Team Zhang Yongzhen: new coronavirus infects human more effectively

category:Hot
 Team Zhang Yongzhen: new coronavirus infects human more effectively


u00b7Through the comparison of genome sequences, it was found that there was gene recombination between the new coronavirus and the SARS like virus infecting human beings or bats, but it could not be presumed that the gene recombination promoted the birth of the new coronavirus.

This paper was written by Professor Zhang Yongzhen of Shanghai public health clinical center and School of public health of Fudan University, who led the team research institute. It was submitted to nature on January 7, and was officially released nearly a month later. Professor Zhang is the first researcher to publish the sequence of the virus, which was published on January 10.

New infectious diseases such as SARS and Zika pose a major threat to public health. In spite of extensive research, how, when and where new diseases appear is still quite uncertain. A serious respiratory disease has recently been reported in Wuhan, Hubei Province, China. Since the first patient was hospitalized on 12 December 2019, at least 1975 cases have been reported as of 25 January 2020. Epidemiological investigation shows that the outbreak is related to the seafood market in Wuhan. We studied a patient working in the market who was admitted to Wuhan Central Hospital on December 26, 2019, suffering from severe respiratory syndrome, including fever, dizziness and cough. A new type of RNA virus of coronaviridae was identified by sequencing the macrogenomic RNA of bronchoalveolar lavage fluid samples, which is called wh-human-1 coronavirus. The phylogenetic analysis of the complete virus genome (29903 nucleotides) shows that the virus is most similar to a group of SARS like coronaviruses (beta coronavirus, sarbecovirus subgenus) (89.1% nucleotide similarity). The outbreak highlights that the ability of the virus to spill from animals is causing serious human diseases.

In order to investigate the possible pathogens associated with the disease, we collected bronchoalveolar lavage fluid (BALF) and sequenced its deep transcriptome. Clinical specimens were processed in the Biosafety Level 3 Laboratory of Shanghai public health clinical center. Through gene comparison test, 2019-ncov is most closely related to bat-sl-covzc45.

In order to better understand the potential of whcv infection in humans, the receptor binding domain (RBD) of its spike protein was compared with that of SARS CoV and SARS like cov in bats. The results show that 2019-ncov can effectively utilize human ACE2 as a cell entry receptor, thus potentially promoting human to human transmission.

At the same time, gene detection also shows that 2019-ncov has gene recombination, but there is no evidence that recombination promotes the emergence of whcv.

New infectious diseases such as SARS and Zika pose a major threat to public health. In spite of extensive research, how, when and where new diseases appear is still quite uncertain. A serious respiratory disease has recently been reported in Wuhan, Hubei Province, China. Since the first patient was hospitalized on 12 December 2019, at least 1975 cases have been reported as of 25 January 2020. Epidemiological investigation shows that the outbreak is related to the seafood market in Wuhan. We studied a patient working in the market who was admitted to Wuhan Central Hospital on December 26, 2019, suffering from severe respiratory syndrome, including fever, dizziness and cough. A new type of RNA virus of coronaviridae was identified by sequencing the macrogenomic RNA of bronchoalveolar lavage fluid samples, which is called wh-human-1 coronavirus. The phylogenetic analysis of the complete virus genome (29903 nucleotides) shows that the virus is most similar to a group of SARS like coronaviruses (beta coronavirus, sarbecovirus subgenus) (89.1% nucleotide similarity). The outbreak highlights that the ability of the virus to spill from animals is causing serious human diseases.

The patient in the study was a 41 year old male with no history of hepatitis, tuberculosis or diabetes. He was hospitalized in Wuhan Central Hospital on December 26, 2019, six days after the onset of the disease. Fever, chest distress, cough, pain and weakness were reported one week after treatment (Table 1). Cardiovascular, abdominal and neurological examinations were normal. Mild lymphocytopenia (less than 900 cells per cubic millimeter) was observed, but WBC and platelet counts were normal on the CBC test. In the blood chemistry test, the C-reactive protein (CRP, blood 41.4mg/l, reference range 0-6mg / L) was increased, and the levels of aspartate transaminase, lactate dehydrogenase and creatine kinase were slightly increased. According to the arterial blood gas (ABG) test, the patient suffered from mild hypoxemia with an oxygen level of 67mmhg. On the first day of admission (the sixth day after the onset of the disease), the chest X-ray picture was abnormal, accompanied by airspace shadow, such as turbid ground glass, and two lungs showed focal consolidation and plaque consolidation (extended data figure 1). CT scan of the chest showed bilateral focal consolidation, lobar consolidation and lamellar consolidation, especially in the lower lung. Chest X-ray showed diffuse patchy and fuzzy shadows on both sides on the 5th day (11th day) after admission. The preliminary etiology investigation excluded the presence of influenza virus, Chlamydia pneumoniae and Mycoplasma pneumoniae through the commercial pathogen antigen detection kit, and confirmed by PCR. Other common respiratory pathogens, including adenoviruses, were also negative by qPCR (extended data figure 2). Despite the combination of antibiotic, antiviral and glucocorticoid therapy, the patient showed respiratory failure and received a high flow of noninvasive oxygen supply. After three days of treatment, the patients condition did not improve and he was sent to intensive care unit (ICU). The patient was transferred to another hospital in Wuhan for further treatment six days after admission.

Table 1:

Extended data figure 1:

An epidemiological survey conducted by the center for Disease Control and Prevention (CDC) in Wuhan showed that the patient worked in the local indoor seafood market. It is worth noting that, in addition to fish and shellfish, a variety of live wild animals, including hedgehogs, badgers, snakes and birds (turtledoves), as well as animal carcasses and meat, are sold on the market before the outbreak. There are no bats for sale. Although the patient may have had contact with wild animals on the market, he recalled that there was no contact with live birds.

In order to investigate the possible pathogens associated with the disease, we collected bronchoalveolar lavage fluid (BALF) and sequenced its deep transcriptome. Clinical specimens were processed in the Biosafety Level 3 Laboratory of Shanghai public health clinical center. The total RNA was extracted from 200 u03bc lbal solution, and a metatranscriptome library was constructed for double terminal (150bp) sequencing, which was used for pair terminal (150bp) sequencing and pair terminal (150bp) sequencing, as mentioned above. In total, we generated 56565928 sequence readings that were assembled from scratch and screened for potential pathogens. Among the 384096 overlapping groups assembled by megahit 8, the longest (30474 nucleotides [NT]) has a high abundance and is closely related to bat SL covzc45 (GenBank accession No. mg772933), which was previously sampled in China, with NT identity of 89.1% (supplementary Tables 1 and 2). The genome sequence and terminal of the new virus were determined by rtpcr9 and 5 / 3RACE kits (Takara), respectively. The new virus was named wh-hunman-1 coronavirus (whcv) (also known as 2019-ncov), and its entire genome sequence (29903nt) has been transmitted to GenBank accession number mn908947. According to the whole genome remapping RNA SEQ data of whcv, 123613 reading segments were assembled, providing 99.99% genome coverage with an average depth of 6.04x (range: 0.01x-78.84x) (extended data figure 3). The viral load in BALF samples was estimated to be 3.95 u00d7 108 copies / ml by quantitative PCR (extended data figure 4).

Extended data figure 3

Extended data figure 4

The genome of whcv is characterized by sequence alignment of two representative members of beta coronavirus: SARS covtor2 (ay274119) and bat SL covzc45 (mg772933).

Based on the sequence alignment and ORF prediction, UTR and ORF of whcv were located. The genome of whcv virus is similar to these two coronaviruses (Fig. 1 and supplementary Table 3), and the gene sequence is 5 - replicase orf1ab-s-envelope (E) - membrane (m) - n-3. Whcv has a typical 5 and 3 terminal sequence of beta coronavirus, with 265nt at the 5 terminal and 229nt at the 3 terminal. The predicted length of orf1ab gene of whcv replicase was 21291nt, including 16 predicted non structural proteins (supplementary Table 4), followed by (at least) 13 downstream ORFs. In addition, whcv and SARS CoV in nsp1 share a highly conserved domain (llrkngnkg: amino acid 122130). The predicted length of S, orf3a, e, m and N genes of whcv was 3822, 828, 228, 669 and 1260nt, respectively. In addition to these ORF regions shared by all members of the sarbecovirus subtype, whcv is similar to SARS CoV in that it carries an ORF8 prediction gene (366nt in length) located between the ORF genes of M and n. The ORF function of whcv is predicted based on known coronaviruses and is given in supplementary table 5. In a way similar to that of SARS covtor2, it is easy to identify a leading transcriptional regulation sequence (TRS) and nine putative main TRS upstream of the ORF, and the putative conservative TRS core sequence appears in two forms - acgaac or cuaaac (supplementary table 6).

Figure 1

In order to determine the evolutionary relationship between whcv and previously identified coronavirus, we estimated phylogenetic tree based on nucleotide sequence of the whole genome sequence, orf1a and 1b of non structural protein gene, and major structural proteins encoded by s, e, m and n (Fig. 2 and extended data Fig. 5). In all phylogeny, the whcv is clustered with members of the sarbecovirus subclass, including SARS CoV, which caused the global SARS pandemic in 2002-2003, and many SARS like coronavirus samples from bats. However, according to the genes used, whcv changed the topological position within the subgenus sarbecovirus, suggesting the past recombination history in this group of viruses (Fig. 2 and extended data Fig. 5). Specifically, in the S-gene tree (extended data, figure 5), whcv is most closely related to bat-sl-covzc45, with amino acid (AA) identity of 82.3% (amino acid identity of 77.2% with SARS CoV; table 3 is supplemented), while in ORF1b phylogeny, whcv falls to the basic position in rotavirus subgenus (figure 2). This topological division was also observed in the system tree for estimating the conserved domain of pp1ab, which may reflect the recombination between bat sarbecovirus viruses (extended data figure 6).

Extended data figure 5

Figure 2

Extended data figure 6

In order to better understand the potential of whcv infection in humans, the receptor binding domain (RBD) of its spike protein was compared with that of SARS CoV and SARS like cov in bats. The RBD sequence of whcv is more similar to that of SARS CoV (73.8% - 74.9% amino acid identity) and SAS like CoV, including rb4874, rs7327 and rs4231 (75.9% - 76.9% amino acid identity), which can be entered into cells by human ACE2 receptor (supplementary table 7). In addition, whcvrbd was only one amino acid longer than SARS covrbd (extended data figure 7a). In contrast, SARS like CoVs in other bats, including RP3 strains that could not use human ACE2, had amino acid deletions at 473-477 and 460-472 positions compared with SARS CoVs (extended data figure 7a). The previously determined crystal structure (pdb2ajf) of SARS covrbd combined with human ACE2 shows that regions 473-477 and 460-472 interact directly with human ACE2, so it may be important in determining species specificity (extended data figure 7b). We predicted the three-dimensional protein structure of whcv, rs4874 and rp3rbd domains by using the SWISS-MODEL server through protein homology modeling, and compared them with the crystal structure of SARS covrbd domain (pdb2ghv) (extended data figure 7c-f). Consistent with sequence alignment, the predicted protein structure of whcv and rs4874rbd domains is closely related to that of SARS CoV, and different from that of RP3. In addition, the N-terminal of whcvs protein is more similar to that of SARS CoV, rather than other human coronaviruses that can bind to sialic acid (hku1 and OC43, extended data figure 8)). The amino acid sequence and predicted protein structure between whcv and SARS covrbd domains indicate that whcv can effectively utilize human ACE2 as cell entry receptor, thus potentially promoting human to human transmission.

Extended data figure 7

Extended data figure 8

In order to further characterize the putative recombination events in the evolutionary history of rotavirus, a recombinant detection program V4 (rdp4) was used to analyze the whole genome sequences of whcv and four representative coronaviruses - covrp3, covzc45, covzxc21 and SARS covtor2. Although the similarity chart shows that there may be recombination events between whcv and SARS CoV or SARS like cov (extended data figure 9), there is no significant evidence that the whole genome has been reconstructed. However, some evidence of past recombination (P < 3.147 u00d7 10-3 to P < 9.198 u00d7 10-9) was detected in the s genes of whcv, SARS CoV and bat SARS like cov (wiv1 and rsshc014). The similarity map suggested that there were recombination breakpoints at nucleotide 1029 and 1652 in the three regions of whcv S gene (Fig. 3). Whcv is closely related to bat-sl-covzc45 and bat-slcovzxc21 in the phylogeny from segments NT1 to 1029 and nt1652 to the end of the sequence, while whcv is closely related to SARS coronavirus that can be directly transmitted by humans and SARS like coronavirus (wiv1 and rsshc014) in bats in nt1030 to 1651 (RBD region). Although these recombination events seem to be common in sarbecovirus, there is no evidence that recombination promotes the emergence of whcv.

Extended data figure 9

Figure 3

(function() {(window. Slotbydup = window. Slotbydup| []). Push ({ID: u5811557, container: ssp_, async: true});}) ()); coronavirus is related to many infectious diseases in human, including SARS in 2002 / 3 and mers in 2012. Four other coronaviruses - human coronaviruses hku1, OC43, nl63 and 229E - are also associated with respiratory diseases. Although SARS like coronavirus 9 has been widely found in mammals including bats in China since 2005, the exact source of human coronavirus infection is still unclear. In this paper, we describe a novel coronavirus, whcv (2019 ncov), from BALF of a patient with severe respiratory disease in Wuhan, China. Phylogenetic analysis shows that whcv is a new type of virus in the genus betacoronavirus (sarbecovirus), so it has some genomic and systemic similarities with SARS CoV, especially in RBD. These genomic and clinical similarities with SARS, as well as their large presence in clinical samples, provide evidence for the association between whcv and ongoing respiratory disease outbreaks in Wuhan. Although isolation of the virus from only one patient is not sufficient to conclude that it causes respiratory symptoms, our findings are independently confirmed in other patients. The identification of several SARS like coronaviruses in bats has led to the assumption that these animals are the natural hosts of these viruses. Although SARS like virus has been widely recognized in bats in China, the same virus as SARS CoV has not been recorded. It is worth noting that whcv is most closely related to bat coronavirus, and even shows 100% amino acid similarity with bat SL covzc45 in nsp7 and E proteins. Therefore, these data suggest that bats may be hosts of whcv. However, since there were several animals on the market at the time of the first report of the disease, more work was needed to determine the natural host and any intermediate host of the whcv. Source: Netease open course Author: Zhang Yongzhen, February 3, 2020, responsible editor of School of public health, Fudan University: Li Zhaoxin ufe63 ns3501

Coronavirus is associated with many outbreaks of infectious diseases in humans, including SARS in 2002 / 3 and mers in 2012. Four other coronaviruses - human coronaviruses hku1, OC43, nl63 and 229E - are also associated with respiratory diseases. Although SARS like coronavirus 9 has been widely found in mammals including bats in China since 2005, the exact source of human coronavirus infection is still unclear. In this paper, we describe a novel coronavirus, whcv (2019 ncov), from BALF of a patient with severe respiratory disease in Wuhan, China. Phylogenetic analysis shows that whcv is a new type of virus in the genus betacoronavirus (sarbecovirus), so it has some genomic and systemic similarities with SARS CoV, especially in RBD. These genomic and clinical similarities with SARS, as well as their large presence in clinical samples, provide evidence for the association between whcv and ongoing respiratory disease outbreaks in Wuhan. Although isolation of the virus from only one patient is not sufficient to conclude that it causes respiratory symptoms, our findings are independently confirmed in other patients.

The identification of several SARS like coronaviruses in bats has led to the assumption that these animals are the natural hosts of these viruses. Although SARS like virus has been widely recognized in bats in China, the same virus as SARS CoV has not been recorded. It is worth noting that whcv is most closely related to bat coronavirus, and even shows 100% amino acid similarity with bat SL covzc45 in nsp7 and E proteins. Therefore, these data suggest that bats may be hosts of whcv. However, since there were several animals on the market at the time of the first report of the disease, more work was needed to determine the natural host and any intermediate host of the whcv.