived small interfering RNAs (vsiRNAs) exhibited typical characteristics of viruses in plant hosts. The evolutionary analysis suggests that SRAV represents a unique class of plant-hosted flavi-like viruses with an unusual genome organization and evolutionary status, distinct from previously identified flavi-like viruses documented to infect plants. The latter shows a close evolutionary relationship to flavi-like viruses primarily found in plant-feeding invertebrates and lacks evidence of triggering host RNA interference (RNAi) responses so far. Moreover, mining the transcriptome shotgun assembly (TSA) database identified two novel viral sequences with a similar genome organization and evolutionary status to SRAV. In summary, our study resolves the disagreement regarding the taxonomic status of SRAV and suggests the potential existence of two distinct clades of plant-hosted flavi-like viruses with independent evolutionary origins. Furthermore, our research provides the first evidence of plant-hosted flavi-like viruses triggering the host's RNAi antiviral response. The widespread occurrence of SRAV underscores its potential ecological significance in alfalfa, a crop of substantial economic importance.

Anelloviruses are a group of small, circular, single-stranded DNA viruses that are found ubiquitously across mammalian hosts. Here, we explored a large number of publicly available human microbiome datasets and retrieved a total of 829 anellovirus genomes, substantially expanding the known diversity of these viruses. The majority of new genomes fall within the three major human anellovirus genera: , and , while we also present new genomes of the under-sampled , and genera. We performed recombination analysis and show evidence of extensive recombination across all human anelloviruses. Interestingly, more than 95% of the detected events are between members of the same genus and only 15 inter-genus recombination events were detected. The breakpoints of recombination cluster in hotspots at the ends and outside of the ORF1 gene, while a recombination coldspot was detected within the gene. Our analysis suggests that anellovirus evolution is governed by homologous recombination; however, events between distant viruses or ones producing chimaeric ORF1s likely lead to nonviable recombinants. The large number of genomes further allowed us to examine how essential genomic features vary across anelloviruses. These include functional domains in the ORF1 protein and the nucleotide motif of the replication loop region, required for the viruses' rolling-circle replication. A subset of the genomes assembled in both this and previous studies are completely lacking these essential elements, opening up the possibility that anellovirus intracellular populations contain nonstandard viral genomes. However, low-read depth of the metagenomically assembled contigs may partly explain the lack of some features. Overall, our study highlights key features of anellovirus genomics and evolution, a largely understudied group of viruses whose potential in virus-based therapeutics is recently being explored.

Despite the increasing burden of dengue in Kenya and Africa, the introduction and expansion of the virus in the region remain poorly understood. The objective of this study is to examine the genetic diversity and evolutionary histories of dengue virus (DENV) serotypes 1 and 3 in Kenya and contextualize their circulation within circulation dynamics in the broader African region. Viral RNA was extracted from samples collected from a cohort of febrile patients recruited at clinical sites in Kenya from 2013 to 2022. Samples were tested by polymerase chain reaction (PCR) for DENV presence. Five DENV-positive samples were serotyped, and complete viral genomes for phylogenetic inference were obtained via sequencing on Illumina platforms. Sequences generated in our study were combined with global datasets of sequences, and Bayesian and maximum likelihood methods were used to infer phylogenetic trees and geographic patterns of spread with a focus on Kenya and Africa as a whole. Four new DENV-1 and one new DENV-3 genomes were successfully sequenced and combined with 328 DENV-1 and 395 DENV-3 genomes from elsewhere for phylogenetic analyses. The DENV-1 sequences from our study formed a monophyletic cluster with an inferred common ancestor in 2019 (most recent common ancestor 2019 and 95% high posterior density 2018-19), which was closely related to sequences from Tanzania. The single DENV-3 sequence clustered with sequences from Tanzania and Kenya, was collected between 2017 and 2019 and was related to recent outbreaks in the region. Phylogenetic trees resolved multiple clades of DENV-1 and DENV-3 concurrently circulating in Africa, introduced in the early-to mid-2000s. Three DENV-1 and four DENV-3 clades are highlighted, introduced between 2000 and 2015. Phylogeographic models suggest frequent, independent importations of DENV lineages into Kenya and Africa from East and South-East Asia via distinct geographic pathways. DENV-1 and DENV-3 evolutionary dynamics in Africa are characterized by the cocirculation of multiple recently introduced lineages. Circulating lineages are introduced via distinct geographic pathways that may be centered around regional nexus locations. Increased surveillance is required to identify key regional locations that drive spread, and dengue interventions should focus on interrupting spread at these locations.

[This corrects the article DOI: 10.1093/ve/vead053.].

A consistent area of interest since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been the sequence composition of the virus and how it has changed over time. Many resources have been developed for the storage and analysis of SARS-CoV-2 data, such as GISAID (Global Initiative on Sharing All Influenza Data), NCBI, Nextstrain, and outbreak.info. However, relatively little has been done to compile codon usage data, codon-level mutation data, and secondary structure data into a single database. Here, we assemble the aforementioned data and many additional virus attributes in a new database entitled SARS-CoV-2 CoCoPUTs. We begin with an overview of the composition and overlap between two of the largest sources of SARS-CoV-2 sequence data: GISAID and NCBI Virus (GenBank). We then evaluate different types of sequence curation strategies to reduce the dataset of millions of sequences to only one sequence per Pango lineage variant. We then performed specific analyses on the coding sequences (CDSs), including calculating codon usage, codon pair usage, dinucleotides, junction dinucleotides, mutations, GC content, effective number of codons (ENCs), and effective number of codon pairs (ENCPs). We have also performed whole-genome secondary RNA structure prediction calculations for each variant, using the LinearPartition software and modified selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) data that are available online. Finally, we compiled all the data into our resource, SARS-CoV-2 CoCoPUTs, and paired many of the resulting statistics with variant proportion data over time in order to derive trends in viral evolution. Although the overall codon usage of SARS-CoV-2 did not change drastically, in line with the previous literature on this subject, we did observe that while overall GC% content decreased, GC% of the third position in the codon was more positive relative to overall GC% content between February 2021 and July 2023. Over the same interval, we noted that both synonymous and nonsynonymous mutations increased in number, with nonsynonymous mutations outpacing synonymous mutations at a rate of 3:1. We noted that the predicted whole-genome secondary structures nearly all contained the previously described virus-activated inhibitor of translation (VAIT) stem loops, validating for the first time their existence in a whole-genome secondary structure prediction for many SARS-CoV-2 variants (as opposed to previous local secondary structure predictions). We also separately produced a synonymous mutation-deprived set of SARS-CoV-2 variant sequences and repeated the secondary structure calculations on this set. This revealed an interesting trend of reduced ensemble free energy compared to the unaltered variant structures, indicating that synonymous mutations play a role in increasing the free energy of viral RNA molecules. These data both validate previous studies describing increases in viral free energy in human viruses over time and indicate a possible role for synonymous mutations in viral biology.

Wild birds are important hosts of influenza A viruses (IAVs) and play an important role in their ecology. The emergence of the A/goose/Guangdong/1/1996 H5N1 (Gs/GD) lineage marked a shift in IAV ecology, leading to recurrent outbreaks and mortality in wild birds from 2002 onwards. This lineage has evolved and diversified over time, with a recent important derivative being the 2.3.4.4b sub-lineage, which has caused significant mortality events in wild bird populations. An H5N1 clade 2.3.4.4b virus was transmitted into North America from Eurasia in 2021, with the first detection being in Newfoundland and Labrador in Atlantic Canada, and this virus and its reassortants then spread broadly throughout North America and beyond. Following the first 2021 detection, there have been three additional known incursions of Eurasian-origin strains into Atlantic Canada, a second H5N1 strain in 2022 and two H5N5 strains in 2023. In this study, we document a fifth incursion in Atlantic Canada that occurred in 2023 by another H5N5 strain. This strain spread throughout Atlantic Canada and into Quebec, infecting numerous species of wild birds and mammals. Genomic analysis revealed mammalian-adaptive mutations in some of the detected viruses (PB2-E627K and PB2-D701N) and mutations in the hemagglutinin (HA) and neuraminidase (NA) genes that are associated with enhanced viral fitness and avian transmission capabilities. Our findings indicate that this virus is continuing to circulate in wildlife, and confirms Atlantic Canada is an important North American entry point for Eurasian IAVs. Continued surveillance and genomic analysis of IAVs detected in the region is crucial to monitor the evolution of these viruses and assess potential risks to wildlife and public health.

In North America, raccoon rabies virus (RRV) is a public health concern due to its potential for rapid spread, maintenance in wildlife, and impact on human and domesticated animal health. RRV is an endemic zoonotic pathogen throughout the eastern USA. In 1991, an outbreak of RRV in Fairfield County, Connecticut, spread through the state and eventually throughout the Northeast and into Canada. Factors that contribute to, or curb, RRV transmission should be explored and quantified to guide targeted rabies control efforts, including the size and location of buffer zones of vaccinated animals. However, population dynamics and potential underlying determinants of rabies virus diversity and circulation in Connecticut have not been fully studied. In this study, we aim to (i) investigate RRV source-sink dynamics between Connecticut and surrounding states and provinces, (ii) explore the impact of the Connecticut River as a natural barrier to transmission, and (iii) characterize the genomic diversity and transmission dynamics in Connecticut. Using RRV whole-genome sequences collected from various host species between 1990 and 2020, we performed comparative genetic and Bayesian phylodynamic analyses at multiple spatial scales. We analyzed 71 whole-genome sequences from Connecticut, including 21 recent RRV specimens collected at the Connecticut Veterinary Medical Diagnostic Laboratory that we sequenced for this study. Our analyses revealed evidence of RRV incursions over the US-Canada border, including bidirectional spread between Quebec and Vermont. Additionally, we highlighted the importance of Connecticut and New York in seeding RRV transmission in eastern North America, including two introduction events from New York to Connecticut that resulted in sustained local transmission. While RRV transmission does occur across the Housatonic and Connecticut Rivers, we demonstrated the distinct presence of spatial structuring in the phylogenetic trees and characterized the directionality of RRV migration. The significantly higher mean transition rates from locations east to west of the Connecticut River, compared to west to east, may be leveraged in directing interventions to fortify these natural barriers. Ultimately, the findings of these international, regional, and state analyses can inform targeted control programs, vaccination efforts, and enhanced surveillance at borders of key viral sources and sinks.

The importance of asymptomatic transmission was a key discovery in our efforts to study and intervene in the COVID-19 pandemic. In (Johns Hopkins University Press, 2024), Joshua Weitz uses this aspect of SARS-CoV-2 natural history to discuss many counterintuitive characteristics of the pandemic. In this essay, I engage the arguments in the book, and discuss why asymptomatic transmission is such a critical dimension of the study of infectious diseases. I explore ideas contained within and connect them to related issues in evolutionary virology and disease ecology, including epistemic uncertainty and the evolution of virulence. Furthermore, I comment on the broader messages in the text, including the gap between scientific knowledge and social understanding.

Hypermutated proviruses, which arise in a single Human Immunodeficiency Virus (HIV) replication cycle when host antiviral APOBEC3 proteins introduce extensive guanine to adenine mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). However, hypermutated sequences are routinely excluded from phylogenetic trees because their extensive mutations complicate phylogenetic inference, and as a result, we know relatively little about their within-host evolutionary origins and dynamics. Using >1400 longitudinal single-genome-amplified HIV sequences isolated from six women over a median of 18 years of follow-up-including plasma HIV RNA sequences collected over a median of 9 years between seroconversion and ART initiation, and >500 proviruses isolated over a median of 9 years on ART-we evaluated three approaches for masking hypermutation in nucleotide alignments. Our goals were to (i) reconstruct phylogenies that can be used for molecular dating and (ii) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the approaches (stripping all positions containing putative APOBEC3 mutations from the alignment or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought -intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for -intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, supporting the use of these corrected trees for molecular dating. Subsequent molecular dating of hypermutated proviruses revealed that these sequences spanned a wide within-host age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection and can persist for decades. In two of the six participants, hypermutated proviruses differed from -intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories reveal insights into their origins and longevity toward a more comprehensive understanding of HIV persistence during ART.

Botswana, like the rest of the world, has been significantly impacted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In December 2022, we detected a monophyletic cluster of genomes comprising a sublineage of the Omicron variant of concern (VOC) designated as B.1.1.529.5.3.1.1.1.1.1.1.74.1 (alias FN.1, clade 22E). These genomes were sourced from both epidemiologically linked and unlinked samples collected in three close locations within the district of Greater Gaborone. In this study, we assessed the worldwide prevalence of the FN.1 lineage, evaluated its mutational profile, and conducted a phylogeographic analysis to reveal its global dispersal dynamics. Among approximately 16 million publicly available SARS-CoV-2 sequences generated by 30 September 2023, only 87 were of the FN.1 lineage, including 22 from Botswana, 6 from South Africa, and 59 from the UK. The estimated time to the most recent common ancestor of the 87 FN.1 sequences was 22 October 2022 [95% highest posterior density: 2 September 2022-24 November 2022], with the earliest of the 22 Botswana sequences having been sampled on 7 December 2022. Discrete trait reconstruction of FN.1 identified Botswana as the most probable place of origin. The FN.1 lineage is derived from the BQ.1.1 lineage and carries two missense variants in the spike protein, S:K182E in NTD and S:T478R in RDB. Among the over 90 SARS-CoV-2 lineages circulating in Botswana between September 2020 and July 2023, FN.1 was most closely related to BQ.1.1.74 based on maximum likelihood phylogenetic inference, differing only by the S:K182E mutation found in FN.1. Given the early detection of numerous novel variants from Botswana and its neighbouring countries, our study underscores the necessity of continuous surveillance to monitor the emergence of potential VOCs, integrating molecular and spatial data to identify dissemination patterns enhancing preparedness efforts.

Influenza A and B viruses represent significant global health threats, contributing substantially to morbidity and mortality rates. However, a comprehensive understanding of the molecular epidemiology of these viruses in Brazil, a continental-size country and a crucial hub for the entry, circulation, and dissemination of influenza viruses within South America, still needs to be improved. This study addresses this gap by consolidating data and samples across all Brazilian macroregions, as part of the Center for Viral Surveillance and Serological Assessment project, together with an extensive number of other Brazilian sequences provided by a public database during the epidemic seasons spanning 2021-23. Phylogenetic analysis of the hemagglutinin segment of influenza A/H1N1pdm09, A/H3N2, and influenza B/Victoria-lineage viruses revealed that in 2021 and in the first semester of 2022, the A/H3N2 2a.3 strain was the predominant circulating strain. Subsequently, the A/H3N2 2b became the prevalent strain until October, when it was substituted by A/H1N1pdm09 5a.2a and 5a.2a.1 lineages. This scenario was maintained during the year of 2023. B/Victoria emerged and circulated at low levels between December 2021 and September 2022 and then became coprevalent with A/H1N1pdm09 5a.2a and 5a.2a.1 lineages. The comparison between the vaccine strain A/Darwin/9/2021 and circulating viruses revealed shared mutations to aspartic acid at residues 186 and 225 across all A/H3N2 lineages from 2021 to 2023, altering the charge in the receptor-binding domain. For A/H1N1pdm09, the 2022 consensus of 5a.2a.1 and the vaccine strain A/Victoria/2570/2019 showed 14 amino acid substitutions. Key residues H180, D187, K219, R223, E224, and T133 are involved in hydrogen interactions with sialic acids, while N130, K142, and D222 may contribute to distance interactions based on docking analyses. Importantly, distinct influenza A lineage frequency patterns were observed across Brazil's macroregions, underscoring the regional variations in virus circulation. This study characterizes influenza A and B viruses circulating in Brazil, providing insights into their circulation patterns and dynamics across Brazilian macroregions. These findings hold significant implications for public health interventions, informing strategies to mitigate transmission risks, optimize vaccination efforts, and enhance outbreak control measures.

Interest in phage therapy-the use of bacterial viruses to treat infections-has increased recently because of the rise of infections with antibiotic-resistant bacteria and the failure to develop new antibiotics to treat those infections. Phages have shown therapeutic promise in recent work, and successful treatment minimally requires giving the patient a phage that will grow on their infecting bacterium. Although nature offers a bountiful and diverse supply of phages, there have been a surprising number of patient infections that could not be treated with phages because no suitable phage was found to kill the patient's bacterium. Here, we develop computational models to analyze an alternative approach to obtaining phages with new host ranges-directed evolution via laboratory propagation of phages to select mutants that can grow on a new host. The models separately explore alternative directed evolution protocols for phage variants that overcome three types of bacterial blocks to phage growth: a block in adsorption, temperate phage immunity to superinfection, and abortive infection. Protocols assume serial transfer to amplify pre-existing, small-effect mutants that are initially rare. Best protocols are sensitive to the nature of the block, and the models provide several insights for enhancing success specific to each case. A common result is that low dilution rates between transfers are beneficial in reducing the mutant growth rate needed to ascend. Selection to overcome an adsorption block is insensitive to many protocol variations but benefits from long selection times between transfers. A temperate phage selected to grow on its lysogens can evolve in any of three phenotypes, but a common protocol favors the desired changes in all three. Abortive infection appears to be the least amenable to evolving phage growth because it is prone to select phages that avoid infection.

HIV-1 Vif's principal function is to counter the antiretroviral activities of DNA-editing APOBEC3 cytidine deaminases. Unconstrained APOBEC3 activity introduces premature stop codons in HIV-1 genes and can lead to viral inactivation. To investigate the evolution and diversification of Vif over the HIV-1 pandemic and document evidence of APOBEC3-mediated pressure, we analyzed 4612 publicly available sequences derived from 10 dominant subtypes and circulating recombinant forms (CRFs) using the Hervé platform. We found widespread evidence of diversifying selection that was convergent across subtypes and CRFs, but remarkable stability in consensus sequences over time. Divergence and selection did not favor APOBEC3-interacting sites. We furthermore found that APOBEC3-induced substitutions in and genes increased over time and were positively associated with diversity. These results suggest that APOBEC3-driven adaptation in Vif is relatively rare and that permissiveness to human APOBEC3-induced substitution as a mechanism for generating diversity may be advantageous to HIV-1 evolution.