RESUMO
Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.
Assuntos
Regiões 3' não Traduzidas , Vírus Chikungunya , Genoma Viral , RNA Viral , Recombinação Genética , Replicação Viral , Vírus Chikungunya/genética , Regiões 3' não Traduzidas/genética , RNA Viral/genética , RNA Viral/metabolismo , Animais , Replicação Viral/genética , Febre de Chikungunya/virologia , Febre de Chikungunya/genética , Febre de Chikungunya/transmissão , Humanos , Aedes/virologia , Aedes/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Linhagem CelularRESUMO
Alphaviruses such as chikungunya and western equine encephalitis viruses are important human pathogens transmitted by mosquitoes that have recently caused large epidemic and epizootic outbreaks. The epidemic potential of alphaviruses is often related to enhanced mosquito transmission. Tissue barriers and antiviral responses impose bottlenecks to viral populations in mosquitoes. Substitutions in the envelope proteins and the presence of repeated sequence elements (RSEs) in the 3'UTR of epidemic viruses were proposed to be specifically associated to efficient replication in mosquito vectors. Here, we discuss the molecular mechanisms that originated RSEs, the evolutionary forces that shape the 3'UTR of alphaviruses, and the significance of RSEs for mosquito transmission. Finally, the presence of RSEs in the 3'UTR of viral genomes appears as evolutionary trait associated to mosquito adaptation and emerges as a common feature among viruses from the alphavirus and flavivirus genera.
Assuntos
Infecções por Alphavirus/transmissão , Vírus Chikungunya/genética , Vírus da Encefalite Equina do Oeste/genética , Infecções por Flavivirus/transmissão , Flavivirus/genética , Genoma Viral , Proteínas do Envelope Viral/genética , Regiões 3' não Traduzidas , Infecções por Alphavirus/virologia , Animais , Vírus Chikungunya/classificação , Vírus Chikungunya/patogenicidade , Culicidae/virologia , Vírus da Encefalite Equina do Oeste/classificação , Vírus da Encefalite Equina do Oeste/patogenicidade , Flavivirus/classificação , Flavivirus/patogenicidade , Infecções por Flavivirus/virologia , Regulação da Expressão Gênica , Humanos , Repetições de Microssatélites , Mosquitos Vetores/virologia , Filogenia , Transdução de Sinais , Proteínas do Envelope Viral/metabolismo , Replicação ViralRESUMO
Chikungunya virus (CHIKV) is a reemerging and rapidly spreading pathogen transmitted by mosquitoes. The emergence of new epidemic variants of the virus is associated with genetic evolutionary traits, including duplication of repeated RNA elements in the 3' untranslated region (UTR) that seemingly favor transmission by mosquitoes. The transmission potential of a given variant results from a complex interplay between virus populations and anatomical tissue barriers in the mosquito. Here, we used the wild-type CHIKV Caribbean strain and an engineered mutant harboring a deletion in the 3' UTR to dissect the interactions of virus variants with the anatomical barriers that impede transmission during the replication cycle of the virus in Aedes mosquitoes. Compared to the 3'-UTR mutant, we observed that the wild-type virus had a short extrinsic incubation period (EIP) after an infectious blood meal and was expectorated into mosquito saliva much more efficiently. We found that high viral titers in the midgut are not sufficient to escape the midgut escape barrier. Rather, viral replication kinetics play a crucial role in determining midgut escape and the transmission ability of CHIKV. Finally, competition tests in mosquitoes coinfected with wild-type and mutant viruses revealed that both viruses successfully colonized the midgut, but wild-type viruses effectively displaced mutant viruses during systemic infection due to their greater efficiency of escaping from the midgut into secondary tissues. Overall, our results uncover a link between CHIKV replication kinetics and the effect of bottlenecks on population diversity, as slowly replicating variants are less able to overcome the midgut escape barrier.IMPORTANCE It is well established that selective pressures in mosquito vectors impose population bottlenecks for arboviruses. Here, we used a CHIKV Caribbean lineage mutant carrying a deletion in the 3' UTR to study host-virus interactions in vivo in the epidemic mosquito vector Aedes aegypti We found that the mutant virus had a delayed replication rate in mosquitoes, which lengthened the extrinsic incubation period (EIP) and reduced fitness relative to the wild-type virus. As a result, the mutant virus displayed a reduced capacity to cross anatomical barriers during the infection cycle in mosquitoes, thus reducing the virus transmission rate. Our findings show how selective pressures act on CHIKV noncoding regions to select variants with shorter EIPs that are preferentially transmitted by the mosquito vector.
Assuntos
Aedes/virologia , Febre de Chikungunya/transmissão , Vírus Chikungunya/patogenicidade , Trato Gastrointestinal/virologia , Interações Hospedeiro-Patógeno , Mosquitos Vetores/virologia , Replicação Viral , Animais , Vírus Chikungunya/genética , Feminino , Humanos , Mutação , Carga ViralRESUMO
The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3'UTR. Sequence repeats are conserved at the CHIKV 3'UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3'UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions.