4- Discussion
The COVID-19 statistics show a high distribution of this disease in Iran and all around the word 7. The SARS-CoV-2 shows a great potential for the transmission due to its highly contagious nature23. Considering the importance of the public knowledge about the SARS-CoV-2 epidemiologic and its phylogenetic analysis, in the present preliminary study in Iran, we aimed to investigate and track the SARS-CoV-2 in Iranian COVID-19 patients using molecular and phylogenetic methods.
The neighbor joining phylogenetic analysis demonstrated the highest similarity between Iranian sequences for nsp-2 and the obtained sequence from Wuhan of Hubei province, China (NC_045512) as well as a familial cluster of SARS-CoV-2 in Hong Kong- Shenzhen Hospital, Shenzhen, Guangdong, China, whose direct relationship with the seafood market of Wuhan of Hubei province, China (MN975262) 21 is confirmed, as illustrated in Figure 2. Also, the neighbor joining phylogenetic analysis showed the most similarity for RdRp with obtained sequence from MN938384 21 and MN985325, which were the first obtained sequences from the USA (Figure 3). Analysis of the S sequence showed a high similarity with NC_045512 and MN938384 sequences (Figure 4). The maximum likelihood phylogenetic indicates that the Bat coronavirus RaTG13 (MN996532), Bat coronavirus Cp/Yunnan2011 (JX993988), and other SARS-related bat coronaviruses have the highest similarity revealing a close relationship among their ancestors considering the RdRp (Figure 5).
As a major consideration, coronaviruses encode six main and variable numbers of accessory open reading frames and in case of the SARS-CoV-2, from 5’ to 3’ ORFs including ORF1a/b, spike protein or surface glycoprotein, membrane , envelope and nucleocapsid main ORFs, and 3a, 6, 7a, 7b, 8, and 10 accessories 24-26. The S glycoprotein is responsible for the attachment of the virus into the host cell. The S glycoprotein is cleaved to S1 and S2 in SARS-CoV and SARS-CoV-2 by host TMPRSS2 serine protease 27,28. The S1 domain is vital for the virus attachment as receptor in host cell and the S2 domain is useful for the membrane fusion 27,28. The SARS-CoV and -CoV-2 use the Angiotensin Converting Enzyme 2 for the attachment 29. The conducted studies reported that the SARS-CoV-2 S protein shows 80% and 76% similarity with bat-SL-CoVZXC21 and SARS-CoV, respectively 30. Regardless of S protein, the ORF 1a/b of the virus encodes 16 nonstructural proteins, which is known as nsp (nonstructural protein). The nsp-12 of the SARS-CoV-2 plays a role as dependent polymerase, while the nsp-2 function is not clear yet 30. Our current phylogenetic study showed that the Iranian isolates have the most similarity with the Chinese isolates. The study conducted by Eden et al. 31 showed that the isolated sequences from the Australian and New Zealand patients who travelled from Iran showed a clade different from those of other circulating strains. They also suggested that three major substitutions in the coding and non-coding regions of the nsp-2 include G1397A, T28688C, and G29742T in this particular clade. The results of the current study did not show any differences or clades in the assessed sequences between Iranian strains and those obtained from other countries in the phylogenetic study of the RdRp, nsp-2, and S proteins. However, it should be noted that the Eden et al. 31phylogenetic study was conducted using the whole genome of the virus in contrast to our study. The MSA assessment of the nsp-2 for the three suggested substitutions by Eden et al. could not be assessed due to differences between our sequencing regions. Furthermore, Bal et al. 9 could find three nucleotide variations in some of the assessed samples in France. They suggested that these variations might be associated with intra host evolution of the virus. In another study, conducted by Sardar et al 32, it is suggested that some of the mutations in isolated viruses could be unique. For instance, a substation in S protein C24351T is unique in Indian isolates.
A major limitation of the current study was the limited region targeted for the nucleotide sequencing. Using high throughput methods and next generation sequencing (NGS) could result in a great advancement in the assessment of these variations 33. These comprehensive methods can also further our knowledge on Iranian SARS-CoV-2 isolates.
In conclusion, the results of the present study indicated that the SARS-CoV-2 sequences from Iran have great similarity with Chinese and reference sequences although limited regions were sequenced and no variations were observed among them. To sum up, we found no identified differences between Iranian isolates and those from other regions. The current investigation was conducted as a preliminary study in Iran; further studies are to be performed to increase our knowledge regarding SARS-CoV-2 isolates.