Coronaviridae (CoVs) are the largest known single-stranded RNA viruses. 1 Human coronaviridae have been discovered for SARS 3 and more recently also for 2019-nCoV, 4 suggesting events of inter-species transmissions. 5 No vaccine for 2019-nCoV has been publicly released, but a World effort has arisen toward the characterization of the molecular determinants and evolutionary features of this novel virus. An initial comparison of 10 genomic sequences from 2019-nCoV specimens has reported a low heterogeneity of this viruses with intersample sequence identity above 99.9%. 6 There are currently 54 2019-nCoV complete genome sequences from the Global Initiative for Sharing all Influenza Data (Gisaid 7 ) and from GenBank, 8 plus two partial sequences obtained by the Spallanzani hospital in Rome, Italy (also from Gisaid).
In this short report, we set out to characterize the heterogeneity of all 2019-nCoV genomes and proteomes available at the moment of the study, comparing them to other representative coronaviridae, specifically SARS, MERS, and BCoV.
We will generate phylogenetic trees of the 2019-nCoV cases and apply entropy-based analyses of position-wise variance and categorical principal component analysis (CATPCA) as an alternative method to estimate the sequence distance between all analyzed viruses.
All genomic sequences were collected on 02 February 2020 from GenBank 8 or Gisaid. 7 MSA was performed using MUSCLE v3.8.31. 9 MSA visualization was generated via Jalview v 2.11.0. 10 Phylogenetic model generation and tree visualization were done using MEGAX v 10.1.7, 11 using the Maximum Likelihood method and Tamura-Nei model. 12 The tree structure was validated by running the analysis on 100 bootstrapped input datasets. 13 CATPCA was performed on R version 3.6.1 using the package FactoMineR. 14 Specifically, an MSA FASTA file from MUSCLE is loaded in R and converted into a categorical matrix, with genomes as rows and nucleotide coordinates as columns. Factors are defined as A, C, G, T, N, or-(gap), as described in results. Then, the FactoMineR multiple correspondence analysis algorithms is run with default parameters and custom R functions are used to plot the component values for each genome.
Pairwise protein identity and coverage were calculated using BLAST protein v2.6.0 15 with BLOSUM62 matrix and default parameters. Nucleotide sequence identity and coverage were calculated using BLAST nucleotide v2.6.0. 15 Prediction of structural protein disorder was performed using GLOBPLOT2, an implementation of the Russell/Linding algorithm. 16 3 | RESULTS
We collected 53 full genomic 2019-nCoV sequences from the Gisaid database (Table S1) Similarly to a previous report with 10 virus specimens, 6 we detected very high conservation between the 56 analyzed 2019-nCoV genomes, with sequence identity above 99%. We found a bat CoV genome (Gisaid EPI_ISL_402131) with 96.2% sequence identity (and query coverage above 99%) to the 2019-nCoV reference sequence (NC_045512.2), while the previously reported closest bat CoV (bat-SL-CoVZC45) has a sequence similarity of 88%. 6 The reference human SARS genome (NC_004718.3) appears more distant from the reference 2019-nCoV, with sequence identity of 80.26% and query coverage of 98%.
We aligned all the 70 coronavirus sequences using MUSCLE 9 and inferred the evolutionary relationships between these sequences with a Tamura-Nei Maximum Likelihood estimation 12 The global multiple sequence alignment (MSA) is available as Supporting Information File S1.
Given the high homogeneity between 2019-nCoV genomes, we developed a novel method to classify genomic sequences, based on CATPCA. 14 On the other hand, position 28151 falls within ORF8 and is characterized by the presence of either a C or a U. This causes a Ser/Leu change in amino acid (aa) 84, which can affect the conformation of the peptide, given that Serine is a polar amino acid, and Leucine is nonpolar.
Aa84 appears to be nonconserved also across other coronaviridae ( Figure 4A , black arrow).
We analyzed the alternative isoforms of 2019-nCoV ORF8-aa84 alternative isoforms, dubbed ORF8-L (Leucine) and ORF8-S (Serine).
Unfortunately, no crystal structures of close homologs to the ORF8 protein are available for a reliable homology modeling to measure the structural impact of this amino acid substitution. The closest 3D model to 2019-nCoV ORF8 available in Protein Data Bank 19 is a short 22 amino acid stretch in the protein entry 6P65, with a nonsignificant E-value of 0.848. We, therefore, employed de novo methods to infer the structural features of ORF8. One important effect we could detect is a significant effect of Serine in ORF8-S in inducing structural disorder in the protein C-terminal portion, which is not predicted to be present in the ORF8-L ( Figure 4B ), using the Russell/Linding algorithm. 16 Moreover, it did not escape our attention that the ORF8-S could theoretically generate a novel phosphorylation target for the mammalian host Serine/Threonine kinases of the host organism. So, we searched for ORF8 homologous substrates in the Mammalia NCBI nr protein database, but could not find matches within the E-value threshold of 1.
We performed a cross-species analysis for all proteins encoded by the 2019-nCoV and its relatives. We, therefore, analyzed the protein sequences encoded by all ORFs in these genomes 