Attenuation of Getah virus by a single amino acid substitution at residue 253 of the E2 protein that might be part of a new heparan sulfate binding site on alphaviruses

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo . Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the 3D structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.

Towards a Mathematical Model for the Viral Progression in the Pharynx

In this work, a comprehensive model for the viral progression in the pharynx has been developed. This one-dimension model considers both Fickian diffusion and convective flow coupled with chemical reactions, such as virus population growth, infected and uninfected cell accumulation as well as virus clearance. The effect of a sterilizing agent such as an alcoholic solution on the viral progression in the pharynx was taken into account and a parametric analysis for the effect of kinetic rate parameters on virus propagation was made. Moreover, different conditions caused by further medical treatment, such as a decrease in virus yield per infected cell, were examined. It is shown that the infection fails to establish by decreasing the virus yield per infected cell. It is believed that this work could be used to further investigate the medical treatment of viral progression in the pharynx.

Impairment of T cells' antiviral and anti-inflammation immunities may be critical to death from COVID-19

Clarifying dominant factors determining the immune heterogeneity from non-survivors to survivors is crucial for developing therapeutics and vaccines against COVID-19. The main difficulty is quantitatively analysing the multi-level clinical data, including viral dynamics, immune response and tissue damages. Here, we adopt a top-down modelling approach to quantify key functional aspects and their dynamical interplay in the battle between the virus and the immune system, yielding an accurate description of real-time clinical data involving hundreds of patients for the first time. The quantification of antiviral responses gives that, compared to antibodies, T cells play a more dominant role in virus clearance, especially for mild patients (96.5%). Moreover, the anti-inflammatory responses, namely the cytokine inhibition and tissue repair rates, also positively correlate with T cell number and are significantly suppressed in non-survivors. Simulations show that the lack of T cells can lead to more significant inflammation, proposing an explanation for the monotonic increase of COVID-19 mortality with age and higher mortality for males. We propose that T cells play a crucial role in the immunity against COVID-19, which provides a new direction–improvement of T cell number for advancing current prevention and treatment.

Data-Driven Analysis of COVID-19 Reveals Persistent Immune Abnormalities in Convalescent Severe Individuals

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of “long COVID-19”, and defines key cells and cytokines that delineate true and quasi-convalescent states.

Characteristics and Prognosis of Antibody Non-Responders with COVID-19

Background：The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading globally. The information regarding the characteristics and prognosis of antibody non-responders with COVID-19 is scarce.Method: In this retrospective, single-center study, we included all the patients with confirmed COVID-19 using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) admitted to the Fire God Mountain hospital from February 3, 2020, to April 14, 2020. A total of 1921 patients were divided into the antibody-negative group (n=94) and antibody-positive group (n=1827), and the 1:1 propensity score matching (PSM) was used to match two groups.Results: In the antibody negative group, 40 patients (42.6%) were male, 54 patients (57.4%) were female, and 49 patients (52.1%) were older than 65 years old. Cough was the most common symptoms in the antibody negative group. White blood cell counts (WBC) 6.6×109/L [5.0, 9.1], Neutrophils 4.3×109/L [3.1, 6.6], C-reactive protein 7.3 mg/L [1.3, 49.0], Procalcitonin (PCT) 0.1 ng/mL [0.0, 0.2], Interleukin-6 (IL-6) 64.2 [1.5, 28.7], Lactate dehydrogenase (LDH) 193.8 U/L [154.9,260.6], Creatine kinase 60.5 U/L [40.5, 103.7], Creatine kinase isoenzyme 10.3 ng/mL [8.2, 14.5], Urea nitrogen 5.3 mmol/L [4.0, 8.7] and Creatinine 77.7 μmol/L [60.6, 98.7] were significantly higher in antibody negative patients than in antibody positive group (P<0.005). The days of nucleic acid negative conversion in the antibody negative group was shorter than that in the antibody positive group (P < 0.001). Meanwhile, the hospitalization time of antibody negative patients was shorter than that of antibody positive patients (8.0 [6.0, 10.0] VS 13.0 [8.2, 23.0], P < 0.001).Conclusion: Some COVID-19 patients without specific antibodies had mild symptoms, but the inflammatory reaction caused by innate clinical immunity was more intense than those with antibodies, and the virus was cleared faster. The production of specific antibodies was unnecessary for SARS-CoV-2 clearance, and non-specific immune responses played an essential role in virus clearance.

Modeling hepatitis C virus kinetics during liver transplantation reveals the role of the liver in virus clearance

While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from 5 liver-transplant patients throughout the anhepatic (absence of liver) phase and for 4 hours post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n=3) or declined (n=2) with t1/2~1h. Immediately post-reperfusion, virus declined in a biphasic manner in 4 patients consisting of a rapid decline (t1/2=5min) followed by a slower decline (t1/2=67min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.

Impact Of Adaptive Natural Killer Cells, KLRC2 Genotype And Cytomegalovirus Reactivation On Late Mortality In Patients With Severe Covid-19 Lung Disease

Objective: COVID-19 infection results in severe lung disease in a small but significant number of infected patients. The etiopathogenesis in a subset of such patients, who continue to have progressive pulmonary disease following virus clearance remains unexplored. Methods: We investigated the role of NKG2C+/NKG2A- adaptive natural killer (ANK) cells, KLRC2 genotype and cytomegalovirus reactivation in 22 such patients. Results: The median duration of virus positivity was 23 days and the median duration of hospitalisation was 48 days. The overall survival at 60 days in this group was 50%. Older age and comorbidities impacted survival negatively. CMV viremia was documented in 11 patients, with a survival of 25% vs 80% in those without viremia with viral load correlating with mortality. ANK cells were markedly depressed in all patients at day 15. However, persistently low ANK cells at 30 days along with an inversely high NKG2C-/NKG2A+ inhibitory NK cells significantly correlated with high CMV viremia as well as mortality, irrespective of KLRC2 genotype. Day 30 ANK cells were significantly lower in KLRC2 deletion group. IFN-gamma and Perforin release were severely compromised in all patients at day +15, with significant improvement in the survivors at day +30, but not in those with adverse outcome. Conclusion: Patients with severe lung disease even after negative SARS-CoV-2 status, with persistently reduced and functionally compromised ANK cells, are more likely to have CMV reactivation and an adverse outcome, independent of KLRC2 genotype.

Effects of Overexpression of the Egyptian Fruit Bat Innate Immune Genes on Filovirus Infections in the Host Cells

Bats constitute a large and diverse group of mammals with unique characteristics. One of these is the ability of bats to maintain various pathogens, particularly viruses, without evidence of disease. The innate immune system has been implicated as one of the important components involved in this process. However, in contrast to the human innate immune system, little data is available for bats. In the present study we generated 23 fusion constructs of innate immune genes of Egyptian fruit bat (Rousettus aegyptiacus) with mCherry as a fluorescent reporter. We evaluated the effects of overexpressing these genes on the replication of Marburg and Ebola viruses in the Egyptian fruit bat cell line R06EJ. Both viruses were substantially inhibited by overexpression of type I, II and III interferons, as well as by DDX58 (RIG-I), IFIH1, and IRF1. Our observations suggest that the broad antiviral activity of these genes reported previously in human cells is conserved in Egyptian fruit bats and these possess anti-filovirus activities that may contribute to the efficient virus clearance.

SARS-CoV-2 blood RNA load predicts outcome in critically ill COVID-19 patients

Background SARS-CoV-2 RNA loads in patient specimens may act as a clinical outcome predictor in critically ill patients with COVID-19. Methods We evaluated the predictive value of viral RNA loads and courses in the blood compared to the upper and lower respiratory tract loads of critically ill COVID-19 patients. Daily specimen collection and viral RNA quantification by RT-qPCR was performed in all consecutive 170 COVID-19 patients between March 2020 and February 2021 during the entire ICU stay (4145 samples analyzed). Patients were grouped according to their 90-days outcome as survivors (n=100) or non-survivors (n=70). Results In non-survivors, blood SARS-CoV-2 RNA loads were significantly higher at the time of admission to the ICU (p=0.0009). Failure of blood RNA clearance was observed in 33/50 (66 %) of the non-survivors compared to 12/64 (19 %) of survivors (p&lt;0.0001). As determined by multivariate analysis, taking sociodemographic and clinical parameters into account, blood SARS-CoV-2 RNA load represents a valid and independent predictor of outcome in critically ill COVID-19 patients (OR [log10]: 0.23 [0.12 – 0.42], p&lt;0.0001) with a significantly higher effect for survival compared to the respiratory tract SARS-CoV-2 RNA loads (OR [log10]: 0.75 [0.66 – 0.85], p&lt;0.0001). Blood RNA loads exceeding 2.51 x 10 3 SARS-CoV-2 RNA copies/ml were found to indicate a 50% probability of death. Consistently, 29/33 (88%) of the non-survivors with failure of virus clearance exceeded this cut-off value constantly. Conclusion Blood SARS-CoV-2 load is an important independent outcome predictor and should be further evaluated for treatment allocation and patient monitoring.