NEW YORK – Researchers at the Chinese Center for Disease Control and Prevention and their collaborators have sequenced the 2019 novel coronavirus (2019-nCoV) pathogen from patient samples and have found it to be genetically distinct from the severe acute respiratory syndrome (SARS) virus that caused an epidemic in 2002 and 2003, as well as from the Middle East respiratory syndrome (MERS) virus that was detected in 2012.
As they described on Wednesday in The Lancet, the researchers sequenced samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan, China. Four samples were sent to BGI for sequencing on the company's DNBSEQ-T7 platform. In addition, Chinese CDC researchers obtained complete and partial 2019-nCoV genome sequences from six of the samples (two from the same patient) using a combination of Sanger, Illumina, and Oxford Nanopore sequencing. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes and the researchers determined the terminal regions by rapid amplification of the cDNA ends.
In all, they developed ten genome sequences of 2019-nCoV from the nine patients, eight of which were complete. They then performed a phylogenetic analysis of the 2019-nCoV genomes and other coronavirus genomes to determine the evolutionary history of the new virus and to help infer its likely origin.
"Notably, 2019-nCoV was closely related (with 88 percent identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79 percent) and MERS-CoV (about 50 percent)," the authors wrote. "Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives, bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV."
SARS emerged in Guangdong, China, in November 2002 and resulted in more than 8,000 human infections and 774 deaths in 37 countries. MERS, which was first detected in Saudi Arabia in 2012, has been responsible for 2,494 laboratory-confirmed cases of infection and 858 fatalities since September 2012, including 38 deaths following a single introduction into South Korea.
The researchers also found that the eight complete 2019-nCoV genomes were more than 99.98 percent identical, which indicated that the virus had very recently emerged into the human population. The largest nucleotide difference was four mutations.
Importantly, they added, 2019-nCoV was sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. And although the phylogenetic analysis suggested that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent the intermediate host that facilitated the emergence of the virus in humans.
"Clearly, this infection is a major public health concern, particularly as this outbreak coincides with the peak of the Chinese Spring Festival travel rush, during which hundreds of millions of people will travel through China," the authors noted. "As a typical RNA virus, the average evolutionary rate for coronaviruses is roughly 10– ⁴ nucleotide substitutions per site per year, with mutations arising during every replication cycle. It is, therefore, striking that the sequences of 2019-nCoV from different patients described here were almost identical, with greater than 99.9 percent sequence identity. This finding suggests that 2019-nCoV originated from one source within a very short period and was detected relatively rapidly."
However, they added, constant surveillance of arising mutations will be needed. Although phylogenetic analysis showed that a bat reservoir exists for coronaviruses in general and for 2019-nCoV in particular, several facts suggest that another animal is acting as an intermediate host between bats and humans. First, the outbreak was first reported in late December 2019, when most bat species in Wuhan are hibernating. Second, no bats were sold or found at the Huanan seafood market. Third, the sequence identity between 2019-nCoV and its close relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21 was less than 90 percent, which is reflected in the relatively long branch between them. Therefore, bat-SL-CoVZC45 and bat-SL-CoVZXC21 are not direct ancestors of 2019-nCoV.
Further, the researchers said, bats acted as the natural reservoir in both SARS-CoV and MERS-CoV, with another animal acting as an intermediate host and humans as terminal hosts. It seems therefore likely that 2019-nCoV might have also been initially hosted by bats, and might have been transmitted to humans via currently unknown animals sold at the Huanan seafood market.
"Our results suggest that recombination events are complex and are more likely occurring in bat coronaviruses than in 2019-nCoV," the investigators concluded. "Hence, despite its occurrence, recombination is probably not the reason for emergence of this virus, although this inference might change if more closely related animal viruses are identified."