A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.Fil: Julve Parreño, Jose Manuel. Universidad Politécnica de Valencia; EspañaFil: Huet, Estefanía. Universidad Politécnica de Valencia; EspañaFil: Fernández del Carmen, Asun. Universidad Politécnica de Valencia; EspañaFil: Segura, Alvaro. Universidad de Costa Rica; Costa RicaFil: Venturi, Micol. Universidad Politécnica de Valencia; EspañaFil: Gandía, Antoni. Universidad Politécnica de Valencia; EspañaFil: Pan, Wei-Song. Universidad Politécnica de Valencia; EspañaFil: Albaladejo, Irene. Universidad Politécnica de Valencia; EspañaFil: Forment, Javier. Universidad Politécnica de Valencia; EspañaFil: Pla, Davinia. Instituto de Biomedicina de Valencia; EspañaFil: Wigdorovitz, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; ArgentinaFil: Calvete, Juan J.. Instituto de Biomedicina de Valencia; EspañaFil: Gutiérrez, Carlos. Universidad de Las Palmas de Gran Canaria; EspañaFil: Gutiérrez, José María. Universidad de Costa Rica; Costa RicaFil: Granell, Antonio. Universidad Politécnica de Valencia; EspañaFil: Orzáez, Diego. Universidad Politécnica de Valencia; Españ

Multiple viral infections in Agaricus bisporus - characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing

Thirty unique non-host RNAs were sequenced in the cultivated fungus, Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3′ motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interactive viral ecosystem with sequence variability ranging over 2 orders of magnitude and evidence of recombination, horizontal gene transfer and variable fragment numbers. Large numbers of viral RNAs were detected in multiple Agaricus samples; up to 24 in samples symptomatic for disease and 8–17 in asymptomatic samples, suggesting adaptive strategies for co-existence. The viral composition of growing cultures was dynamic, with evidence of gains and losses depending on the environment and included new hypothetical viruses when compared with the current transcriptome and EST databases. As the non-cellular transmission of mycoviruses is rare, the founding infections may be ancient, preserved in wild Agaricus populations, which act as reservoirs for subsequent cell-to-cell infection when host populations are expanded massively through fungiculture

Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro

[EN] Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlappingThis work was supported by grants BFU2012-30805 (SFE) and BFU2012-36346 (MAF) from the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica and by an EMBO Short-Term Fellowship and the Mentoring Program from the Foundation for Polish Science (BHJ).Hasiów-Jaroszewska, B.; Fares Riaño, MA.; Elena Fito, SF. (2014). Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro. 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Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector

Mieszane infekcje wirusowe roślin: współdziałanie czy rywalizacja terytorialna między wirusami?

Mieszane infekcje wirusowe powstają w wyniku zróżnicowanego w czasie (nadkażenie) bądź równoczesnego (koinfekcja) wniknięcia do komórek gospodarza cząstek różnych wirusów lub różnych szczepów tego samego wirusa. Interakcje między wirusami w roślinie gospodarzu klasyfikowane są najczęściej jako synergistyczne lub antagonistyczne. Zjawisko synergizmu zachodzi wówczas, gdy obecność jednego wirusa wpływa stymulująco na dynamikę replikacji drugiego. Ma miejsce, gdy wirus obecny w roślinie tłumi, za pośrednictwem indukowanego przez siebie białka, potranskrypcyjne wyciszanie genu (PTGS), czyli naturalną reakcję obronną rośliny, która może zostać skierowana przeciwko innemu wirusowi, wnikającemu do rośliny. Wzrostowi akumulacji wirusów w roślinie zwykle towarzyszy zaostrzenie objawów chorobowych, w porównaniu z objawami na roślinach porażonych tymi wirusami oddzielnie. Poważne zaostrzenie procesu chorobowego towarzyszące interakcji synergistycznej może doprowadzić do przedwczesnej śmierci rośliny. W przeciwieństwie do synergizmu, istotą oddziaływań antagonistycznych jest hamowanie czynności życiowych jednego wirusa przez drugi wirus. Typowym przykładem interakcji antagonistycznej jest odporność (ochrona) krzyżowa, zjawisko wykorzystywane w ochronie roślin przed porażeniem przez wirulentne szczepy wirusów. Przeważa hipoteza, że ten typ odporności opiera się na zjawisku wyciszania RNA wirusowego. W odpowiedzi na infekcję wirusem, w roślinie indukowany jest sygnał uruchamiający mechanizm PTGS. W sytuacji, gdy do tkanki rośliny wnika kolejny wirus, o dużym stopniu homologii z wirusem już w niej obecnym, strategia obronna rośliny ukierunkowuje się na zdegradowanie jego RNA. Ochrona krzyżowa może wiązać się z wystąpieniem w roślinie zjawiska tzw. oddzielenia przestrzennego. W spektakularnej formie zachodzi ono wtedy, gdy homologiczne wirusy równocześnie wnikają do rośliny. Wówczas ich podpopulacje odseparowują się od siebie, zasiedlając różne komórki w tkance gospodarza. W opinii wirusologów, poznanie interakcji między wirusami jest istotne dla zrozumienia ewolucji i patogenezy tych pasożytów.Mixed infections occur when two or more viruses or strains of the same species invade the host at different times (super-infection) or simultaneously (co-infection). Interactions between plant viruses in mixed infections are generally categorized as synergistic or antagonistic. Synergism refers to a situation in which the presence and activity of one virus stimulates the replication dynamics of the second virus. The phenomenon occurs when the virus present in the host plant suppresses, by a specific viral protein, post-transcriptional gene silencing (PTGS), i. e. a natural plant defense response, and may target another virus entering the plant cells. The increase in virus accumulation is usually associated with an enhanced severity of disease symptoms exhibited by the plant, as compared with those shown by singly infected plants. Severe response to the multiple infection may lead to the premature death of the plant. Unlike synergism, antagonistic interactions rely on the inhibition of life functions of one virus by a second virus. A typical example of antagonistic interaction is cross-protection, the phenomenon used to protect cultivated plants against infection by virulent strains of viruses. According to the currently prevailing hypothesis, behind this phenomenon is the mechanism of viral RNA silencing. In response to infection, a signal is induced in the plant which initiates PTGS to degrade RNA of a homologous virus. Cross-protection can be manifested in the host tissue by the occurrence of spatial separation between the viral subpopulations. In its spectacular form this phenomenon occurs when two or more homologous viruses invade the plant simultaneously. Then their subpopulations separate from each other, thus colonizing different cells in the host tissue. Virologists believe that good knowledge of within-host interactions between viruses will contribute to a better understanding of viral evolution and pathogenesis