Drugs targeting structural and nonstructural proteins of the chikungunya virus: A review.

Chikungunya virus (CHIKV) is a single positive-stranded RNA virus of the Togaviridae family and Alphavirus genus, with a typical lipid bilayer envelope structure, and is the causative agent of human chikungunya fever (CHIKF). The U.S. Food and Drug Administration has recently approved the first chikungunya vaccine, Ixchiq; however, vaccination rates are low, and CHIKF is prevalent owing to its periodic outbreaks. Thus, developing effective anti-CHIKV drugs in clinical settings is imperative. Viral proteins encoded by the CHIKV genome play vital roles in all stages of infection, and developing therapeutic agents that target these CHIKV proteins is an effective strategy to improve CHIKF treatment efficacy and reduce mortality rates. Therefore, in the present review article, we aimed to investigate the basic structure, function, and replication cycle of CHIKV and comprehensively outline the current status and future advancements in anti-CHIKV drug development, specifically targeting nonstructural (ns) proteins, including nsP1, nsP2, nsP3, and nsP4 and structural proteins such as capsid (C), E3, E2, 6K, and E1.

Co-expression analysis suggests lncRNA-mRNA interactions enhance antiviral immune response during acute Chikungunya fever in whole blood of pediatric patients.

Chikungunya virus is an arbovirus that causes the neglected tropical disease chikungunya fever, common in tropical areas worldwide. There is evidence that arboviruses alter host transcriptome and modulate immune response; this modulation may involve transcriptional and post-transcriptional control mechanisms mediated by long non-coding RNAs (lncRNAs). Herein, we employed bioinformatic analysis to evaluate co-expression of lncRNAs and their putative target mRNAs in whole blood during natural Chikungunya infection in adolescent boys. Sequencing data from GSE99992 was uploaded to the Galaxy web server, where data was aligned with HISAT2, gene counts were estimated with HTSeq-count, and differential expression was run with DESeq2. After gene classification with Biomart, Pearson's correlation was applied to identify potential interactions between lncRNAs and mRNAs, which were later classified into cis and trans according to genomic location (FEELnc) and binding potential (LncTar), respectively. We identified 1,975 mRNAs and 793 lncRNAs that were differentially expressed between the acute and convalescent stages of infection in the blood. Of the co-expressed lncRNAs and mRNAs, 357 potentially interact in trans and 9 in cis; their target mRNAs enriched pathways related to immune response and viral infections. Out of 52 enriched KEGG pathways, the RIG-I like receptor signaling is enriched by the highest number of target mRNAs. This pathway starts with the recognition of viral pathogens, leading to innate immune response mediated by the production of IFN-I and inflammatory cytokines. Our findings indicate that alterations in lncRNA expression in adolescent boys, induced by acute Chikungunya infection, potentially modulate mRNAs that contribute to antiviral immune responses.

A structural and functional analysis of opal stop codon translational readthrough during Chikungunya virus replication.

Chikungunya virus (CHIKV) is an alphavirus, transmitted by Aedes species mosquitoes. The CHIKV single-stranded positive-sense RNA genome contains two open reading frames, coding for the non-structural (nsP) and structural proteins of the virus. The non-structural polyprotein precursor is proteolytically cleaved to generate nsP1-4. Intriguingly, most isolates of CHIKV (and other alphaviruses) possess an opal stop codon close to the 3' end of the nsP3 coding sequence and translational readthrough is necessary to produce full-length nsP3 and the nsP4 RNA polymerase. Here we investigate the role of this stop codon by replacing the arginine codon with each of the three stop codons in the context of both a subgenomic replicon and infectious CHIKV. Both opal and amber stop codons were tolerated in mammalian cells, but the ochre was not. In mosquito cells all three stop codons were tolerated. Using SHAPE analysis we interrogated the structure of a putative stem loop 3' of the stop codon and used mutagenesis to probe the importance of a short base-paired region at the base of this structure. Our data reveal that this stem is not required for stop codon translational readthrough, and we conclude that other factors must facilitate this process to permit productive CHIKV replication.

Influence of host genetic polymorphisms involved in immune response and their role in the development of Chikungunya disease: a review.

Chikungunya virus (CHIKV) is transmitted by the bite of infected mosquitoes and can cause significant pathogenicity in humans. Moreover, its importance has increased in the Americas since 2013. The primary vectors for viral delivery are the mosquito species Aedes aegypti and Aedes albopictus. Several factors, including host genetic variations and immune response against CHIKV, influence the outcomes of Chikungunya disease. This work aimed to gather information about different single nucleotide polymorphisms (SNPs) in genes that influence the host immune response during an infection by CHIKV. The viral characteristics, disease epidemiology, clinical manifestations, and immune response against CHIKV are also addressed. The main immune molecules related to this arboviral disease elucidated in this review are TLR3/7/8, DC-SIGN, HLA-DRB1/HLA-DQB1, TNF, IL1RN, OAS2/3, and CRP. Advances in knowledge about the genetic basis of the immune response during CHIKV infection are essential for expanding the understanding of disease pathophysiology, providing new genetic markers for prognosis, and identifying molecular targets for the development of new drug treatments.

Midgut transcriptomic responses to dengue and chikungunya viruses in the vectors Aedes albopictus and Aedes malayensis.

Dengue (DENV) and chikungunya (CHIKV) viruses are among the most preponderant arboviruses. Although primarily transmitted through the bite of Aedes aegypti mosquitoes, Aedes albopictus and Aedes malayensis are competent vectors and have an impact on arbovirus epidemiology. Here, to fill the gap in our understanding of the molecular interactions between secondary vectors and arboviruses, we used transcriptomics to profile the whole-genome responses of A. albopictus to CHIKV and of A. malayensis to CHIKV and DENV at 1 and 4 days post-infection (dpi) in midguts. In A. albopictus, 1793 and 339 genes were significantly regulated by CHIKV at 1 and 4 dpi, respectively. In A. malayensis, 943 and 222 genes upon CHIKV infection, and 74 and 69 genes upon DENV infection were significantly regulated at 1 and 4 dpi, respectively. We reported 81 genes that were consistently differentially regulated in all the CHIKV-infected conditions, identifying a CHIKV-induced signature. We identified expressed immune genes in both mosquito species, using a de novo assembled midgut transcriptome for A. malayensis, and described the immune architectures. We found the JNK pathway activated in all conditions, generalizing its antiviral function to Aedines. Our comprehensive study provides insight into arbovirus transmission by multiple Aedes vectors.

DNA Damage Response Signaling Is Crucial for Effective Chikungunya Virus Replication.

Viruses utilize a plethora of strategies to manipulate the host pathways and hijack host machineries for efficient replication. Several DNA and few RNA viruses are reported to interact with proteins involved in DNA damage responses (DDRs). As the DDR pathways have never been explored in alphaviruses, this investigation intended to understand the importance of the DDR pathways in chikungunya virus (CHIKV) infection in vitro, in vivo, and ex vivo models. The study revealed that CHIKV infection activated the Chk2 and Chk1 proteins associated with the DDR signaling pathways in Vero, RAW264.7, and C2C12 cells. The comet assay revealed an increase in DNA damage by 95%. Inhibition of both ATM-ATR kinases by the ATM/ATR kinase inhibitor (AAKi) showed a drastic reduction in the viral particle formation in vitro. Next, the treatment of CHIKV-infected C57BL/6 mice with this drug reduced the disease score substantially with a 93% decrease in the viral load. The same was observed in human peripheral blood mononuclear cell (hPBMC)-derived monocyte-macrophage populations. Additionally, silencing of Chk2 and Chk1 reduced viral progeny formation by 91.2% and 85.5%, respectively. Moreover, CHIKV-nsP2 was found to interact with Chk2 and Chk1 during CHIKV infection. Furthermore, CHIKV infection induced cell cycle arrest in G1 and G2 phases. In conclusion, this work demonstrated for the first time the mechanistic insights regarding the induction of the DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in the future. IMPORTANCE Being intracellular parasites, viruses require several host cell machineries for effectively replicating their genome, along with virus-encoded enzymes. One of the strategies involves hijacking of the DDR pathways. Several DNA and few RNA viruses interact with the cellular proteins involved in the DDR pathways; however, reports regarding the involvement of Chk2 and Chk1 in alphavirus infection are limited. This is the first study to report that modulation of DDR pathways is crucial for effective CHIKV infection. It also reveals an interaction of CHIKV-nsP2 with two crucial host factors, namely, Chk2 and Chk1, for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest the cell cycle in G1 and G2 phases for efficient viral infection. This information might facilitate the development of effective therapeutics for controlling CHIKV infection in the future.

Modeling the impact of genetically modified male mosquitoes in the spatial population dynamics of Aedes aegypti.

The mosquito Aedes aegypti is the primary vector of diseases such as dengue, Zika, chikungunya, and yellow fever. Improving control techniques requires a better understanding of the mosquito's life cycle, including spatial population dynamics in endemic regions. One of the most promising techniques consists of introducing genetically modified male mosquitoes. Several models proposed to describe this technique present mathematical issues or rely on numerous parameters, making their application challenging to real-world situations. We propose a model describing the spatial population dynamics of the Aedes aegypti in the presence of genetically modified males. This model presents some mathematical improvements compared to the literature allowing deeper mathematical analysis. Moreover, this model relies on few parameters, which we show how to obtain or estimate from the literature. Through numerical simulations, we investigate the impacts of environmental heterogeneity, the periodicity of genetically modified male releases, and released genetically modified males quantity on the population dynamics of Aedes aegypti. The main results point to that the successful application of this vector control technique relies on releasing more than a critical amount of modified males with a frequency exceeding a specific critical value.

Re-Cellularised Kidney Scaffold for Chikungunya Virus Propagation: A Novel Approach.

BACKGROUND: Re-emerging viral attacks are catastrophic for health and economy. It is crucial to grasp the viral life cycle, replication and mutation policies and attack strategies. It is also absolute to fathom the cost-efficient antiviral remedies earliest possible. METHODS: We propose to use a lab-grown organ (re-cellularized scaffold of sheep kidney) for viral culture and understand its interaction with extra-cellular matrices of the host tissue. RESULTS: Our findings showed that the chikungunya virus (CHIKV) could be better replicated in tissue-engineered bio models than cell culture. A decrease in ds-DNA levels emphasized that CHIKV propagates within the re-cellularized and cell culture models. There was an increase in the viral titres (pfu/ml) in re-cellularized scaffolds and control groups. The lipid peroxidation levels were increased as the infection was progressed in cell culture as well as re-cellularized and control groups. The onset and progress of the CHIKV attacks (cellular infection) lead to transmembrane domain fatty acid peroxidation and DNA breakdown, landing in cellular apoptosis. Simultaneously cell viability was inversely proportional to non-viability, and it decreased as the infection progressed in all infected groups. Histological findings and extracellular matrix evaluation showed the impairment in medullary, cortex regions due to propagation of CHIKV and plaques generations. CONCLUSION: This method will be a breakthrough for future virus culture, drug interaction and to study its effect on extracellular matrix alterations. This study will also allow us to investigate the correct role of any vaccine or antiviral drugs and their effects on re-engineered organ matrices before moving towards the animal models.

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA's physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA's bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Can presence of HLA type I and II alleles be associated with clinical spectrum of CHIKV infection?

Host immune response and virulence factors are key to disease susceptibility. However, there are no known association studies of human leukocyte antigen (HLA) class I and II alleles with chikungunya virus (CHIKV) infection in the Latin American population. Here, we aimed to identify HLA alleles present in patients with CHIKV infection versus healthy controls as well as the allelic association with the clinical spectrum of the disease. We conducted a cross-sectional analysis of a community cohort and included patients aged 18 years and older with serologically confirmed CHIKV infection. HLA typing of HLA-A, HLA-B, and HLA-DRB1 alleles was performed. Two-by-two tables were used to establish associations between allele presence and clinical characteristics. Data from 65 patients with confirmed CHIKV infection were analyzed for HLA typing. CHIKV infection was significantly associated with the presence of HLA-A*68 [p = .005; odds ratio (OR): 8.90; 95% confidence interval (CI): 1.88-42.13], HLA-B*35 (p = .03; OR: 2.01; 95% CI: 1.06-3.86), HLA-DRB*01 (p <.001; OR: 5.70; 95% CI: 1.95-16.59), HLA-DRB1*04 (p <.001; OR: 7.37; 95% CI: 3.33-16.30), and HLA-DRB1*13 (p = .004; OR: 3.75; 95% CI: 1.50-9.39) alleles in patients versus healthy subjects. A statistically significant relationship was found between the presence of a rash on the face or abdomen and the presence of HLA-DRB1*04 (p = .028; OR: 3.2; 95% CI: 1.11-9.15 and p = .007; OR: 4.33; 95% CI: 1.45-12.88, respectively). Our study demonstrated that, in our cohort, HLA type I and type II alleles are associated with CHIKV infection, and an HLA type II allele is associated with dermatological symptoms. Further research is needed to establish a path for future investigation of genes outside the HLA system to improve knowledge of the pathophysiology of CHIKV infection and its host-pathogen interaction.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

Prevalence and Influence of the -174 G/C Polymorphism in the Interleukin-6 Gene in Arboviruses Infections.

Dengue virus and Chikungunya virus are arboviruses that affect thousands of people worldwide annually. The mechanisms involved in viral pathogenesis still need to be better understood. Single nucleotide polymorphisms (SNPs) in immune genes may be involved in the protection, susceptibility, and/or progression of these diseases. This study was performed to investigate the SNP -174 G/C in the interleukin-6 (IL-6) gene in patients with dengue or chikungunya from Northeastern Brazil. A total of 581 blood samples were analyzed, of which 244 were part of the negative control group, genomic DNA was extracted, and the SNP was genotyped using real-time polymerase chain reaction (PCR). The data obtained were used to conduct statistical analyses of the genotype and allele frequencies. We suggest that the G/C genotype and C allele of the SNP -174 G/C in the IL-6 gene are related to protection against dengue in the studied population. No significant differences were observed in chikungunya patients. This is the first study that assessed the association of the SNP -174 G/C in patients with chikungunya. We identified the presence of the C allele as a protective factor against dengue in the studied population.

BHK-21 Cell Clones Differ in Chikungunya Virus Infection and MXRA8 Receptor Expression.

Baby hamster kidney-21 (BHK-21) cells are widely used to propagate and study many animal viruses using infection and transfection techniques. Among various BHK-21 cell clones, the fibroblast-like BHK-21/C-13 line and the epithelial-like BHK-21/WI-2 line are commonly used cell clones for alphavirus research. Here we report that BHK-21/WI-2 cells were significantly less susceptible to primary infection by the alphavirus chikungunya virus (CHIKV) than were BHK-21/C-13 cells. The electroporation efficiency of alphavirus RNA into BHK-21/WI-2 was also lower than that of BHK-21/C-13. The growth of CHIKV was decreased in BHK-21/WI-2 compared to BHK-21/C-13, while primary infection and growth of the alphavirus Sindbis virus (SINV) were equivalent in the two cell lines. Our results suggested that CHIKV entry could be compromised in BHK-21/WI-2. Indeed, we found that the mRNA level of the CHIKV receptor MXRA8 in BHK-21/WI-2 cells was much lower than that in BHK-21/C-13 cells, and exogenous expression of either human MXRA8 or hamster MXRA8 rescued CHIKV infection. Our results affirm the importance of the MXRA8 receptor for CHIKV infection, and document differences in its expression in two clonal cell lines derived from the original BHK-21 cell cultures. Our results also indicate that CHIKV propagation and entry studies in BHK-21 cells will be significantly more efficient in BHK-21/C-13 than in BHK-21/WI-2 cells.

Facile method for delivering chikungunya viral replicons into mosquitoes and mammalian cells.

Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and inefficient replicon delivery methods. This paper demonstrates the use of a baculovirus to facilitate the efficient delivery of autonomous CHIKV replicons into mosquito and mammalian cells in vitro as well as adult mosquitoes in vivo. The efficacy of this approach was verified via co-localization among an eGFP reporter, nsP1, and dsRNA as well as through the inhibition of an RNA-dependent RNA polymerase (RdRp) null mutation (DDAA) in nsP4, or the treatment of a known antiviral compound (6-azauridine). We also investigated the correlation between CHIKV replicon-launched eGFP expression and the effectiveness of CHIKV replicon variants in inducing IFN-ß expression in human cell lines. This delivery method based on a single vector is applicable to mosquito and mammalian cells in seeking to decipher the mechanisms underlying CHIKV replication, elucidate virus-host interactions, and develop antivirals. This study presents an effective alternative to overcome many of the technological issues related to the study and utilization of autonomous arbovirus replicons.

Dynamic Molecular Epidemiology Reveals Lineage-Associated Single-Nucleotide Variants That Alter RNA Structure in Chikungunya Virus.

Chikungunya virus (CHIKV) is an emerging Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.

Chikungunya Virus Infection Outcome: A Systematic Review of Host Genetics.

Background: Chikungunya virus (CHIKV) is a global concern, inducing chikungunya fever and trigging an arthritogenic chronic phase beyond some severe forms. Outcomes of CHIKV infections in humans are dependent on genetic variations. Here, a systematic review was performed to show evidence of genetic variations on infection outcomes of patients. Methods: Searches were performed in Scopus, SciELO, MEDLINE/PubMed, Web of Science, OneFile (GALE), Periódicos CAPES and ScienceDirect Journals databases. The PICOS approach was used to assess the eligibility of records. A meta-analysis was also conducted to show an association between described alleles/genes and CHIKV infection outcome. Results: Reviews of genetic variants were conducted on genes: CD 209, OAS1, OAS2, OAS3, MIF, TLR-3, TLR-7, TLR-8, MYD-88, KIR, HLA-B; HLA-C; DRB1 and DQB1. Studies were performed on Gabon, Singapore, and India, including Indians, Malay, Gabonese and Chinese ethnicities and published between 2009-2017. The meta-analysis was performed with DRB1 *01; *03; *04; *07; *10; *11; *13; *14 and *15 and DQB1 *02; *03; *05 and *06 alleles with Indian population sample. Sampling power was >80% and a significant positive association between DRB1*14 and CHIKV infection was found (OR = 1.67, 95% CI = 1.04-2.67; p = .03). Conclusion: Majority of the studies were conducted in India. Meta-analysis suggests that DRB1*14 is related to the susceptibility of symptomatic CHIKV infection in Indian population. The literature about CHIKV infection and genetic variations is scarce. The precise role of genetic variation in CHIKV is not clear yet. Further studies are necessary to provide more concrete evidences.

Epidemiologic and clinical investigations during a chikungunya outbreak in Rio Grande do Norte State, Brazil.

The first autochthonous case of chikungunya virus (CHIKV) infection in Brazil was in September 2014 in the State of Amapá, and from there it rapidly spread across the country. The present study was conducted in 2016 in the state of Rio Grande do Norte, and the aims were to describe the epidemiological and the clinical aspects of the CHIKV outbreak. Biological samples from 284 chikungunya suspected cases were screened for CHIKV and Flavivirus (FV) RNA using qRT-PCR. Negative PCR samples were also screened for anti-CHIKV and anti-FVIgM by ELISA. CHIKV RNA were detected in 125 samples mostly occurring from January through March (46%), mainly affecting adults and older adults. We found a gradual decrease in viral RNA over the disease time. Anti-CHIKV IgM was found in 47.5% after negative CHIKV qRT-PCR. Interestingly, 45.0% simultaneously had positive results for CHIKV and FV IgM, suggesting the occurrence of virus co-circulation. The most frequent symptom was fever (91%). Women presented more chance to develop nausea and abdominal pain compared to men. Our data described and allows us to better understand the clinical and epidemiological aspects of the 2016 chikungunya outbreak in Rio Grande do Norte and can help in the early clinical diagnosis of the virus.

Role of MicroRNAs in Bone Pathology during Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is an alphavirus, transmitted by mosquitoes, which causes Chikungunya fever with symptoms of fever, rash, headache, and joint pain. In about 30%-40% of cases, the infection leads to polyarthritis and polyarthralgia. Presently, there are no treatment strategies or vaccine for Chikungunya fever. Moreover, the mechanism of CHIKV induced bone pathology is not fully understood. The modulation of host machinery is known to be essential in establishing viral pathogenesis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate major cellular functions by modulating gene expression. Fascinatingly, recent reports have indicated the role of miRNAs in regulating bone homeostasis and altered expression of miRNAs in bone-related pathological diseases. In this review, we summarize the altered expression of miRNAs during CHIKV pathogenesis and the possible role of miRNAs during bone homeostasis in the context of CHIKV infection. A holistic understanding of the different signaling pathways targeted by miRNAs during bone remodeling and during CHIKV-induced bone pathology may lead to identification of useful biomarkers or therapeutics.

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression.

Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.

Identification of cellular inhibitors against Chikungunya virus replication by a cDNA expression cloning combined with MinION sequencing.

cDNA expression cloning has been shown to be a powerful approach in the search for cellular factors that control virus replication. In this study, cDNA library screening using a pool of cDNA derived from interferon-treated human cells was combined with the MinION sequencer to identify cellular genes inhibiting Chikungunya virus (CHIKV) replication. Challenge infection of CHIKV to Vero cells transduced with the cDNA library produced virus-resistant cells. Then, the MinION sequence of cDNAs extracted from the surviving cells revealed that the open reading frames of TOM7, S100A16, N-terminally truncated form of ECI1 (ECI1ΔN59), and RPL29 were inserted in many of the cells. Importantly, the transient expression of TOM7, S100A16, and ECI1ΔN59 was found to inhibit the replication of CHIKV in Huh7 cells, indicating that these cellular factors were potentially anti-CHIKV molecules. Thus, our study demonstrated that cDNA expression cloning combined with the MinION sequencer allowed a rapid and comprehensive detection of cellular inhibitors against CHIKV.