SELEX based aptamers with diagnostic and entry inhibitor therapeutic potential for SARS-CoV-2.

Frequent mutation and variable immunological protection against vaccination is a common feature for COVID-19 pandemic. Early detection and confinement remain key to controlling further spread of infection. In response, we have developed an aptamer-based system that possesses both diagnostic and therapeutic potential towards the virus. A random aptamer library (~ 1017 molecules) was screened using systematic evolution of ligands by exponential enrichment (SELEX) and aptamer R was identified as a potent binder for the SARS-CoV-2 spike receptor binding domain (RBD) using in vitro binding assay. Using a pseudotyped viral entry assay we have shown that aptamer R specifically inhibited the entry of a SARS-CoV-2 pseudotyped virus in HEK293T-ACE2 cells but did not inhibit the entry of a Vesicular Stomatitis Virus (VSV) glycoprotein (G) pseudotyped virus, hence establishing its specificity towards SARS-CoV-2 spike protein. The antiviral potential of aptamers R and J (same central sequence as R but lacking flanked primer regions) was tested and showed 95.4% and 82.5% inhibition, respectively, against the SARS-CoV-2 virus. Finally, intermolecular interactions between the aptamers and the RBD domain were analyzed using in silico docking and molecular dynamics simulations that provided additional insight into the binding and inhibitory action of aptamers R and J.

Vector derived artificial miRNA mediated inhibition of West Nile virus replication and protein expression.

West Nile virus (WNV) has been found to be a common cause of neuroinvasive arboviral disease worldwide in human and horses. The process of RNA interference induced by small RNA molecules, like small interfering RNA (siRNA) and microRNA (miRNA), proved to be a novel approach for preventing viral infections. So far there is no published data for inhibition of West Nile virus by vector delivered artificial miRNA which believed to have more inhibitory potential than small interfering (siRNA). In the present study, we designed two artificial miRNA (amiRNAs) targeting the conserved NS5 and NS2A genomic regions of West Nile virus. These amiRNAs oligos were cloned in to miRNA based vector having murine miR-155 backbone which allows the high expression of amiRNAs in green fluorescent protein (GFP) tagged form. Vero cells were transiently transfected by cytomegalovirus (CMV) promoter derived vector expressing amiRNAs transcribed by RNA Pol II. Efficacy of amiRNA targeting the NS5 and NS2A regions of WNV was determined in highly virulent WNV Eg101 strain in Vero cells. The result indicated that both amiRNA effectively inhibit West Nile virus replication. The concatenated amiRNA having dual pre-amiRNA expression cassette showed better efficacy. amiRNA targeting NS5 showed best protection against WNV infection and percentage reduction of WNV titer was observed at 96 hpi is 97.11%. Further study for inhibition of WNV replication was assessed by plaque assay, quantitative reverse transcriptase PCR (qRT-PCR) assay, Immunofluorescence assay and Western blot analysis. Present study concludes that amiRNA (NS5) targeting conserved region of gene significantly reduced the virus replication as determined by plaque assay. Similarly, reduction was also observed at RNA and protein level through real-time RT-PCR and Western blot analysis directly correlate with the inhibition of WNV replication. Here, we describe our current understanding of the role of miRNAs in host defense response against West Nile virus, as well as their potential as new therapeutic approaches.

Inhibition of West Nile virus Replication by Bifunctional siRNA Targeting the NS2A and NS5 Conserved Region.

BACKGROUND: The West Nile Virus (WNV) has emerged as one of the most significant arboviral infection in many parts of the world and is associated with the encephalitis affecting mainly human and horses. In spite of the fact that the WNV is threat for the public health, there is no vaccine or therapeutic available for the treatment of WNV. METHODS: In this study, we tested a novel RNA interference based technique to inhibit WNV replication in Vero cells. Two siRNAs were designed against the NS2A and NS5 regions of WNV which are highly conserved among Flaviviruses as it play important role in apoptosis and in viral replication respectively. In addition to this, dual functional siRNA is designed by joining an immunostimulatroy motif with the NS2A and NS5 specific siRNA. The antiviral activity was evaluated by detecting both the infectious virus and its genome. RESULTS: The bifunctional siRNA resulted in significant reduction of virus titre in siRNA transfected cells as compared to controls. The antiviral efficacy was most effective at 48hr post infection. These results were in accordance with the quantitative RT-PCR assay revealing similar reduction in WNV genomic RNA. The expression of housekeeping gene was not affected by the siRNA indicating no off target effect and non-interference in cellular mechanism. CONCLUSION: Thus, this bifunctional siRNA intervention paves the new way for therapeutic treatment of WNV disease.

Enhanced production and immunological characterization of recombinant West Nile virus envelope domain III protein.

West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation. Wet biomass obtained after batch and fed-batch cultivation processes was 11.2 g and 84 g/L of culture respectively. Protein yield after affinity purification was 5.76 mg and 5.81 mg/g wet cell weight after batch and fed-batch processes respectively. The purified WNV EDIII elicited specific antibodies in rabbits, confirming its immunogenicity. Moreover, the antibodies were able to neutralize WNV in vitro. These results established that the refolded and purified WNV EDIII could be a potential vaccine candidate.

Emergence of influenza A (H1N1)pdm09 genogroup 6B and drug resistant virus, India, January to May 2015.

To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.