Implementation of blood-brain barrier on microfluidic chip: Recent advance and future prospects.

The complex structure of the blood-brain barrier (BBB) hinders its modeling and the treatment of brain diseases. The microfluidic technology promotes the development of BBB-on-a-chip platforms, which can be used to reproduce the complex brain microenvironment and physiological reactions. Compared with traditional transwell technology, microfluidic BBB-on-a-chip shows great technical advantages in terms of flexible control of fluid shear stress in the chip and fabrication efficiency of the chip system, which can be enhanced by the development of lithography and three-dimensional (3D) printing. It is convenient to accurately monitor the dynamic changes of biochemical parameters of individual cells in the model by integrating an automatic super-resolution imaging sensing platform. In addition, biomaterials, especially hydrogels and conductive polymers, solve the limitations of microfluidic BBB-on-a-chip by compounding onto microfluidic chip to provide a 3D space and special performance on the microfluidic chip. The microfluidic BBB-on-a-chip promotes the development of basic research, including cell migration, mechanism exploration of neurodegenerative diseases, drug barrier permeability, SARS-CoV-2 pathology. This study summarizes the recent advances, challenges and future prospects of microfluidic BBB-on-a-chip, which can help to promote the development of personalized medicine and drug discovery.

In Silico Modeling of Virus Particle Propagation and Infectivity along the Respiratory Tract: A Case Study for SARS-COV-2 (preprint)

Respiratory viruses including Respiratory syncytial virus (RSV), influenza virus and cornaviruses such as Middle Eastern respiratory virus (MERS) and SARS-CoV-2 infect and cause serious and sometimes fatal disease in thousands of people annually. It is critical to understand virus propagation dynamics within the respiratory system because new insights will increase our understanding of virus pathogenesis and enable infection patterns to be more predictable in vivo, which will enhance targeting of vaccines and drug delivery. This study presents a computational model of virus propagation within the respiratory tract network. The model includes the generation network branch structure of the respiratory tract, biophysical and infectivity properties of the virus, as well as air flow models that aid the circulation of the virus particles. The model can also consider the impact of the immune response aim to inhibit virus replication and spread. The model was applied to the SARS-CoV-2 virus by integrating data on its life-cycle, as well as density of Angiotensin Converting Enzyme (ACE2) expressing cells along the respiratory tract network. Using physiological data associated with the respiratory rate and virus load that is inhaled, the model can improve our understanding of the concentration and spatiotemporal dynamics of virus.

SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier (preprint)

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

Covidex: an ultrafast and accurate tool for virus subtyping (preprint)

Covidex is an open-source, alignment-free machine learning subtyping tool for viral species. It is a shiny app that allows a fast and accurate classification in pre-defined clusters for SARS-CoV-2 and FMDV genome sequences. The user can also build its own classification models with the Covidex model generator. AvailabilityCovidex is open-source, cross-platform compatible, and is available under the terms of the GNU General Public License v3 (http://www.gnu.org/licenses/gpl.txt). Covidex is available via SourceForge https://sourceforge.net/projects/covidex or the web application https://cacciabue.shinyapps.io/shiny2/ Contactcacciabue.marco@inta.gob.ar; marcocacciabue@yahoo.com

Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay (preprint)

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

Ubiquitous Forbidden Order in R-group classified protein sequence of SARS-CoV-2 and other viruses (preprint)

Each amino acid in a polypeptide chain has a distinctive R-group associated with it. We report here a novel method of species characterization based upon the order of these R-group classified amino acids in the linear sequence of the side chains associated with the codon triplets. In an otherwise pseudo-random sequence, we search for forbidden combinations of kth order. We applied this method to analyze the available protein sequences of various viruses including SARS-CoV-2. We found that these ubiquitous forbidden orders (UFO) are unique to each of the viruses we analyzed. This unique structure of the viruses may provide an insight into viruses chemical behavior and the folding patterns of the proteins. This finding may have a broad significance for the analysis of coding sequences of species in general.

First study on surveillance of SARS-CoV-2 RNA in wastewater systems and related environments in Wuhan: Post-lockdown (preprint)

Wastewater-based epidemiology (WBE) has emerged as an effective environmental surveillance tool in monitoring fecal-oral pathogen infections within a community. Congruently, SARS-CoV-2 virus, the etiologic agent of COVID-19, has been demonstrated to infect the gastrointestinal tissues, and be shed in feces. In the present study, SARS-CoV-2 RNA was concentrated from wastewater, sludge, surface water, ground water, and soil samples of municipal and hospital wastewater systems and related environment in Wuhan during the COVID-19 middle and low risk periods, and the viral RNA copies quantified using RT-qPCR. From the findings of this study, during the middle risk period, one influent sample and three secondary treatment effluents collected from Waste Water Treatment Plant 2 (WWTP2), as well as two influent samples from wastewater system of Hospital 2 were SARS-CoV-2 RNA positive. One sludge sample collected from Hospital 4; which was obtained during low risk period, was positive for SARS-CoV-2 RNA. These study findings demonstrate the significance of WBE in continuous surveilling and monitoring of SARS-CoV-2 at the community level, even when the COVID19 prevalence is low. Therefore, the application of WBE is principally useful in tracking the level of infections in communities and the risk assessment of the secondary environment.

Targeting pentose phosphate pathway for SARS-CoV-2 therapy (preprint)

It becomes more and more obvious that deregulation of host metabolism play an important role in SARS-CoV-2 pathogenesis with implication for increased risk of severe course of COVID-19. Furthermore, it is expected that COVID-19 patients recovered from severe disease may experience long-term metabolic disorders. Thereby understanding the consequences of SARS-CoV-2 infection on host metabolism can facilitate efforts for effective treatment option. We have previously shown that SARS-CoV-2-infected cells undergo a shift towards glycolysis and that 2-deoxy-D-glucose (2DG) inhibits SARS-CoV-2 replication. Here, we show that also pentose phosphate pathway (PPP) is remarkably deregulated. Since PPP supplies ribonucleotides for SARS-CoV-2 replication, this could represent an attractive target for an intervention. On that account, we employed the transketolase inhibitor benfooxythiamine and showed dose-dependent inhibition of SARS-CoV-2 in non-toxic concentrations. Importantly, the antiviral efficacy of benfooxythiamine was further increased in combination with 2DG.

Temporal landscape of mutation accumulation in SARS-CoV-2 genomes from Bangladesh: possible implications from the ongoing outbreak in Bangladesh (preprint)

Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. Thus the analysis of genomic mutations of these circulatory strains may give an insight into the molecular basis of SARS-CoV-2 pathogenesis and evolution favoring the development of effective treatment and containment strategies. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA 7, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains in the country belong to B and/or L type among cluster A to Z and strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in Nonspecific protein 2 (NSP2), NSP3, RNA dependent RNA polymerase (RdRp), Helicase, Spike, ORF3a, and Nucleocapsid (N) protein were common in the circulating strains with varying degrees and the most unique mutations(UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, E (Envelope), NSP7a, ORF 6, and ORF 7b suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed an association with sex and age of the COVID-19 positive cases. So, identification of these mutational accumulation patterns may greatly facilitate drug/ vaccine development deciphering the age and the sex dependent differential susceptibility to COVID-19.