Virus isolation and genome sequencing
Within a record time of less than a month, the novel coronavirus was identified, isolated and sequenced by three groups of Chinese scientists. A large consortium coordinated by W. Tan from the Chinese Centers for Disease Control and Prevention (Chinese CDC) obtained eight complete viral genome sequences from nine patients either by direct RNA isolation and sequencing from bronchoalveolar lavage fluid (BALF) or from classical virus isolation on human airway epithelial cells (Lu et al., 2020). The eight genomes showed sequence identity of 99.98%, i.e. only four nucleotides differed out of the 30 000 nucleotide‐long single‐stranded RNA genomes. This level of viral genomic identity isolated from different human subjects is unusual for an RNA virus that has been circulating for a long time in the human population. This observation suggests a recent single spill‐over event from an animal source into humans. Geneticists estimated that this event might have occurred in November 2019. The closest relatives of the new virus are coronaviruses isolated from bats in Eastern China, but they shared only 87.6% sequence identity with the new isolates. Therefore, viral taxonomists consider the novel coronavirus as a new viral species belonging to clade 2 of the Sarbecovirus group, acronym for SARS comprising betacoronaviruses. With the SARS virus, the new isolates shared even less, namely 79% sequence identity. While bats are still considered the most likely source for this novel coronavirus, bats were already hibernating at the time of onset of this epidemic and no bats were sold at the Huanan food market in Wuhan, suggesting an intermediate animal host where adaptation to human transmission might have occurred. Live hedgehogs, badgers, snakes and turtledoves were sold at the Huanan market. Claims were made, but were not (yet) substantiated that snakes or pangolins were intermediate hosts for creating the coronavirus by recombination events. W. Tan and colleagues, who now constitute the China Novel Coronavirus Investigating and Research Team, described subsequently the isolation of further coronaviruses from three patients in Wuhan who tested negative for 18 viral and four bacterial respiratory pathogens. These viruses were closely related to those of the earlier nine patients. In human airway epithelial cells, the cytopathic effect of this virus was a lack of cilium beating (Zhu et al., 2020).
Another group led by Y.‐Z. Zhang from the zoonosis group of the Chinese CDC in Beijing determined the genome of a closely related coronavirus by deep meta‐transcriptomic sequencing of the bronchoalveolar fluid of a patient working at the seafood market of Wuhan (Wu et al., 2020c). Analysis of the cell receptor interacting viral S (spike) gene showed indications of a recombination event. Protein modelling suggested the human angiotensin‐converting enzyme II (ACE2) protein as receptor.
Z.‐L. Shi from the Wuhan Institute of Virology, and collaborators, presented a detailed characterization of the viruses detected in seven patients from Wuhan suffering from severe pneumonia. Six of them were professionally associated with the local food market (Zhou et al., 2020). Metagenomics of BALF yielded coronaviruses that shared 99.9% sequence identity between the patients and the other coronavirus isolates from Wuhan. Using primers corresponding to a region in the S gene, these scientists developed a sensitive and outbreak‐specific PCR test providing a diagnostic tool for the virus detection in BALF or alternatively oral swabs of patients. For one patient, the researchers demonstrated an IgM seroconversion followed by an IgG seroconversion when using ELISA technique with the viral nucleocapsid as target antigen. Three patients showed specific IgM responses indicating an acute infection. This serological evidence is important since the full set of Koch’s postulates for the identification of a new pathogen has not yet been fulfilled. From one patient, they isolated a virus that caused cytopathic effects on the established Vero cell line after 3 days of incubation. The virus‐infected cells were stained with specific antibodies and were observed by fluorescence microscopy, allowing the establishment of a virus neutralization test. All patients developed neutralizing serum antibodies. While HeLa cells could not be infected with the novel coronavirus, this cell line became susceptible when expressing the ACE2 protein of human, bat and civet (but not of mouse) origin indicating that this novel coronavirus uses this protein as a cell receptor, as did the SARS coronavirus from the 2002 epidemic.
Zhang et al. (2020a) compared 27 novel coronavirus genomes isolated from patients in three Chinese cities (Wuhan, Zhejiang and Guangdong) and Thailand, all of whom had contact to Wuhan. The genomes were very similar but could still be classified into six genogroups, suggesting that mutations had accumulated within patients during the current outbreak. Curiously, the most basal genogroups were detected in Guangdong, and not Wuhan, isolates. Another group did an in‐depth annotation of the genomes of the novel coronaviruses with SARS virus and identified 380 amino acid substitutions affecting all predicted proteins of the viral genome (Wu, Peng et al., 2020b).