Highly infectious, less pathogenic and antibody resistant Omicron XBB.1, XBB.1.5 and XBB.1.5.1-XBB.1.5.39 subvariant Coronaviruses do not produce ORF8 protein due to 8th codon GGA=TGA Termination Codon Mutation (preprint)

RNA viruses are very mutation prone. We recently reported SARS-CoV-2 ORF8 gene CAA = TAA and AAA = TAA Termination Codon Mutations in B.1.1.7 variants with no production of viable ORF8 protein. We described here another GGA = TGA termination codon in the 8th codon of ORF8 gene located exclusively in XBB.1 (XBB.1.16 and XBB.1.22) and XBB.1.5 subvariants (XBB.1.5.1 to XBB.1.5.39) but not in XBB.2 variant or Alpha, Beta, Gamma, Delta and Omicron BA.1, BA.2, BA.4, BA.5, BF.7 and BQ.1 subvariants. However, G > T mutation at 27915 also created an alternate ATG codon but the protein product was short due to preceding TAG and TGA termination codons. The originally located following ATG codons were there but in alternate reading frames and ultimately no ORF8 protein was formed in XBB.1.5 subvariants which were spreading highly now over BA.2.75, BA.4.6, BA.5.2.1, BF.7 and BQ.1.1 subvariants. This is a vivid example of three termination codon mutations in the coronavirus ORF8 protein which was implicated as target for many human proteins regulating interferon production, chromosome instability, antibody production, pathogenicity and virus clearance. The 30nt deletion in the 3’-UTR, including 24LPP, and 145Y deletions in Spike protein as well as N-protein 31ERS and ORF1ab polyprotein 3675SGF deletions made XBB.1.5 Omicron coronavirus weak and less pathogenic so that WHO declared coronaviruses as non-emergency pathogen.

The 249RWMD spike protein insertion in Omicron BQ.1 subvariant compensates the 24LPP and 69HV deletions and may cause severe disease than BF.7 and XBB.1 subvariants (preprint)

Alarming antibody evasion properties were documented for new BF, BQ and XBB Omicron subvariants. Most immune-drugs were inactive neutralizing those COVID-19 subvariants and viral titers were exceptionally low as compared to deadly B.1.1.7, B.1.617.2 and B.1.1.529 variants with D614G, N501Y and L452R mutations in spike. The 91% nucleotides changes in spike protein of BQ.1 were resulted in AA changes whereas only 52% nucleotides changes resulted in AAs changes in ORF1ab. The N460K and K444T mutations in BQ.1 may be important driving force for immune-escape similar to F486S and N480K mutations in BA.2.75 subvariant and related XBB.1 subvariant. Further, the R346T mutation as found in BA.4.6 and BF.7, was regained in BQ.1.1 and BA.2.75.2 to enhance immune escape and infectivity (> 80%). The L452R and F486V mutations in spike were main drivers of Omicron BA.2 conversion to BA.4 and BA.5 in presence of 69HV deletion. Whereas 24LPP spike deletion and 3675SGF ORF1ab protein deletion were found in all Omicron viruses including BQ.1 and XBB.1. Interestingly, we found about 211 COVID-19 sequences with four amino acids (249RWMD) insertion near the RBD domain of Omicron viruses similar to 215EPE three amino acids insertion in Omicron BA.1 variant. Such sequences first detected in California and extended to Florida, Washington and Michigan as well as other adjoining US states. An one amino acid deletion (140Y) in spike was also found in BA.4.6, BQ.1.5, BQ.1.8, BQ.1.14, BQ.1.1.5, XBB.1 as well as related AZ.3, BU.1, BW.1, CR.2, CP.1 and CQ.1 subvariants but was not detected in BA.2.75, BF.7, XBD, BQ.1, BQ.1.1, BQ.1.2, BQ.1.6, BQ.1.10, BQ.1.12, BQ.1.16, BQ.1.19, BQ.1.22, BQ.1.1.1, BQ.1.1.4, BQ.1.1.12 and related BK.1, BN.1, BM.1.1.1, BR.2, BU.1, CA.1, CD.2, CH.1.1 subvariants. Thus, BQ.1 insertion was compensated the other deletions and would be more infectious than BA.2.75, BF.7 and XBB.1 subvariants even there was a 26nt deletion in the 3’-UTR. The spike protein R341T one amino acid change in BQ.1.1 and BQ.1.1.1 might be important but no 249RWMD insertion.

A Method of Identification of SARS-CoV-2 Variant Using NCBI BLAST-2 100% Homology Search with Specific Oligonucleotides Selected at the Deletion Boundaries of S, N, ORF7a, ORF8 and ORF1ab Proteins (preprint)

Genomic sequencing of many SARS-CoV-2 variants with higher transmission and immune-escape were reported due to point mutations and deletions. Thus, whether a newly sequenced SARS-CoV-2 belongs to Alpha, Beta, Gamma, Delta, or Omicron (BA.1, BA.2, BA.4 and BA.5) variants must be known. We multi-aligned the different Spike, ORF1ab and Nucleocapsid proteins of those corona virus variants and detected different lineage specific deletions and point mutations. Different COVID-19 sequences were aligned with CLUSTAL Omega software and oligonucleotides from deletion boundary were selected. BLAST search using those oligonucleotides clearly predicted the specific variant type with 100% homology and was very useful for new corona virus sequence characterization. Selection of sub-variants were done by oligonucleotides selected at the specific point mutation boundaries leading to amino acid change. COVID-19 variant status was not reported in most published corona virus sequences and this method would be very useful application to understand the nature of expected prognosis of corona virus infected patients in less technology-equipped countries.

Ribosomal Proteins Homologies and Methylation of Mitoribosome by Corona Virus Nsp9, Nsp10 and Previously Described Nsp13-16 Proteins Suggested Their Roles in the Inhibition of Host Protein Synthesis: Discovery of New Therapeutic Targets against Corona Virus Infections (preprint)

Multi-Alignment method coupled with phylogenetic analysis we disclosed the Nsp9 and Nsp10 non-structural proteins of Corona Virus as rRNA RlmH/K methyltransferases with similarities with bin recombinase and int-core integrase fold. Further, Nsp9 has similarities to S8 ribosomal protein and Nap10 has similarity to S10 ribosomal protein. Previously, we showed Nsp13, Nsp14, Nsp15 and Nsp16 are also different types of rRNA RlmE/N and Cfr-like methyltransferases-ribonuclease with RNA helicase domains. Two domains of Nsp13 astonishingly have similarities to ribosomal proteins L6 and L9. Taken together, Nsp9/10 and Nsp13-16 proteins could mimic host ribosome assembly and also could methylate rRNA of mitobibosome preventing mitochondrial protein synthesis and oxidative phosphorylation. Low ATP synthesis causes lowering blood pressure following coma but very ATP concentration (1-10nM) surely induces platelets aggregation through vWA, collagen and GpIIb/IIIa proteins followed by fibrin formation and blood clotting as recently have seen in the lung of many Corona virus infected patients. We have also postulated that two polyproteins itself resemble like 28S and 38S mitoribosome subunits and compete with rRNAs inhibiting the ribosome turnover and new protein synthesis due to their similarities with many ribosomal proteins. Such finding may be valuable in computer-based novel drug design against Corona virus.

Coronavirus ORF1ab Polyprotein Associated Nsp16 Protein is a RlmE Methyltransferase and May Methylate 21S Mitochondrial rRNA of Host Cells Inhibiting Protein Synthesis (preprint)

Covid-19 infections are rapidly spreading worldwide with more than 100000 death and thus understanding the molecular mechanism of tropism of human cells is an urgent need for drug design. We have described here a bioinformatics approach to predict the functional aspects of non-structural nsp16 protein of Corona virus. The covid-19 7098 AA large polyprotein was degraded into sixteen proteins and last nsp16 protein was found an RlmE type rRNA methyltransferase. Nsp16 has no similarity to bacterial RlmABCD but has 25 percent similarity to the bacterial RlmE protein which methylates the U2551 2-hydroxy group of Ribose. The nsp16 proteins of different corona viruses like covid-19, bat-coronavirus, SARS and MERS have strong homology. Mrm2 and Dim1 like yeast and mammalian rRNA methyltransferases have 26-33 percent homologies but not with 2-O-capping MTase as reported previously. Rrp8 MTases also has no similarity to nsp16. We postulated that mitochondrial rRNA methylation of bronchial cells were mediated by the nsp16 protein causing inhibition of protein synthesis due to poor assembly of aminoacyl-tRNA or mRNA and peptidyl transferase at the PTC. This is one of the new molecular mechanism of corona virus cellular tropism and different than ACE-2 mediated blockage of cellular signalling to inhibit aldesterone biosynthesis with abnormal Na+ ions in cells. We also designed primers based on nsp16 cDNA sequence (nt 20659-21552, accession no MT121215) specific for Covid-19 diagnosis by RT-PCR.