Laboratory Methods for the Diagnosis of SARS-Cov-2

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.

Influence of AFP On Surgical Outcomes in Non-B Non-C Patients With Curative Resection For Hepatocellular Carcinoma

Objective: To study the clinical and prognostic features of non-B non-C alpha-fetoprotein (AFP) (-)-hepatocellular carcinoma (HCC) (NBNC-AFP(-)-HCC), and the relationship between the prognostic features of HCC and hepatitis B virus surface antigen (HBsAg) status and AFP. Methods: We enrolled 227 patients underwent hepatic resection for HCC between January 1998 and December 2007 in Sun Yat-Sen University Cancer Center, all of them were diagnosed with HCC by pathology. All patients were stratified into one of four groups (B-AFP(+)-HCC, B-AFP(-)-HCC, NBNC-AFP(+)-HCC, and NBNC-AFP(-)-HCC) according to AFP levels and HBsAg status. The clinicopathologic and survival characteristics of NBNC-AFP(-)-HCC patients were compared with all other three groups. Results: Out of the 105 NBNC-HCC patients, 43 patients (40.9%) were AFP-negative HCC. There were some differences in factors between the B-AFP(+) and NBNC-AFP(-) patients, such as age, body mass index (BMI), diabetes, and ALT (P<0.05). On univariate analysis, tumor size, secondary tumor, and portal invasion were prognostic factors for overall survival (OS) and disease-free survival (DFS) (P<0.05). Cox multivariate regression analysis suggested that tumor size and tumor number (P<0.05) were independent predictors. In addition, compared with that in the B-AFP(+)-HCC, B-AFP(-)-HCC, and NBNC-AFP(+)-HCC groups, the NBNC-AFP(-)-HCC patients had the best DFS (P<0.05). Compared with that in the B-AFP(+)-HCC and NBNC-AFP(+)-HCC groups, the NBNC-AFP(-)-HCC patients had better OS(P<0.05), and survival rates were similar to those of B-AFP(-)-HCC patients. Conclusion: NBNC-AFP(-)-HCC patients had a relatively favorable prognosis. It can serve as a useful marker in predicting the risk of tumor recurrence in the early stages.

Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600–680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50–500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

Size- and concentration-dependence of antiviral and virucidal activity of Au nanoparticles against adenoviruses and influenza viruses H1N1

Over the past 10 years, many scientific groups have experimentally shown that non-functionalized nanoparticles show a pronounced antiviral and antimicrobial action against different pathogens. In order to understand the mechanism of nanoparticles action it is important to know its peculiarities, i.e. dependences on different nanoparticles and pathogen properties.In this work we studied how Au nanoparticles act on the viruses outside and inside the cell, and compare this action for two sizes of nanoparticles and two types of the viruses. The study has been conducted for adenovirus and H1N1 influenza virus, and nanoparticles of 5 nm and 20 nm diameter.Virucidal and antiviral actions were observed experimentally for both types of nanoparticles against both viruses. It has been shown that intensity of virucidal action depends on the nanoparticles concentration non-monotonically for adenovirus. It has also been shown with electron microscopy that the viruses are destructed after 5 nm nanoparticles adsorption on their surface; and that the viruses change their shape after 20 nm nanoparticles adsorption on their surface. The model of physical adsorption of nanoparticles on the virus surface due to near-field interaction proposed in previous works may explain observed results on virucidal action of nanoparticles.

COVID 19 breakthrough infection risk: a simple physical model describing the dependence on antibody concentration

The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface: namely that the probability of protection is the probability of antibody binding to more than a critical number of the spike proteins protruding from the virus. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of breakthrough infection given measured antibody concentration

Hepatitis B Immunoglobulin Inhibits The Secretion of HBV Via Antigen-Antibody Precipitation in The Multivesicular Body

Although the main action of human hepatitis B immunoglobulin (HBIG) of neutralizing the hepatitis B virus surface antigen (HBsAg) in the serum is known, HBIG is known to be localized in the cell. However, the effect of intracellularly located HBIG is not well elucidated due to the low purity of conventional plasma-derived HBIG (cHBIG). We attempted to clarify the mechanism of action of internalized HBIG using recombinant HBIG (lenvervimab). We used HBsAg cell lines, non-HBsAg cell lines and human HBsAg-producing hepatocytes. Autophagosome lysis pathway related proteins and Rab5, calnexin, giantin, and Rab7 were used to localize HBsAg and anti-HBs-IgG in the cytoplasm with Western blot and confocal microscopy.Intracellular anti-HBs-IgG (lenvervimab and cHBIG) transported by Fc receptor-mediated endocytosis increased the autophagosomes, but there was no change in autolysis. HBsAg and anti-HBs-IgG precipitated in the cytoplasm and co-localized in the multivesicular body. HBsAg secretion in the culture medium was decreased after lenvervimab. Simultaneously, the amount of cellular HBsAg increased in the cell lines but decreased in the human hepatocytes. Furthermore, intracellular lenvervimab was not easily washed out only in the HBsAg cell lines.Lenvervimab decreases the secretion of HBsAg, and HBsAg-antibody precipitation in the multivesicular body might play an important role.

COVID 19 breakthrough infection risk: a simple physical model describing the dependence on antibody concentration

The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface: namely that the probability of protection is the probability of antibody binding to more than a critical number of the spike proteins protruding from the virus. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of breakthrough infection given measured antibody concentration

Silicon Nanowire Field-Effect Transistor as Label-Free Detection of Hepatitis B Virus Proteins with Opposite Net Charges

The prevalence of hepatitis B virus (HBV) is a global healthcare threat, particularly chronic hepatitis B (CHB) that might lead to hepatocellular carcinoma (HCC) should not be neglected. Although many types of HBV diagnosis detection methods are available, some technical challenges, such as the high cost or lack of practical feasibility, need to be overcome. In this study, the polycrystalline silicon nanowire field-effect transistors (pSiNWFETs) were fabricated through commercial process technology and then chemically functionalized for sensing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus X protein (HBx) at the femto-molar level. These two proteins have been suggested to be related to the HCC development, while the former is also the hallmark for HBV diagnosis, and the latter is an RNA-binding protein. Interestingly, these two proteins carried opposite net charges, which could serve as complementary candidates for evaluating the charge-based sensing mechanism in the pSiNWFET. The measurements on the threshold voltage shifts of pSiNWFETs showed a consistent correspondence to the polarity of the charges on the proteins studied. We believe that this report can pave the way towards developing an approachable tool for biomedical applications.