Absorbed plant MIR2911 in honeysuckle decoction inhibits SARS-CoV-2 replication and accelerates the negative conversion of infected patients

Dear Editor, The Coronavirus disease 2019 (COVID-19) pandemic is one of the most serious global public health crises to date. As of July 12, 2020, more than 12.6 million cases of COVID-19 infection with 0.56 million deaths were confirmed worldwide. Since there are no effective therapeutics to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the causative virus of COVID-19) infection so far, the pandemic is rapidly spreading worldwide. It is urgent to develop effective therapies, not only to treat infected patients but also to control the pandemic. Our previous studies have demonstrated that a plant microRNA, MIR2911, which is enriched in honeysuckle decoction (HD), directly targets influenza A viruses (IAV), including H1N1, H5N1, and H7N9 subtypes by binding to their mRNA and blocking protein translation. Oral administration of HD can prevent IAV infection and reduce H5N1-induced mouse death. Subsequent studies have shown that MIR2911 also directly inhibits the replication of various viruses in addition to IAVs. Upon dietary uptake, these microRNAs self-assemble into exosomes and are then secreted into the circulation and delivered into target tissues or specific cells, including the liver, lung, spleen, pancreas, and T cells. Given the unique GC-enriched nucleotide composition of MIR2911 (GGCCGGGGGACGGACUGGGA) and after we analyzed the genome sequence of SARS-CoV-2, it is most likely that the virus genome contains MIR2911-binding sites and that MIR2911 can inhibit SARS-CoV-2 replication directly. In the present study, we assessed the inhibitory effect of absorbed MIR2911 in HD on SARS-CoV-2 replication and conducted a clinical study to investigate the efficacy of HD in COVID-19 patients. By using bioinformatics analysis, we predicted that there are 179 putative MIR2911-binding sites in the SARSCoV-2 genome. Twenty-eight binding sites (Supplementary Table S1) were confirmed by classic luciferase assay (Supplementary Fig. S1), which are distributed widely in the virus genome (Fig. 1a), indicating that MIR2911 may be able to inhibit the translation of almost all the proteins of SARS-CoV-2. In order to assess the direct effect of absorbed MIR2911 on SARS-CoV-2 replication, cellular exosomes were collected from culture medium of HEK293T cells transfected with synthetic MIR2911 or control non-coding RNA (ncRNA), as similar method to previous report (Fig. 1b). Isolated cellular exosomes with/without MIR2911 were separately pre-incubated with 5 × 10 Vero E6 cells (ATCC1586) in 0.25ml cell medium for 8 h. After changing the culture medium, the cells were infected with SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019) at a multiplicity of infection (MOI) of 0.01. Efficacies were evaluated by quantification of viral copy numbers in the cell supernatant via quantitative real-time RT-PCR (qRT-PCR) at 24 h post infection (p.i.) (Fig. 1b). As shown in Fig. 1c,


miRNA target prediction and Luciferase report assay
SARS-CoV-2 genome sequence was acquired from the NCBI database (Refseq ID: MN908947). RNAhybrid 2 was employed to predict potential MIR2911 binding sites on SARS-COV-2 genome.
To test the binding efficiency of MIR2911 to the SARS-CoV-2 genes, a plasmid carrying the luciferase gene linked to a fragment of the target gene 3'-UTR harboring putative MIR2911 binding sites was co-transfected into HEK293T cells along with synthetic NC or MIR2911 (GenePharm, China). HEK293T cells were cultured in DMEM containing 10% FBS and seeded in 24-well plates. At 24 h after plating, 0.2 µg of firefly luciferase reporter plasmid, 0.1 µg of βgalactosidase expression vector (Ambion, CA, USA) and equal amounts (20 pmol) of synthetic MIR2911 were transfected into cells with Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instructions. The β-galactosidase plasmid was used as a transfection control. At 24 h post-transfection, the cells were analysed using a luciferase assay kit (Promega, WI, USA). All experiments were performed in triplicate wells for each condition independently.

Human serum Sample Collection
To assess the function of exosomal MIR2911 in serum, this study enrolled 3 donors. Every donor was fed 200 ml honeysuckle decoction, which was prepared from 30 g dried honeysuckle. 10 ml Serum samples were collected before and 2 hours after oral administrating honeysuckle decoction.

Exosome isolation
For exosome collection from culture cells, HEK293T cells were transfected with synthetic MIR2911 or ncRNA by using Lipofectomine 2000 (Invitrogen, US). After 36 h, exosomes were isolated from the cell culture medium using a Total Exosome Isolation Reagent (from cell culture medium, Invitrogen, US) according to the manufacturer's instructions. Briefly, after cell debris and shedding vesicles were removed by centrifugation at 2000×g for 30 min and then at 10000×g for 1 h, the supernatant was mixed with isolation reagent and incubated at 4°C overnight. The solution was centrifuged at 3000×g for 1 h (all steps were performed at 4°C). Exosome pellets were collected and re-suspended in PBS.
For human serum exosome collection, exosomes were isolated from human serum samples using a Serum Exosome Isolation Kit (Vazyme, China) according to the manufacturer's instructions. Exosome pellets were collected and re-suspended in DMEM.

RNA isolation and quantitative RT-PCR assays
Small RNAs (<100 nt) from honeysuckle decoction were extracted using the Universal Plant MicroRNA Kit (Biotech, China) according to manufacturer's instructions. Total RNAs from human exosome were extracted using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions.
To detect MIR2911 level, quantitative RT-PCR was performed using TaqMan miRNA probes (Applied Biosystems, USA) using an LC96 PCR machine according to the manufacturer's instructions. A series of synthetic MIR2911 oligonucleotides at known concentrations was reverse transcribed and amplified to build standard curve. The absolute amount of MIR2911 was then calculated in reference to the standard curve.

Evaluation of antiviral activities of exosomes
The cell viabilities of serum exosomes on Vero E6 Cells were determined by CCK8 assays (GLPBio, USA). To test the antiviral efficacy of serum exosome from donors, Vero E6 cells were cultured overnight in 48-well cell-culture petridish with a density of 5 × 10 4 cells/well. Cells were pretreated with cellular exosomes isolated from 1.5 ml cell medium or serum exosomes isolated from 62.5 μl serum from different doners for 8 hours. Subsequently, treated Vero E6 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. After 1 h of incubation, the virus-exosome mixture was removed and cells were washed with warm PBS and incubated in fresh medium. At 24 hours p.i., the cell supernatant was collected and lysed. The viral RNA extraction and quantitative real time PCR (RT-PCR) analysis was described in our previous study 3 .

Clinical Study
Moderate type patients infected by SARS-CoV-2 virus in Nanjing Second Hospital from January 2020 to March 2020 were included to this study. As MIR2911 were abundant in honeysuckle decoction (10.5 pmol /30g honeysuckle) and undetectable in Traditional Chinese Medicine (TCM) mixture, patients were divided into two groups, the MIR2911 + group (patients received routine anti-viral therapy plus honeysuckle decoction,10.5pmol MIR2911/oral 100ml, twice daily/30g dry honeysuckle/per patient for 14 consecutive days) and MIR2911group (patients received routine anti-viral therapy plus TCM mixture). All 6 patients from a clinical study (Chinese Clinical Study Register number, ChiCTR2000029822) were enrolled in MIR2911 + group. Other 69 patients received anti-viral therapy plus TCM mixture treated at the same hospital were enrolled in MIR2911group.
All enrolled patients have pneumonia confirmed by chest computed tomography imaging. COVID-19 infection was determined by pharyngo swab quantitative RT-qPCR assay which performed by either local Chinese Center for Disease Control and Prevention (CDC) or the designated diagnostic laboratory in the hospital.

COVID-19 Diagnosis
RNA was extracted from pharyngo swab samples by nucleic acid isolation kit (magnetic beads) supplied by Bioperfectus Technologies. Quantitative RT-PCR was performed by ABI 7500 with Novel Coronavirus 2019-nCoV Nucleic Acid Testing Kit in accordance with the instructions of the manufacturer (BGI BIOTECHNOLOGY, WUHAN, Lot number:6020200217). RT-qPCR results can be reported as positive when it fulfilled either of the following criteria: 1. Ct value ≤38 with a "s" shape amplification curve; 2. Double testing amplification curves are both "s" shape if Ct value >38.

Outcome Measures
The primary endpoint was SARS-CoV-2 negative conversion rate at the 7 th day, defined as the percentage of enrolled patients converted to SARS-CoV-2 PCR-negative at the 7 th day. The day patients received first dose of honeysuckle decoction or TCM mixture was recorded as 1 st day. The second outcomes were times taken to become SARS-CoV-2 PCR-negative after honeysuckle decoction/TCM mixture or since diagnosis as Covid-19 infection. Adverse events (AE) were classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0(CTCAE 5.0).

Statistics
Data is presented as mean ± SEM. When comparing the antiviral effect and cell viabilities of exosomes from two groups, P values are calculated using two-tailed students' t test. When comparing TTN and HD-TTN of MIR2911 + and MIR2911groups, P values are calculated using Cox regression with the adjustment of sex factor. When comparing HD-TTN of male or female patients, P values are calculated using log-rank test and Mantel-Haenszel method.