Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

BACKGROUND: Success of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown. METHODOLOGY AND PRINCIPAL FINDINGS: we have constructed four mutant FMDVS that encode only one VPG: either VPg(1), VPg(3), or two chimeric versions containing part of VPg(1) and VPg(3). All mutants, except that encoding only VPg(1), were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and detach BHK-21 cells from the monolayer, the first stage of cell killing. CONCLUSIONS: The results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses pathogenesis are discussed

Counteracting Quasispecies Adaptability: Extinction of a Ribavirin-Resistant Virus Mutant by an Alternative Mutagenic Treatment

[Background] Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, nonmutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin.[Methodology and Principal Findings] Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis[Conclusions] (i) When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii) A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii) Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a high dose of the same mutagen, followed by a combination of another mutagen with an antiviral inhibitor.Work supported by grants BFU2005-00863, BFU2008-02816/BMC, Proyecto Intramural de Frontera del CSIC 200820FO191, FIPSE 36558/06, and Fundacio´n Ramo´n Areces. CIBERehd is funded by Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

Genetic and Anatomic Determinants of Enzootic Venezuelan Equine Encephalitis Virus Infection of Culex (Melanoconion) taeniopus

Venezuelan equine encephalitis (VEE) is a re-emerging, mosquito-borne viral disease with the potential to cause fatal encephalitis in both humans and equids. Recently, detection of endemic VEE caused by enzootic strains has escalated in Mexico, Peru, Bolivia, Colombia and Ecuador, emphasizing the importance of understanding the enzootic transmission cycle of the etiologic agent, VEE virus (VEEV). The majority of work examining the viral determinants of vector infection has been performed in the epizootic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus. Based on the fundamental differences between the epizootic and enzootic cycles, we hypothesized that the virus-vector interaction of the enzootic cycle is fundamentally different from that of the epizootic model. We therefore examined the determinants for VEEV IE infection in the enzootic vector, Culex (Melanoconion) taeniopus, and determined the number and susceptibility of midgut epithelial cells initially infected and their distribution compared to the epizootic virus-vector interaction. Using chimeric viruses, we demonstrated that the determinants of infection for the enzootic vector are different than those observed for the epizootic vector. Similarly, we showed that, unlike A. taeniorhynchus infection with subtype IC VEEV, C. taeniopus does not have a limited subpopulation of midgut cells susceptible to subtype IE VEEV. These findings support the hypothesis that the enzootic VEEV relationship with C. taeniopus differs from the epizootic virus-vector interaction in that the determinants appear to be found in both the nonstructural and structural regions, and initial midgut infection is not limited to a small population of susceptible cells

Mosquitoes Put the Brake on Arbovirus Evolution: Experimental Evolution Reveals Slower Mutation Accumulation in Mosquito Than Vertebrate Cells

Like other arthropod-borne viruses (arboviruses), mosquito-borne dengue virus (DENV) is maintained in an alternating cycle of replication in arthropod and vertebrate hosts. The trade-off hypothesis suggests that this alternation constrains DENV evolution because a fitness increase in one host usually diminishes fitness in the other. Moreover, the hypothesis predicts that releasing DENV from host alternation should facilitate adaptation. To test this prediction, DENV was serially passaged in either a single human cell line (Huh-7), a single mosquito cell line (C6/36), or in alternating passages between Huh-7 and C6/36 cells. After 10 passages, consensus mutations were identified and fitness was assayed by evaluating replication kinetics in both cell types as well as in a novel cell type (Vero) that was not utilized in any of the passage series. Viruses allowed to specialize in single host cell types exhibited fitness gains in the cell type in which they were passaged, but fitness losses in the bypassed cell type, and most alternating passages, exhibited fitness gains in both cell types. Interestingly, fitness gains were observed in the alternately passaged, cloned viruses, an observation that may be attributed to the acquisition of both host cell–specific and amphi-cell-specific adaptations or to recovery from the fitness losses due to the genetic bottleneck of biological cloning. Amino acid changes common to both passage series suggested convergent evolution to replication in cell culture via positive selection. However, intriguingly, mutations accumulated more rapidly in viruses passed in Huh-7 cells than in those passed in C6/36 cells or in alternation. These results support the hypothesis that releasing DENV from host alternation facilitates adaptation, but there is limited support for the hypothesis that such alternation necessitates a fitness trade-off. Moreover, these findings suggest that patterns of genetic evolution may differ between viruses replicating in mammalian and mosquito cells

Variabilidad genómica entre cepas del herpes virus bovino tipo I aisladas de casos clínicos de IBR

En este trabajo se ha secuenciado un fragmento de la glicoproteína de infección gB de cepas del herpes virus bovino tipo 1 (BHV-1) aisladas de casos clínicos de IBR, en España y otros países. En este sentido, un total de 19 cepas del virus BHV-1, han sido sometidos a amplificación y secuenciación por PCR. Se amplificó un fragmento de 511 pb comprendido entre las regiones 1302 hasta 1813 del gen de la gB. Los resultados muestran una homología de un 100% entre 16 cepas secuenciadas, sin embargo, fueron detectadas diferencias en tres cepas (dos cepas españolas) clasificadas como BHV-1, 1, aisladas de casos con entidad clínica respiratoria. La frecuencia de mutación es del orden de 3,6 x 10 elevado -4 y las modificaciones en la secuencia nucleotídica con mayor frecuencia dan lugar a cambios aminoacídicos. No se puede determinar el grado de importancia de estos cambios, pero se puede suponer que ellos estan relacionados el pasaje del vírus por los animales huéspedes y / o los distintos grados de patogenicidad de las cepas

Variabilité et evolution des populations virales: bilan et implications

National audienceRNA virus populations consist of complex distributions of closely related but not identical genomes known as viral quasi-species. The quasi-species concept describes the dynamics of these genomes subjected to a continuous process of variation, competition, and selection. Quasi-species dynamics has broad implications not only in the understanding of the molecular mechanisms underlying adaptation of RNA viruses but also in the design of strategies for control and prevention of viral disease. Viral load and genetic heterogeneity have a determinant influence on the adaptation of RNA virus to their environment. Vaccines designed to control diseases caused by highly variable viruses must contain several B and T epitopes to provide an ample and diversified immune response. Similarly, antiviral drugs should be used in combination therapy to minimize selection of resistant viruses. The theoretical model of quasi-species has opened the way for new antiviral therapies based on augmentation of the mutation rate during replication of viral RNA. Finally the quasi-species concept provides the basis for defining the selective factors that could influence the evolution of RNA virus and promote the emergence or reemergence of viral diseases