Spike protein amino acid variants increased miRNA binding and hence reduced virulence of SARS-CoV-2 in Jordan, Middle East

Initial epidemiological studies inform the central COVID-19 disease prophet. Some papers have been studying miRNA and viral RNA interaction target predictions over the past few years. In this work, via the miRDB database, we determined the target scores of predicted miRNA to bind with the ss-RNA of SARS-CoV-2 in general and spike gene in speci�c. Our predicted miRNA targets of the ss-RNA of SARS-CoV-2 might destabilize and hence inhibit the ss-RNA translation of SARS-CoV-2 and prevent viral replication that has been established by more than 80% of asymptomatic infected cases in Jordan due to host miRNA interactions. In respiratory epithelial cells, the high scoring miRNAs prediction covers the RNA from 5' to 3' that explain successful antiviral defenses against ss-RNA of SARS-CoV-2 and lead to new nucleotide deletion mechanisms. The exciting �nding here that the substitution 1841A>G at the viral genomic RNA level or the D614G at spike protein level showed a change in the predicted miRNA sequence and an increase in the target score (from 91 to 92) (hsa-miR-4793-5p to hsa-miR-3620-3p).


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused an outbreak in Wuhan city and characterized as a pandemic by the World Health Organization WHO [1].The rst case of SARS-CoV-2 was reported to the Jordanian Ministry of Health on March 2, 2020, for a Jordanian citizen who was in Italy.To the date of this report, there are 522 con rmed cases, 387 recovered and nine deaths of COVID-19 in Jordan, according to the Jordanian Ministry of Health o cial via its web site launched as a uni ed source of information about the preventive measurements and symptoms of corona virus-2019 (https://corona.moh.gov.jo/en).
In addition, 18 years ago, severe coronavirus acute breathing syndrome (SARS-CoV) appeared in China.It has spread to over 30 countries, infecting around 8,000 people, killing young people 10%, and aging 50%.No SARS CoV or other up-and-coming CoV vaccines or antivirals such as Middle East respiratory CoV (MERS-CoV) are approved to date [2].The molecular mechanisms of viral pathogenesis will provide thoughtful help in the search for effective and secure therapeutic strategies against new and well-known human CoVs.
MicroRNAs (miRNAs) are non-coding RNAs that control many function targets within a cell by controlling protein levels through binding to mRNA translation process or mRNA abundance.Many evidence shows that miRNAs container the RNA virus replication and pathogenesis through direct binding to the RNA virus-mediated and changes in the host transcriptome.Proof of host miRNAs can bind to a wide-ranging of RNA viruses, straight adaptable their pathogenesis through mimics' cellular mRNAs tolerating direct binding of the miRNA to the viral RNA.Theoretically, the regulation is analogous to that of host mRNAs [3,4].
Many miRNA targeted in In uenza viral RNA segments were linked with the activity of host miRNA-induced antiviral defense.This represents potential Treatment with a combination of the ve miRNAs through agomir delivery to suppressed viral replication and effectively improved protection against lethal challenge with PR8 in mice [5].
In previous study, severe acute coronavirus syndrome (SARS-CoV) causes human fatal disease and reaction and extensive pulmonary disease.The signi cance of small non-coding RNAs for SARS-CoV pathologies, sequenced lung RNAs of infected mouse, and three 18 to 22nt small viral RNAs (SvRNAs), were discovered.The three svRNAs originated from the SARS-CoV genomic regions nsp3 (svRNA-nsp3.1 and -nsp3.2),and N (svRNA-N).CoV svRNAs were autonomous, cell type and host species from RNase III; but the extent of viral replication was dependent.In vivo lung pathology and pro-in ammatory cytokine release, antagomir-mediated inhibition of svRNA-N signi cantly decreased.This indicates that SvRNAs contribute to the pathogenesis of SARS-CoV and demonstrates the potential for antagomers of svRNA-N as antivirals [6].
To understand the early steps of COVID-19 infection, we predicted miRNAs targeting the submitted 29903 bp ss-RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 sequence), the isolate of Wuhan-Hu-1, complete genome.A predicted miRNAs targeting region at 3822 bp ss-RNA of the spike glycoprotein of SARS-CoV-2 was revealed.Also, we predicted miRNAs targeting a variable region of the ss-RNA spike glycoprotein of SARS-CoV-2 sequences from 20 positive nasopharyngeal specimens.These specimens were collected in Jordan and sequenced by Biolab Diagnostic Laboratories (Jordan) & Andersen lab at Scripps Research (USA), who deposited the sequences in GISAID, a maintained global database based in Germany.The perception in this work might help scientists to understand the molecular mechanisms of viral pathogenesis.In addition, it might support the research for effective, safe therapeutic strategies against known human CoVs and new emergent strains with a focus on miRNA-induced antiviral human body defense, which could be a potential treatment development for SARS-CoV-2.

Methods
The miRDB is an online http:/mirdb.org/custom.htmldatabase for the target and working annotations of miRNA.All targets in miRDB were anticipated by MirTarget, a bioinformatics tool that was developed through the study of thousands of miRNA target interactions from high-performance research [7,8].Using http:/biomodel.uah.es/en/lab/cybertory/analysis/trans.htm to convert sequences from miRNA to DNA to demonstrate genome sequence alignment using ChromosPros Version 2.1.9.Wuhan-Hu-1, full NC 045512.2accession genome sequence number and MIRNAs targeting area 3822 bp ss-RNA-spike SARs-CoV-2 Spike.We predicted miRNA targets 29903 bp ss-RNA (SARS-CoV-2 sequence) isolate Wuhan-Hu-1.In addition, miRNAs are predicted to target region on the SARs-CoV-2 sequence Accession no YP 009724390 ss-RNA spike glycoprotein variability from Jordanian positive nasopharyngeal specimens sequenced by Biolab Diagnostic Laboratories (Jordan) & Andersen lab at Scripps Research (USA) who published sequences retrieved from GISAID (https://corona.moh.gov.jo/en)(https://gisaid.org).
Moreover, in our study here we predicted the score of miRNAs targeting regions on many ss-RNA spike glycoprotein of SARS-CoV-2 sequence from Jordanian samples with amino acid substitutions (NCBI Reference Sequence: NC_045512.2region: 21563-25384 and Nomenclature sequence, Amino Acid Variant (SAV) and annotation used the accession number YP_009724390.1).
The original sequence of the del 432TTA, and the del Y144 have the same miRNA with a target score of 91.The original 1841A target miRNA score (91, 64 and 56) for (hsa-miR-4793-5p, hsa-miR-143-5p and hsa-miR-3133) respectively.The interesting nding here that the 1841A>G, and D614G showed a change in the predicted miRNA and an increase in the target score (from 91 to 92) (hsa-miR-4793-5p to hsa-miR-3620-3p).However, the original and 3415G>T D1139Y showed the same sequence of the miRNA (hsa-miR-548g-3p) and an increase in the target score of 80 to 81.The last substitution of 3499 G>A G1167S showed the same the miRNA sequence of (hsa-miR-155-5p) and a decrease in the target score from 73 to 72.One of the record genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV-1) isolated from humans after transmission human to human was the acquisition of a speci c 29-nucleotide deletion occurred in open reading frame 8 (ORF8).Three top target score of miRNAs prediction (hsa-miR-497-5p, hsa-miR-195-5p and hsa-miR-21-3p) showed on Table 12, 16 and 24 have an expression in the respiratory epithelial cells and effective antiviral defenses against the ss-RNA of SARS-CoV-2 and lead to new mechanisms interaction binding miRNA and SARS-CoV-2 of nucleotide deletion [5][6]& [9].

Conclusion
Over the past few years, some articles studied the target prediction of miRNA and viral RNA interaction.In our predictions, more than 80% asymptomatic of the disease raised due to the host miRNA interactions, which have identi ed the target where the genome replication of the ss-RNA of SARS-CoV-2 has changed to inhibit the translation of the ss-RNA and hence preventing viral replication and stabilization by subsequent generations.The top target score of miRNAs prediction cover from 5' to 3' in the respiratory epithelial cells that might be the reason for an effective antiviral defense against the ss-RNA of SARS-CoV-2 and lead to new mechanisms of nucleotide deletion in the coding region of a protein.The miRNAs found in all tissues have target gene functions, which lead to an identi cation of novel cellular pathways to block RNA viral replication or even host cell-speci c targeting the regulation for the ss-RNA of SARS-CoV-2.

Table 17 :
Original and del 432 Y144 have the same miRNA and target score

Table 18 :
Original 1841 A target miRNA score

Table 19 :
1841A>G showed a change the sequence of the miRNA and increase in the target score from 90 to 91 on variable ss-RNA glycoprotein of SARS-COV-2 sequence from Jordanian samples

Table 21 :
3415G>T D1139Y show same the miRNA and increase the target score 80 to 81 on variation ss-RNA spike glycoprotein of SARS-COV-2 sequence from Jordanian samples

Table 23 :
3499 G>A G1167S show same the miRNA and decrease the score 73 to 72 on variation ss-RNA spike glycoprotein of SARS-COV-2 sequence Jordanian samples

Table 24 :
Predicted miRNAs targeting on variation ss-RNA spike glycoprotein of SARS-COV-2 sequence from Jordanian samples