Dispersal patterns and influence of air travel during the global expansion of SARS-CoV-2 variants of concern.

The Alpha, Beta, and Gamma SARS-CoV-2 variants of concern (VOCs) co-circulated globally during 2020 and 2021, fueling waves of infections. They were displaced by Delta during a third wave worldwide in 2021, which, in turn, was displaced by Omicron in late 2021. In this study, we use phylogenetic and phylogeographic methods to reconstruct the dispersal patterns of VOCs worldwide. We find that source-sink dynamics varied substantially by VOC and identify countries that acted as global and regional hubs of dissemination. We demonstrate the declining role of presumed origin countries of VOCs in their global dispersal, estimating that India contributed <15% of Delta exports and South Africa <1%-2% of Omicron dispersal. We estimate that >80 countries had received introductions of Omicron within 100 days of its emergence, associated with accelerated passenger air travel and higher transmissibility. Our study highlights the rapid dispersal of highly transmissible variants, with implications for genomic surveillance along the hierarchical airline network.

The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Detection of a SARS-CoV-2 variant of concern in South Africa.

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus1,2. Here we describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by eight mutations in the spike protein, including three substitutions (K417N, E484K and N501Y) at residues in its receptor-binding domain that may have functional importance3-5. This lineage was identified in South Africa after the first wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be determined, the genomic data-which show rapid expansion and displacement of other lineages in several regions-suggest that this lineage is associated with a selection advantage that most plausibly results from increased transmissibility or immune escape6-8.

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus.

IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Establishment of five new genera in the family Geminiviridae: Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus.

Geminiviruses are plant-infecting, circular single-stranded DNA viruses that have a geminate virion morphology. These viruses infect both cultivated and non-cultivated monocotyledonous and dicotyledonous plants and have a wide geographical distribution. Nine genera had been established within the family Geminiviridae (Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus) as of 2020. In the last decade, metagenomics approaches have facilitated the discovery and identification of many novel viruses, among them numerous highly divergent geminiviruses. Here, we report the establishment of five new genera in the family Geminiviridae (Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus) to formally classify twelve new, divergent geminiviruses.

Virion-Associated Nucleic Acid-Based Metagenomics: A Decade of Advances in Molecular Characterization of Plant Viruses.

Over the last decade, viral metagenomic studies have resulted in the discovery of thousands of previously unknown viruses. These studies are likely to play a pivotal role in obtaining an accurate and robust understanding of how viruses affect the stability and productivity of ecosystems. Among the metagenomics-based approaches that have been developed since the beginning of the 21st century, shotgun metagenomics applied specifically to virion-associated nucleic acids (VANA) has been used to disentangle the diversity of the viral world. We summarize herein the results of 24 VANA-based studies, focusing on plant and insect samples conducted over the last decade (2010 to 2020). Collectively, viruses from 85 different families were reliably detected in these studies, including capsidless RNA viruses that replicate in fungi, oomycetes, and plants. Finally, strengths and weaknesses of the VANA approach are summarized and perspectives of applications in detection, epidemiological surveillance, environmental monitoring, and ecology of plant viruses are provided. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Evolutionary Analyses of Base-Pairing Interactions in DNA and RNA Secondary Structures.

Pairs of nucleotides within functional nucleic acid secondary structures often display evidence of coevolution that is consistent with the maintenance of base-pairing. Here, we introduce a sequence evolution model, MESSI (Modeling the Evolution of Secondary Structure Interactions), that infers coevolution associated with base-paired sites in DNA or RNA sequence alignments. MESSI can estimate coevolution while accounting for an unknown secondary structure. MESSI can also use graphics processing unit parallelism to increase computational speed. We used MESSI to infer coevolution associated with GC, AU (AT in DNA), GU (GT in DNA) pairs in noncoding RNA alignments, and in single-stranded RNA and DNA virus alignments. Estimates of GU pair coevolution were found to be higher at base-paired sites in single-stranded RNA viruses and noncoding RNAs than estimates of GT pair coevolution in single-stranded DNA viruses. A potential biophysical explanation is that GT pairs do not stabilize DNA secondary structures to the same extent that GU pairs do in RNA. Additionally, MESSI estimates the degrees of coevolution at individual base-paired sites in an alignment. These estimates were computed for a SHAPE-MaP-determined HIV-1 NL4-3 RNA secondary structure. We found that estimates of coevolution were more strongly correlated with experimentally determined SHAPE-MaP pairing scores than three nonevolutionary measures of base-pairing covariation. To assist researchers in prioritizing substructures with potential functionality, MESSI automatically ranks substructures by degrees of coevolution at base-paired sites within them. Such a ranking was created for an HIV-1 subtype B alignment, revealing an excess of top-ranking substructures that have been previously identified as having structure-related functional importance, among several uncharacterized top-ranking substructures.

ICTV Virus Taxonomy Profile: Geminiviridae 2021.

The family Geminiviridae includes viruses with mono- or bipartite single-stranded, circular DNA genomes of 2.5-5.2 kb. They cause economically important diseases in most tropical and subtropical regions of the world. Geminiviruses infect dicot and monocot plants and are transmitted by insect vectors. DNA satellites are associated with some geminiviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Geminiviridae which is available at ictv.global/report/geminiviridae.

Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses.

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

Compartmentalization and Clonal Amplification of HIV-1 in the Male Genital Tract Characterized Using Next-Generation Sequencing.

Compartmentalization of HIV-1 between the systemic circulation and the male genital tract may have a substantial impact on which viruses are available for sexual transmission to new hosts. We studied compartmentalization and clonal amplification of HIV-1 populations between the blood and the genital tract from 10 antiretroviral-naive men using Illumina MiSeq with a PrimerID approach. We found evidence of some degree of compartmentalization in every study participant, unlike previous studies, which collectively showed that only ∼50% of analyzed individuals exhibited compartmentalization of HIV-1 lineages between the male genital tract (MGT) and blood. Using down-sampling simulations, we determined that this disparity can be explained by differences in sampling depth in that had we sequenced to a lower depth, we would also have found compartmentalization in only ∼50% of the study participants. For most study participants, phylogenetic trees were rooted in blood, suggesting that the male genital tract reservoir is seeded by incoming variants from the blood. Clonal amplification was observed in all study participants and was a characteristic of both blood and semen viral populations. We also show evidence for independent viral replication in the genital tract in the individual with the most severely compartmentalized HIV-1 populations. The degree of clonal amplification was not obviously associated with the extent of compartmentalization. We were also unable to detect any association between history of sexually transmitted infections and level of HIV-1 compartmentalization. Overall, our findings contribute to a better understanding of the dynamics that affect the composition of virus populations that are available for transmission.IMPORTANCE Within an individual living with HIV-1, factors that restrict the movement of HIV-1 between different compartments-such as between the blood and the male genital tract-could strongly influence which viruses reach sites in the body from which they can be transmitted. Using deep sequencing, we found strong evidence of restricted HIV-1 movements between the blood and genital tract in all 10 men that we studied. We additionally found that neither the degree to which particular genetic variants of HIV-1 proliferate (in blood or genital tract) nor an individual's history of sexually transmitted infections detectably influenced the degree to which virus movements were restricted between the blood and genital tract. Last, we show evidence that viral replication gave rise to a large clonal amplification in semen in a donor with highly compartmentalized HIV-1 populations, raising the possibility that differential selection of HIV-1 variants in the genital tract may occur.

A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.

BACKGROUND AIMS: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. METHODS: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. RESULTS: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. CONCLUSIONS: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

How virulent are emerging maize-infecting mastreviruses?

Maize streak disease (MSD) is one of the most significant biotic constraints on the production of Africa's most important cereal crop. Until recently, the only virus known to cause severe MSD was the A-strain of maize streak virus (MSV/A), a member of the genus Mastrevirus, family Geminiviridae. However, over the past decade, two other mastreviruses, MSV/C and maize streak Réunion virus (MSRV), have been repeatedly found in the absence of MSV/A in maize plants displaying severe MSD symptoms. Here, we report on infectious clones of MSV/C and MSRV and test their ability to cause severe MSD symptoms. Although cloned MSV/C and MSRV genomes could cause systemic symptomatic infections in MSD-sensitive maize genotypes, these infections yielded substantially milder symptoms than those observed in the field. The MSV/C and MSRV isolates that we have examined are therefore unlikely to cause severe MSD on their own. Furthermore, mixed infections of MSRV and MSV/C with other mild MSV strains also consistently yielded mild MSD symptoms. It is noteworthy that MSRV produces distinctive striate symptoms in maize that are similar in pattern, albeit not in severity, to those seen in the field, showing that this virus may contribute to the severe MSD symptoms seen in the field. Therefore, despite not fulfilling Koch's postulates for MSV/C and MSRV as causal agents of severe MSD, we cannot exclude the possibility that these viruses could be contributing to currently emerging maize diseases.

Taxonomy update for the family Alphasatellitidae: new subfamily, genera, and species.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded DNA molecules (~1-1.4 kb) that encode a replication-associated protein and have commonly been associated with some members of the families Geminiviridae, Nanoviridae, and Metaxyviridae (recently established). Here, we provide a taxonomy update for the family Alphasatellitidae following the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021. The taxonomic update includes the establishment of the new subfamily Petromoalphasatellitinae. This new subfamily includes three new genera as well as the genus Babusatellite, which previously belonged to the subfamily Nanoalphasatellitinae. Additionally, three new genera and 14 new species have been established in the subfamily Geminialphasatellitinae, as well as five new species in the subfamily Nanoalphasatellitinae.

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

Heterogeneity of the rice microbial community of the Chinese centuries-old Honghe Hani rice terraces system.

The Honghe Hani rice terraces system (HHRTS) is a traditional rice cultivation system where Hani people cultivate remarkably diverse rice varieties. Recent introductions of modern rice varieties to the HHRTS have significantly increased the severity of rice diseases within the terraces. Here, we determine the impacts of these recent introductions on the composition of the rice-associated microbial communities. We confirm that the HHRTS contains a range of both traditional HHRTS landraces and introduced modern rice varieties and find differences between the microbial communities of these two groups. However, this introduction of modern rice varieties has not strongly impacted the overall diversity of the HHRTS rice microbial community. Furthermore, we find that the rice varieties (i.e. groups of closely related genotypes) have significantly structured the rice microbial community composition (accounting for 15%-22% of the variance) and that the core microbial community of HHRTS rice plants represents less than 3.3% of all the microbial taxa identified. Collectively, our study suggests a highly diverse HHRTS rice holobiont (host with its associated microbes) where the diversity of rice hosts mirrors the diversity of their microbial communities. Further studies will be needed to better determine how such changes might impact the sustainability of the HHRTS.

Evidence for both Intermittent and Persistent Compartmentalization of HIV-1 in the Female Genital Tract.

HIV-1 has been shown to evolve independently in different anatomical compartments, but studies in the female genital tract have been inconclusive. Here, we examined evidence of compartmentalization using HIV-1 subtype C envelope (Env) glycoprotein genes (gp160) obtained from matched cervicovaginal lavage (CVL) and plasma samples over 2 to 3 years of infection. HIV-1 gp160 amplification from CVL was achieved for only 4 of 18 acutely infected women, and this was associated with the presence of proinflammatory cytokines and/or measurable viremia in the CVL. Maximum likelihood trees and divergence analyses showed that all four individuals had monophyletic compartment-specific clusters of CVL- and/or plasma-derived gp160 sequences at all or some time points. However, two participants (CAP177 and CAP217) had CVL gp160 diversity patterns that differed from those in plasma and showed restricted viral flow from the CVL. Statistical tests of compartmentalization revealed evidence of persistent compartment-specific gp160 evolution in CAP177, while in CAP217 this was intermittent. Lastly, we identified several Env sites that distinguished viruses in these two compartments; for CAP177, amino acid differences arose largely through positive selection, while insertions/deletions were more common in CAP217. In both cases these differences contributed to substantial charge changes spread across the Env. Our data indicate that, in some women, HIV-1 populations within the genital tract can have Env genetic features that differ from those of viruses in plasma, which could impact the sensitivity of viruses in the genital tract to vaginal microbicides and vaccine-elicited antibodies.IMPORTANCE Most HIV-1 infections in sub-Saharan Africa are acquired heterosexually through the genital mucosa. Understanding the properties of viruses replicating in the female genital tract, and whether these properties differ from those of more commonly studied viruses replicating in the blood, is therefore important. Using longitudinal CVL and plasma-derived sequences from four HIV-1 subtype C-infected women, we found fewer viral migrations from the genital tract to plasma than in the opposite direction, suggesting a mucosal sieve effect from the genital tract to the blood compartment. Evidence for both persistent and intermittent compartmentalization between the genital tract and plasma viruses during chronic infection was detected in two of four individuals, perhaps explaining previously conflicting findings. In cases where compartmentalization occurred, comparison of CVL- and plasma-derived HIV sequences indicated that distinct features of viral populations in the CVL may affect the efficacy of microbicides and vaccines designed to provide mucosal immunity.

Sorghum mastrevirus-associated alphasatellites: new geminialphasatellites associated with an African streak mastrevirus infecting wild Poaceae plants on Reunion Island.

Nine complete nucleotide sequences of geminialphasatellites (subfamily Geminialphasatellitinae, family Alphasatellitidae) recovered from the wild Poaceae Sorghum arundinaceum collected in Reunion are described and analyzed. While the helper geminivirus was identified as an isolate of maize streak virus (genus Mastrevirus, family Geminiviridae), the geminialphasatellite genomes were most closely related to, and shared ~63% identity with, clecrusatellites. Even though the geminialphasatellite molecules lack an adenine rich-region, they have the typical size of geminialphasatellites, encode a replication-associated protein in the virion sense, and have probable stem-loop structures at their virion-strand origins of replication. According to the proposed geminialphasatellite species and genus demarcation thresholds (88% and 70% nucleotide identity, respectively), the genomes identified here represent a new species (within a new genus) for which we propose the name "Sorghum mastrevirus-associated alphasatellite" (genus "Sorgasalphasatellite").