The beginning of the year 2020 is marked by the emergence of the COVID-19 coronavirus that causes respiratory diseases. However, coronaviruses have been quite prevalent considering the high number of coronaviruses that are known to infect a variety of animals including hedgehog, camel, rat, mink, mouse and bird. For humans, the earliest coronavirus capable of infecting children and adults was identified in 1960s (Kahn et al., 2005). Of the 7 human coronaviruses that have been identified, 4 strains (OC43, HKU1, NL63, 229E) cause relatively harmless common cold. The SARS–CoV and MERS-CoV coronaviruses cause more severe respiratory disorders with mortality rates of ~9% and >30%, respectively.
In the case of COVID-19, individuals with weakened immunity show greater susceptibility. These include individuals whose immunity is compromised due to cancer therapy (ex. chemotherapy), acquired immune deficiency syndrome (ex. HIV), or transplantation (ex. bone marrow, organ) requiring immune suppressing drugs. Some report has estimated that the risk of COVID-19 infection may increase two-fold for cancer patients (Al-Shamsi et al., 2020). Further, exacerbating medical conditions such as chronic lung disease with serious asthma, diabetes, chronic kidney patients requiring dialysis, and liver disorders also constitute risk factors. Fatality rates differ considerably depending on the countries (Oke et al., 2020).
As COVID-19 was recently identified, viral proteins encoded by its genome have not been fully characterized. The following information is based on the genomic analysis of COVID-19, and prior knowledge regarding coronaviruses in general and its most closely related SARS coronavirus (Knoops et al., 2008). Coronaviruses are medium-sized (~120 nm diameter) RNA viruses and their genome consists of positive sense (translated) single stranded RNA that are among the largest (26-32 kb). Replication of coronavirus involves the generation of negative strand intermediates (Fehr et al., 2015). This is mediated by RNA replicase, ‘RNA-dependent RNA polymerase’ (RdRP), which catalyzes RNA synthesis using RNA template. In addition to generating full-length negative stranded RNA template (for replication), it may generate discontinuous shorter negative stranded templates (for gene expression). The multiple subgenomic mRNAs produced are unique as they contain sequences found at both ends of the genome. Exactly how the full-length RNA is synthesized (while generating a nested set of RNAs with common polyadenylated 3’-ends) is not completely understood.
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For gene expression, COVID-19 virus utilizes both transcriptional and post-transcriptional mechanisms, and its genome may encode multiple open reading frames (ORFs) and other accessory gene products (Malik et al., 2020). In the case of SARS virus, replicase gene is encoded by ORF-1a and 1b, with the latter being translated via ‘ribosomal frameshifting’ occurring near the 3’ end of ORF-1a RNA. For COVID-19 virus, ORF-1 and -2 spanning 2/3 of 5’ terminal genome may encode polyproteins pp1a and pp1b, which undergo further cleavage by proteases to yield 11 and 16 distinct proteins, respectively, to enable virus replication and genome maintenance. Like other coronaviruses, the 3’-proximal domain of the genome may encode spike (S), membrane protein (M), envelope protein (E) and nucleocapsid (N).
Coronaviruses are mutation-prone, and known to undergo genetic recombination (ex. when multiple viruses infect the same cell), which may contribute to evolutionary divergence. Following the isolation of COVID-19 strain, its genomic sequence was compared with other known coronaviruses. The COVID-19 sequence aligned closely with Bat-SARS-like coronavirus exhibiting 88.2% identity (Malik et al, 2020), which closely parallels 86.9% identity reported by Zhu et al., 2020. Further, investigators at Chinese Academy of Sciences (China) uncovered that the full-length genome of COVID-19 exhbited 96.2% identity with bat coronavirus BatCoV RaTG13 isolated from intermediate horseshoe bat Rhinolophus affinis from Yunnan province (Zhou et al., 2020) located to the southwest of (ca. >600 miles) Wuhan city., shedding light on its potential origin. Figure denotes the proposed taxonomic distribution based on the alignments. However, further research is being done to determine if other host(s) may have played a role in the evolution/transmission of COVID-19 to humans.
Critical to preventing epidemic is the ability to diagnose the infected early on to preempt further propagation. For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay. Bio-Synthesis, Inc. also specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA). A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as conjugate to peptides. It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides. To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade. Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer. The BNA technology that we offer provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance. It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone. Its single-mismatch discriminating power was especially useful for diagnosis (ex. FISH using DNA probe). More importantly, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides for clinical application.
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References
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Oke J, Heneghan C. Global Covid-19 Case Fatality Rates. https://www.cebm.net/global-covid-19-case-fatality-rates/
Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270-273 (2020). PMID: 32015507 doi: 10.1038/s41586-020-2012-7.
Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R. et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 382:727-733 (2020) doi:10.1056/NEJMoa2001017