How the Chinese Academy of Medical Sciences used 5 cases to find a new coronavirus

How was the “cause” of the new coronavirus pneumonia identified? A paper published in the English edition of the Chinese Medical Journal on January 30th by the Chinese Academy of Medical Sciences and other scientific research institutes documented the discovery process. The research team collected clinical data and bronchial alveoli irrigation (BAL) samples from 5 patients with severe pneumonia in Wuhan Jinyintan Hospital, and used next-generation sequencing techniques to detect nucleic acids extracted from irrigation lotions.

Finally, the team isolated the virus and established the most likely system development tree.

How the Chinese Academy of Medical Sciences used 5 cases to find a new coronavirus

Sequencing results showed that each of the five samples had a previously unknown beta-coronavirus (SARS, MERS is also a genus).

The five virus sequences are highly homogenetoted, with nucleotide similarity of 99.8% to 99.9%. They are similar to nucleotides of SARS coronaviruses and MERS coronaviruses at 79.0% and 51.8%, respectively, and may use the same receptors as SARS viruses.

From the point of view of system development, this new coronavirus is the closest to the SARS-like coronavirus found in the body of Zhejiang Zhoushan chrysanthemum manta ray in 2005, with nucleotide similarity of 87.6% to 87.7%, but in different branches. In addition, they all include a complete open reading box 8 gene region, further suggesting that the new virus may have originated in bats.

Samples from the deep lung of 5 patients

The five patients, who were admitted between 15 December and 22 December 2019, were admitted to Jinyintan Hospital between 18 and 29 December 2019, showing fever, cough, and different white blood cell counts, but the number of lymphocytes was generally low. The patient’s condition progressed rapidly, as of the completion of the paper there were 4 cases of acute respiratory distress syndrome, 2 cases of in vitro membrane pulmonary oxygenation, 1 case discharged from hospital, 1 case of death. Of these, 2 cases did not report contact with the South China Seafood Market.

Specifically, case 1 is 65-year-old male, South China seafood market merchants, when the disease coughless sputum, sustained fever, 16 days after the development of severe shortness of breath.

Case 2 is a 49-year-old woman, south China seafood market employees, the onset of high fever, dry cough, 5 days after the emergence of breathing difficulties.

Case 3 was a 52-year-old woman with a fever, a cough, a glassy, only case with out acute respiratory distress syndrome, and was discharged from the hospital on 8 January (17 days after the onset of symptoms).

Case 4 was a 41-year-old male with high fever and dry cough at the onset of the disease, and developed acute respiratory distress syndrome after 7 days.

Case 5 was a 61-year-old male who worked at a seafood market in South China, was hospitalized after 7 days with fever, cough and breathing difficulties, combined with chronic liver disease and celiac mucus tumor, and died of multiple organ failure.

From December 24, 2019 to January 1, 2020, the researchers sampled the five patients as part of a public health emergency survey approved by the National Health And Reform Commission.

Stitch out the whole genome of the virus

Each sample contained 200 microliters of bronchial alveoli irrigation, from which the researchers extracted nucleic acids and obtained 50 microliters of elute.

The raw sequencing data was filtered to obtain high-quality data from 12.0% to 92.0%, of which sample No. 5 had the highest viral read ratio. Almost all virus-read data belongs to coronaviruses. Stitching, the researchers came up with the entire genome sequence of the virus. The proportion of mapping of whole genome sequences for each sample ranged from 0.27% of sample 4 to 85% of patients no. 5.

In addition, the sample satout almost no known bacterial pathogens, including streptococcus, Baumabacillus and pseudomonas.

The nucleotide similarity of the five stitched-up coronavirus genome-wide sequences was as high as 99.8% to 99.9%. The genomic structure is 5′-ORF1ab-S-E-M-N-3′ similar to the most famous bat SARS-like coronavirus. In addition, they have an open reading box with sarbecovirus sub-characteristics.

New virus! It could have originated in bats.

Homologous analysis showed that these whole genome sequences were similar to SARS coronaviruses and MERS coronaviruses at 79.0% and 51.8%, respectively.

The most similar is the SARS-like coronavirus (SL-CoV ZC45 and ZXC21) carried by two chrysanthemum bats found in Zhoushan in 2005. This is 87.6% to 87.7%, but surprisingly, on the most conservative RNA polymerase sequence of coronaviruses, the sample virus and the bat virus SL-CoV ZC45 showed only 89.0% consistency.

According to the standards of the International Committee on Virus Classification, consistency of less than 90% can be determined as a new virus.

The system development tree that was constructed shows that the new coronary virus is closely related to bat SL-CoV ZC45 and ZXC21 viruses, but belongs to an independent evolutionary branch. The new virus contains ORF3 and the complete ORF8 gene region, which is the characteristic sequence of bat-derived coronaviruses. All indications are that it may well have originated in bats.

In addition, it may have a receptor binding region for amino acid sequences similar to the SARS virus, perhaps using the same receptor.

The National Health and Reform Commission’s Key Laboratory of Pathogen Systems Biology and the Christopher Merrier Laboratory of the Chinese Academy of Medical Sciences/Beijing Concord Medical College are the first units of the paper.

On January 12, the World Health Organization provisionally named the new coronavirus 2019nCoV, making it the first infectious coronavirus in history.

On January 26, the whole genome sequence of the five new 2019 coronaviruses was included in the National Genome Science Data Center (NGDC).