Potyviruses (family Potyviridae, genus Potyvirus) are the result of an initial radiation event that occurred 6,600 years ago. The genus currently consists of 167 species that infect monocots or dicots, including domesticated and wild plants. Potyviruses are transmitted in a non-persistent way by more than 200 species of aphids. As indicated by their wide host range, worldwide distribution, and diversity of their vectors, potyviruses have an outstanding capacity to adapt to new hosts and environments. However, factors that confer adaptability are poorly understood. Viral RNA-dependent RNA polymerases introduce nucleotide substitutions that generate genetic diversity. We hypothesized that selection imposed by hosts and vectors creates a footprint in areas of the genome involved in host adaptation. Here, we profiled genomic and polyprotein variation in all species in the genus Potyvirus. Results showed that the potyviral genome is under strong negative selection. Accordingly, the genome and polyprotein sequence are remarkably stable. However, nucleotide and amino acid substitutions across the potyviral genome are not randomly distributed and are not determined by codon usage. Instead, substitutions preferentially accumulate in hypervariable areas at homologous locations across potyviruses. At a frequency that is higher than that of the rest of the genome, hypervariable areas accumulate non-synonymous nucleotide substitutions and sites under positive selection. Our results show, for the first time, that there is correlation between host range and the frequency of sites under positive selection. Hypervariable areas map to the N terminal part of protein P1, N and C terminal parts of helper component proteinase (HC-Pro), the C terminal part of protein P3, VPg, the C terminal part of NIb (RNA-dependent RNA polymerase), and the N terminal part of the coat protein (CP). Additionally, a hypervariable area at the NIb-CP junction showed that there is variability in the sequence of the NIa protease cleavage sites. Structural alignment showed that the hypervariable area in the CP maps to the N terminal flexible loop and includes the motif required for aphid transmission. Collectively, results described here show that potyviruses contain fixed hypervariable areas in key parts of the genome which provide mutational robustness and are potentially involved in host adaptation.

The polerovirus (family Solemoviridae, genus Polerovirus) genome consists of single, positive strand RNA organized in overlapping open reading frames (ORFs) that, in addition to others, code for protein 0 (P0, a gene silencing suppressor), a coat protein (CP, ORF3) and a read-through domain (ORF5) that is fused to the CP to form a CP-RT protein. The genus Polerovirus contains 26 virus species that infect a wide variety of plants from cereals to cucurbits, to peppers. Poleroviruses are transmitted by a wide range of aphid species in the genera Rhopalosiphum, Stiobion, Aphis, and Myzus. Aphid transmission is mediated both by the CP and the CP-RT. In viruses, mutational robustness and structural flexibility are necessary for maintaining functionality in genetically diverse sets of host plants and vectors. Under this scenario, within a virus genome, mutations preferentially accumulate in areas that are determinants of host adaptation or vector transmission. In this study, we profiled genomic variation in poleroviruses. Consistent with their multifunctional nature, single nucleotide variation and selection analyses showed that ORFs coding for P0 and the read-through domain within the CP-RT are the most variable and contain the highest frequency of sites under positive selection. An order/disorder analysis showed that protein P0 is not disordered. In contrast, proteins CP-RT and VPg contain areas of disorder. Disorder is a property of multifunctional proteins with multiple interaction partners. Results described here suggest that using contrasting mechanisms, P0, VPg and CP-RT mediate adaptation to host plants, to vectors, and are contributors to the broad host and vector range of poleroviruses. Profiling genetic variation across the polerovirus genome has practical applications in diagnostics, breeding for resistance, identification of susceptibility genes, and contributes to our understanding of virus interactions with their host, vectors, and environment.

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<p><em>Severe acute respiratory syndrome coronavirus 2</em> (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. Two mRNA vaccines based on the spike protein S have been authorized by the Food and Drug Administration. Antibody-based diagnostic test detect antibodies developed against protein S. Mutations in the genome of SARS-CoV-2 might compromise the precision of diagnostic tests and the efficacy of vaccines and antiviral drugs. We recently profiled genomic variation in human coronaviruses SARS[1]CoV, SARS-CoV-2, and <em>Middle East respiratory syndrome coronavirus</em> (MERS-CoV). As in all species of the genus Betacoronavirus, the genome is hyper variable, and mutations are not random. The most variable cistron codes for the spike S protein. Hyper variation in protein S has the potential to affect the efficacy of vaccines, the reliability of antibody-based diagnostic test, and predicts potential for repeated SARS-CoV-2 infections. Here we review the basics of coronavirus biology and genomic variation, and link them to diagnostic tests, vaccines, and antiviral drugs.</p>

Virus evolution is the change in the genetic structure of a viral population over time and results in the emergence of new viral variants, strains, and species with novel biological properties, including adaptation to new hosts. There are host, vector, environmental, and viral factors that contribute to virus evolution. To achieve or fine tune compatibility and successfully establish infection, viruses adapt to a particular host species or to a group of species. However, some viruses are better able to adapt to diverse hosts, vectors, and environments. Viruses generate genetic diversity through mutation, reassortment, and recombination. Plant viruses are exposed to genetic drift and selection pressures by host and vector factors, and random variants or those with a competitive advantage are fixed in the population and mediate the emergence of new viral strains or species with novel biological properties. This process creates a footprint in the virus genome evident as the preferential accumulation of substitutions, insertions, or deletions in areas of the genome that function as determinants of host adaptation. Here, with respect to plant viruses, we review the current understanding of the sources of variation, the effect of selection, and its role in virus evolution and host adaptation.

RNA viruses exist as populations of genome variants. Virus-infected plants accumulate 21-24 nucleotide small interfering RnAs (siRnAs) derived from viral RnA (virus-derived siRnAs) through gene silencing. This paper describes the profile of mutations in virus-derived siRNAs for three members of the family Potyviridae: Turnip mosaic virus (tuMV), Papaya ringspot virus (pRSV) and Wheat streak mosaic virus (WSMV). for tuMV in Arabidopsis thaliana, profiles were obtained for mechanically inoculated rosette leaves and systemically infected cauline leaves and inflorescence. Results are consistent with selection pressure on the viral genome imposed by local and systemic movement. By genetically removing gene silencing in the plant and silencing suppression in the virus, our results showed that antiviral gene silencing imposes selection in viral populations. Mutations in siRnAs derived from a PRSV coat protein transgene in the absence of virus replication showed the contribution of cellular RnA-dependent RnA polymerases to the generation of mutations in virus-derived siRnAs. collectively, results are consistent with two sources of mutations in virus-derived siRNAs: viral RNA-dependent RNA polymerases responsible for virus replication and cellular RnA-dependent RnA polymerases responsible for gene silencing amplification. In plants virus infection triggers antiviral gene silencing that results in the accumulation of virus-derived small interfering RNAs (siRNAs) that are normally 21-24 nucleotides long and associate with argonaute proteins to form RNA-induced silencing complexes and guide translational repression or cleave of viral RNA targets based on sequence complementarity 1-3. Virus-derived siRNAs are generated by dicer-like proteins (DCL), mainly DCL4 (21-nt) and DCL2 (22-nt), using dsRNA substrates that include virus replication intermediates and fold-back structures in single stranded RNA 4-7. Additionally, siRNAs are silencing signals and move cell-to-cell, root to shoot, and in some cases between plants and fungi 8. Plant virus infection depends on the ability of the virus to replicate in infected cells, move cell-to-cell via plasmodesmata and systemically 9. Genetic heterogeneity in combination with genetic robustness allows viruses to adapt to diverse hosts, vectors, and environments 10-12. In infected plants, viruses exists as diverse populations of genomes forming a quasispecies 12. A critical source of variation that drives virus evolution is the viral RNA-dependent RNA polymerases which lack proofreading activity and introduce mutations (nucleotide substitutions, insertions, or deletions) during virus replication. As a result, each replication cycle might produce new variants containing nucleotide substitutions, insertions, or deletions 11-15. Selection eliminates unfit variants and favors variants with a competitive advantage, resulting in one or few dominant genetic variants surrounded by a large number of low-frequency, less-fit, or emerging variants 12,15,16. High-throughput sequencing has expanded our ...

Computational skills are increasingly important for conducting research in the agricultural and natural sciences. However, barriers to training availability and accessibility have left many life scientists under-prepared. To address these challenges, we developed a workshop series led by graduate students to cover topics relevant to research needs among life scientists. Our workshops incorporated guided practice to facilitate a student-centered learning environment. Further, in response to changes mandated by the COVID-19 pandemic, the workshop series was converted to a virtual format and extended to life science researchers beyond our home institution. We highlight how to effectively structure workshops to develop computational skills and adapt in-person activities to the virtual environment. Survey data shows our workshops reached a highly diverse group of scientists, representing more than eight departments, in both in-person and virtual renditions. Further, we demonstrate an increase in self-reported confidence in student abilities to apply concepts to their own research following content delivery. This increase was facilitated in both virtual and in-person environments, showing that adapting content for online delivery is capable of supporting student learning gains. The success of this workshop series shows the importance of hands-on, guided practice in developing computational skills to fill gaps in training in the agricultural and natural sciences.

The Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 originated in bats and adapted to infect humans. Several SARS-CoV-2 strains have been identified. Genetic variation is fundamental to virus evolution, and in response to selection pressure, is manifested as the emergence of new strains and species adapted to different hosts or with novel pathogenicity. The combination of variation and selection forms a genetic footprint on the genome, consisting of the preferential accumulation of mutations in particular areas. Properties of betacoronaviruses contributing to variation and the emergence of new strains and species are beginning to be elucidated. To better understand their variation, we profiled the accumulation of mutations in all species in the genus Betacoronavirus , including SARS-CoV-2 and two other species that infect humans: SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Variation profiles identified both genetically stable and variable areas at homologous locations across species within the genus Betacoronavirus . The S glycoprotein is the most variable part of the genome and is structurally disordered. Other variable parts include proteins 3, 7, and ORF8, which participate in replication and suppression of antiviral defense. In contrast, replication proteins in ORF 1b are the least variable. Collectively, our results show that variation and structural disorder in the S glycoprotein is a general feature of all members of the genus Betacoronavirus , including SARS-CoV-2. These findings highlight the potential for the continual emergence of new species and strains with novel biological properties and indicate that the S glycoprotein has a critical role in host adaptation. IMPORTANCE Natural infection with SARS-CoV-2 and vaccines trigger the formation of antibodies against the S glycoprotein, which are detected by antibody-based diagnostic tests. Our analysis showed that variation in the S glycoprotein is a general feature of all species in the genus Betacoronavirus including three species that infect humans: SARS-CoV, SARS-CoV-2, and MERS-CoV. The variable nature of the S glycoprotein provides an explanation for the emergence of SARS-CoV-2, the differentiation of SARS-CoV-2 into strains, and the probability of SARS-CoV-2 repeated infections in people. Variation of the S glycoprotein also has important implications for the reliability of SARS-CoV-2 antibody-based diagnostic tests and the design and deployment of vaccines and antiviral drugs. These findings indicate that adjustments to vaccine design and deployment and to antibody-based diagnostic tests are necessary to account for S glycoprotein variation.