Calcium signaling: an emerging player in plant antiviral defense.

Calcium is a universal messenger in different kingdoms of living organisms and regulates most physiological processes, including defense against pathogens. The threat of viral infections in humans has become very clear in recent years, and this has triggered detailed research into all aspects of host-virus interactions, including the suppression of calcium signaling in infected cells. At the same time, however, the threat of plant viral infections is underestimated in society, and research in the field of calcium signaling during plant viral infections is scarce. Here we highlight an emerging role of calcium signaling for antiviral protection in plants, in parallel with the known evidence from studies of animal cells. Obtaining more knowledge in this domain might open up new perspectives for future crop protection and the improvement of food security.

Plant Protection against Viruses: An Integrated Review of Plant Immunity Agents.

Plant viruses are an important class of pathogens that seriously affect plant growth and harm crop production. Viruses are simple in structure but complex in mutation and have thus always posed a continuous threat to agricultural development. Low resistance and eco-friendliness are important features of green pesticides. Plant immunity agents can enhance the resilience of the immune system by activating plants to regulate their metabolism. Therefore, plant immune agents are of great importance in pesticide science. In this paper, we review plant immunity agents, such as ningnanmycin, vanisulfane, dufulin, cytosinpeptidemycin, and oligosaccharins, and their antiviral molecular mechanisms and discuss the antiviral applications and development of plant immunity agents. Plant immunity agents can trigger defense responses and confer disease resistance to plants, and the development trends and application prospects of plant immunity agents in plant protection are analyzed in depth.

The rising threat of geminiviruses: molecular insights into the disease mechanism and mitigation strategies.

BACKGROUND: Geminiviruses are among the most threatening emerging plant viruses, accountable for a huge loss to agricultural production worldwide. These viruses have been responsible for some serious outbreaks during the last few decades across different parts of the world. Sincere efforts have been made to regulate the disease incidence by incorporating a multi-dimensional approach, and this process has been facilitated greatly by the advent of molecular techniques. But, the mixed infection due to the polyphagous nature of vectors results in viral recombination followed by the emergence of novel viral strains which thus renders the existing mitigation strategies ineffective. Hence, a multifaceted insight into the molecular mechanism of the disease is really needed to understand the regulatory points; much has been done in this direction during the last few years. The present review aims to explore all the latest developments made so far and to organize the information in a comprehensive manner so that some novel hypotheses for controlling the disease may be generated. METHODS AND RESULTS: Starting with the background information, diverse genera of geminiviruses are listed along with their pathological and economic impacts. A comprehensive and detailed mechanism of infection is elaborated to study the interactions between vector, host, and virus at different stages in the life cycle of geminiviruses. Finally, an effort isalso made to analyze the progress made at the molecular level for the development of various mitigation strategies and suggest more effective and better approaches for controlling the disease. CONCLUSION: The study has provided a thorough understanding of molecular mechanism of geminivirus infection.

Rhizomania: Hide and Seek of Polymyxa betae and the Beet Necrotic Yellow Vein Virus with Beta vulgaris.

The molecular interactions between Polymyxa betae, the protist vector of sugar beet viruses, beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania, and Beta vulgaris have not been extensively studied. Here, the transmission of BNYVV to sugar beet by P. betae zoospores was optimized using genetically characterized organisms. Molecular interactions of aviruliferous and viruliferous protist infection on sugar beet were highlighted by transcriptomic analysis. P. betae alone induced limited gene expression changes in sugar beet, as a biotrophic asymptomatic parasite. Most differentially expressed plant genes were down-regulated and included resistance gene analogs and cell wall peroxidases. Several enzymes involved in stress regulation, such as the glutathione-S-transferases, were significantly induced. With BNYVV, the first stages of the P. betae life cycle on sugar beet were accelerated with a faster increase of relative protist DNA level and an earlier appearance of sporangia and sporosori in plants roots. A clear activation of plant defenses and the modulation of genes involved in plant cell wall metabolism were observed. The P. betae transcriptome in the presence of BNYVV revealed induction of genes possibly involved in the switch to the survival stage. The interactions were different depending on the presence or absence of the virus. P. betae alone alleviates plant defense response, playing hide-and-seek with sugar beet and allowing for their mutual development. Conversely, BNYVV manipulates plant defense and promotes the rapid invasion of plant roots by P. betae. This accelerated colonization is accompanied by the development of thick-walled resting spores, supporting the virus survival. [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.

Differential gene expression in aphids following virus acquisition from plants or from an artificial medium.

BACKGROUND: Poleroviruses, such as turnip yellows virus (TuYV), are plant viruses strictly transmitted by aphids in a persistent and circulative manner. Acquisition of either virus particles or plant material altered by virus infection is expected to induce gene expression deregulation in aphids which may ultimately alter their behavior. RESULTS: By conducting an RNA-Seq analysis on viruliferous aphids fed either on TuYV-infected plants or on an artificial medium containing purified virus particles, we identified several hundreds of genes deregulated in Myzus persicae, despite non-replication of the virus in the vector. Only a few genes linked to receptor activities and/or vesicular transport were common between the two modes of acquisition with, however, a low level of deregulation. Behavioral studies on aphids after virus acquisition showed that M. persicae locomotion behavior was affected by feeding on TuYV-infected plants, but not by feeding on the artificial medium containing the purified virus particles. Consistent with this, genes potentially involved in aphid behavior were deregulated in aphids fed on infected plants, but not on the artificial medium. CONCLUSIONS: These data show that TuYV particles acquisition alone is associated with a moderate deregulation of a few genes, while higher gene deregulation is associated with aphid ingestion of phloem from TuYV-infected plants. Our data are also in favor of a major role of infected plant components on aphid behavior.

Silencing suppressors of rice black-streaked dwarf virus and rice stripe virus hijack the 26S proteasome of Laodelphax striatellus to facilitate virus accumulation and transmission.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) is transmitted by small brown planthopper (Laodelphax striatellus [L. striatellus]) and causes devastating disease in rice. P9-1 has silencing suppression activity and is the key protein for viroplasm formation in RBSDV-infected plants and insects; however, its exact function is poorly understood. RESULTS: In this study, the P9-1 of RBSDV interacted with L. striatellus 26S proteasome subunit RPN8. RBSDV accumulation in L. striatellus increased after the 26S proteasome was disrupted by silencing the RPN8 expression. This finding indicated that L. striatellus 26S proteasome played a defense role against RBSDV infection by regulating RBSDV accumulation. Further investigations revealed that P9-1 could competitively bind to RPN8 with RPN7, thereby disrupting the assembly of 26S proteasome in L. striatellus and promoting the infection of RBSDV in insect vectors, and further affecting the transmission of the virus to rice by insect vectors. Similar to P9-1, rice stripe virus (RSV) NS2, a weak silencing suppressor, regulated virus accumulation and transmission by hijacking RPN8 to interfere with the function of 26S proteasome in L. striatellus. CONCLUSION: These results suggest that viruses promote their own infection via interfering with ubiquitination pathway of insect vectors, and this mechanism might be of universal importance. These findings provide a new insight into the mechanism of virus transmission in insect vectors. © 2022 Society of Chemical Industry.

Effector-mediated plant-virus-vector interactions.

Hemipterans (such as aphids, whiteflies, and leafhoppers) are some of the most devastating insect pests due to the numerous plant pathogens they transmit as vectors, which are primarily viral. Over the past decade, tremendous progress has been made in broadening our understanding of plant-virus-vector interactions, yet on the molecular level, viruses and vectors have typically been studied in isolation of each other until recently. From that work, it is clear that both hemipteran vectors and viruses use effectors to manipulate host physiology and successfully colonize a plant and that co-evolutionary dynamics have resulted in effective host immune responses, as well as diverse mechanisms of counterattack by both challengers. In this review, we focus on advances in effector-mediated plant-virus-vector interactions and the underlying mechanisms. We propose that molecular synergisms in vector-virus interactions occur in cases where both the virus and vector benefit from the interaction (mutualism). To support this view, we show that mutualisms are common in virus-vector interactions and that virus and vector effectors target conserved mechanisms of plant immunity, including plant transcription factors, and plant protein degradation pathways. Finally, we outline ways to identify true effector synergisms in the future and propose future research directions concerning the roles effectors play in plant-virus-vector interactions.

Flotillin 2 Facilitates the Infection of a Plant Virus in the Gut of Insect Vector.

Most plant viruses require insect vectors for transmission. One of the key steps for the transmission of persistent-circulative plant viruses is overcoming the gut barrier to enter epithelial cells. To date, little has been known about viral cofactors in gut epithelial cells of insect vectors. Here, we identified flotillin 2 as a plasma membrane protein that facilitates the infection of rice stripe virus (RSV) in its vector, the small brown planthopper. Flotillin 2 displayed a prominent plasma membrane location in midgut epithelial cells. The nucleocapsid protein of RSV and flotillin 2 colocalized on gut microvilli, and a nanomolar affinity existed between the two proteins. Knockout of flotillin 2 impeded the entry of virions into epithelial cells, resulting in a 57% reduction of RSV levels in planthoppers. The knockout of flotillin 2 decreased disease incidence in rice plants fed by viruliferous planthoppers from 40% to 11.7%. Furthermore, flotillin 2 mediated the infection of southern rice black-streaked dwarf virus in its vector, the white-backed planthopper. This work implies the potential of flotillin 2 as a target for controlling the transmission of rice stripe disease. IMPORTANCE Plant viral diseases are a major threat to world agriculture. The transmission of 80% of plant viruses requires vector insects, and 54% of vector-borne plant viruses are persistent-circulative viruses, which must overcome the barriers of gut cells with the help of proteins on the cell surface. Here, we identified flotillin 2 as a membrane protein that mediates the cell entry of rice stripe virus in its vector insect, small brown planthopper. Flotillin 2 displays a prominent cellular membrane location in midgut cells and can specifically bind to virions. The loss of flotillin 2 impedes the entry of virions into the midgut cells of vector insects and substantially suppresses viral transmission to rice. Therefore, flotillin 2 may be a promising target gene for manipulation in vector insects to control the transmission of rice stripe disease and perhaps that of other rice virus diseases in the future.

Identification and stress function verification of the HAK/KUP/KT family in Gossypium hirsutum.

The potassium transporter family HAK/KUP/KT is a large group of proteins that are important in plant potassium transport and play a crucial role in plant growth and development. The members of the family play an important role in the response of plants to abiotic stress by maintaining osmotic balance. However, the function of the family in cotton is unclear. In this study, whole genome identification and characterization of the HAK/KUP/KT family from upland cotton (Gossypium hirsutum) were carried out. Bioinformatics methods were used to identify HAK/KUP/KT family members from the G. hirsutum genome and to analyse the physical and chemical properties, basic characteristics, phylogeny, chromosome location and expression of HAK/KUP/KT family members. A total of 41 HAK/KUP/KT family members were identified in the G. hirsutum genome. Phylogenetic analysis grouped these genes into four clusters (I, II, III, IV), containing 6, 10, 3 and 22 genes, respectively. Chromosomal distribution, gene structure and conserved motif analyses of the 41 GhHAK genes were subsequently performed. The RNA-seq data and qRT-PCR results showed that the family had a wide range of tissue expression patterns, and they responded to certain drought stresses. Through expression analysis, seven HAK/KUP/KT genes involved in drought stress were screened, and four genes with obvious phenotypes under drought stress were obtained by VIGS verification, which laid a theoretical foundation for the function of the cotton HAK/KUP/KT family.

Selection and Validation of Reliable Reference Genes for Gene Expression Studies in Different Genotypes and TRV-Infected Fruits of Peach (Prunus persica L. Batsch) during Ripening.

Real-time quantitative PCR (RT-qPCR) is a powerful tool to detect and quantify transcription abundance, and the stability of the reference gene determines its success. However, the most suitable reference gene for different genotypes and tobacco rattle virus (TRV) infected fruits was unclear in peach (Prunus persica L. Batsch). In this study, 10 reference genes were selected and gene expression was characterized by RT-qPCR across all samples, including different genotypes and TRV-infected fruits during ripening. Four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) were used to calculate the stability of 10 reference genes. The geNorm analysis indicated that two suitable reference genes should be used for gene expression normalization. In general, the best combination of reference genes was CYP2 and Tua5 for TRV-infected fruits and CYP2 and Tub1 for different genotypes. In 18S, GADPH, and TEF2, there is an unacceptable variability of gene expression in all experimental conditions. Furthermore, to confirm the validity of the reference genes, the expression levels of PpACO1, PpEIN2, and PpPL were normalized at different fruit storage periods. In summary, our results provide guidelines for selecting reliable reference genes in different genotypes and TRV-infected fruits and lay the foundation for accurate evaluation of gene expression for RT-qPCR analysis in peach.

Small RNAs Participate in Plant-Virus Interaction and Their Application in Plant Viral Defense.

Small RNAs are significant regulators of gene expression, which play multiple roles in plant development, growth, reproductive and stress response. It is generally believed that the regulation of plants' endogenous genes by small RNAs has evolved from a cellular defense mechanism for RNA viruses and transposons. Most small RNAs have well-established roles in the defense response, such as viral response. During viral infection, plant endogenous small RNAs can direct virus resistance by regulating the gene expression in the host defense pathway, while the small RNAs derived from viruses are the core of the conserved and effective RNAi resistance mechanism. As a counter strategy, viruses evolve suppressors of the RNAi pathway to disrupt host plant silencing against viruses. Currently, several studies have been published elucidating the mechanisms by which small RNAs regulate viral defense in different crops. This paper reviews the distinct pathways of small RNAs biogenesis and the molecular mechanisms of small RNAs mediating antiviral immunity in plants, as well as summarizes the coping strategies used by viruses to override this immune response. Finally, we discuss the current development state of the new applications in virus defense based on small RNA silencing.

A viral protein disrupts vacuolar acidification to facilitate virus infection in plants.

Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.

Phytosterol metabolism in plant positive-strand RNA virus replication.

The genome of most plant viruses consists of a single positive-strand of RNA (+ ssRNA). Successful replication of these viruses is fully dependent on the endomembrane system of the infected cells, which experiences a massive proliferation and a profound reshaping that enables assembly of the macromolecular complexes where virus genome replication occurs. Assembly of these viral replicase complexes (VRCs) requires a highly orchestrated interplay of multiple virus and co-opted host cell factors to create an optimal microenvironment for efficient assembly and functioning of the virus genome replication machinery. It is now widely accepted that VRC formation involves the recruitment of high levels of sterols, but the specific role of these essential components of cell membranes and the precise molecular mechanisms underlying sterol enrichment at VRCs are still poorly known. In this review, we intend to summarize the most relevant knowledge on the role of sterols in ( +)ssRNA virus replication and discuss the potential of manipulating the plant sterol pathway to help plants fight these infectious agents.

Effects of an Alphasatellite on the Life Cycle of the Nanovirus Faba Bean Necrotic Yellows Virus.

Nanoviruses are plant viruses with a multipartite single-stranded DNA (ssDNA) genome. Alphasatellites are commonly associated with nanovirus infections, but their putative impact on their helper viruses is unknown. In this study, we investigated the role of subterranean clover stunt alphasatellite 1 (here named SCSA 1) on various important traits of Faba bean necrotic yellows virus (FBNYV) in its host plant Vicia faba and aphid vector Acyrthosiphon pisum, including disease symptoms, viral accumulation, and viral transmission. The results indicate that SCSA 1 does not affect the severity of symptoms nor overall FBNYV accumulation in V. faba, but it does change the relative amounts of its different genomic segments. Moreover, the association of SCSA 1 with FBNYV increases the rate of plant-to-plant transmission by a process seemingly unrelated to the simple increase of viral accumulation in the vector. These results represent the first study on the impact of an alphasatellite on the biology of its helper nanovirus. They suggest that SCSA 1 may benefit FBNYV, but the genericity of this conclusion is discussed and questioned. IMPORTANCE Alphasatellites are circular single-stranded DNA molecules frequently found in association with natural isolates of nanoviruses and some geminiviruses, the two ssDNA plant-infecting virus families. While the implications of alphasatellite presence in geminivirus infections are relatively well documented, comparable studies on alphasatellites associated with nanoviruses are not available. Here, we confirm that subterranean clover stunt alphasatellite 1 affects different traits of its helper nanovirus, Faba bean necrotic yellows virus, both in the host plant and aphid vector. We show that the frequencies of the virus segments change in the presence of alphasatellite, in both the plant and the vector. We also confirm that although within-plant virus load and symptoms are not affected by alphasatellite, the presence of alphasatellite decreases within-aphid virus load but significantly increases virus transmission rate, and thus it may confer a possible evolutionary advantage for the helper virus.

Plant Viruses Can Alter Aphid-Triggered Calcium Elevations in Infected Leaves.

Alighting aphids probe a new host plant by intracellular test punctures for suitability. These induce immediate calcium signals that emanate from the punctured sites and might be the first step in plant recognition of aphid feeding and the subsequent elicitation of plant defence responses. Calcium is also involved in the transmission of non-persistent plant viruses that are acquired by aphids during test punctures. Therefore, we wanted to determine whether viral infection alters calcium signalling. For this, calcium signals triggered by aphids were imaged on transgenic Arabidopsis plants expressing the cytosolic FRET-based calcium reporter YC3.6-NES and infected with the non-persistent viruses cauliflower mosaic (CaMV) and turnip mosaic (TuMV), or the persistent virus, turnip yellows (TuYV). Aphids were placed on infected leaves and calcium elevations were recorded by time-lapse fluorescence microscopy. Calcium signal velocities were significantly slower in plants infected with CaMV or TuMV and signal areas were smaller in CaMV-infected plants. Transmission tests using CaMV-infected Arabidopsis mutants impaired in pathogen perception or in the generation of calcium signals revealed no differences in transmission efficiency. A transcriptomic meta-analysis indicated significant changes in expression of receptor-like kinases in the BAK1 pathway as well as of calcium channels in CaMV- and TuMV-infected plants. Taken together, infection with CaMV and TuMV, but not with TuYV, impacts aphid-induced calcium signalling. This suggests that viruses can modify plant responses to aphids from the very first vector/host contact.

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage.

Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.

Modelling interference between vectors of non-persistently transmitted plant viruses to identify effective control strategies.

Aphids are the primary vector of plant viruses. Transient aphids, which probe several plants per day, are considered to be the principal vectors of non-persistently transmitted (NPT) viruses. However, resident aphids, which can complete their life cycle on a single host and are affected by agronomic practices, can transmit NPT viruses as well. Moreover, they can interfere both directly and indirectly with transient aphids, eventually shaping plant disease dynamics. By means of an epidemiological model, originally accounting for ecological principles and agronomic practices, we explore the consequences of fertilization and irrigation, pesticide deployment and roguing of infected plants on the spread of viral diseases in crops. Our results indicate that the spread of NPT viruses can be i) both reduced or increased by fertilization and irrigation, depending on whether the interference is direct or indirect; ii) counter-intuitively increased by pesticide application and iii) reduced by roguing infected plants. We show that a better understanding of vectors' interactions would enhance our understanding of disease transmission, supporting the development of disease management strategies.

Genome-Wide Identification of GDSL-Type Esterase/Lipase Gene Family in Dasypyrum villosum L. Reveals That DvGELP53 Is Related to BSMV Infection.

GDSL-type esterase/lipase proteins (GELPs) characterized by a conserved GDSL motif at their N-terminus belong to the lipid hydrolysis enzyme superfamily. In plants, GELPs play an important role in plant growth, development and stress response. The studies of the identification and characterization of the GELP gene family in Triticeae have not been reported. In this study, 193 DvGELPs were identified in Dasypyrum villosum and classified into 11 groups (clade A-K) by means of phylogenetic analysis. Most DvGELPs contain only one GDSL domain, only four DvGELPs contain other domains besides the GDSL domain. Gene structure analysis indicated 35.2% DvGELP genes have four introns and five exons. In the promoter regions of the identified DvGELPs, we detected 4502 putative cis-elements, which were associated with plant hormones, plant growth, environmental stress and light responsiveness. Expression profiling revealed 36, 44 and 17 DvGELPs were highly expressed in the spike, the root and the grain, respectively. Further investigation of a root-specific expressing GELP, DvGELP53, indicated it was induced by a variety of biotic and abiotic stresses. The knockdown of DvGELP53 inhibited long-distance movement of BSMV in the tissue of D. villosum. This research provides a genome-wide glimpse of the D. villosum GELP genes and hints at the participation of DvGELP53 in the interaction between virus and plants.

Top Three Strategies of ss(+)RNA Plant Viruses: Great Opportunists and Ecosystem Tuners with a Small Genome.

ss(+)RNA viruses represent the dominant group of plant viruses. They owe their evolutionary superiority to the large number of mutations that occur during replication, courtesy of RNA-dependent RNA polymerase. Natural selection rewards successful viral subtypes, whose effective tuning of the ecosystem regulates the interactions between its participants. Thus, ss(+)RNA viruses act as shuttles for the functionally important genes of the participants in symbiotic relationships within the ecosystem, of which the most common ecological triad is "plant-virus-insect". Due to their short life cycle and large number of offspring, RNA viruses act as skillful tuners of the ecosystem, which benefits both viruses and the system as a whole. A fundamental understanding of this aspect of the role played by viruses in the ecosystem makes it possible to apply this knowledge to the creation of DNA insecticides. In fact, since the genes that viruses are involved in transferring are functionally important for both insects and plants, silencing these genes (for example, in insects) can be used to regulate the pest population. RNA viruses are increasingly treated not as micropathogens but as necessary regulators of ecosystem balance.

A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens.

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting Physcomitrium patens that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named Physcomitrium patens amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of P. patens, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus-host coevolution.