By studying virus samples from new coronary patients from late January to February, the team from Shanghai found that the new coronavirus could be divided into two branches, evolving independently from a common ancestor. However, there was no significant difference in clinical lysome effects between the two branches of the virus. The determinants of the severity of COVID-19 disease development are mainly related to patient age, underlying diseases, lymphocyte reduction and associated cytokine storms.
The study comes from a study published online may 20 in Nature, a leading academic journal, entitled “Viral and host factors to the clinic of COVID-19”. The study was carried out in collaboration with several Shanghai scientific and medical institutions, including the Shanghai Public Health Clinical Center affiliated with Fudan University, the Ruijin Hospital affiliated with Shanghai Jiaotong University, the Shanghai Institute of Hematology, the National Key Laboratory of Medical Genomics, the Institute of Plant Physiology and Ecology of the Chinese Academy of Sciences, the Pasteur Institute in Shanghai and the Shanghai Institute of Nutrition and Health. The team analyzed samples of SARS-CoV-2 virus in 326 new coronal patients admitted to the Shanghai Public Health Clinical Center between late January and February.
The authors are Professor Lu Hongzhou, Party Secretary of Shanghai Public Health Clinical Center, Professor of The Chinese Academy of Engineering, Professor of Shanghai Jiaotong University School of Medicine, Director of the Shanghai Institute of Hematology, Chen Saiyue, and Researcher Wang Shengyue of Shanghai Jiaotong University.
The team analyzed clinical, molecular and immunological data from 326 confirmed cases of new coronary pneumonia admitted by the Shanghai Public Health Clinical Center between January 20 and February 25. Since the first samples were received on January 27, the Critical Disease Genome Research Platform for Major Diseases in the Translational Medical National Critical Infrastructure of Ruijin Hospital has completed high-quality sequencing of the SARS-CoV-2 genome in 112 cases in more than a month. These sequences are similar to the genome sequences in the Global Influenza Data Sharing Program (GISAID) and show a more stable evolutionary process, suggesting that the virus may have adapted to human host transmission in the early stages of the outbreak.
The study also found that there were two major virus branches with different exposure histories in the early stages of the outbreak, but there were no significant differences in the propagation, pathogenicity and clinical manifestations of the two branches, and no sequence of viral mutations was found to be significantly related to patients with COVID-19.
From clinical and immunological studies, it was found that the lymphocytes in patients with COVID-19 decreased significantly, especially in patients with severe illness, CD4 and CD8-T cells showed a significant downward trend, and inflammatory cytokines IL-6 and IL-8 increased significantly.
The team believes that the determinants of the severity of COVID-19 disease development are mainly related to patient age, underlying disease, lymphocyte reduction, and associated cytokine storms, which are key early warning factors for the disease’s transition to severe illness. Early targeted interventions based on the above clinical analysis will help prevent the disease from developing to severe illness.
New coronavirus esstable evolution may have adapted to transmission in human hosts early in the outbreak
The study defined four types of infection. The five patients were asymptomatic patients with no apparent fever, respiratory symptoms, or radiology. The majority of patients (293 cases) were mild patients with fever and pneumonia. 12 cases of severe illness had symptoms of breathing difficulties and enlarged glass-like shadows in the lungs within 24-48 hours. A further 16 patients were treated to acute respiratory distress syndrome (ARDS) and required mechanical ventilation or in vitro membrane oxygenation (ECMO), which was critically ill.
As of 1 April, 315 patients (96.63%) had been discharged from hospital and 6 (1.84%) had died.
The team sequenced 112 samples (sputum, pharynx swabs) and identified 66 synonymous variants and 103 nonsymporyed variants in nine protein coding regions, compared to the world’s first published genome (Wuhan-Hu-1). The replacement rates of ORF1ab, S, ORF3a, E, M, and ORF7a are similar (approximately 3.5 x 10?-4 per site per year), while ORF8 (9.51 x 10?-4 per site per year) and N (1.05 x 10?-3 per site per year) have higher variability rates.
Mutations that recur in the genome of samples from Shanghai are consistent with the virus sequences published in the GISAID database and show a more stable evolutionary process, suggesting that the virus may have adapted to human host transmission in the early stages of the outbreak.
Two branches of the new crown virus, the earliest outbreak is not limited to the South China seafood market
The team then used 94 of these virus genomes and 221 SARS-CoV-2 sequences in the GISAID database for systematic developmental analysis. The study identified the virus as being primarily two branches, both of which contained cases diagnosed in early December 2019; branch I included several sub-branches, and branch II and branch I differ from two related variants ORF8:p.84L?gt;S (28144T?gt;C) and ORF1ab:p.2839S (8782C-T;T).
The sequence of the Shanghai sample was found in both major branches and sub-branches, and studies suggest that this indicates that the new coronary cases in Shanghai have multiple sources of the virus. No significant branch/sub-branch expansion was observed in Shanghai.
In addition, the six patients confirmed to have a history of exposure to the South China seafood market were concentrated in one branch (branch I), while the other 3 cases diagnosed during the same period and the cases of South China seafood market without contact history were branch II, indicating that the outbreak was not limited to the South China seafood market.
The paper points out that these two major monosizes may represent two genealogy that evolved independently from a common ancestor in Wuhan in early December 2019, with only one (branch I) associated with contact with the South China seafood market, where dense stalls, vendors and customers may promote human-to-human transmission.
Consistentwith, a epidemiological survey of the earliest cases in Wuhan before December 18 found that two patients were associated with the South China seafood market and five were not associated with them. Their systematic developmental analysis suggests that the outbreak could occur as early as late November 2019.
The team also compared the clinical performance of patients infected with branch I or branch II virus. They found no statistical differences in disease severity (p-0.88), lymphocyte count (p-0.79), CD3 T cell count (p-0.21), C-reactive protein (p-0.83) or D-dipolymer (p-0.19) and the duration of the disease (p-0.79). As a result, the team believes that the two branches of the virus show similar pathogenic effects despite changes in genome sequences.
Similarly, no significant correlation was found between the severity of the disease and the 13 most common variants (synonymous and non-synonymous).
Host factors associated with the severity of COVID-19 disease development
A notable feature of the study was that some infected people (5 cases, 1.53%) were able to detect significant detox, but did not show significant symptoms. In contrast, one-sided and bi-sided turbidity of the lungs was observed in mild and critical cases, and critically ill patients deteriorated rapidly in just two days.
The team further analyzed the patient’s immunobiochemical indicators. A notable feature of COVID-19 is the reduction of performing lymphocytes, especially in critical and critical conditions (preliminary post-hospital test results, p-6 x 10-6). A detailed analysis of the lymphocyte subtypes showed that CD3-T cells were most affected (p?lt;10?-6), cd4-plus and CD8-T cells had similar trends (CD4-T cells, p?lt;10-6; CD8-T cells, p-1 x 10-5).
It is worth noting that the changes in T lymphocytes are not only statistically significant in critical cases, but also in three other categories (asymptomatic, mild and severe) (CD3-T cells, p-0.013; CD8-T cells, p-0.004). In contrast, cd19-B cells, although significantly decreased in critically ill patients (p-1 x 10-5), there was no significant change in asymptomatic, mild, severe cases (p-0.47).
After further examining each set of data, the team concluded that as the disease worsened, CD3-T lymphocytes (tested for 7, 8, 11, 14-18, 22-25, 28, 29 days after onset) showed a gradual downward trend (p-lt;0.05), and cd4 plus CD8-T cells showed a similar trend. However, there is no such phenomenon in NK cells (CD16 plus, CD56 plus) or B cells (CD19 plus).
Next, the study compared the clinical parameters of the co-disease group and found that although the median age of the co-disease group was higher (p-0.02), the risk of further development of the disease in the co-disease group did increase significantly (p-0.01). In fact, the single variable logic regression analysis showed that age (p?lt; 0.0001), lymphocyte count (p?lt; 0.0001), comorbidity (p s 0.01) and sex (p s 0.014) were all major factors associated with the severity of the new crown. Multivariate analysis shows that age (p s 0.002) and lymphocyte reduction (p s 0.002) are two main independent factors, and the combination is not statistically significant.
During hospitalization and treatment, the team tested the levels of 11 cytokines (IFN-alpha, IFN-thium, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, and IL-17) in the patient’s serum. Among them, the changes of IL-6 (p-lt;10?6) and IL-8 (p-1 x 10-5) were the most significant. It is worth noting that these two cytokines are negatively correlated with lymphocytes counts.
In addition, the team combined longitudinal data from each group of cytokines to map their patterns of fluctuations with the time after onset. They collected data on the highest IL-6 for the 6th to 10th days after each patient became ill, and compared critically ill patients with non-critical patients. The level of IL-6 in the critically ill group increased significantly (p-0.001). There were similar significant differences in patient sylweding IL-8 levels from the 16th to 20th day after onset of the disease (p-0.006).
The study suggests that there is a strong link between inflammatory cytokines and the pathogenesis of SARS-CoV-2 infection.
The study further confirmed that CD3-T cells were the main inhibitory cell type in infected patients, while CD19-B cells and CD16-CD56-NK cells were less inhibited. In fact, a decrease in lymphocytes, especially cd4/CD8 cells, is also a major manifestation of SARS-CoV-2 infection.
In addition, longitudinal monitoring of major cytokines showed that IL-6 and IL-8 were negatively correlated with lymphocyte counts, and that IL-6 dynamics were highly correlated with disease severity.
Of course, the current relationship between viral activity, cytokine release and lymphocytes reduction is unclear. The team hypothesized that immune pathology responses to SARS-CoV-2, including “cytokine storms” and CD3-T lymphocytes reduction, may at least partly constitute the underlying mechanisms for disease progression and death.