APOBEC3G Polymorphism as a Selective Barrier to Cross-Species Transmission and Emergence of Pathogenic SIV and AIDS in a Primate Host

Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary “arms-race” between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary “arms-race” hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.     

Pliant pathogens: Estimating viral spread when confronted with new vector,host, and environmental conditions

1. Pathogen spread rates are determined, in part, by the performance of pathogens under altered environmental conditions and their ability to persist while switching among hosts and vectors.
2. To determine the effects of new conditions (host, vector, and nutrient) on pathogen spread rate, we introduced a vector‐borne viral plant pathogen, Barley Yellow Dwarf Virus PAV (BYDV‐PAV) into hosts, vectors, and host nutrient supplies that it had not encountered for thousands of viral generations. We quantified pathogen prevalence over the course of two serial inoculations under the new conditions. Using individual‐level transmission rates from this experiment, we parameterized a dynamical model of disease spread and projected spread across host populations through a growing season.
3. A change in nutrient conditions (increased supply of phosphorus) reduced viral transmission whereas shifting to a new vector or host species had no effect on infection prevalence. However, the reduction in the new nutrient environment was only temporary; infection prevalence recovered after the second inoculation.
4. Synthesis. These results highlight how robust the pathogen, BYDV‐PAV, is to changes in its biotic and abiotic environment. Our study also highlights the need to quantify longitudinal infection information beyond snapshot assessments to project disease risk for pathogens in new environments.

     

Under siege: virus control in plant meristems and progeny

In the arms race between plants and viruses, two frontiers have been utilized for decades to combat viral infections in agriculture. First, many pathogenic viruses are excluded from plant meristems, which allows the regeneration of virus-free plant material by tissue culture. Second, vertical transmission of viruses to the host progeny is often inefficient, thereby reducing the danger of viral transmission through seeds. Numerous reports point to the existence of tightly linked meristematic and transgenerational antiviral barriers that remain poorly understood. In this review, we summarize the current understanding of the molecular mechanisms that exclude viruses from plant stem cells and progeny. We also discuss the evidence connecting viral invasion of meristematic cells and the ability of plants to recover from acute infections. Research spanning decades performed on a variety of virus/host combinations has made clear that, beside morphological barriers, RNA interference (RNAi) plays a crucial role in preventing—or allowing—meristem invasion and vertical transmission. How a virus interacts with plant RNAi pathways in the meristem has profound effects on its symptomatology, persistence, replication rates, and, ultimately, entry into the host progeny.

We review what is known about the biological mechanisms regulating virus exclusion from—or invasion of—plant host meristems and progeny, including possible consequences and implications of these phenomena.     

Vertically transmitted viral endosymbionts of insects: do sigma viruses walk alone?

Insects are host to a wide range of vertically transmitted bacterial endosymbionts, but we know relatively little about their viral counterparts. Here, we discuss the vertically transmitted viral endosymbionts of insects, firstly examining the diversity of this group, and then focusing on the well-studied sigma viruses that infect dipterans. Despite limited sampling, evidence suggests that vertically transmitted viruses may be common in insects. Unlike bacteria, viruses can be transmitted through sperm and eggs, a trait that allows them to rapidly spread through host populations even when infection is costly to the host. Work on Drosophila melanogaster has shown that sigma viruses and their hosts are engaged in a coevolutionary arms race, in which the spread of resistance genes in the host population is followed by the spread of viral genotypes that can overcome host resistance. In the long-term, associations between sigma viruses and their hosts are unstable, and the viruses persist by occasionally switching to new host species. It therefore seems likely that viral endosymbionts have major impacts on the evolution and ecology of insects.     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as “Cluster 5” that contained mutations in the spike protein. However, the functional properties of these “Cluster 5” mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.     

Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles

Many host cell surface proteins, including viral receptors, are incorporated into enveloped viruses. To address the functional significance of these host proteins, murine leukemia viruses containing the cellular receptors for Rous sarcoma virus (Tva) or ecotropic murine leukemia virus (MCAT-1) were produced. These receptor-pseudotyped viruses efficiently infect cells expressing the cognate viral envelope glycoproteins, with titers of up to 10⁵ infectious units per milliliter for the Tva pseudotypes. Receptor and viral glycoprotein specificity and functional requirements are maintained, suggesting that receptor pseudotype infection recapitulates events of normal viral entry. The ability of the Tva and MCAT-1 pseudotypes to infect cells efficiently suggests that, in contrast to human immunodeficiency virus type 1 entry, neither of these retroviral receptors requires a coreceptor for membrane fusion. In addition, the ability of receptor pseudotypes to target infected cells suggests that they may be useful therapeutic reagents for directing infection of viral vectors. Receptor-pseudotyped viruses may be useful for identifying new viral receptors or for defining functional requirements of known receptors. Moreover, this work suggests that the production of receptor pseudotypes in vivo could provide a mechanism for expanded viral tropism with potential effects on the pathogenesis and evolution of the virus.     

Reviewing the ecological evidence base for management of emerging tropical zoonoses: Kyasanur Forest Disease in India as a case study

Zoonoses disproportionately affect tropical communities and are associated with human modification and use of ecosystems. Effective management is hampered by poor ecological understanding of disease transmission and often focuses on human vaccination or treatment. Better ecological understanding of multi-vector and multi-host transmission, social and environmental factors altering human exposure, might enable a broader suite of management options. Options may include “ecological interventions” that target vectors or hosts and require good knowledge of underlying transmission processes, which may be more effective, economical, and long lasting than conventional approaches. New frameworks identify the hierarchical series of barriers that a pathogen needs to overcome before human spillover occurs and demonstrate how ecological interventions may strengthen these barriers and complement human-focused disease control. We extend these frameworks for vector-borne zoonoses, focusing on Kyasanur Forest Disease Virus (KFDV), a tick-borne, neglected zoonosis affecting poor forest communities in India, involving complex communities of tick and host species. We identify the hierarchical barriers to pathogen transmission targeted by existing management. We show that existing interventions mainly focus on human barriers (via personal protection and vaccination) or at barriers relating to Kyasanur Forest Disease (KFD) vectors (tick control on cattle and at the sites of host (monkey) deaths). We review the validity of existing management guidance for KFD through literature review and interviews with disease managers. Efficacy of interventions was difficult to quantify due to poor empirical understanding of KFDV–vector–host ecology, particularly the role of cattle and monkeys in the disease transmission cycle. Cattle are hypothesised to amplify tick populations. Monkeys may act as sentinels of human infection or are hypothesised to act as amplifying hosts for KFDV, but the spatial scale of risk arising from ticks infected via monkeys versus small mammal reservoirs is unclear. We identified 19 urgent research priorities for refinement of current management strategies or development of ecological interventions targeting vectors and host barriers to prevent disease spillover in the future.     

Transferrin receptor 1 in the zoonosis and pathogenesis of New World hemorrhagic fever arenaviruses

At least five New World arenaviruses cause severe human hemorrhagic fevers. These viruses are transmitted to humans through contact with their respective South American rodent hosts. Each uses human transferrin receptor 1 (TfR1) as its obligate receptor. Accidental similarities between human TfR1 and TfR1 orthologs of arenaviral host species enable zoonoses, whereas mice and rats are not infectable because they lack these TfR1 determinants of infection. All pathogenic New World arenaviruses bind to a common region of the apical domain of TfR1. The ability of a New World arenavirus to use human TfR1 is absolutely predictive of its ability to cause hemorrhagic fevers in humans. Nonpathogenic arenaviruses, closely related to hemorrhagic fever arenaviruses, cannot utilize human TfR1 but efficiently enter cells through TfR1 orthologs of their native rodent hosts. Mutagenesis studies suggest that minor changes in the entry glycoproteins of these nonpathogenic viruses may allow human transmission. TfR1 is upregulated as a result of iron sequestration during the acute-phase response to infection, and the severity of disease may result from amplification of viral replication during this response.     

Viral component of the human genome

Relationships between viruses and their human host are traditionally described from the point of view taking into consideration hosts as victims of viral aggression, which results in infectious diseases. However, these relations are in fact two-sided and involve modifications of both the virus and host genomes. Mutations that accumulate in the populations of viruses and hosts may provide them advantages such as the ability to overcome defense barriers of host cells or to create more efficient barriers to deal with the attack of the viral agent. One of the most common ways of reinforcing anti-viral barriers is the horizontal transfer of viral genes into the host genome. Within the host genome, these genes may be modified and extensively expressed to compete with viral copies and inhibit the synthesis of their products or modulate their functions in other ways. This review summarizes the available data on the horizontal gene transfer between viral and human genomes and discusses related problems.     

West Nile virus genetic diversity is maintained during transmission by Culex pipiens quinquefasciatus mosquitoes

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.     

Apoptosis Induction Influences Reovirus Replication and Virulence in Newborn Mice

Apoptosis is a type of controlled cell death that is essential for development and tissue homeostasis. It also serves as a robust host response against infection by many viruses. The capacity of neurotropic viruses to induce apoptosis strongly correlates with virulence. However, the precise function of apoptosis in viral infection is not well understood. Reovirus is a neurotropic virus that induces apoptosis in a variety of cell types, including central nervous system neurons, leading to fatal encephalitis in newborn mice. To determine the effect of apoptosis on reovirus replication in the host, we generated two otherwise isogenic viruses that differ in a single amino acid in viral capsid protein μ1 that segregates with apoptotic capacity. Apoptosis-proficient and apoptosis-deficient viruses were compared for replication, dissemination, tropism, and tissue injury in newborn mice and for the capacity to spread to uninfected littermates. Our results indicate that apoptotic capacity enhances reovirus replication in the brain and consequent neurovirulence but reduces transmission efficiency. The replication advantage of the apoptosis-proficient strain is limited to the brain and correlates with enhanced infectivity of neurons. These studies reveal a new cell type-specific determinant of reovirus virulence.     

Biosecurity Implications of New Technology and Discovery in Plant Virus Research

Human activity is causing new encounters between viruses and plants. Anthropogenic interventions include changing land use, decreasing biodiversity, trade, the introduction of new plant and vector species to native landscapes, and changing atmospheric and climatic conditions. The discovery of thousands of new viruses, especially those associated with healthy-appearing native plants, is shifting the paradigm for their role within the ecosystem from foe to friend. The cost of new plant virus incursions can be high and result in the loss of trade and/or production for short or extended periods. We present and justify three recommendations for plant biosecurity to improve communication about plant viruses, assist with the identification of viruses and their impacts, and protect the high economic, social, environmental, and cultural value of our respective nations'' unique flora: 1) As part of the burden of proof, countries and jurisdictions should identify what pests already exist in, and which pests pose a risk to, their native flora; 2) Plant virus sequences not associated with a recognized virus infection are designated as “uncultured virus” and tentatively named using the host plant species of greatest known prevalence, the word “virus,” a general location identifier, and a serial number; and 3) Invest in basic research to determine the ecology of known and new viruses with existing and potential new plant hosts and vectors and develop host-virus pathogenicity prediction tools. These recommendations have implications for researchers, risk analysts, biosecurity authorities, and policy makers at both a national and an international level.     

The pollen virome: A review of pollen-associated viruses and consequences for plants and their interactions with pollinators

The movement of pollen grains from anthers to stigmas, often by insect pollinator vectors, is essential for plant reproduction. However, pollen is also a unique vehicle for viral spread. Pollen-associated plant viruses reside on the outside or inside of pollen grains, infect susceptible individuals through vertical or horizontal infection pathways, and can decrease plant fitness. These viruses are transferred with pollen between plants by pollinator vectors as they forage for floral resources; thus, pollen-associated viral spread is mediated by floral and pollen grain phenotypes and pollinator traits, much like pollination. Most of what is currently known about pollen-associated viruses was discovered through infection and transmission experiments in controlled settings, usually involving one virus and one plant species of agricultural or horticultural interest. In this review, we first provide an updated, comprehensive list of the recognized pollen-associated viruses. Then, we summarize virus, plant, pollinator vector, and landscape traits that can affect pollen-associated virus transmission, infection, and distribution. Next, we highlight the consequences of plant–pollinator–virus interactions that emerge in complex communities of co-flowering plants and pollinator vectors, such as pollen-associated virus spread between plant species and viral jumps from plant to pollinator hosts. We conclude by emphasizing the need for collaborative research that bridges pollen biology, virology, and pollination biology.     

Multiple infection dynamics has pronounced effects on the fitness of RNA viruses

Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two‐fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka‐Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed.     

The viral era: New biotechnologies give humans an unprecedented control over Nature and require appropriate safeguards

New biotechnologies such as gene drives and engineered viruses herald a viral era that would give humans exceptional power over any organism at the level of the genotype. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business, Ecology

We are entering a new phase in our relationship with nature: after mechanization, automation and digitalization, a new era of autonomous technical objects is dawning. The most advanced of these technologies are characterized by viral behaviour. The COVID‐19 pandemic has again aptly demonstrated the power of viral systems: not only because of the SARS‐CoV‐2 virus'' ability to jump into and rapidly spread among the human population while wreaking havoc with human societies, but also because some of the vaccines developed against the virus are themselves based on viruses. Both developments give us some ideas of the possible impact of new biotechnologies that aim to create artefacts with viral behaviour in order to shape and control our natural environment. In this essay, the focus is on the use of genetically engineered organisms and the genetic manipulation of wild species. This change has a more direct relationship to our natural environment than autonomous software artefacts such as computer apps or digital viruses that “live” in their artificial “ecosystems” of information‐processing devices. The development of artificial biological systems will therefore require new methods for monitoring and intervention given their potential to autonomously spread within natural ecosystems.     

Cytopathogenesis and Inhibition of Host Gene Expression by RNA Viruses

Many viruses interfere with host cell function in ways that are harmful or pathological. This often results in changes in cell morphology referred to as cytopathic effects. However, pathogenesis of virus infections also involves inhibition of host cell gene expression. Thus the term “cytopathogenesis,” or pathogenesis at the cellular level, is meant to be broader than the term “cytopathic effects” and includes other cellular changes that contribute to viral pathogenesis in addition to those changes that are visible at the microscopic level. The goal of this review is to place recent work on the inhibition of host gene expression by RNA viruses in the context of the pathogenesis of virus infections. Three different RNA virus families, picornaviruses, influenza viruses, and rhabdoviruses, are used to illustrate common principles involved in cytopathogenesis. These examples were chosen because viral gene products responsible for inhibiting host gene expression have been identified, as have some of the molecular targets of the host. The argument is made that the role of the virus-induced inhibition of host gene expression is to inhibit the host antiviral response, such as the response to double-stranded RNA. Viral cytopathogenesis is presented as a balance between the host antiviral response and the ability of viruses to inhibit that response through the overall inhibition of host gene expression. This balance is a major determinant of viral tissue tropism in infections of intact animals.     

The Evolution of Resistance to Simian Immunodeficiency Virus (SIV): A Review

Examining how pathogens cross species boundaries, spread within species, and persist within their hosts is an essential part of understanding the factors that underpin the evolution of virulence and host resistance. Here, we review current knowledge about the genetic diversity, molecular epidemiology, prevalence, and pathogenicity of simian immunodeficiency viruses (SIVs). SIVs have crossed species boundaries from simian hosts to humans on at least 12 separate occasions, one of which led to the global HIV–AIDS crisis. Though SIVs infect a wide range of primates, scientists have only recently begun to describe the natural history of SIV infection in their natural hosts. Several new studies reveal how both viral and host factors are responsible for the transmission to, and adaptation in, new hosts. These studies also suggest that the spread of the virus may be affected by host-specific traits, including social structure, mating system and demographic history. These studies challenge the traditional view that SIV is relatively benign in its natural host, and instead suggest that a highly dynamic relationship exists between SIV and its simian hosts.     

Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.     

Biological and molecular events associated with simultaneous transmission of plant viruses by invertebrate and fungal vectors

Viruses are likely to be the most dangerous parasites of living organisms because of their widespread occurrence, possible deleterious effects on their hosts and high rates of evolution. Virus host‐to‐host transmission is a critical step in the virus life cycle, because it enables survival in a given environment and efficient dissemination. As hosts of plant viruses are not mobile, these pathogens have adopted diverse transmission strategies involving various vector organisms, mainly arthropods, nematodes, fungi and protists. In nature, plants are often infected with more than one virus at a time, thereby creating potential sources for vectors to acquire and transmit simultaneously two or more viruses. Simultaneous transmission can result in multiple infections of new host plants, which become subsequent potential sources of the viruses, thus enhancing the spread of the diseases caused by these pathogens. Moreover, it can contribute to the maintenance of viral genetic diversity in the host communities. However, despite its possible significance, the problem of the simultaneous transmission of plant viruses by vectors has not been investigated in detail. In this review, the current knowledge on multiple viral transmissions by aphids, whiteflies, leafhoppers, planthoppers, nematodes and fungi is outlined.