key: cord-0994199-yhnp384h authors: Rizvi, Zaigham Abbas; Tripathy, Manas Ranjan; Sharma, Nishant; Goswami, Sandeep; Srikanth, N; Sastry, J L N; Mani, Shailendra; Surjit, Milan; Awasthi, Amit; Dikshit, Madhu title: Effect of prophylactic use of intra-nasal oil formulations in the hamster model of Covid-19 date: 2021-06-28 journal: bioRxiv DOI: 10.1101/2021.06.25.449990 sha: 72b1bf161f2d71b994989b34c985283daf947c39 doc_id: 994199 cord_uid: yhnp384h Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection initiates with viral entry in upper respiratory tract leading to coronavirus disease 2019 (Covid-19). Severe Covid-19 is characterized by pulmonary pathologies associated with respiratory failure. Thus, therapeutics aimed at inhibiting entry of the virus or its internalization in the upper respiratory tract, are of interest. Herein, we report the prophylactic application of two intra-nasal formulations provided by the National Medicinal Plant Board (NMPB), Anu oil and Til tailya in SARS-CoV2 infection hamster model. Prophylactic nasal instillation of these oil formulations exhibited reduced viral load in lungs, and resulted in reduced body weight loss and pneumonitis. In line with reduced viral load, histopathlogical analysis revealed a reduction in lung pathology in Anu oil group as compared to the control infected group. However, Til tailya group did not show a significant reduction in lung pathology. Furthermore, molecular analysis using mRNA expression profiling indicated reduced expression of pro-inflammatory cytokines genes, including Th1 and Th17 cytokines for both the intra-nasal formulations as a result of decreased viral load. Together, the prophylactic intra-nasal application of Annu oil seems to be useful in limiting both the viral load and disease severity disease in SARS-CoV2 infection in hamster model. i.e. 4 days post infection (dpi), as presented schematically in the study design (Fig. 1A) . In line 109 with the earlier published reports, a decrease in body weight of SARS-CoV2-infected hamsters 110 was observed with 5-8% body weight reduction on 4 days dpi. Hamsters receiving TT or AO, 111 before SARS-CoV2 infection, did not lose body weight as was observed in the SARS-CoV2-112 infected group (Fig. 1B & 1C) . SARS-CoV2 infection in hamster model is characterized by 113 lung inflammation, pneumonitis, and cytokines release 1, 23, 24 . To further understand the other 114 parameters related to lung-associated pathologies, gross morphological changes were 115 compared between healthy, SARS-CoV2 -infected and SARS-CoV2 infected plus oil 116 formulations groups. Results obtained indicated a reduction in the regions of pneumonitis in the excised lungs of AO 118 group, but not TT group, as compared to infection control ( Fig. 1D & 1E) . As reported earlier, 119 splenomegaly is one of the critical parameters indicative of active infection 20 . Thus we tested 120 the splenomegaly between different groups and found that AO, but not TT, showed inhibition 121 in splenomegaly as compared to the SARS-CoV2 infected hamsters ( Fig. 1F and 1G ). We 122 also evaluated the lung viral load at 4 dpi and calculated the fold reduction in viral load in AO 123 and TT treated groups as compared to the SARS-CoV2 infected groups. Our 124 23 125 data indicate that compared to the SARS-CoV2 infected group, viral loads in AO and TT 126 treated groups were ~3 and ~2 fold less respectively (Fig. 1H) . Together, these data indicated hamsters as compared to SARS-CoV2 infected hamsters. There was no sign of bronchitis in 137 SARS-CoV2 infected-AO treated hamsters with overall significant mitigation in disease score 138 as compared to SARS-CoV2 infected hamsters ( Fig. 2A and 2B ). Hamsters treated with TT, 139 however, showed little or no improvement in lung injury and overall disease score as compared 140 to the infected control ( Fig. 2A and 2B) . Together, lung pathology associated with SARS- CoV2 was found to be resolved in AO group but not in TT group with around 1.5 fold rescue 142 in disease index in AO group as compared to the infected control group. Fig. 3A and 3B) . Prophylactic use of AO in SARS-CoV2 infected hamsters did not 159 reduce PAI-1 expression (Fig. 3C) . In contrast to AO, TT data showed no reduction in lung 160 injury as compared to infected control (Fig. 3A) . Surprisingly, there was a marked increase in 161 mast cell markers in TT group as compared to the infected control (Fig. 3B) . Overall, consistent 162 with our lung histology data, a profound reduction in the expression of lung injury genes and 163 mast cell markers as compared to the infected control was observed in AO treated groups. Th17 cell cytokines together with pro-inflammatory cytokines expression (Fig. 4A, 4B and 175 4D). However, surprisingly only TT but not AO was able to reduce the Th2 cytokines gene 176 expression ( Fig. 4A and 4C) . Further, we found an elevated IFNy secretion in both AO and 177 TT treated animals ( Fig. 4A and 4C) . Together, we show that AO and TT reduced the pro-178 inflammatory cytokines. HGPRT GATAGATCCACTCCCATAACTG TACCTTCAACAATCAAGACATTC tryptase β2 TCGCCACTGTATCCCCTGAA CTAGGCACCCTTGACTTTGC chymase ATGAACCACCCTCGGACACT AGAAGGGGGCTTTGCATTCC muc1 CGGAAGAACTATGGGCAGCT GCCACTACTGGGTTGGTGTAAG Sftpd TGAGCATGACAGACGTGGAC GGCTTAGAACTCGCAGACGA Eotaxin ATGTGCTCTCAGGTCATCGC TCCTCAGTTGTCCCCATCCT PAI-1 CCGTGGAACCAGAACGAGAT ACCAGAATGAGGCGTGTCAG IFNy TGTTGCTCTGCCTCACTCAGG AAGACGAGGTCCCCTCCATTC TNFa AGAATCCGGGCAGGTCTACT TATCCCGGCAGCTTGTGTTT IL13 AAATGGCGGGTTCTGTGC AATATCCTCTGGGTCTTGTAGATGG IL17A ATGTCCAAACACTGAGGCCAA GCGAAGTGGATCTGTTGAGGT IL10 GGTTGCCAAACCTTATCAGAA ATG TTCACCTGTTCCACAGCCTTG IL6 GGACAATGACTATGTGTTGTTAGAA AGGCAAATTTCCCAATTGTATCCAG Histology 298 The lung of the euthanized animals was fixed in 10% formalin solution and then embedded in 299 paraffin. Samples embedded paraffin blocks were then cut into 3 µm fine sections and then 300 mounted on silane-coated glass slides. 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We acknowledge IDRF (THSTI) for the support at ABSL3 facility Diwedi for their support in conducting ABSL3 animal experiments 440 and Jitender Chandel for providing technical help. Small animal facility and Immunology Core 441 for providing support in experimentation. Hamsters were procured from CDRI ILBS for support in histological analysis and assessment. The following reagent was deposited 443 by the Centers for Disease Control and Prevention and obtained through BEI Resources, 444 NIAID, NIH: SARS Related Coronavirus ABSL3 procedures: ZAR, MRT, NS; qPCR primer designing and analysis: ZAR 450 Revised the manuscript The authors declare no competing interest.