Chikungunya is a self-limiting disease that caused by chik virus. Chik virus is Alphavirus group in Togaviridae family. This disease is signed with acute fever, pain in articulation especially at knee joint, wrist, finger and toe, as well as spinal column with an eruption of the skin. On Mei , in Tanah Raja occurred Chikungunya outbreaks that attack citizen.
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Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus CHIKV , a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity or antimutator polymerase variant, CY, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant.
We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis.
Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus SINV , which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent.
Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.
Chikungunya CHIKV is a re-emerging mosquito-borne virus that constitutes a major and growing human health burden.
Therefore, a single virus generates millions of viral progeny that carry a multitude of distinct mutations in their genomes. In this study, we isolated CHIKV mutators strains that make more errors than the wildtype virus , to study how higher mutation rates affect fitness in arthropod-borne viruses arboviruses. CHIKV mutators have reduced virulence in mice and severe replication defects in Aedes mosquito cells.
However, these replication defects result in selective pressure for reversion of mutators to a wildtype polymerase in mosquito hosts. To examine how mutators would behave in an insect model in absence of this genetic instability, we isolated mutators of a related virus, Sindbis virus SINV.
This work shows proof of principle that arbovirus mutators can exhibit attenuation in both mammalian and insect hosts, and may remain a viable vaccine strategy. PLoS Pathog 10 1 : e Editor: Laura D. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. During replication, RNA viruses generate approximately 1 error per 10 4 nucleotides copied, giving rise to an immense population of genetically distinct but closely related variants  ,  ,  , . However, this lack of information on genetic diversity has obscured crucial aspects of virus biology.
Although RNA-dependent RNA polymerases RdRp have a high intrinsic error rate, their mutation rates can be altered to generate both higher and lower fidelity variants antimutators and mutators, respectively  ,  ,  ,  ,  ,  ,  ,  ,  , .
Thus far, antimutator variants are thought to replicate more slowly, making fewer genomes with greater accuracy; in contrast, mutator variants have been shown to replicate more quickly, synthesizing more viral genomes but introducing many errors during the replication process  ,  ,  ,  , . Despite this, overall growth and titers of polymerase fidelity variants are not significantly different when grown in isolation in cell culture; for mutators the negative effects of accumulating deleterious mutations are only noticeable after several rounds of replication  , .
In recent works, these variants have been useful in exploring how the course of viral infection is affected by either restricted or expanded population diversity  , .
Current evidence indicates that mutation frequencies of RNA viruses have been optimized over time to be neither too accurate nor too erroneous  ,  ,  ,  ,  ,  , . It is thought that error-prone replication allows the virus to explore sequence space to gain adaptability and accumulate potentially advantageous mutations.
For several RNA viruses, limiting viral population diversity has fitness costs in vivo. Despite similar in vitro growth phenotypes, variants that make fewer errors have reduced titers and exhibit restricted tropism in animal models  ,. This restriction in tropism may be due to cooperative inter-variant interactions or beneficial minority variants that are missing in a situation with restricted population diversity .
It is also proposed that high mutation rates of influenza A may contribute to altered tropism, allowing infection of new hosts .
Therefore, it seems that the relatively high error rates of RNA viruses generate a level of diversity that facilitates adaptive fitness advantages. In contrast, there is also an upper threshold to mutation frequencies; if crossed, extreme error rates lead to the accumulation of deleterious mutations and loss of genetic integrity. Evidence for this is demonstrated by treatment of numerous RNA viruses with nucleoside analog mutagens, which increase mutation frequencies and result in extinction by lethal mutagenesis  ,  ,  ,  , .
Although thus far RdRp mutators have not exhibited growth defects in isolation in vitro , a recent paper showed that HIV mutator and antimutator strains were less fit than wildtype in competition assays . In addition, several studies recently report in vivo attenuation of mutator strains: Coxsackie virus B3 mutator strains present reduced viral titers in key organs and fail to establish persistent infections in mice  , and a severe acute respiratory syndrome SARS coronavirus mutator strain exhibits reduced pathogenesis in several mouse models .
Antimutator and mutator variants are valuable tools to study where the threshold of advantageous polymerase error exists for viruses facing different selective pressures.
In this respect, arboviruses represent a special evolutionary position due to their need to replicate in disparate hosts, which is accompanied by distinct selective pressures. Arbovirus fitness is not necessarily reduced due to obligate host-cycling alternating passages of CHIKV did not limit viral fitness , yet it has been shown that evolvability may be reduced due to these evolutionary constraints  ,  ,  ,  ,  , .
For alphaviruses, evidence suggests that viral diversity is most restricted in the insect host, due to more stringent population bottlenecks and selective pressures  ,  ,  ,  ,  , .
Since minority variants are thought to play important roles in arbovirus pathogenesis, transmission, and emergence  ,  ,  ,  ,  ,  , the implications of altered polymerase fidelity and mutation rates merit further study. Recently, this question was partially addressed using a chikungunya virus antimutator variant .
In most cases, functions of these proteins are putative in CHIKV and have only been shown in related model viruses, such as Semliki forest virus and Sindbis virus  ,  ,  , . Nsp4 is the RdRp, responsible for nucleotide incorporation during replication .
Ribavirin causes nucleotide misincorporation by the RdRp, adding selective pressure for an intrinsically more faithful polymerase  ,  , . This antimutator strain harbored a single amino acid change Y in nsp4. Although Y showed no growth defects in vitro , the variant was moderately attenuated in vivo in both mammalian and mosquito hosts . However, no arbovirus mutators have been isolated thus far. To this end, we mutated the conserved cysteine residue at position to obtain several mutators in the arboviruses CHIKV and SINV, confirming this position's importance in determining alphavirus fidelity.
We used these novel mutator strains to examine how increased polymerase error affects arbovirus fitness in vitro and in vivo ; interestingly, mutator strains presented distinct cell- and host-specific phenotypes.
All newly generated DNA plasmids were Sanger sequenced in full GATC Biotech to confirm mutagenesis of position and to ensure no second-site mutations were introduced. Cells 0. Cells were allowed to recover for 10 minutes at room temperature then mixed with 6 ml of pre-warmed media and placed into a T flask.
Following incubation, cells were overlaid with 0. At passage 3, all viruses used were fully sequenced to ensure no second site mutations. Virus was harvested at 72 hours and mean titers were obtained by TCID Viruses were serially diluted in 8 ten-fold dilutions in DMEM. To determine mutation frequencies, all mutants were electroporated in tandem into BHK cells. Supernatants were collected 48 hours later and viral RNA was extracted. Modified products were cloned using the TopoTA cloning kit Invitrogen , and single colonies were picked for sequencing.
Mutation frequencies were determined as previously described . Mutation frequencies from mouse muscle were determined using RNA extracted from homogenized muscle samples from mice that most closely represented the median titer for that variants. For estimating in vivo mutation frequencies, a minimum of 50 clones were sampled per population.
PCRs were fragmented Fragmentase , multiplexed, clustered, sequenced in the same lane with Illumina cBot and GAIIX technology and analyzed with established deep sequencing data analysis tools and in house scripts. Briefly, per-base Phred quality scores were utilized to trim bases with error probabilities higher than 0.
Once the pileup is done, an in-house script collects the data per-position and calculates the variance at each nucleotide position by root mean square deviation RMSD and determines the mean variance and standard error across the whole genome . To estimate population diversity in a phenotypic assay, we performed neutralization assays using viruses which had been passaged 3 times on BHK cells, using the n Neutralizing antibody CHK a kind gift from Dr.
Diamond . Assays were then overlayed with agarose and developed as described above for a plaque assay.
Plaques were counted and normalized to the untreated control for each virus. We obtained similar titers by standard TCID 50 and CPE assay, indicating that viruses amplified on mosquito cells were still equally infectious when titered on Vero cells. Forty-eight hours post-transfection, supernatant containing progeny virus was collected. Eight-day old CD-1 litters were inoculated with pfu of wildtype or mutant SINV strains in the same fashion, and monitored for symptoms of hind limb paralysis and survival.
Seven days post infection, mosquitoes were dissected to obtain legs and wings, and saliva was obtained by in vitro transmission assay; in brief, mosquitoes were salivated for 30—45 min by placing the proboscis in a pipette tip containing FBS.
Following salivation, bodies were frozen. To confirm ingestion, a sample of engorged mosquitoes was immediately homogenized at time 0. Three- to four-day-old female flies were injected with 50 nL of a virus dilution containing pfu in 10 mM Tris-HCl pH 7. Fly mortality at day 1 was attributed to damage produced by the injection, and these flies were excluded from further analyses.
Mortality was monitored daily for 10 days, and every 3—4 days flies were transferred to fresh vials. In all experiments, 30—60 flies per genotype group were injected. Homogenates of individual flies were titrated on by plaque assay on Vero cells, as described above. All experiments were performed in triplicate unless noted otherwise. Since Coxsackie virus B3 mutator strains are situated in a structurally analogous area, we hypothesized that this position plays important roles in modulating intrinsic CHIKV RdRp fidelity .
After three passages in BHK cells, viruses were Sanger sequenced to determine genetic stability. Of the 19 substitutions, 12 were viable and genetically stable Table 1. This high number of viable variants indicates that position has structural plasticity and can tolerate a wider range of substitutions than in previous attempts at generating fidelity variants of other RNA viruses  , .
Interestingly, unstable viruses did not readily revert to wildtype, but mutated to other variants, including the antimutator form of the protein, Y Table 1. The only strict biochemical requirement we observed was a necessity for uncharged residues, as all variants with charged residues D, E, H, K, or R were unstable or not recoverable. In addition, we observed a general correlation between hydrophobicity of the substituted amino acid and stability or viability of the variant, where hydrophobic amino acids were preferred.
Finally, as a first characterization of virus fitness, we measured the mean size of plaques. Because polymerase fidelity variants have altered intrinsic rates of in correct nucleotide incorporation, they have often been identified by their relative resistance or sensitivity to nucleoside analog RNA mutagens  ,  , . Therefore, we addressed the sensitivity of all 12 genetically stable variants to ribavirin Table 1 and Figure 1A.
We expect antimutator variants such as Y to demonstrate resistance, and mutator variants to demonstrate sensitivity when compared to wildtype. Though these variants presented small plaque phenotypes, virus stocks reached wildtype-like titers, with the exception of N and Q Table 1. ND, not determined.
Case Report: Myocarditis Following Recent Chikungunya and Dengue Virus Coinfection: A Case Report
In in the District Gandapura number of Chikungunya cases there were 37 cases. The purpose of this study was to determine the factors associated with Chikungunya fever in the Work Area Health Center Gandapura bireuen District in This type of study is a cross sectional analytic. The population in this study is the head of a family with a number of 72 samples. The results showed no significant association between the presence of water with events Shelter p.
Manifestasi Klinis Infeksi Virus Chikungunya Pada Kejadian Luar Biasa Di Indonesia
Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus CHIKV , a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity or antimutator polymerase variant, CY, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo.