key: cord-259152-pwvcwlh8 authors: Ji, Wei; Wang, Wei; Zhao, Xiaofang; Zai, Junjie; Li, Xingguang title: Cross‐species transmission of the newly identified coronavirus 2019‐nCoV date: 2020-02-19 journal: J Med Virol DOI: 10.1002/jmv.25682 sha: doc_id: 259152 cord_uid: pwvcwlh8 The current outbreak of viral pneumonia in the city of Wuhan, China, was caused by a novel coronavirus designated 2019‐nCoV by the World Health Organization, as determined by sequencing the viral RNA genome. Many initial patients were exposed to wildlife animals at the Huanan seafood wholesale market, where poultry, snake, bats, and other farm animals were also sold. To investigate possible virus reservoir, we have carried out comprehensive sequence analysis and comparison in conjunction with relative synonymous codon usage (RSCU) bias among different animal species based on the 2019‐nCoV sequence. Results obtained from our analyses suggest that the 2019‐nCoV may appear to be a recombinant virus between the bat coronavirus and an origin‐unknown coronavirus. The recombination may occurred within the viral spike glycoprotein, which recognizes a cell surface receptor. Additionally, our findings suggest that 2019‐nCoV has most similar genetic information with bat coronovirus and most similar codon usage bias with snake. Taken together, our results suggest that homologous recombination may occur and contribute to the 2019‐nCoV cross‐species transmission. China has been the epicenter of emerging and re-emerging viral infections that continue to stir a global concern. In the last 20 years, China has witnessed several emerging viral diseases, including an avian influenza in 1997, 1 and other wildlife animals were also sold. 4 On 3 January 2020, WMHC updated the number of cases to a total of 44 with 11 of them in critical condition. On 5 January, the number of cases increased to 59 with 7 critically ill patients. The viral pneumonia outbreak was not caused by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), influenza virus, or adenovirus as determined by laboratory tests. 4 On 10 January, it was reported that a novel coronavirus designated 2019-nCoV by the World Health Organization (WHO) 5 was identified by high-throughput sequencing of the viral RNA genome, which was released through virological.org. More significantly, the newly identified 2019-CoV has also been isolated from one patient. The availability of viral RNA sequence has made it All authors contributed equally to this work. possible to develop reverse-transcription polymerase chain reaction (RT-PCR) methods for the detection of viral RNA in samples from patients and potential hosts. 6 As a result, 217 patients were confirmed to be infected with the 2019-nCoV, and 9 patients died as of 20 January 2020. Several patients from Wuhan were also reported in Thailand, Singapore, Hong Kong, South Korea, and Japan. Highthroughput sequencing of viral RNA from patients' samples has identified a novel coronavirus designated 2019-nCoV by the World Health Organization. Currently, a total of 14 full-length sequences of the 2019-nCoV were released to GISAID and GeneBank. The coronavirinae family consists of four genera based on their genetic properties, including genus Alphacoronavirus, genus Betacoronavirus, genus Gammacoronavirus, and genus Deltacoronavirus. 7 The coronavirus RNA genome (ranging from 26 to 32 kb) is the largest among all RNA viruses. 8 Coronavirus can infect humans and many different animal species, including swine, cattle, horses, camels, cats, dogs, rodents, birds, bats, rabbits, ferrets, mink, snake, and other wildlife animals. 7, 9 Many coronavirus infections are subclinical. 7, 9 SARS-CoV and MERS-CoV belong to the Betacoronavirus genus and are zoonotic pathogens that can cause severe respiratory diseases in humans. 7 The outbreak of viral pneumonia in Wuhan is associated with history of exposure to virus reservoir at the Huanan seafood wholesale market, suggesting a possible zoonosis. The seafood market also sold live animals such as snakes, marmots, birds, frogs, and hedgehogs. Currently, there is no evidence suggesting a specific wildlife host as a virus reservoir. Studies of relative synonymous codon usage (RSCU) bias between viruses and their hosts suggested that viruses tends to evolve codon usage bias that is comparable to their hosts. 10, 11 Results from our analysis suggest that 2019-nCoV has most similar genetic information with bat coronovirus and has most similar codon usage bias with snake. More interestingly, an origin-unknown homologous recombination may occured within the spike glycoprotein of the 2019-nCoV, 5 which may explain its cross-species transmission, and limited person-person spread. The newly sequenced Beta-coronavirus (MN908947) genome was downloaded from the GenBank database. Five hundred closely related sequences were also downloaded from GenBank. Out of them, 271 genome sequences (>19 000 bp in length) were used in this study together with the above-described Beta-coronavirus (2019-nCoV, MN908947) genome sequence (Table S1 ). The geographic origins of the sequences were from Bulgaria (n = 1), Canada (n = 2), China (n = 67), Germany (n = 1), Hong Kong (n = 5), Italy (n = 1), Kenya (n = 1), Russia (n = 1), Singapore (n = 24), South Korea (n = 1), Taiwan (n = 11), United Kingdom (n = 2), United States of America (n = 67), and unknown (n = 88). Sequences were aligned using MAFFT v7.222, 12 followed by manual adjustment using BioEdit v7.2.5. 13 Phylogenetic trees were constructed using maximum-likelihood methods and general time-reversible model of nucleotide substitution with gamma-distributed rates among sites (GTR+G substitution model) in RAxML v8.0.9. 14 Support for the inferred relationships was evaluated by a bootstrap analysis with 1000 replicates and trees were midpoint-rooted. To investigate the putative parents of the 2019-nCoV, we performed Similarity and Bootscanning plot analyses based on the Kimura two-parameter model with a window size of 500 bp, step size of 30 bp using SimPlot v.3.5.1. 15 We divided our data set into four clades, the newly discovered 2019-nCoV sequence was grouped as the query sequence. The closest relative coronaviruses (bat-SL- To estimate the RSCU bias of the 2019-nCoV and its potential host(s), reported. 18 A heat map of RSCU was drawn with MeV 4.9.0 software. 19 The coronavirus and their potential hosts were clustered using a Euclidean distance method. 23 and classical swine fever virus. 18 Similarity plot analysis of the 2019-nCoV revealed that homologous recombination may occurred between Clade A strains (bat-coronaviruses) and the origin-unknown isolates, located within the spike glycoprotein that recognizes cell surface receptor (Figure 2 ). These characteristics indicate that cross-species transmission may be caused by homologous recombination. As parasitic microorganism, virus codon usage pattern resembles its host to some extent. The RSCU bias shows that the 2019-nCoV, bat-SL-CoVZC45, and snakes from China have similar synonymous codon usage bias ( Figure 3A , Table 1 Huanan Seafood Wholesale Market where many patients worked or had a history of exposure to wildlife or farm animals. In this study, we have performed an evolutionary analysis using The host range of some animal coronaviruses was promiscuous. 7 They caught our attention only when they caused human diseases such as SARS, MERS, and 2019-nCoV pneumonia. 4, 9, 28 It is critical to determine the animal reservoir of the 2019-nCoV to understand the molecular mechanism of its cross-species spread. Homologous recombination within viral structural proteins between coronaviruses from different hosts may be responsible for "cross-species" transmission. 27 Information obtained from RSCU analysis provides some insights to the question of wildlife animal reservoir although it requires further validation by experimental studies in animal models. Currently, the 2019-nCoV has not been isolated from animal species although it was obtained from one patient. Identifying and characterizing the animal reservoir for 2019-nCoV will be helpful for investigation of the recombination and for a better understanding of its person-to-person spread among human populations. 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Writing: WJ and XL. Data collection: JZ, WW, and XZ. Data analysis: WJ and XL. http://orcid.org/0000-0002-0818-5578