NoncompetitiveFluorescence Polarization Aptamer Assayfor Detecting SARS-CoV‑2 Variants

Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic which not only created a situation of dealing with public health emergency but also triggered the financial crisis of international concern. The current situation demands rapid, convenient and reliable diagnosis of the disease to downregulate its spread. Primary method of diagnosis presently being used, such as nucleic acid testing (RT-PCR), CT scans etc. involve time-consuming advanced machinery for imaging/ RNA replication and highly skilled technicians which could be only done in a laboratory set-up. A rapid, simple yet selective naked eye detection methodology that does not require any advanced instrumental techniques is highly desirable. In this study, we report computational results which could form the basis of a simple and rapid strategy for the detection of SARS-Cov-2 using peptide (screened from angiotensin-converting enzyme 2 (ACE2) receptor of host cell) functionalized gold nanoparticles (GNPs). This is based on the preferential binding of viral spike (S) protein to ACE2 receptor situated on the surface of the host cell membrane by which the virus gains access to the host cell. The interaction of peptide coated GNPs with spike protein has been investigated using coarse grained molecular dynamic simulations. The potential of mean force calculation of spike protein confirmed strong binding between peptide and receptor binding domain (RBD) of spike protein. The results presented here demonstrate the potential of this peptide coated GNPs-based system in the development of convenient sensors for the clinical diagnosis.

Test strips have been widely used in various rapid on-site biochemical sensing due to its simple and rapid detections. Unfortunately, many test strips only support qualitative sensing mainly because their detecting results are often determined by visual inspection. In order to solve the problem, we design a handheld fluorescence test strip reader, which can collect fluorescence emission from test strip thus quantify target under detection. The proposed handheld fluorescence test strip reader composes of rather compact optical system including a light emitting diode (LED) for excitation, an optical bandpass filter for fluorescence signal filtering and a smartphone for fluorescence signal collection and data analysis. All the optical elements are integrated using 3-D printed structures and coupled with the smartphone to support on-site detection. In addition, a smartphone application is also designed to automatically quantify fluorescence emission intensity, and it definitely can support quantitative target detection. Proved by a series of applications especially on rapid on-site detections, the handheld fluorescence test strip reader can detect target automatically, rapidly and accurately. Therefore, the proposed handheld fluorescence test strip reader is a potential tool for rapid on-site biochemical detection in various applications including agricultural, biomedical, and environmental fields.

A Palm Germ-Radar (PaGeR) for rapid and simple COVID-19 detection by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation

Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed

7-α methoxy substitution of cefuroxime, cephamandole, cephapirin and cephalosporin 87/359 was found to have a marked effect upon affinity of the β-lactam antibiotics for the penicillin-binding proteins of Escherichia coli K12. The 7-α methoxy (cephainycin) derivatives had no affinity for PBP2, a reduced affinity for PBP3 and, with the exception of cefoxitin, (7-α methoxy 87/359), a reduced affinity for PBPs la/lb, compared to their non-7-α methoxy counterparts. Also, the 7-α methoxy substitution greatly enhanced the binding of the antibiotics to PBPs 5/6, which correlated with their increased inhibitory activity for membrane bound model transpeptidase activity in E. coli K12. These results are discussed in relation to the antibacterial activity of the compounds against a wild-type strain and an isogenic permeability mutant of E. coli K12.

The growth and release of several alphaviruses, including several strains of Sindbis virus (the wild-type strain, the large plaque and small plaque variants of the HR strain, and the HR mutant ts103), Semliki Forest virus(SFV) and Middelburg virus, and of the unrelated rhabdovirus, vesicular stomatitis virus (VSV), have been compared in chick cells and in BHK-21 cells as a function of the culture conditions for the host cell and the ionic strength of the medium. The small plaque strain of Sindbis HR, as well as SFV, grew better in BHK cells, whereas the large plaque strain of Sindbis HR showed a preference for chick cells. Wild-type Sindbis and VSV grew equally well in either cell. The optimum ionic strength for virus production as well as inhibition of virus release into the medium at low ionic strength depended upon both the virus and the host cell. Thus, VSV grown in medium of low ionic strength gave no additional release of virus on incubation with hypertonic medium (minimum effect), whereas ts103 released very little virus without exposure to hypertonic conditions (maximum effect). The viruses could be ordered as follows: minimum effect = vesicular stomatitis virus < Middelburg virus < Semliki Forest virus < Sindbis wt < Sindbis HR (large plaque) < Sindbis HR (small plaque) < Sindbis ts103 = maximum effect. After several passages in culture, chick cells required hypertonic conditions for optimum production and release of Sindbis virus. Furthermore, BHK cells cultured in different media responded differently to ionic strength for virus production and release. These results suggest that there is a charge-dependent stop in the maturation of alpha-viruses, possibly a configurational rearrangement of glycoprotein E2 upon its formation from the precursor PE2, which is sensitive to the ionic strength of the medium, to the composition of the host plasmalemma and to differences in the virus glycoproteins.

The choline analog, N-isopropylethanolamine (IPE), inhibits the growth of both Chinese hamster ovary CHO-K1 and mouse L-M cells by two kinetically distinct mechanisms; I, a reversible and concentration-dependent reduction in the logarithmic population doubling rate and the saturation density of cultures by low IPE levels in the media; and II, an irreversible and time-dependent killing of cells by high IPE concentrations. Both types of inhibition are independent of media depletion, cell density, or the time of treatment after cell plating; however, the actual IPE concentration that is necessary to elicit Type I or Type II inhibition in each cell line is dependent on the choline level of the media. Ethanolamine, methionine, or betaine have no effect on IPE-induced growth inhibition. From a mutagenized population of CHO-K1 cells we isolated variant cell strains that are resistant to the lethal effect of IPE. It was determined that with both the wild type and variant strains the sensitivity of cells to growth inhibition by IPE (both Type I and Type II) was proportional to the degree by which choline uptake was inhibited by the analog. Retinoic acid, which inhibits the growth of some fibroblast and epithelial cell lines by a concentration-dependent reduction in population doubling rate and saturation density, behaves synergistically with IPE to inhibit the growth of CHO-K1 cells. Dibutyryl cyclic AMP, on the other hand, causes only an additive increase in the growth inhibition of CHO-K1 populations that also are treated with IPE.

Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose alpha-chain. The gamma-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl alpha-chain even though previous reports suggested that the presence of an extended alpha-chain hinders C3 PEA substitution of Hep II. Approximately 50% of gamma-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and gamma-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.

While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naïve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naïve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naïve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naïve counterparts.

The Omicron(B.1.1.529) variant of SARS-CoV-2 emerged in November 2021 and is rapidly spreading among the human population1. Although recent reports reveal that the Omicron variant robustly escapes vaccine-associated and therapeutic neutralization antibodies2-10, the pathogenicity of the virus remains unknown. Here we show that the replication of Omicron is substantially attenuated in human Calu3 and Caco2 cells. Further mechanistic investigations reveal that Omicron is inefficient in its use of transmembrane serine protease 2 (TMPRSS2) compared with wild-type SARS-CoV-2(HKU-001a) and previous variants, which may explain its reduced replication in Calu3 and Caco2 cells. The replication of Omicron is markedly attenuated in both the upper and lower respiratory tracts of infected K18-hACE2 mice compared with that of the wild-type strain and Delta(B.1.617.2) variant, resulting in its substantially ameliorated lung pathology. Compared with wild-type SARS-CoV-2 and the Alpha(B.1.1.7), Beta(1.351) and Delta variants, infection by Omicron causes the lowest reduction in body weight and the lowest mortality rate. Overall, our study demonstrates that the replication and pathogenicity of the Omicron variant of SARS-CoV-2 in mice is attenuated compared with the wild-type strain and other variants.

ObjectivesKnowledge is limited on how changing SARS-CoV-2 variants may translate into different characteristics and affect the prognosis of patients with long COVID, especially following Omicron variants. We compared long-term prognosis of patients in a Danish Post-COVID Clinic infected with wild-type strain, Alpha, Delta, or Omicron variants as well as the pre-Omicron compared to the Omicron period. MethodsAt enrollment, a Post-COVID symptom Questionnaire (PCQ), and standard health scores, were registered and repeated four times until 1.5 years after infection. PCQ was the primary outcome to assess the severity of long COVID, and Delta PCQ to assess failure to improve. ResultsA total of 806 patients were enrolled. Patients infected with Omicron and Delta variants presented with more severe long COVID (median PCQ 43 in Delta vs 38 in wild-type, P = 0.003) and health scores (EuroQol five-dimension five-level-index was 0.70 in Omicron vs 0.76 in wild-type, P = 0.009 and 0.78 pre-Omicron, P = 0.006). At 1.5 years after infection, patients had no clinically meaningful decline in severity of long COVID, and 57% (245/429) of patients failed to improve 1.5 years after infection, with no differences between variants. ConclusionMore than half of patients referred to a Post-COVID Clinic failed to improve in long COVID severity 1.5 years after infection regardless of variants of SARS-CoV-2.

Hepatitis B virus (HBV) DNA levels and liver histological necroinflammation grades are correlated with the antiviral efficacy. It is necessary to clarify the relationship between HBV replication levels apportioned by the same hepatic parenchyma cell volume and severity of liver histological necroinflammation grades in both hepatitis B e antigen (HBeAg)-positive and HBeAg-negative chronic hepatitis B. The serum HBV DNA levels apportioned by the same hepatic parenchyma cell volume were compared between HBeAg-positive and HBeAg-negative chronic hepatitis B as well as among liver histological necroinflammation grades 1, 2, 3 and 4, respectively. There were no differences in the serum HBV DNA levels between HBeAg-positive and HBeAg-negative chronic hepatitis B as well as among liver histological necroinflammation grades 1, 2, 3 and 4. However, there were differences in the serum HBV DNA levels apportioned by the same hepatic parenchyma cell volume among liver histological necroinflammation grades 1, 2, 3 and 4 in both HBeAg-positive and HBeAg-negative chronic hepatitis B, respectively. There were no differences in HBV DNA levels with the same liver histological necroinflammation grade activated by HBV wild-type and variant strains. After the differences in hepatic parenchyma cell volume for HBV replication of the same liver histological necroinflammation grade accompanied by different hepatic fibrosis stages were adjusted, the serum HBV DNA level apportioned by the same hepatic parenchyma cell volume was correlated with the severity of liver histological necroinflammation grade.

Even though extreme containment and mitigation strategies were implemented by numerous governments around the world to slow down the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the number of critically ill patients and fatalities keeps rising. This crisis has highlighted the socioeconomic disparities of health care systems within and among countries. As new CoVID policies and responses are implemented to lessen the impact of the virus, it is imperative (1) to consider additional mitigation strategies critical for the development of effective countermeasures, (2) to promote long-term policies and strict regulations of the trade of wildlife and live animal markets, and (3) to advocate for necessary funding and investments in global health, specifically for the prevention of and response to natural and manmade pandemics. This document considers some of these challenges.