Biology subjects

Mamidi, P.

Publications and source records attributed to Mamidi, P..

5 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

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↗

bioRxiv2021

MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MK2 and MK3 were selected for further analysis. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation (a) inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during the late phase of CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of this drug against CHIKV. Additionally, CMPD1 also inhibited HSV1 and SARS CoV2-19 infection in vitro. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as key targets for developing effective anti-CHIKV strategies in future. Author summaryChikungunya virus has been a dreaded disease from the first time it occurred in 1952 Tanzania. Since then it has been affecting the different parts of the world at different time periods in large scale. It is typically transmitted to humans by bites of Aedes aegypti and Aedes albopictus mosquitoes. Although, studies have been undertaken to combat the disease still there are no effective strategies like vaccines or antivirals against it. Therefore it is essential to understand the virus and host interaction to overcome this hurdle. In this study two host factors MK2 and MK3 have been taken into consideration to see how they regulate the multiplication of the virus. The in vitro experiments demonstrated that inhibition of MK2 and MK3 restricted viral infection Further, it was observed that this is due to the blocking of lamellipodium formation by modifying the factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Besides, decreased disease score as well as better survivability was noticed in the in vivo experiments with mice. Therefore, MK2 and MK3 could be considered as the key targets for controlling CHIKV infection.

microbiology↗

bioRxiv2019

Molecular epidemiology of Japanese Encephalitis Virus in pig population of Odisha, Assam and Manipur states of India

Japanese encephalitis virus (JEV) comes under the family Flaviviridae and genus flavivirus. It predominantly infects the children under the age of 10 years and the case fatality rate can stretch out as high as 30%. Pigs act as reservoir and amplifying intermediate host for JEV. Recent report suggested longer persistence of JEV in tonsil than in circulation of experimentally infected pigs. The current investigation was conducted to understand the prevalence and molecular epidemiology of JEV infection in pigs in three different geographical sites in India (Odisha, Assam and Manipur). Serum samples were tested by ELISA and RT-PCR for detection of JEV, while only RT-PCR was done in case of tonsils tissues collected from pigs slaughtered in abattoir. Prevalence of JEV was highest in Manipur (25.45% in serum and 10.08% in tonsil) but lower in Assam (3.75% in serum and 0% in tonsils) and Odisha (1.49% in serum and 3.7% in tonsils). The percentage of sero-positivity was found to be 3.75% of IgM and 9.9% of IgG in Assam and Odisha respectively. Genotype III (GIII) of JEV was the dominant genotype and sporadic mutations of S83G, H76P, E78Q, C55S, and S64W along with two consistent mutations V46S and V51I were observed in all the GIII strains. Analysis of the E gene sequence revealed a single mutation, S118N in the GI strain. Older pigs (above 7 months) were found to be infected relatively more (8.6%) than younger pigs (age group 3-7 months). In conclusion, the high JE virus infection rate of pig in the current locations suggests the need for continuous surveillance of this virus in pigs which will ultimately help to adopt an effective control strategy to prevent the spread of JE infection to human.\n\nAuthor summaryJapanese encephalitis is one of the contributing factors in acute encephalitis syndrome cases reported across India as well as Asia. Primarily young naive human population are affected with JEV. The death rate can be as high as 30% and in about 30%-50% surviving population paralysis, brain damage or other serious permanent sequelae may be observed. The viral load gets amplified in pigs and thus plays a crucial role in transmitting the infection in human communities living in close proximity to pig dwelling. The current study was conducted to demonstrate prevalence of JEV in pig population of three geographical regions of India viz. the States of Odisha, Assam and Manipur that have reported JE outbreaks in human population. The current study demonstrates that the rate of infection is 3.28% among pigs in Manipur followed by Assam and Odisha. GIII was found to be the most predominant JEV genotype, while only one GI genotype strain was detected from Odisha region. These findings suggested the need of continuous surveillance of this virus in pigs and proper implementation of human and animal vaccination programme to control the infection.

microbiology↗