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The recent detection of both Nova virus (NVAV) and Bruges virus (BRGV) in European moles (Talpa europaea) in Belgium and Germany prompted a search for related hantaviruses in the Iberian mole (Talpa occidentalis). RNAlater®-preserved lung tissue from 106 Iberian moles, collected during January 2011 to June 2014 in Asturias, Spain, were analyzed for hantavirus RNA by nested/hemi-nested RT-PCR. Pairwise alignment and comparison of partial L-segment sequences, detected in 11 Iberian moles from four parishes, indicated the circulation of genetically distinct hantaviruses. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, demonstrated three distinct hantaviruses in Iberian moles: NVAV, BRGV, and a new hantavirus, designated Asturias virus (ASTV). Of the cDNA from seven infected moles processed for next generation sequencing using Illumina HiSeq1500, one produced viable contigs, spanning the S, M and L segments of ASTV. The original view that each hantavirus species is harbored by a single small-mammal host species is now known to be invalid. Host-switching or cross-species transmission events, as well as reassortment, have shaped the complex evolutionary history and phylogeography of hantaviruses such that some hantavirus species are hosted by multiple reservoir species, and conversely, some host species harbor more than one hantavirus species.
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Infecções por Hantavirus , Toupeiras , Orthohantavírus , Animais , Filogenia , Espanha , Orthohantavírus/genética , Teorema de Bayes , Infecções por Hantavirus/veterináriaRESUMO
Background: Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas. There are no approved vaccines or antivirals. TC-83 and V3526 are the best-characterized vaccine candidates for VEEV. Both are live-attenuated vaccines and have been associated with safety concerns, albeit less so for V3526. A previous attempt to improve the TC-83 vaccine focused on further attenuating the vaccine by adding mutations that altered the error incorporation rate of the RNA-dependent RNA polymerase (RdRp). Methods: The research presented here examines the impact of these RdRp mutations in V3526 by cloning the 3X and 4X strains, assessing vaccine efficacy against challenge in adult female CD-1 mice, examining neutralizing antibody titers, investigating vaccine tissue tropism, and testing the stability of the mutant strains. Results: Our results show that the V3526 RdRp mutants exhibited reduced tissue tropism in the spleen and kidney compared to wild-type V3526, while maintaining vaccine efficacy. Illumina sequencing showed that the RdRp mutations could revert to wild-type V3526. Conclusions: The observed genotypic reversion is likely of limited concern because wild-type V3526 is still an effective vaccine capable of providing protection. Our results indicate that the V3526 RdRp mutants may be a safer vaccine design than the original V3526.
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Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfections in mosquito cells. Negev virus (NEGV) isolates were also used to express green fluorescent protein (GFP) and anti-chikungunya virus (CHIKV) antibody fragments during coinfections with CHIKV. NEGV expressing anti-CHIKV antibody fragments was able to further reduce replication of CHIKV during coinfections, while reductions of CHIKV with NEGV expressing GFP were similar to titers with wild-type NEGV alone. These results are the first to show that negeviruses induce superinfection exclusion of arboviruses and to demonstrate a novel approach to deliver antiviral antibody fragments with paratransgenic ISVs. The ability to inhibit arbovirus replication and express exogenous proteins in mosquito cells makes negeviruses a promising platform for control of arthropod-borne pathogens. IMPORTANCE Negeviruses are a group of insect-specific viruses (ISVs), viruses known to infect only insects. They have been discovered over a wide geographical and species range. Their ability to infect mosquito species that transmit dangerous arboviruses makes negeviruses a candidate for a pathogen control platform. Coinfections of mosquito cells with a negevirus and an alphavirus demonstrated that negeviruses can inhibit the replication of alphaviruses. Additionally, modifying Negev virus (NEGV) to express a fragment of an anti-CHIKV antibody further reduced the replication of CHIKV in coinfected cells. This is the first evidence to demonstrate that negeviruses can inhibit the replication of important arboviruses in mosquito cells. The ability of a modified NEGV to drive the expression of antiviral proteins also highlights a method for negeviruses to target specific pathogens and limit the incidence of vector-borne diseases.
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Alphavirus/fisiologia , Vírus de Insetos/fisiologia , Replicação Viral , Aedes/virologia , Animais , Células Cultivadas , Vírus Chikungunya/fisiologia , Chlorocebus aethiops , Culex/virologia , Vírus O'nyong-nyong/fisiologia , Vírus da Floresta de Semliki/fisiologia , Células VeroRESUMO
Transmission of arthropod-borne viruses (arboviruses) involves infection and replication in both arthropod vectors and vertebrate hosts. Nearly all arboviruses are RNA viruses with high mutation frequencies, which leaves them vulnerable to genetic drift and fitness losses owing to population bottlenecks during vector infection, dissemination from the midgut to the salivary glands and transmission to the vertebrate host. However, despite these bottlenecks, they seem to avoid fitness declines that can result from Muller's ratchet. In addition, founder effects that occur during the geographic introductions of human-amplified arboviruses, including chikungunya virus and Zika virus, can affect epidemic and endemic circulation, as well as virulence. In this Review, we discuss the role of genetic drift following population bottlenecks and founder effects in arboviral evolution and spread, and the emergence of human disease.
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Infecções por Arbovirus/transmissão , Infecções por Arbovirus/virologia , Arbovírus/genética , Culicidae/virologia , Doenças Transmitidas por Vetores/virologia , Animais , Deriva Genética , Genômica , HumanosRESUMO
Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.
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Vírus da Encefalite Equina Venezuelana/genética , Deleção de Genes , Genes Reporter/genética , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Química Click/métodos , Cavalos , RNA/genética , RNA Viral/genética , Recombinação Genética/genética , Análise de Sequência de RNA , Vacinas Atenuadas , Células VeroRESUMO
Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3D56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3D56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.
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Alphavirus/genética , Mutação , Vírion/genética , Replicação Viral/genética , Alphavirus/fisiologia , Animais , Chlorocebus aethiops , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Variação Genética , Genoma Viral/genética , Taxa de Mutação , Vacinas Atenuadas/genética , Células Vero , Proteínas do Envelope Viral/genética , Vacinas Virais/genéticaRESUMO
In recent years, it has become evident that a generational gap has developed in the community of arbovirus research. This apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. On the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. This paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. In this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. To this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead.
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Infecções por Arbovirus/virologia , Arbovírus/fisiologia , Animais , Infecções por Arbovirus/epidemiologia , Infecções por Arbovirus/história , Arbovírus/isolamento & purificação , Arbovírus/ultraestrutura , Genoma Viral , Genômica/métodos , Saúde Global , História do Século XX , História do Século XXI , Humanos , Pesquisa/históriaRESUMO
The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.
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Culicidae/virologia , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Mutação , RNA Polimerase Dependente de RNA/genética , Vertebrados/virologia , Replicação Viral/genética , Animais , Culicidae/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Fenótipo , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Vertebrados/genéticaRESUMO
RNA viruses replicate with low fidelity due to the error-prone nature of the RNA-dependent RNA polymerase, which generates approximately one mutation per round of genome replication. Due to the large population sizes produced by RNA viruses during replication, this results in a cloud of closely related virus variants during host infection, of which small increases or decreases in replication fidelity have been shown to result in virus attenuation in vivo, but not typically in vitro. Since the discovery of the first RNA virus fidelity mutants during the mid-aughts, the field has exploded with the identification of over 50 virus fidelity mutants distributed amongst 7 RNA virus families. This review summarizes the current RNA virus fidelity mutant literature, with a focus upon the definition of a fidelity mutant as well as methods to confirm any mutational changes associated with the fidelity mutant. Due to the complexity of such a definition, in addition to reports of unstable virus fidelity phenotypes, the future translational utility of these mutants and applications for basic science are examined.
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Evolução Biológica , Mutação , Infecções por Vírus de RNA/virologia , Vírus de RNA/fisiologia , Replicação Viral , Animais , Humanos , Biossíntese de Proteínas , Estabilidade de RNA , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica , Replicação Viral/genéticaRESUMO
The myxoma virus (a microparasite) reduced wild rabbit numbers worldwide when introduced in the 1950s, and is known to interact with co-infecting helminths (macroparasites) causing both increases and decreases in macroparasite population size. In the 1990s Rabbit Haemorrhagic Disease Virus (RHDV) infected rabbits and also significantly reduced rabbit numbers in several countries. However, not much is known about RHDV interactions with macroparasites. In this study, we compare prevalence and intensity of infection for three gastrointestinal nematode species (Trichostrongylus retortaeformis, Graphidium strigosum and Passalurus ambiguus) before and after RHDV spread across host populations in Scotland and New Zealand. During one common season, autumn, prevalence of T. retortaeformis was higher after RHDV spread in both locations, whereas it was lower for G. strigosum and P. ambiguus after RHDV arrived in New Zealand, but higher in Scotland. Meanwhile, intensity of infection for all species decreased after RHDV arrived in New Zealand, but increased in Scotland. The impact of RHDV on worm infections was generally similar across seasons in Scotland, and also similarities in seasonality between locations suggested effects on infection patterns in one season are likely similar year-round. The variable response by macroparasites to the arrival of a microparasite into Scottish and New Zealand rabbits may be due to differences in the environment they inhabit, in existing parasite community structure, and to some extent, in the relative magnitude of indirect effects. Specifically, our data suggest that bottom-up processes after the introduction of a more virulent strain of RHDV to New Zealand may affect macroparasite co-infections by reducing the availability of their shared common resource, the rabbits. Clearly, interactions between co-infecting micro- and macroparasites vary in host populations with different ecologies, and significantly impact parasite community structure in wildlife.
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During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy.
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Viral diversity is theorized to play a significant role during virus infections, particularly for arthropod-borne viruses (arboviruses) that must infect both vertebrate and invertebrate hosts. To determine how viral diversity influences mosquito infection and dissemination Culex taeniopus mosquitoes were infected with the Venezuelan equine encephalitis virus endemic strain 68U201. Bodies and legs/wings of the mosquitoes were collected individually and subjected to multi-parallel sequencing. Virus sequence diversity was calculated for each tissue. Greater diversity was seen in mosquitoes with successful dissemination versus those with no dissemination. Diversity across time revealed that bottlenecks influence diversity following dissemination to the legs/wings, but levels of diversity are restored by Day 12 post-dissemination. Specific minority variants were repeatedly identified across the mosquito cohort, some in nearly every tissue and time point, suggesting that certain variants are important in mosquito infection and dissemination. This study demonstrates that the interaction between the mosquito and the virus results in changes in diversity and the mutational spectrum and may be essential for successful transition of the bottlenecks associated with arbovirus infection.
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Working with virological samples requires validated inactivation protocols for safe handling and disposal. Although many techniques exist to inactivate samples containing viruses, not all procedures have been properly validated or are compatible with subsequent assays. To aid in the development of inactivation protocols for Alphaviruses, and specifically Venezuelan equine encephalitis virus (VEEV), a variety of methods were evaluated for their ability to completely inactivate a high titer sample of the vaccine strain VEEV TC-83. The methods evaluated include reagents used in RNA extraction, fixation, treatment with a detergent, and heat inactivation. Most methods were successful at inactivating the sample; however, treatment with only Buffer AVL, SDS, and heat inactivation at 58⯰C for one hour were not capable of complete inactivation of the virus in the sample. These results provide a substantial framework for identifying techniques that are safe for complete inactivation of Alphaviruses and to advise protocol implementation.
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Desinfetantes/farmacologia , Desinfecção , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos da radiação , Temperatura Alta , Animais , Linhagem Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Efeito Citopatogênico Viral/efeitos da radiação , Desinfecção/métodos , Células VeroRESUMO
Venezuelan equine encephalitis (VEE) complex alphaviruses are important re-emerging arboviruses that cause life-threatening disease in equids during epizootics as well as spillover human infections. We conducted a comprehensive analysis of VEE complex alphaviruses by sequencing the genomes of 94 strains and performing phylogenetic analyses of 130 isolates using complete open reading frames for the nonstructural and structural polyproteins. Our analyses confirmed purifying selection as a major mechanism influencing the evolution of these viruses as well as a confounding factor in molecular clock dating of ancestors. Times to most recent common ancestors (tMRCAs) could be robustly estimated only for the more recently diverged subtypes; the tMRCA of the ID/IAB/IC/II and IE clades of VEE virus (VEEV) were estimated at ca. 149-973 years ago. Evolution of the IE subtype has been characterized by a significant evolutionary shift from the rest of the VEEV complex, with an increase in structural protein substitutions that are unique to this group, possibly reflecting adaptation to its unique enzootic mosquito vector Culex (Melanoconion) taeniopus. Our inferred tree topologies suggest that VEEV is maintained primarily in situ, with only occasional spread to neighboring countries, probably reflecting the limited mobility of rodent hosts and mosquito vectors.
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Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/epidemiologia , Evolução Molecular , Doenças dos Cavalos/virologia , América , Sequência de Aminoácidos , Animais , Culex/virologia , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Doenças dos Cavalos/epidemiologia , Cavalos/virologia , Humanos , Insetos Vetores/virologia , FilogeniaRESUMO
Since chikungunya virus (CHIKV) was introduced into the Americas in 2013, its geographic distribution has rapidly expanded. Of 119 serum samples collected in 2014 from febrile patients in southern Mexico, 79% were positive for CHIKV or IgM against CHIKV. Sequencing results confirmed CHIKV strains closely related to Caribbean isolates.
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Febre de Chikungunya/epidemiologia , Vírus Chikungunya/patogenicidade , Surtos de Doenças/estatística & dados numéricos , Febre de Causa Desconhecida/etiologia , Animais , Anticorpos Antivirais/sangue , Febre de Chikungunya/patologia , Culicidae/virologia , Febre de Causa Desconhecida/epidemiologia , Humanos , Insetos Vetores/patogenicidade , Insetos Vetores/virologia , México/epidemiologia , Dados de Sequência MolecularRESUMO
In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions.
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Infecções por Alphavirus/epidemiologia , Alphavirus/genética , Evolução Biológica , Biota/imunologia , Surtos de Doenças , Alphavirus/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Filogenia , Venezuela/epidemiologiaRESUMO
Chikungunya virus (CHIKV) has a long history of emergence into urban transmission cycles from its ancestral, enzootic, sylvatic foci in Sub-Saharan Africa, most recently spreading to the Americas beginning in 2013. Since 2004, reemergence has resulted in millions of cases of severe, debilitating and often chronic arthralgia on five continents. Here, we review this history based on phylogenetic studies, and discuss probable future spread and disease in the Americas. We also discuss a series of mutations in the recently emerged Indian Ocean Lineage that has adapted the virus for transmission for the first time by the Aedes albopictus urban mosquito vector, and compare CHIKV to other arboviruses with and without similar histories of urbanization. This article forms part of a symposium in Antiviral Research on "Chikungunya discovers the New World."
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Febre de Chikungunya/epidemiologia , Febre de Chikungunya/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Epidemias , Evolução Molecular , Saúde Global , Humanos , Epidemiologia Molecular , Filogenia , População Rural , População UrbanaRESUMO
To generate the most diverse phylogenetic dataset for the flaviviruses to date, we determined the genomic sequences and phylogenetic relationships of 14 flaviviruses, of which 10 are primarily associated with Culex spp. mosquitoes. We analyze these data, in conjunction with a comprehensive collection of flavivirus genomes, to characterize flavivirus evolutionary and biogeographic history in unprecedented detail and breadth. Based on the presumed introduction of yellow fever virus into the Americas via the transatlantic slave trade, we extrapolated a timescale for a relevant subset of flaviviruses whose evolutionary history, shows that different Culex-spp. associated flaviviruses have been introduced from the Old World to the New World on at least five separate occasions, with 2 different sets of factors likely to have contributed to the dispersal of the different viruses. We also discuss the significance of programmed ribosomal frameshifting in a central region of the polyprotein open reading frame in some mosquito-associated flaviviruses.
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Evolução Molecular , Flavivirus/genética , Genoma Viral , Fases de Leitura Aberta , Filogenia , Sequência de Bases , Flavivirus/classificação , Flavivirus/fisiologia , Mudança da Fase de Leitura do Gene Ribossômico , Dados de Sequência Molecular , FilogeografiaRESUMO
The term arbovirus denotes viruses that are transmitted by arthropods, such as ticks, mosquitoes, and other biting arthropods. The infection of these vectors produces a certain set of evolutionary pressures on the virus; involving migration from the midgut, where the blood meal containing the virus is processed, to the salivary glands, in order to transmit the virus to the next host. During this process the virus is subject to numerous bottlenecks, stochastic events that significantly reduce the number of viral particles that are able to infect the next stage. This article reviews the latest research on the bottlenecks that occur in arboviruses and the way in which these affect the evolution and fitness of these viruses. In particular we focus on the latest research on three important arboviruses, West Nile virus, Venezuelan equine encephalitis virus and Chikungunya viruses and compare the differing effects of the mosquito bottlenecks on these viruses as well as other evolutionary pressures that affect their evolution and transmission.
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Infecções por Arbovirus/transmissão , Arbovírus/genética , Culicidae/virologia , Insetos Vetores/virologia , Animais , Infecções por Arbovirus/virologia , Arbovírus/fisiologia , Biodiversidade , Evolução Biológica , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Vírus do Nilo Ocidental/genéticaRESUMO
BACKGROUND: The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes. FINDINGS: Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d'Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 - 588 nt) of unknown function in the 5' region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. CONCLUSIONS: In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.