Characterization and Vector Competence Studies of Chikungunya Virus Lacking Repetitive Motifs in the 3' Untranslated Region of the Genome.
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ABSTRACT:
SUBMITTER: Karliuk Y
PROVIDER: S-EPMC7999577 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3' untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (DeltaII; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37 degrees C). In a few of the DeltaII transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 microg per 3 x 10(6) cells). Viruses from two DeltaII-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the DeltaII deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3D(pol)), and the other was localized to the 3' UTR, upstream of stem I.
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Project description:The mosquito Aedes albopictus is one of the most dangerous invasive species. Its worldwide spread has created health concerns as it is a major vector of arboviruses of public health significance such as chikungunya (CHIKV). Dynamics of different genetic backgrounds and admixture events may have impacted competence for CHIKV in adventive populations. Using microsatellites, we infer the genetic structure of populations across the expansion areas that we then associate with their competence for different CHIKV genotypes. Here we show that the demographic history of Ae. albopictus populations is a consequence of rapid complex patterns of historical lineage diversification and divergence that influenced their competence for CHIKV. The history of adventive populations is associated with CHIKV genotypes in a genotype-by-genotype interaction that impacts their vector competence. Thus, knowledge of the demographic history and vector competence of invasive mosquitoes is pivotal for assessing the risk of arbovirus outbreaks in newly colonized areas.
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Project description:BackgroundFrom October 2014 to March 2015, French Polynesia experienced for the first time a chikungunya outbreak. Two Aedes mosquitoes may have contributed to chikungunya virus (CHIKV) transmission in French Polynesia: the worldwide distributed Ae. aegypti and the Polynesian islands-endemic Ae. polynesiensis mosquito.MethodsTo investigate the vector competence of French Polynesian populations of Ae. aegypti and Ae. polynesiensis for CHIKV, mosquitoes were exposed per os at viral titers of 7 logs tissue culture infectious dose 50%. At 2, 6, 9, 14 and 21 days post-infection (dpi), saliva was collected from each mosquito and inoculated onto C6/36 mosquito cells to check for the presence of CHIKV infectious particles. Legs and body (thorax and abdomen) of each mosquito were also collected at the different dpi and submitted separately to viral RNA extraction and CHIKV real-time RT-PCR.ResultsCHIKV infection rate, dissemination and transmission efficiencies ranged from 7-90%, 18-78% and 5-53% respectively for Ae. aegypti and from 39-41%, 3-17% and 0-14% respectively for Ae. polynesiensis, depending on the dpi. Infectious saliva was found as early as 2 dpi for Ae. aegypti and from 6 dpi for Ae. polynesiensis. Our laboratory results confirm that the French Polynesian population of Ae. aegypti is highly competent for CHIKV and they provide clear evidence for Ae. polynesiensis to act as an efficient CHIKV vector.ConclusionAs supported by our findings, the presence of two CHIKV competent vectors in French Polynesia certainly contributed to enabling this virus to quickly disseminate from the urban/peri-urban areas colonized by Ae. aegypti to the most remote atolls where Ae. polynesiensis is predominating. Ae. polynesiensis was probably involved in the recent chikungunya outbreaks in Samoa and the Cook Islands. Moreover, this vector may contribute to the risk for CHIKV to emerge in other Polynesian islands like Fiji, and more particularly Wallis where there is no Ae. aegypti.
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Project description:Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.
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Project description:The 3' untranslated genome region (UTR) of arthropod-borne viruses is characterized by enriched direct repeats (DRs) and stem-loop structures. Despite many years of theoretical and experimental study, on-going positive selection on the 3'UTR had never been observed in 'real-time,' and the role of the arbovirus 3'UTR remains poorly understood. We observed a lineage-specific 3'UTR sequence pattern in all available Asian lineage of the mosquito-borne alphavirus, chikungunya virus (CHIKV) (1958-2009), including complicated mutation and duplication patterns of the long DRs. Given that a longer genome is usually associated with less efficient replication, we hypothesized that the fixation of these genetic changes in the Asian lineage 3'UTR was due to their beneficial effects on adaptation to vectors or hosts. Using reverse genetic methods, we examined the functional importance of each direct repeat. Our results suggest that adaptation to mosquitoes, rather than to mammalian hosts, is a major evolutionary force on the CHIKV 3'UTR. Surprisingly, the Asian 3'UTR appeared to be inferior to its predicted ancestral sequence for replication in both mammals and mosquitoes, suggesting that its fixation in Asia was not a result of directional selection. Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia. We propose that this introduction of a 3'UTR with deletions led to genetic drift and compensatory mutations associated with the loss of structural/functional constraints, followed by two independent beneficial duplications and fixation due to positive selection. Our results provide further evidence that the limited epidemic potential of the Asian CHIKV strains resulted from founder effects that reduced its fitness for efficient transmission by mosquitoes there.
| S-EPMC3757053 | biostudies-literature
Project description:Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes debilitating musculoskeletal pain and inflammation and can persist for months to years after acute infection. Although studies of humans and experimentally infected animals suggest that CHIKV infection persists in musculoskeletal tissues, the mechanisms for this remain poorly understood. To evaluate this further, we isolated CHIKV from the serum of persistently infected Rag1-/- mice at day 28. When inoculated into naive wild-type (WT) mice, this persistently circulating CHIKV strain displayed a capacity for earlier dissemination and greater pathogenicity than the parental virus. Sequence analysis revealed a nonsynonymous mutation in the E2 glycoprotein (E2 K200R) and a deletion within the 3' untranslated region (3'-UTR). The introduction of these changes into the parental virus conferred enhanced virulence in mice, although primary tropism for musculoskeletal tissues was maintained. The E2 K200R mutation was largely responsible for enhanced viral dissemination and pathogenicity, although these effects were augmented by the 3'-UTR deletion. Finally, studies with Irf3/Irf7-/- and Ifnar1-/- mice suggest that the E2 K200R mutation enhances viral dissemination from the site of inoculation independently of interferon regulatory factor 3 (IRF3)-, IRF7-, and IFNAR1-mediated responses. As our findings reveal viral determinants of CHIKV dissemination and pathogenicity, their further study should help to elucidate host-virus interactions that determine acute and chronic CHIKV infection.IMPORTANCE CHIKV is a globally spreading, mosquito-transmitted virus that causes debilitating acute and chronic musculoskeletal disease in humans. The viral genetic determinants that dictate the severity of acute and chronic diseases are not understood. To improve our understanding of CHIKV pathogenesis, we evaluated a CHIKV strain isolated from the serum of chronically infected immunocompromised mice. Sequence analysis of this persistent CHIKV strain identified two mutations, an amino acid change in the E2 viral attachment protein and a deletion within the 3'-UTR of the viral genome. We identified roles for these mutations in the enhancement of viral dissemination from the inoculation site and in disease severity. These data improve our understanding of the viral determinants of CHIKV pathogenesis and adaptive changes that occur during viral persistence.
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Project description:Mutations in the untranslated regulatory regions of genes may result in abnormal gene expression or transcriptional regulation. In this study, we characterize the ornithine transcarbamylase (OTC) mRNA isoforms of the X-linked OTC gene involved in the urea formation in the liver. Our data revealed that two major transcripts (OTC-t1 and OTC-t2) are more highly expressed than any of the other isoforms in all the tissues analyzed, though a longer transcript (OTC-t3) was also isolated and characterized from the brain sample. The OTC-t2 sequence fully matches the OTC mRNA reference sequence (NM_000531.5). All three isoforms use a canonical AAUAAA hexamer that is predicted to fold into a hairpin secondary structure which might be exposed to the cleavage and polyadenylation specificity factor. In addition, we observed that the OTC-t1 and OTC-t2 transcripts display heterogeneity at the cleavage sites in a tissue-dependent manner. Taken together, our data demonstrate that several mRNA isoforms are transcribed from the OTC gene, thereby indicating a wide degree of variability in post-transcriptional regulation.
| S-EPMC3322402 | biostudies-literature
Project description:BackgroundLike many countries from the Americas, Cuba is threatened by Aedes aegypti-associated arboviruses such as dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. Curiously, when CHIKV was actively circulating in the region in 2013-2014, no autochthonous transmission of this virus was detected in Havana, Cuba, despite the importation of chikungunya cases into this city. To investigate if the transmission ability of local mosquito populations could explain this epidemiological scenario, we evaluated for the first time the vector competence of two Ae. aegypti populations (Pasteur and Párraga) collected from Havana for dengue virus type 1 (DENV-1), CHIKV, and ZIKV.Methodology/principal findingsMosquito populations were fed separately using blood containing ZIKV, DENV-1, or CHIKV. Infection, dissemination, and transmission rates, were estimated at 3 (exclusively for CHIKV), 7, and 14 days post exposure (dpe) for each Ae. aegypti population-virus combination. Both mosquito populations were susceptible to DENV-1 and ZIKV, with viral infection and dissemination rates ranging from 24-97% and 6-67% respectively. In addition, CHIKV disseminated in both populations and was subsequently transmitted. Transmission rates were low (<30%) regardless of the mosquito population/virus combination and no ZIKV was detected in saliva of females from the Pasteur population at any dpe.Conclusions/significanceOur study demonstrated the ability of Ae. aegypti from Cuba to transmit DENV, ZIKV, and CHIKV. These results, along with the widespread distribution and high abundance of this species in the urban settings throughout the island, highlight the importance of Ae. aegypti control and arbovirus surveillance to prevent future outbreaks.
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Project description:The Asian tiger mosquito (Aedes albopictus) is an important vector of a number of arboviruses, including Zika (ZIKV), dengue (DENV), and chikungunya (CHIKV) viruses, and has recently expanded its range in the eastern United States to southern New England and New York. Given the recent establishment and proliferation of Ae. albopictus in this region and the increasing amount of international travel between the United States and endemic countries, there is a need to elucidate the public health risk posed by this mosquito species in the Northeast. Accordingly, we evaluated the competence of four Ae. albopictus populations from Connecticut and New York, for two strains each of ZIKV, DENV serotype 2 (DENV-2), and CHIKV, currently circulating in the Americas, to evaluate the local transmission risk by this vector. We found that local Ae. albopictus populations are susceptible to infection by all three viruses but are most capable of transmitting CHIKV. Variation in competence was observed for ZIKV and CHIKV, driven by the virus strains and mosquito population, whereas competence was more homogeneous for the DENV-2 strains under evaluation. These results suggest that under optimal circumstances, Ae. albopictus could support localized transmission of these viruses and emphasize the importance of maintaining mosquito surveillance and control programs to suppress Ae. albopictus populations and limit further range expansion of this species.
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