Antiviral Activity of Belladonna During Japanese Encephalitis Virus Infection via Inhibition of Microglia Activation and Inflammation Leading to Neuronal Cell Survival
Authors: Karuna Shanker
Srinivasulu Gadugu
Debadatta Nayak
Vimal K Maurya
Russell Kabir
Swatantra Kumar
Shailendra K Saxena
Anil Khurana
Raj K Manchanda
Journal: ACS Chemical Neuroscience (2020)
Discipline: NEUROSCIENCE & BEHAVIOR
Keywords: Japanese encephalitis virus; NS3 NTPase/helicase; antiviral; atropine; belladonna
Affiliations: Centre for Advanced Research (CFAR), Faculty of Medicine, King George’s Medical University (KGMU), Lucknow 226003, India, School of Allied Health, Faculty of Health, Education, Medicine, and Social Care, Anglia Ruskin University, Chelmsford CM1 1SQ, United Kingdom, CCRH, Ministry of Ayush, Janakpuri, New Delhi 110058, India, Department of Medicine, JSPS Government Medical College, Hyderabad 500013, India, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India

Abstract:  Japanese encephalitis virus (JEV) is the main cause of viral encephalitis resulting in more than 68 000 clinical cases every year with case fatality rate as high as 30-40% for which no specific treatments are available. We have recently exhibited belladonna may be widely applicable for the treatment of various neurological disorders. Therefore, we developed a hydroalcoholic formulation of belladonna (B200) consisting of atropine and scopolamine and showed its antiviral efficacy against JEV infection. B200 treatment increases neuronal cell survival by reducing JEV induced cytopathic effects which were evident from significant reduction in necrotic cell population by flow-cytometry analysis and caspase 3 and 8 enzymatic activities. B200 treatment was found to reduce the intracellular JEV level observed by significant reduction in JEV-fluorescein isothiocyanate (FITC) expression in both neurons and microglia. Because microglia plays a crucial role in JEV pathogenesis, we further investigated the anti-JEV effects of B200 on human microglia cells and elucidated the mechanism of action by performing whole-transcriptome sequencing. Gene expression analysis revealed that B200 reduces the pro-apoptotic and inflammatory gene expression observed by significant reduction in BAD, BAX, CASP3, CASP8, IL1B, and CXCL10 and increase in IL10 responsive gene expression. Interestingly, our molecular docking analysis revealed that atropine and scopolamine interact with the His288 residue of NS3 protein, a crucial residue for RNA unwinding and ATPase activity that was further confirmed by degradation of NS3 protein. Drug likeness, ADME (absorption, distribution, metabolism, and excretion), and toxicity analysis further suggests that atropine and scopolamine both cross the blood-brain barrier, which is crucial for effective treatment of Japanese encephalitis (JE). 

Retracted: No

PubPeer comments:
 In this paper the authors have used a highly infectious strain of Japanese Encephalitis virus. This paper does not have a statement on compliance with ethical standards. For the responsible conduct of research, ethical declarations are an integral part of any scientific communication. It is important for the authors to add a line that necessary ethical and biosafety clearances were taken from the host institute university to carry out this study. , 
  Interestingly, our molecular docking analysis revealed that atropine and scopolamine interact with the His288 residue of NS3 protein, a crucial residue for RNA unwinding and ATPase activity that was further confirmed by degradation of NS3 protein. Drug likeness, ADME (absorption, distribution, metabolism, and excretion), and toxicity analysis further suggests that atropine and scopolamine both cross the bloodu2013brain barrier, which is crucial for effective treatment of Japanese encephalitis (JE). I am superbly confused as to why the authors decided on whole plant** instead of purified atropine and why they did not also screen synthetic M1 antagonists like Telenzepine or Oxybutynin ** Whole plant extract is quite toxic as it contains a synergistic toxin mixture of atropine and solanine and as such, medicinal grade atropine does not contain solanine. , 
 ![file] Fig. 1 A: How did they characterized the B200 preparation: B200 was found to contain 87.76% of atropine (39.22) and 6.31% of scopolamine (55.45). How these values were determined without mentioning a standard curve of Atropine and Scopalamine?? Cell viability data: statistical analysis? AM+_SD? no error bars? How many replicates were used in this experiment and how many times the experiment was repeated. Most significantly, about the B200 concentrations used, there is a clear discrepancy in the X axis of figure and the B200 concentrations mentioned in the figure legend. Fig.1B: Which cell line was used in this experiment representing neurons and Microglia. Was this experiment repeated thrice in triplicates, no Error bars?? No statistics? Fig.1C: Again, no error bars, Were experiments repeated three times in triplicates? What is the duration of experiment? in the beginning of the experiment, there are only 15-20% viable cells, does it mean that the rest of the cells in the preparation were dead?? If there are 20% viable cells why the line of regression says that no viable cell was present in the beginning of the experiment?? Fig.1D: Which cell line was used, According to Fig 1B, at 2 ug g hardly any nerve cell Migroglia survive, However in this figure, 7ug ml was used and still no loss in cell viability? How this discrepancy can be explained. And, again reproducibility not confirmed, Author say that the experiment was done in triplicates; Were these experiments repeated three times?? ![file] Fig 2 A. Authors did not provide methodology of this experiment. Which cell line was used, Which kit was used and duration of the experiment, staining protocol, data analysis? In this figure and in the manuscript authors conclude that B200 reduces apoptosis in JEV-infected cells. What is the % of cell statistics of each quadrant. It can be appreciated that there is increased early apoptosis (Annexin V positive events) clearly contradicting the essence of this manuscript that B200 is neuroprotective during JEV infection. Authors plotted the necrotic cell quantitation. It is very much possible that what authors are perceiving as necrotic cells could just well be doublets. It is known that Necrosis and Apoptosis are two entirely different modes of cell death. Authors plotted necrotic cell data when an assay for apoptosis was performed as mentioned in the title of the figure. Figure 2 C D: The authors here show the caspase enzyme activity. What is the catalogue number of the kit from Sigma. Authors have used colorimetric assay and are able to detect Caspase-3 activity at picomole levels. Whereas during Caspase analysis there is corresponding increase in the caspase-8 activity even in control. How it can be explained. ![file] Fig 3A: The data of JEV decreases after B200 is not reliable, microscopy is semiquantitative the method of indirect Immunofluorescence gives a lot of background fluorescence. The images are taken at a very low magnification (magnification is not mentioned). Fig. 3B: Was experiment was repeated three times? No error bars. no statistical analysis Fig 3C. About microglia data, the data analysis is missing that authors are mentioning in the figure legend. ![file] ![file] ![file] Fig. 7E and F: the main finding of their research paper is the role of IL10 in neuronal cell survival. Transcriptome findings were validated only in the case of IL-10 and not in the case of other propapototic genes. Authors did not use a control of B200 treated neuronal cells? This is a pivotal control in this experiment. The B200 control is missing only in this figure panel. value are also not shown in the histogram? ![file] Figure 8A: The authors do not mention that what is the affinity of Atropine and Sclopamine to NS3, what is their Dissociation constant. Just superimposing theses structure have no meaning until we know if their strength of the interaction is significant. Figure 8F: There is no negative control for NS3 expression analysis. This kind of negative control is pivotal to rule out the possibility that NS3 WB band is specific to only cells that are infected with JEV. The first lane is very confusing. The cell lysate is from JEV positive or JEV negative? Figure 8G: In the quantitation data also, no control of non JEV sample. ![file] ,