key: cord-0956424-sirtp2gi authors: Hassan, Sk. Sarif; Choudhury, Pabitra Pal; Basu, Pallab; Jana, Siddhartha Sankar title: Molecular conservation and differential mutation on ORF3a gene in Indian SARS-CoV2 genomes date: 2020-06-12 journal: Genomics DOI: 10.1016/j.ygeno.2020.06.016 sha: 712c92eb4ae1ea6dd4e06d290d00eec9d9330639 doc_id: 956424 cord_uid: sirtp2gi Abstract A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory protein in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty-two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2's virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservation of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2. Since December 2019, the coronavirus disease (COVID- 19) due to the severe acute respiratory syndrome (SARS) originating from Wuhan, China, has been causing a pandemic across the world [1, 2] . The causative virus, SARS-CoV2 is a positive-stranded RNA virus with genome size approximately of 30000 bases. The genome of SARS-CoV2 contains twenty-nine open reading frames (ORFs) [3, 4] . Among the twenty-nine ORFs, there are sixteen nonstructural proteins (nsps), four structural proteins (E, M, N, S), and six or seven accessory proteins such as ORF3a, ORF6, ORF7a, ORF7b, ORF8 and ORF10 [5, 6, 7] . SARS-CoV2 has been thought to be evolved due to rapid mutation, and recombination with existing other coronavirus in the body. They can alter tissue tropism, cross the species barrier and adapt to different epidemiological situations [8] . Sequence similarity based phylogeny infers that the SARS-CoV2 forms a distinct lineage with Bat-SARS-like coronaviruses that belong to the genus Beta-coronavirus (  -CoVs) [9] . The [25] . Note that, except the genomes MT012098, MT050493 all the other thirty genomes belong to the L-type as per classification made in the article [26] . A set of brief remarks on the accessory protein coding genes across the thirty-two genomes from the Indian patients is given in Table 1 . The proteins ORF7a, ORF6 and ORF10 are 100% conserved in the thirty-two SARS-CoV2 genomes of Indian origin. It is noteworthy that after some days (while the manuscript was under-review) on May 24, 2020, in the Indian genomes, some missense mutations ove r the protein ORF7a, ORF7b and ORF8 are found as reported in the article [27] . However, there are four different types of ORF3a genes that are found based on single-point mutations. Table 1 : Accessory proteins coding genes with associated remarks based on the thirty two genomes from India Accessory Gene Remarks Based on the thirty two Indian Genomes Three single-point mutations ( viz. G to T and C to T) are found in ORF3a J o u r n a l P r e -p r o o f gene across the thirty genomes. 100% identical across all the thirty two genomes. 100% identical across all the thirty two genomes. 100% identical except in the genome MT435082. From 318th onwards 20 ambiguous base N are placed. 100% identical across all the thirty genomes. 100% identical except in the genomes MT435081 and MT435082. Note that MT435081 and MT35082 contain the truncated ORF8 gene. In the truncated genes there is a point mutation from C to T . Note that ORF8 and ORF7a are exactly of same length but it does not have any significant similarity. In Indian patients, we found twenty-two ORF3a-Type-1 and seven ORF3a-Type-2 genomes among the thirty-two genomes of the Indian patients. The rest of the two types of mutations (we have seen 2+1=3 genomes) are Indian patients specific and have only one base difference with ORF3a-Type-2 and two bases differences from the 50 ORF3a-Type-1. We named these two Indian groups as ORF3a-Type-3 and ORF3a-Type-4 (refer to Table 2 ). The nucleotide frequencies, length and some associated remarks of the four types of ORF3a genes of SARS-CoV2 genomes of the Indian patients including the ORF3a genes of the pangolin and Bat CoV are presented in Table 2 . Clearly, in the present SARS-CoV2 genomes, the one amino acid E, Glutamic acid is inserted after 240th aa of the ORF3 protein sequence into the ORF3a protein sequence which is shown in the The Fig.3 follows that the ORF3a-Type-3 is obtained by two single point mutation (G to T) Table 4 and 5. In order to determine the molecular level conservation and descriptions of the ORF3a genes across different hosts as mentioned, some methods are discussed [28, 29, 30, 31, 32, 33] , which would be subsequently used. Shannon entropy is a measure of the amount of information (measure of uncertainty). Conservation of each of the four nucleotides has been determined using Shannon entropy [34, 35] . Note that it is assumed In addition to the different conservation SEs, some basic derivative features such as nucleotide frequency and density, frequency of all possible sixteen dimers, frequency of codon usages, frequency of amino acids in the protein seq uence encoded by the ORF3a gene, GC content, pyrimidine density are obtained for a given ORF3a gene sequence [28, 30] . It is worth noting that the first positive frame has been considered to determine codons and double nucleotides over a given gene. The counts of the nucleotide bases, length, GC content and pyrimidine density (py density) and the conservation Shannon entropy (ConV_SE) of the seventeen ORF3a genes across three different hosts are tabulated in Table 6 . The density of each nucleotide bases across the seventeen ORF3a genes are plotted in the Table 6, respectively. In each ORF3a gene the density of T is maximum and G is minimum. Also, it is noted the density of C dominates that of G over all the ORF3a genes of three different hosts. The ORF3a genes are pyrimidine-rich with percentage approximately 53% across different genomes as mentioned in the Table 6 . Also, the ORF3a possesses the highest GC content across the Bat CoV genomes and which is ranging from 39.68% to 41.34%. After a single mutation, the GC content of ORF3a-Type-2 is slightly reduced to 39.5% from that of the ORF3a-Type-1 gene. The GC content of Pangolin CoVs is turned out to be minimum and that is 39.13%. The GC content of ORF3a-Type-2 gene and ORF3a of MT040333 is identical though the density of G and C are slightly different in the respective sequences. The ORF3a genes across fifteen different genomes of CoV of the three hosts are highly conserved with equally likely presence of the four nucleotide bases as the Conv_SE for all the genes are turned out to be approximately 1. Based on features of the ORF3a gene across the seventeen CoV genomes, as mentioned in the Table 6 , a phylogeny has been developed as shown in Fig.6 . All possible words consisting letters from the set { , , , } A T C G of length two are commonly known as dimers. The frequency of dimers and conservation Shannon entropy of dimers (Dim_SE) over the seventeen ORF3a genes across various genomes of CoV are presented in the Table 7 . Also, a graphical representation of the frequency of the dimers of four types (dimers start with A, T, C and G) are given in Fig.7 . Based on the frequency of dimers across the ORF3a genes over the geno mes the following phylogeny is made in Fig.8 . The frequency usages of all the codons over the ORF3a genes across the SARS-CoV2 genomes of Indian patients including genomes of Pangolin and Bat CoVs are given in Table 8 The Codon_SE of ORF3a genes across the genomes are ranging from 0.9111 to 0.9289 and this emerges to a certain degree of uncertainty of codon conservation over the gene. The following phylogeny of the seven genomes is made by using the frequency of codon usages over the gene ORF3a, as shown in Fig.9 . The frequency of amino acids over the gene ORF3a across the genome of Indian patients, Pangolin and Bat are presented in the Table 9 . All the twenty amino acids are present over the gene ORF3a across all the genomes and it is turned out that the ORF3a protein is Luicine-rich with percentage approximately 10%. It is worth mentioning that the ORF3a gene of SARs-CoV genomes was Table 9 . Based on the frequency distribution of amino acids the following phylogeny (Fig.11) Among all the accessory proteins of SARS-CoV2, ORF3a is found to be very much important in SH conceived the problem. SH, PPC, PB and SSJ analysed the data and result. SH wrote the initial draft which was checked and edited by all other authors to generate the final version. The authors do not have any conflicts of interest to declare. 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We also acknowledge the anonymous reviewers and editors for their careful reading the manuscript and putting constructive suggestions.J o u r n a l P r e -p r o o f Journal Pre-proof