Chinese researchers mobilize to classify 2019-nCoV

By Samantha Black, PhD, The Science Advisory Board staff writer

February 3, 2020 -- In response to a recent outbreak of the novel coronavirus (2019-CoV), scientists in China have uncovered genetic similarities with severe acute respiratory syndrome (SARS) coronaviruses by analyzing patient samples from the source of the outbreak. Their findings provide crucial evidence that will aid in the classification and identification of 2019-CoV, according to a new report published in Nature on February 3.

Coronaviruses have been the source of several infectious disease epidemics in humans, such as SARS and Middle East respiratory syndrome (MERS). The current 2019-CoV outbreak began in December 2019, when an outbreak of respiratory illness developed, with patients presenting with symptoms of fever, dry cough, dyspnea, headache, and pneumonia. Most early 2019-CoV cases were in people who visited the Huanan seafood market; however, the spread of the disease has now progressed to human-to-human transmission worldwide.

Samples from seven patients with severe pneumonia (six of whom were seafood market workers) were sent to a laboratory at the Wuhan Institute of Virology (WIV) for pathogen diagnosis and were analyzed by researchers from the WIV, the Center for Biosafety Mega-Science, and the Chinese Academy of Sciences. Initial analysis included pan-CoV polymerase chain reaction (PCR) primers, which resulted in five PCR-positive samples.

A sample of bronchoalveolar lavage fluid went through metagenomic analysis using next-generation sequencing (NGS) to identify potential etiological agents. Of the 10,038,758 total reads, or 1,582 total reads obtained after human genome filtering, 1,378 (87.1%) matched sequences of SARSr-CoV. The researchers used the 29,891-bp genome of SARS-CoV BJ01, sharing 79.5% sequence identity, as the reference for remapping in NGS. The team identified the full-length genome sequences of four additional samples that were more than 99.9% identical to each other using NGS and PCR. The sequences were submitted to the Global Initiative on Sharing All Influenza Data (GISAID).

Upon first phylogenetic analysis, evidence appeared to indicate that 2019-nCoV and SARS-CoV belong to the same species. However, 2019-nCoV genes shared less than 80% sequence identity with SARS-CoV. The virus genome contains six major open reading frames common to coronaviruses and other accessory genes. There were seven conserved replicase domains in the open reading frames that were 94.6% sequence identical between 2019-nCoV and SARS-CoV. So while the two CoVs are clearly related, it may not be as close of a relationship as previously thought.

Upon further analysis, the researchers found a short RNA-dependent RNA polymerase (RdRp) region from a bat coronavirus, termed BatCoV RaTG13, found in China that showed high sequence identity to 2019-nCoV. There was a 96.2% sequence identity between the two samples. Phylogenetic analysis of the full-length genomes, RdRp, and receptor-binding protein spike (S) gene sequences show that RaTG13 is the closest relative of 2019-nCoV and that the two coronaviruses have a distinct lineage from other SARS-CoVs.

The S gene was highly divergent from other CoVs, with less than 75% sequence identity shared with all previously described SARS-CoVs except a 93.1% identity to RaTG13. The close phylogenetic relationship to RaTG13 provides evidence for a bat origin of 2019-nCoV. The major differences between 2019-nCoV and other SARS-CoVs are short insertions in the N-terminal domain and residue changes in the receptor-binding motif. These changes may confer sialic acid binding activity like MERS-CoV, but this hypothesis needs further testing and confirmation.

Antibodies isolated from patients infected with 2019-nCoV have the potential to neutralize the virus. A previously identified horse antibody against SARS-CoV also neutralizes the virus at a low serum dilution, but whether anti-SARS-CoV antibodies cross-react with 2019-nCoV or not needs to be confirmed using serum from humans who have convalesced from SARS-CoV infection.

The team also determined that 2019-nCoV likely enters via the same route as SARS CoVs: the cell receptor angiotensin-converting enzyme II (ACE2). The researchers conducted virus infectivity studies using HeLa cells expressing or not expressing ACE2 proteins in Chinese bats, civets, pigs, and mice. They found that 2019-nCoV can use ACE2 as an entry receptor in the ACE2-expressing cells but not in the ACE2-negative cells, indicating that it is likely a receptor for the virus.

While this report helps explain the origin of the virus, many unanswered questions regarding the epidemic remain, including the transmission routine of the virus among hosts, virulence, treatment options, and how to actively surveil coronaviruses in the future.

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