Genome analysis of alphacoronavirus and betacoronavirus species from South Korean bats

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Jun 28 2022

In a recent study published in Viruses, researchers performed genomic comparisons between alphacoronaviruses (αCoVs) and betacoronaviruses (βCoVs) originating from South Korean Bats.

Background

CoVs are a diverse family of viruses with a wide spectrum of hosts. Ever since the severe acute respiratory syndrome (SARS) outbreak, several bat-associated CoVs have been detected in multiple countries; however, they are not representative of all geographical sites of their hosts. Bat CoVs (BatCoVs) were detected in South Korea based on their ribonucleic acid (RNA)-dependent RNA polymerase (RdRp) genetic sequences. To date, only two genomes of BatCoVs have been reported, including SARS-like BatCoV and BatCoV HCQD-2020. Therefore, further research is required to elucidate the complete BatCoV genome evolution and characteristics.

About the study

In the present study, researchers compared between αCoVs and βCoVs originating from South Korean bats at a genomic level using RNA sequencing. They analyzed BatCoV genome features to determine their evolutionary associations with known viruses.

CoVs were detected in the feces of South Korean bats between 2018 and 2021 using pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) and RNA sequencing. Positive feces samples were initially treated with the deoxyribonuclease I (DNAse I) enzyme, following which RNA extraction was performed. The total RNA was determined, and subsequently, complementary DNA (cDNA) libraries were constructed, and the entire length of BatCoV genomes recently detected in South Korea was sequenced by RNA sequencing.

Specific cytochrome b gene primers were used to identify the bat species. BatCoV genomes were analyzed using phylogenetic analysis based on their genomic structures, conserved replicase polyprotein domains, nucleocapsid (N), open reading frame 1a polyprotein/1b polyprotein (ORF1ab), and spike (S) genes. For the BatCov genome, the probable ORFs were estimated and aligned with the closest genetic sequences identified using Geneious Prime and the basic local alignment search tool (BLAST). In addition, recombination analysis was performed.

Results

Full-length genome sequences of αCoVs such as B20-97 (Myotis petax), B20-177 (Myotis macrodactylus), and B20-104 (Rhinolophus ferrumequinum)-1 and 2 were characterized by 28kb-genome lengths and their genomic assembly demonstrated classical CoV organization of the structural protein genes and the replicase gene.

The transcription regulatory sequence leader (TRS-L) motif was conserved among all detected genomic sequences. Every ORF was preceded by the TRS-body, including ORF8, which codes for unknown functional protein molecules. ORF3 was situated between the envelope (E) gene and the S gene and coded for an accessory protein. The BatCoV B20-97 ORF3 encoded a 225 amino acid (aa)-long protein identical to the porcine coronaviruses (PEDV) accessory membrane protein. ORF3s of the B20-104-1 strain and the B20-104-1 encoded an identical uncharacterized protein as BtMr-SAX2011 ORF3, with estimated proteins comprising 220 aa and 208 aa, respectively.

B20-177 (Myotis macrodactylus) non-structural protein 3 (nsp3), a 226 aa-long protein was recognized as the BatCoV-512 strain. Among all αCoVs, ORF8 was detected subsequential to the N gene and was estimated to code for a 112 to 113 aa-long protein. The B20-97 strain was identical to the nearly-complete JTAC2 sequence of Chinese bats (Murina leucogaster), with 99% similar RdRp aa identities. However, the B20-97 strain shared 67.4% nucleotide (nt) identity values with PEDV in the full-length genome and 56% and 59% aa identities in the N gene and S gene sequences, respectively. The findings indicated that BatCoV B20-97 was similar (<90% nt identities) to BatCoV-512 and PEDV.

B20-104-1 and B20-104-2 sequences shared 72% nt identity values, and 71% and 63% aa identities in the N gene and S gene proteins, respectively. The B20-104-1 and B20-104-2 strains closely matched with the Anlong-57 strain and the BtMr-SAX2011 strain, respectively (71 to 81% nt identities). Sequence identities of B20-104-2 and B20-104-1 and Myotis ricketti were 90% and 95%, respectively.

B20-177 belonged to the αCoV K1 group of highest abundance in Korea, and its sequence most closely matched to an isolate of BatCoV-Jingmen (Jingmen Miniop-terus schreibersii αCoV 2) sharing 72% of nt identity values and 69% and 75% aa similarity with the N and S gene aa sequences, respectively. Both the B20-180 and B20-50 strains were obtained from Rhinolophus ferrumequinum bat species. The genome of SARS-related CoV B20-50 comprised 29,612 nt and was similar to known SARS-related CoVs such as TRS-B and TRS-L.

B20-50 strain shared 98% nt identities with BatCoV 16BO133. In addition, the aa identities were 99% and ~100% similar in the replicase RdRp gene and the S gene, respectively. The B20-50 strain also shared 75% and 70% aa identities with the SARS-CoV genome and the SARS-CoV-2 genome, respectively. Two genomic deletions comprising five aa and 14 aa were noted in the receptor binding motif (RBM) of B20-50, and nine nt deletions were noted for the ORF7b gene.

No ORF was detected among 316 nt-long genome length following the ORF7b gene and preceding the N gene, wherein ORF8 of SARS-related CoVs is situated usually. Multiple alignment analysis demonstrated that 16BO133 and B20-50 lacked an ORF, but the nt identity values with SARS-CoV Tor2 were high. Further, B20-180 genomic sequence was similar to that of MERS-CoV.

B20-180 demonstrated 76% nt identity values with the BtCoV/li/GD/2014-422 sequence and was ≤74% similar to MERS-CoV and other Merbecoviruses. B20-180 RdRp gene sequences shared 94% aa identity values with MERS-CoV and B20-180 S shared 68% to 77% aa identity values with MERS-CoV and BatCoV HKU4,5,25,422. B20-180 RBD shared 70%, 34%, and 61% aa identity values with BatCoV HKU4, HKU5, and MERS-CoV, respectively. In B20-180 RBM, five conserved binding residues for human dipeptidyl peptidase 4 (hDPP4) were found. BatCoV B20-50 was not likely to utilize the host angiotensin-converting enzyme 2 (hACE2) as its receptor with a deficiency in the open reading frame 8 (ORF8) gene region.

Conclusion

Overall, the study findings highlighted the evolutionary associations and the genomic diversity of CoVs among Korean bats with data on the evolution of BatCoVs in relation to the ORF8 gene. The study findings could aid in estimating the pathogenic potential of BatCoVs and understanding their evolution to prevent BatCoV transmission to human beings.