The Ups and Downs of an Out-of-the-Box Scientist with a Curious Mind.

My early life was challenging, and not conducive to the study of science, but my first introduction to viruses was an epiphany for me. I spent the whole of my career dedicated to understanding viruses, driven largely by curiosity. This led me down many different avenues of study, and to work with many wonderful colleagues, most of whom remain friends. Some highlights of my career include the discovery of a mutualistic three-way symbiosis involving a virus, a fungus, and a plant; genetic mapping of a pathogenicity gene in tomato; uncovering a virus in 1,000-year-old corncobs; exploring virus biodiversity in wild plants; and establishing a system to use a fungal virus to understand the epidemiology of its host.

Evaluation of Virus-Free and Wild-Type Isolates of Pseudogymnoascus destructans Using a Porcine Ear Model.

White-nose syndrome (WNS), responsible for the mass mortality of North American bats, lacks economically viable and practical in vitro models for Pseudogymnoascus destructans infection, the causative agent of WNS. Not only are many susceptible North American insectivorous bats nearing extinction and, thus, scarce for experimental studies, but they are difficult to care for and maintain in captivity because of their specialized habitats and diets. In this study, we explored porcine ears as a potential substrate for studying infection development and the dynamics of P. destructans growth in the laboratory. Porcine ear skin shares many tissue-level similarities with bat skin and is a readily available resource. We found the porcine ear model provided a substrate faithfully mimicking external P. destructans colony morphology and internal histology similar to what is seen with P. destructans infections in bat wing membranes. This model provided a major advance by distinguishing virulence attributes between a wild-type Pseudogymnoascus destructans strain harboring a partitivirus common to all North American strains of the fungus and an isogenic virus-cured P. destructans strain. ImageJ analysis showed that the cured P. destructans strain was reduced significantly in ability to produce hyphal cover and showed less spore production on porcine skin. Taken together, these results strengthen our previous finding that the partitivirus infection has a role in WNS and provides a valuable model host tool in understanding P. destructans virulence factors for therapeutic application. IMPORTANCE This work describes an important insight into the role of Pseudogymnoascus destructans partitivirus in fungal biology and provides a model system for studying white-nose syndrome in bats, which has decimated North American populations.

RdRp or RT, That is the Question.

The RNA-dependent RNA polymerase (RdRp) of all known double-stranded RNA viruses is located within the viral particle and is responsible for the transcription and replication of the viral genome. Through an RT-PCR assay, we determined that purified virions, in vitro translated RdRp proteins, and purified recombinant RdRp proteins of partitiviruses also have reverse transcriptase (RT) function. We show that partitivirus RdRps 1) synthesized DNA from homologous and heterologous dsRNA templates; 2) are active using both ssRNA and dsRNA templates; and 3) are active at lower temperatures compared to an optimal reaction temperature of commercial RT enzymes. This finding poses an intriguing question: why do partitiviruses, with dsRNA genomes, have a polymerase with RT functions? In comparison, 3Dpol, the RdRp of poliovirus, did not show any RT activity. Our findings lead us to propose a new evolutionary model for RNA viruses where the RdRp of dsRNA viruses could be the ancestor of RdRps.

Phylogeographic analysis of Pseudogymnoascus destructans partitivirus-pa explains the spread dynamics of white-nose syndrome in North America.

Understanding the dynamics of white-nose syndrome spread in time and space is an important component for the disease epidemiology and control. We reported earlier that a novel partitivirus, Pseudogymnoascus destructans partitivirus-pa, had infected the North American isolates of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats. We showed that the diversity of the viral coat protein sequences is correlated to their geographical origin. Here we hypothesize that the geographical adaptation of the virus could be used as a proxy to characterize the spread of white-nose syndrome. We used over 100 virus isolates from diverse locations in North America and applied the phylogeographic analysis tool BEAST to characterize the spread of the disease. The strict clock phylogeographic analysis under the coalescent model in BEAST showed a patchy spread pattern of white-nose syndrome driven from a few source locations including Connecticut, New York, West Virginia, and Kentucky. The source states had significant support in the maximum clade credibility tree and Bayesian stochastic search variable selection analysis. Although the geographic origin of the virus is not definite, it is likely the virus infected the fungus prior to the spread of white-nose syndrome in North America. We also inferred from the BEAST analysis that the recent long-distance spread of the fungus to Washington had its root in Kentucky, likely from the Mammoth cave area and most probably mediated by a human. The time to the most recent common ancestor of the virus is estimated somewhere between the late 1990s to early 2000s. We found the mean substitution rate of 2 X 10-3 substitutions per site per year for the virus which is higher than expected given the persistent lifestyle of the virus, and the stamping-machine mode of replication. Our approach of using the virus as a proxy to understand the spread of white-nose syndrome could be an important tool for the study and management of other infectious diseases.

Viruses in the phytobiome.

The phytobiome, defined as plants and all the entities that interact with them, is rich in viruses, but with the exception of plant viruses of crop plants, most of the phytobiome viruses remain very understudied. This review focuses on the neglected portions of the phytobiome, including viruses of other microbes interacting with plants, viruses in the soil, viruses of wild plants, and relationships between viruses and the vectors of plant viruses.

Impact of Cultivated Hosts on the Recombination of Cucumber Mosaic Virus.

Cucumber mosaic virus (CMV) is one of the most successful viruses known, infecting over 1,200 species of plants. Like other single-stranded RNA viruses, CMV is known to have a high potential for population diversity due to error-prone replication and short generation times. Recombination is also a mechanism that allows viruses to adapt to new hosts. Host genes have been identified that impact the recombination of RNA viruses by using single-cell yeast systems. To determine the impact that the natural plant host has on virus recombination, we used a high-recombination-frequency strain of CMV, LS-CMV, which belongs to subgroup II, in three different cultivated hosts: Capsicum annuum cv. Marengo (pepper), Nicotiana tabacum cv. Xanthi nc (tobacco), and Cucurbita pepo cv. Black Beauty (zucchini). The recombination frequency was calculated by using an RNA 3 reporter carrying restriction enzyme sites created by introducing silent mutations. Our results show that the recombination frequency of LS-CMV is correlated with the infected host. The recombination events in pepper were 1.8-fold higher than those in tobacco and 5-fold higher than those in zucchini. Furthermore, we observed the generation of defective RNAs in inoculated pepper plants, but not in tobacco or zucchini. These results indicate that the host is involved in both intra- and intermolecular recombination events and that hosts like pepper could foster more rapid evolution of the virus. In addition, we report for the first time the production of defective RNAs in a CMV subgroup II isolate.IMPORTANCE Recombination is an important mechanism used by viruses for their diversification and to adapt to diverse hosts. Understanding the host role in the mechanisms of evolution is important for virus disease management and controlling the emergence of new strains. This study shows the impact that cultivated hosts are playing in the evolution of CMV. Furthermore, our results and previous studies show how some specific hosts could be an ideal environment for the emergence of new viral strains.

Manipulation of Aphid Behavior by a Persistent Plant Virus.

Plants are frequently infected with cytoplasmic RNA viruses that persist for many generations through nearly 100% vertical transmission without producing any symptoms. Movement between plant cells and horizontal transmission have not been observed with these viruses; instead, they are distributed to all host cells through host cell division. Jalapeño peppers (Capsicum annuum) are all infected with Pepper cryptic virus 1 (PCV-1; family Partitiviridae). We compared the effect of odor cues from PCV-1-infected (J+) and virus-free (J-) jalapeño peppers on the aphid Myzus persicae, a common vector of acute plant viruses. Pairwise preference experiments showed a stark contrast to insect-plant interactions in acute virus infections-that is, the virus-infected plants deterred aphids. The acute plant virus Cucumber mosaic virus (CMV) manipulates its host's volatile emissions to attract aphid vectors and facilitate its transmission. We inoculated J+ and J- plants with CMV. Volatiles of J+ and J- CMV-infected plants were more attractive to aphids than those of J+ and J- mock-inoculated plants. However, in pairwise preference experiments with J+ CMV- and J- CMV-infected plants, aphids preferred the J- CMV volatile blend. Aphid reproduction on J+ and J- plants was measured as an indicator of the effect of PCV-1 on host quality for aphids. Aphid reproduction on J+ plants was more than 2-fold lower than that on J- plants.IMPORTANCE This study demonstrates that a persistent plant virus can manipulate aphid behavior. This manipulation is in stark contrast to previously described effects of acute viruses on their hosts that facilitate their transmission. This study demonstrates a positive relationship between Pepper cryptic virus 1 and jalapeño pepper (Capsicum annuum) plants wherein the virus protects the plants from the vector of acute viruses and reduces aphid herbivory. This work reveals an important implication of persistent plant viruses for pest and pathogen management in agriculture.

A 1,000-Year-Old RNA Virus.

Only a few RNA viruses have been discovered from archaeological samples, the oldest dating from about 750 years ago. Using ancient maize cobs from Antelope house, Arizona, dating from ca. 1,000 CE, we discovered a novel plant virus with a double-stranded RNA genome. The virus is a member of the family Chrysoviridae that infect plants and fungi in a persistent manner. The extracted double-stranded RNA from 312 maize cobs was converted to cDNA, and sequences were determined using an Illumina HiSeq 2000. Assembled contigs from many samples showed similarity to Anthurium mosaic-associated virus and Persea americana chrysovirus, putative species in the Chrysovirus genus, and nearly complete genomes were found in three ancient maize samples. We named this new virus Zea mays chrysovirus 1. Using specific primers, we were able to recover sequences of a closely related virus from modern maize and obtained the nearly complete sequences of the three genomic RNAs. Comparing the nucleotide sequences of the three genomic RNAs of the modern and ancient viruses showed 98, 96.7, and 97.4% identities, respectively. Hence, in 1,000 years of maize cultivation, this virus has undergone about 3% divergence.IMPORTANCE A virus related to plant chrysoviruses was found in numerous ancient samples of maize, with nearly complete genomes in three samples. The age of the ancient samples (i.e., about 1,000 years old) was confirmed by carbon dating. Chrysoviruses are persistent plant viruses. They infect their hosts from generation to generation by transmission through seeds and can remain in their hosts for very long time periods. When modern corn samples were analyzed, a closely related chrysovirus was found with only about 3% divergence from the ancient sequences. This virus represents the oldest known plant virus.

Evolutionary and ecological links between plant and fungal viruses.

Contents Summary 86 I. Introduction 86 II. Lineages shared by plant and fungal viruses 87 III. Virus transmission between plants and fungi 90 IV. Additional plant virus families identified in fungi by metagenomics 91 Acknowledgements 91 References 91 SUMMARY: Plants and microorganisms have been interacting in both positive and negative ways for millions of years. They are also frequently infected with viruses that can have positive or negative impacts. A majority of virus families with members that infect fungi have counterparts that infect plants, and in some cases the phylogenetic analyses of these virus families indicate transmission between the plant and fungal kingdoms. These similarities reflect the host relationships; fungi are evolutionarily more closely related to animals than to plants but share very few viral signatures with animal viruses. The details of several of these interactions are described, and the evolutionary implications of viral cross-kingdom interactions and horizontal gene transfer are proposed.

Characterizing Mycoviruses.

A variety of methods for the detection and characterization of fungal viruses are available. For many years, serological and biological assays were used for virus detection. Today, more sensitive methods like polymerase chain reaction, together with sequencing, are widely used to study viruses. Extracting double-stranded (ds) RNA can be a useful approach to detect and study mycoviruses from fungal tissues, as dsRNAs accumulate in infected cells as copies of viral genomes or as replicative intermediates of single-stranded RNA genomes. Here we present a basic protocol for growing fungal strains and isolating dsRNA using cellulose chromatography, followed by molecular diagnostic methods including cDNA synthesis, sequencing, and determination of 5' ends by primer ligation.

Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein.

The effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5' or 3' half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5' mutants were serially passaged in N. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5' mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCE Recently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

Coevolution of a Persistent Plant Virus and Its Pepper Hosts.

There are many nonpathogenic viruses that are maintained in a persistent lifestyle in plants. Plant persistent viruses are widespread, replicating in their hosts for many generations. So far, Endornaviridae is the only family of plant persistent viruses with a single-stranded RNA genome, containing one large open reading frame. Bell pepper endornavirus (BPEV), Hot pepper endornavirus, Capsicum frutescens endornavirus 1 (CFEV 1) have been identified from peppers. Peppers are native to Central and South America and, as domesticated plants, human selection accelerated their evolution. We investigated the evolution of these endornaviruses in different peppers including Capsicum annuum, C. chacoense, C. chinense, C. frutescens, C. baccutum, and C. pubescens using two fragments from the viral helicase (Hel) and RNA dependent RNA polymerase (RdRp) domains. In addition, using single nucleotide polymorphisms, we analyzed the pepper host populations and phylogenies. The endornaviruses phylogeny was correlated with its Capsicum species host. In this study, BPEV was limited to C. annuum species, and the RdRp and Hel phylogenies identified two clades that correlated with the host pungency. No C. annuum infected with CFEV 1 was found in this study, but the CFEV 1 RdRp fragment was recovered from C. chinense, C. frutescens, C. baccutum, and C. pubescens. Hence, during pepper speciation, the ancestor of CFEV 1 may have evolved as a new endornavirus, BPEV, in C. annuum peppers.

Analysis of quasispecies variation in single and mixed viral infection.

Population diversity was examined in individual and natural mixed infections of Cowpea chlorotic mottle virus (CCMV) and Cucumber mosaic virus (CMV) isolates in two systemic hosts, cowpea and Nicotiana benthamiana. Isolates of CCMV and CMV obtained from a cowpea field in Arkansas were separated biologically in cowpea and tobacco plants, respectively. After separation, individual and mixed cultures of both viruses were serially passaged ten times by mechanical inoculation in cowpea and N. benthamiana. High-fidelity reverse transcriptase-polymerase chain reaction (HiFi RT-PCR) of RNA 3, followed by cDNA cloning and sequence analysis was used to assess the quasispecies cloud size of CCMV and CMV populations in passages zero and ten of each host species. The levels of population variation were generally higher in individual infections of CCMV-Car1 and-Car2 isolates, and the CMV-Car2 isolate compared with mixed infections, in both host species, although the significance of the differences varied depending on how mutations were counted. There were no significant differences in the levels of population variation in individual and mixed infections of the CMV-Car1 isolate. Partially fixed mutations were observed in both individual and mixed infections of the CCMV-Car2 isolate in N. benthamiana and CMV-Car1 and-Car2 isolates in both cowpea and N. benthamiana.

Symbiosis: Viruses as Intimate Partners.

Viruses must establish an intimate relationship with their hosts and vectors in order to infect, replicate, and disseminate; hence, viruses can be considered as symbionts with their hosts. Symbiotic relationships encompass different lifestyles, including antagonistic (or pathogenic, the most well-studied lifestyle for viruses), commensal (probably the most common lifestyle), and mutualistic (important beneficial partners). Symbiotic relationships can shape the evolution of the partners in a holobiont, and placing viruses in this context provides an important framework for understanding virus-host relationships and virus ecology. Although antagonistic relationships are thought to lead to coevolution, this is not always clear in virus-host interactions, and impacts on evolution may be complex. Commensalism implies a hitchhiking role for viruses-selfish elements just along for the ride. Mutualistic relationships have been described in detail in the past decade, and they reveal how important viruses are in considering host ecology. Ultimately, symbiosis can lead to symbiogenesis, or speciation through fusion, and the presence of large amounts of viral sequence in the genomes of everything from bacteria to humans, including some important functional genes, illustrates the significance of viral symbiogenesis in the evolution of all life on Earth.

Consensus statement: Virus taxonomy in the age of metagenomics.

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Deep sequencing for discovery and evolutionary analysis of plant viruses.

The advent of next generation sequencing (NGS), or deep sequencing, has allowed great advances to be made in discovery, diagnostics, and evolutionary studies in plant viruses. Various methods have been used for enrichment for virus-specific nucleic acids, each of which have some drawbacks. Many novel viruses have been discovered in plants by NGS technologies, and there is a good deal of promise for more comprehensive studies in virus evolution. However, each aspect of using NGS has its caveats that need to be considered, and there is still a need for better tools of analysis, as well as method for validation of sequence variation.

Using a Novel Partitivirus in Pseudogymnoascus destructans to Understand the Epidemiology of White-Nose Syndrome.

White-nose syndrome is one of the most lethal wildlife diseases, killing over 5 million North American bats since it was first reported in 2006. The causal agent of the disease is a psychrophilic filamentous fungus, Pseudogymnoascus destructans. The fungus is widely distributed in North America and Europe and has recently been found in some parts of Asia, but interestingly, no mass mortality is observed in European or Asian bats. Here we report a novel double-stranded RNA virus found in North American isolates of the fungus and show that the virus can be used as a tool to study the epidemiology of White-nose syndrome. The virus, termed Pseudogymnoascus destructans partitivirus-pa, contains 2 genomic segments, dsRNA 1 and dsRNA 2 of 1.76 kbp and 1.59 kbp respectively, each possessing a single open reading frame, and forms isometric particles approximately 30 nm in diameter, characteristic of the genus Gammapartitivirus in the family Partitiviridae. Phylogenetic analysis revealed that the virus is closely related to Penicillium stoloniferum virus S. We were able to cure P. destructans of the virus by treating fungal cultures with polyethylene glycol. Examination of 62 isolates of P. destructans including 35 from United States, 10 from Canada and 17 from Europe showed virus infection only in North American isolates of the fungus. Bayesian phylogenetic analysis using nucleotide sequences of the viral coat protein geographically clustered North American isolates indicating fungal spread followed by local adaptation of P. destructans in different regions of the United States and Canada. This is the first demonstration that a mycovirus potentially can be used to study fungal disease epidemiology.