Chikungunya virus (CHIKV) has re-emerged as a global public health threat. The inflammatory pathways of RAS and PPAR-γ are usually involved in viral infections. Thus, Telmisartan (TM) with known capacity to block AT1 receptor and activate PPAR-γ, 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 15.34-20.89μM in the Vero and RAW 264.7 cells respectively). Viral RNA and proteins were reduced remarkably. 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-γ increased CHIKV infection suggesting TM’s anti-viral potential by modulating host factors. Besides, reduced activation of all major MAPKs, NF-κB (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-γ/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.

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.

PurposeThe current global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the investigation with clinical, biochemical, immunological, and genomic characterization from patients to understand the pathophysiology of viral infection.MethodsSamples were collected from six asymptomatic and six symptomatic SARS-CoV-2-confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, and treatment regimen were collected from a hospital; viral load was determined by RT-PCR; and the levels of cytokines and circulating antibodies in plasma were assessed by Bio-Plex and isotyping, respectively. In addition, whole-genome sequencing of viral strains and mutational analysis were carried out.ResultsAnalysis of the biochemical parameters highlighted the increased levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT, and ferritin in symptomatic patients. Symptomatic patients were mostly with one or more comorbidities, especially type 2 diabetes (66.6%). The virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. On the other hand, viral load was higher in plasma and serum samples of symptomatic patients, and they develop sufficient amounts of antibodies (IgG, IgM, and IgA). The levels of seven cytokines (IL-6, IL-1α, IP-10, IL-8, IL-10, IFN-α2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO, and MDC) were remarkably higher in asymptomatic patients. The whole-genome sequence analysis revealed that the current isolates were clustered with 19B, 20A, and 20B clades; however, 11 additional changes in Orf1ab, spike, Orf3a, Orf8, and nucleocapsid proteins were acquired. The D614G mutation in spike protein is linked with higher virus replication efficiency and severe SARS-CoV-2 infection as three patients had higher viral load, and among them, two patients with this mutation passed away.ConclusionsThis is the first comprehensive study of SARS-CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and thereby advance the implementation of effective disease control strategies.

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, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. 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 inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late 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. 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 the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.

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Chikungunya virus (CHIKV) has re-emerged as a global public health threat. The inflammatory pathways of RAS and PPAR-γ are usually involved in viral infections. Thus, Telmisartan (TM) with known capacity to block AT1 receptor and activate PPAR-γ, 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 15.34-20.89µM 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-γ increased CHIKV infection suggesting TM’s anti-viral potential by modulating host factors. Besides, reduced activation of all major MAPKs, NF-κB (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-γ/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.

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (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 the establishment of research infrastructure to facilitate the rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, SARS-CoV-2 strains were isolated from patient swab samples collected during the first 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. 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 major goal was to isolate and adapt SARS-CoV-2 viruses in cell culture with minimum modifications to facilitate research activities involved in the understanding of the molecular virology, host–virus interactions, drug discovery, and animal challenge models that eventually contribute toward the development of reliable therapeutics.

Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and in vivo mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, in vitro. Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, in vitro. These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, in vitro and supported by molecular docking analysis, in silico. TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.