Biology subjects

Keshry, S. S.

Publications and source records attributed to Keshry, S. S..

4 recordsLinked to original sources

bioRxiv2025

Chikungunya virus susceptibility increases in the absence of mk2b and mk3 genes in zebrafish model

Chikungunya virus (CHIKV) infection imposes a significant socio-economic burden due to the absence of effective antiviral treatments and vaccines. Host factors are essential for the CHIKV life cycle, making them promising targets for antiviral therapy. Previous studies have identified Mitogen-activated protein kinase activated protein kinase 2 (MK2) and Mitogen-activated protein kinase activated protein kinase 3 (MK3) as key host factors in CHIKV infection; however, their role in an animal model remain unclear. This study highlights the critical roles of MK2 and MK3 host factors in an animal model following CHIKV infection. To investigate their functions, mk2b (mk2b-/-), mk3 (mk3-/-), and mk2b-mk3 (mk2b-/-mk3-/-) double knockout zebrafish were generated using the CRISPR-Cas9 technique. A significant high viral titer of CHIKV was observed in case of all knockout groups compared to the wild-type (WT) control using plaque assay, RT-qPCR and immunofluorescence assay. Among the knockout groups, mk3-/- displayed the highest susceptibility to CHIKV, followed by mk2b-/-. In contrast, the mk2b-/-mk3-/- double knockout exhibited the lowest susceptibility to CHIKV infection. Additionally, severe symptoms such as bent body, impaired response to physical stimuli, and increased mortality were most pronounced in mk3-/- larvae compared to other knockouts and the WT. The expression levels of inf{phi}1 and rsad2 were also elevated in all knockout groups during the early days of infection indicating higher interferon response in the absence of mk2b and mk3 during CHIKV infection. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling the CHIKV infection in organism level and subsequently may contribute in designing antiviral therapeutics in future. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections. Author SummaryCHIKV, transmitted by Aedes aegypti and Aedes albopictus mosquitoes, causes febrile illness and has spread across Africa, Asia, Europe, and America. Despite extensive research, effective antiviral drugs and vaccines are yet to be commercially available. This study examines the role of mk2b and mk3 host factors following CHIKV infection. For this purpose, mk2b and mk3 single as well as double knockout have been generated in zebrafish using the CRISPR-Cas-9 technique. Findings suggest that zebrafish exhibit high CHIKV susceptibility in the absence of mk2b and mk3, confirming its importance during infection. Moreover, these three knockout models could serve as a valuable platform for examine the role of mk2b and mk3 in the presence of other viral infection.

molecular biology↗

bioRxiv2022

DNA damage response signaling is crucial for effective Chikungunya virus replication

Viruses utilize a plethora of strategies to manipulate the host pathways and hijack its machineries for efficient replication. Several DNA as well as handful of RNA viruses are reported to interact with proteins involved in DNA damage responses (DDR). As the DDR pathways have never been explored in Alphaviruses, this investigation intended to determine the importance of the DDR pathways in CHIKV infection through in vitro, in vivo and ex vivo models. The study reveals that CHIKV infection activates the Chk2 and Chk1 proteins associated with DDR signaling pathways and increases DNA damage by 95%. Inhibition of both ATM-ATR kinases by ATM/ATR kinase inhibitor (AAKi) shows drastic reduction in viral particle formation in vitro. Next, the treatment of mice with this drug has been shown to reduce the disease score substantially in CHIKV-infected C57BL/6 mice with 71% decrement in the viral copy and the same has been established in hPBMC-derived monocyte-macrophage populations. Additionally, gene silencing of Chk2 and Chk1 reduces viral progeny formation around 73.7% and 78% respectively. Moreover, it has been demonstrated that CHIKV-nsP2 interacts with Chk2 and Chk1 during CHIKV infection and docking analysis depicts the specific amino acids responsible for these interactions. Further, the data suggests that CHIKV infection induces cell cycle arrest in G1 and G2 phases. In conclusion, this work demonstrated for the first time the mechanistic insight of the induction of DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in future. IMPORTANCEViruses being intra-cellular parasite, need several host cell machineries so as to achieve effective replication of their own genome, along with virus-encoded enzymes. One of the strategies is to hijack the DDR pathways. Several DNA as well as handful of RNA viruses interact with the cellular proteins involved in DDR pathways, however, reports with respect to the association of Chk2 and Chk1 in alphavirus infection are scanty. Hence, this study is amongst the first to report that modulation of DDR pathways is crucial for effective CHIKV infection. This work also shows that there is interaction of CHIKV-nsP2 with two crucial host factors, Chk2 and Chk1 for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest cell cycle in G1 and G2 phases to facilitate viral infection. This information might facilitate to develop effective therapeutics for the control of the CHIKV infection in future.

microbiology↗

bioRxiv2021

Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India.

Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.

microbiology↗

bioRxiv2021

Telmisartan restricts Chikungunya virus infection in vitro and in vivo through the AT1/ PPAR-γ/MAPKs pathways

Chikungunya virus (CHIKV) has re-emerged as a global public health threat. The inflammatory pathways of RAS and PPAR-{gamma} are usually involved in viral infections. Thus, Telmisartan (TM) with known capacity to block AT1 receptor and activate PPAR-{gamma}, was investigated against CHIKV. The anti-CHIKV effect of TM was investigated in vitro (Vero, RAW 264.7 cells and hPBMCs) and in vivo (C57BL/6 mice). TM was found to abrogate CHIKV infection efficiently (IC50 of 15.34-20.89{micro}M in the Vero and RAW 264.7 cells respectively). Viral RNA and proteins were reduced remarkably with the TM driven modulation of host m-TOR signaling. Additionally, TM interfered in the early and late stages of CHIKV life cycle with efficacy in both pre and post-treatment assay. Moreover, the agonist of AT1 receptor and antagonist of PPAR-{gamma} increased CHIKV infection suggesting TMs anti-viral potential by modulating host factors. Besides, reduced activation of all major MAPKs, NF-{kappa}B (p65) and cytokines by TM through the inflammatory axis supported the fact that the anti-CHIKV efficacy of TM is partly mediated through the AT1/PPAR-{gamma}/MAPKs pathways. Interestingly, at the human equivalent dose, TM abrogated CHIKV infection and inflammation significantly leading to reduced clinical score and complete survival of C57BL/6 mice. Additionally, TM reduced infection in hPBMC derived monocyte-macrophage populations in vitro. Hence, TM was found to reduce CHIKV infection by targeting both viral and host factors. Considering its safety and in vivo efficacy, it can be a suitable candidate in future for repurposing against CHIKV.

microbiology↗