Virus Eradication and Synthetic Biology: Changes with SARS-CoV-2?

The eradication of infectious diseases has been achieved only once in history, in 1980, with smallpox. Since 1988, significant effort has been made to eliminate poliomyelitis viruses, but eradication is still just out of reach. As the goal of viral disease eradication approaches, the ability to recreate historically eradicated viruses using synthetic biology has the potential to jeopardize the long-term sustainability of eradication. However, the emergence of the severe acute respiratory syndrome-coronavirus (SARS-CoV)-2 pandemic has highlighted our ability to swiftly and resolutely respond to a potential outbreak. This virus has been synthetized faster than any other in the past and is resulting in vaccines before most attenuated candidates reach clinical trials. Here, synthetic biology has the opportunity to demonstrate its truest potential to the public and solidify a footing in the world of vaccines.

Persistent brainwave disruption and cognitive impairment induced by acute sarin surrogate sub-lethal dose exposure.

Warfare neurotoxicants such as sarin, soman or VX, are organophosphorus compounds which irreversibly inhibit cholinesterase. High-dose exposure with nerve agents (NA) is known to produce seizure activity and related brain damage, while less is known about the effects of acute sub-lethal dose exposure. The aim of this study was to characterize behavioral, brain activity and neuroinflammatory modifications at different time points after exposure to 4-nitrophenyl isopropyl methylphosphonate (NIMP), a sarin surrogate. In order to decipher the impacts of sub-lethal exposure, we chose 4 different doses of NIMP each corresponding to a fraction of the median lethal dose (LD50). First, we conducted a behavioral analysis of symptoms during the first hour following NIMP challenge and established a specific scoring scale for the intoxication severity. The intensity of intoxication signs was dose-dependent and proportional to the cholinesterase activity inhibition evaluated in mice brain. The lowest dose (0.3 LD50) did not induce significant behavioral, electrocorticographic (ECoG) nor cholinesterase activity changes. Animals exposed to one of the other doses (0.5, 0.7 and 0.9 LD50) exhibited substantial changes in behavior, significant cholinesterase activity inhibition, and a disruption of brainwave distribution that persisted in a dose-dependent manner. To evaluate long lasting changes, we conducted ECoG recording for 30 days on mice exposed to 0.5 or 0.9 LD50 of NIMP. Mice in both groups showed long-lasting impairment of theta rhythms, and a lack of restoration in hippocampal ChE activity after 1-month post-exposure. In addition, an increase in neuroinflammatory markers (IBA-1, TNF-α, NF-κB) and edema were transiently observed in mice hippocampus. Furthermore, a novel object recognition test showed an alteration of short-term memory in both groups, 1-month post-NIMP intoxication. Our findings identified both transient and long-term ECoG alterations and some long term cognitive impairments following exposure to sub-lethal doses of NIMP. These may further impact morphopathological alterations in the brain.

Characterization of four BCHE mutations associated with prolonged effect of suxamethonium.

Butyrylcholinesterase (BChE) deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report the characterization of four BCHE mutations associated with prolonged effect of suxamethonium (amino acid numbering based on the matured enzyme): p.20delValPheGlyGlyThrValThr, p.Leu88His, p.Ile140del and p.Arg386Cys. Expression of recombinant BCHE mutants, kinetic analysis and molecular dynamics were undertaken to understand how these mutations induce BChE deficiency. Three of the mutations studied (p.20delValPheGlyGlyThrValThr, p.Ile140del and p.Arg386Cys) lead to a "silent" BChE phenotype. Recombinant BCHE expression studies for these mutants revealed BChE activity levels comparable to untransfected cells. Only the last one (hBChE-L88H) presented BChE activity in the transfected cell culture medium. This BChE mutant (p.Leu88His) is associated with a lower kcat value compare to the wild-type enzyme. Molecular dynamics simulations analyses suggest that a destabilization of a structure implicated in enzyme activity (Ω-loop) can explain the modification of the kinetic parameter of the mutated protein.

Efficacy of intermittent versus standard resuscitative endovascular balloon occlusion of the aorta in a lethal solid organ injury model.

BACKGROUND: High-grade solid organ injury is a major cause of mortality in trauma. Use of resuscitative endovascular balloon occlusion of the aorta (REBOA) can be effective but is limited by ischemia-reperfusion injury. Intermittent balloon inflation/deflation has been proposed as an alternative, but the safety and efficacy prior to operative hemorrhage control is unknown. METHODS: Twenty male swine underwent standardized high-grade liver injury, then randomization to controls (N = 5), 60-min continuous REBOA (cR, n = 5), and either a time-based (10-minute inflation/3-minute deflation, iRT = 5) or pressure-based (mean arterial pressure<40 during deflation, iRP = 5) intermittent schedule. Experiments were concluded after 120 minutes or death. RESULTS: Improved overall survival was seen in the iRT group when compared to cR (p < 0.01). Bleeding rate in iRT (5.9 mL/min) was significantly lower versus cR and iRP (p = 0.02). Both iR groups had higher final hematocrit (26% vs. 21%) compared to cR (p = 0.03). Although overall survival was lower in the iRP group, animals surviving to 120 minutes with iRP had decreased end organ injury (Alanine aminotransferase [ALT] 33 vs. 40 in the iRT group, p = 0.03) and lower lactate levels (13 vs. 17) compared with the iRT group (p = 0.03). No differences were seen between groups in terms of coagulopathy based on rotational thromboelastometry. CONCLUSION: Intermittent REBOA is a potential viable adjunct to improve survival in lethal solid organ injury while minimizing the ischemia-reperfusion seen with full REBOA. The time-based intermittent schedule had the best survival and prolonged duration of tolerable zone 1 placement. Although the pressure-based schedule was less reliable in terms of survival, when effective, it was associated with decreased acidosis and end-organ injury.

Crystalloid fluid suspension results in decreased adhesion burden when compared to bioresorbable membranes in a rat model.

INTRODUCTION: Adhesion formation represents a major cause of long-term morbidity. Suspension of intra-abdominal contents in fluid medium may effectively prevent adhesion formation. We compare saline hydro-flotation (NS) to hyaluronate bioresorbable membranes (HBM) for adhesion prevention following surgery. METHODS: Animals were randomized to four groups: sham (no injury, n = 5), control (injury without intervention, n = 5), HBM (n = 20) or 10 cc NS (n = 21). Interventions were administered after standardized surgical trauma to the cecum and abdominal wall. Necropsies at two weeks were completed to compare adhesion burden using a customary scoring algorithm. RESULTS: Significant adhesion burden was noted in all rats. HBM sustained a more significant adhesion burden with higher total adhesion scores (HBM = 10 vs NS = 8.1/15, p = 0.02). Gross adhesion scores were lower with NS (5.6/9) compared to HBM (7.1/9, p = 0.01). Neo-vascularity was more common in HBM at 2.6/3 versus 1.9/3 with NS (p = 0.01). Percent of the cecum encased with adhesion was higher with HBM (42%) compared to NS (31%, p = 0.05). DISCUSSION: Fluid based anti-adhesion methods should be considered for abdominal adhesion formation prevention.

Extending the golden hour for Zone 1 resuscitative endovascular balloon occlusion of the aorta: Improved survival and reperfusion injury with intermittent versus continuous resuscitative endovascular balloon occlusion of the aorta of the aorta in a porcine severe truncal hemorrhage model.

BACKGROUND: Noncompressible hemorrhage can be controlled using resuscitative endovascular balloon occlusion of the aorta (REBOA). Prolonged ischemia limits REBOA application during Zone 1 deployment. Intermittent inflation/deflation may effectively mitigate this problem. METHODS: A lethal abdominal vascular injury was created in 28 swines. Animals were randomized to controls (n = 7), 60 minutes full REBOA (FR, n = 5), time-based intermittent REBOA (iRT, n = 7), and pressure-based REBOA (iRP, n = 9). Intermittent groups had an initial inflation for 15 minutes, followed by 10-minute inflation: 3-minute deflation cycles (iRT), or an inflate/deflate schedule based on mean arterial pressure (MAP) less than 40 mm Hg (iRP). Experiments were concluded after 120 minutes or death (MAP < 20 mm Hg). RESULTS: Intermittent REBOA animals all survived to 120 minutes versus 15 minutes for controls and 63 minutes for FR (p < 0.001). After 60 minutes, FR animals were more hypotensive (MAP 20 mm Hg vs. 80 mm Hg [iRP] and 100 mm Hg [iRT]; p < 0.001), had lower cardiac output (1.06 mL/min vs. 5.1 L/min [iRP] and 8.2 L/min [iRT]; p < 0.001), higher lactate (12.5 mg/dL vs. 8.5 mg/dL [iRP], p = 0.02), and decreased clot firmness on rotational thromboelastometry than iRP/T (64 mm vs. 69 mm [iRP] and 69 mm [iRT], p = 0.04). Acidosis was worse in iRT versus iRP at 120 minutes (pH 7.28 vs. pH 7.12; p = 0.02), improved lactate (11.9 mg/dL vs. 16.3 mg/dL; p = 0.04), and decreased whole blood resuscitation (452 mL vs. 646 mL, p = 0.05). Blood loss (clot weight) was higher in controls (2.0 kg) versus iRT and iRP (1.16 kg and 1.23 kg; p < 0.01) and not different from FR (0.87 kg; p = 0.10). CONCLUSION: Intermittent REBOA can maintain supraceliac hemorrhage control while decreasing distal ischemia in a swine model. Prolonged survival times, decreased acidosis, and lower resuscitation requirements indicate that this technique could potentially extend Zone 1 REBOA deployment times. Schedules based on MAP may be superior to time-based regimens.

HIV-1 targets L-selectin for adhesion and induces its shedding for viral release.

CD4 and chemokine receptors mediate HIV-1 attachment and entry. They are, however, insufficient to explain the preferential viral infection of central memory T cells. Here, we identify L-selectin (CD62L) as a viral adhesion receptor on CD4+ T cells. The binding of viral envelope glycans to L-selectin facilitates HIV entry and infection, and L-selectin expression on central memory CD4+ T cells supports their preferential infection by HIV. Upon infection, the virus downregulates L-selectin expression through shedding, resulting in an apparent loss of central memory CD4+ T cells. Infected effector memory CD4+ T cells, however, remain competent in cytokine production. Surprisingly, inhibition of L-selectin shedding markedly reduces HIV-1 infection and suppresses viral release, suggesting that L-selectin shedding is required for HIV-1 release. These findings highlight a critical role for cell surface sheddase in HIV-1 pathogenesis and reveal new antiretroviral strategies based on small molecular inhibitors targeted at metalloproteinases for viral release.

Human-Derived Amniotic Membrane Is Associated With Decreased Postoperative Intraperitoneal Adhesions in a Rat Model.

BACKGROUND: Complications from adhesions after intra-abdominal surgery accounts for ~6% of hospital admissions. Currently, hyaluronate/carboxymethylcellulose represents the main option to prevent postoperative adhesion formation. Human amniotic membrane contains inherent anti-inflammatory properties that mitigate adhesion formation. OBJECTIVE: This study aimed to evaluate adhesion generation after surgical trauma with amniotic membranes compared with standard intraperitoneal adhesion barriers. DESIGN: This study is a double-blinded, prospective evaluation. SETTING: This study was conducted at an animal research facility. ANIMALS: Forty male rats were studied. INTERVENTION: Laparotomy was performed with peritoneal disruption to the cecum. Animals were randomly assigned to 1 of 5 groups: sham, control, saline, hyaluronic acid membrane, or amniotic membrane. Animals were euthanized at 14 days. MAIN OUTCOME MEASURES: Independent gross and histological assessments of adhesions were analyzed between groups by using adhesion scoring and microscopy. Scoring was based on the percentage of the cecum involved (0-4), vascularity of adhesions (0-3), strength (0-3), inflammation (0-3), and fibrosis (0-3). Adhered tissue was harvested for polymerase chain reaction analysis for gene regulation activity. RESULTS: All rats survived 14 days. Adhesions were observed in all animals. There were significantly fewer adhesions in the amniotic membrane group (2) versus hyaluronic acid (3) group (p = 0.01). The percentage of adhesion to the cecum was lower in the amniotic membrane group (29%) than in the hyaluronic acid group (47%, p = 0.04). Histological examination showed no significant difference between or within the 3 groups for inflammation or fibrosis. Genetic analysis of adhered tissues supported high rates of epithelialization and inhibition of fibrosis in the amniotic membrane group. LIMITATIONS: We are limited by the small sample size and the preclinical nature of the study. CONCLUSION: Human-derived amniotic membrane is effective at reducing intraperitoneal adhesion after surgical trauma and is superior to the current antiadhesion barriers. Amniotic membranes are well absorbed and demonstrate short-term safety. See Video Abstract at http://links.lww.com/DCR/A554.

Espirito Santo virus: a new birnavirus that replicates in insect cells.

Espirito Santo virus (ESV) is a newly discovered virus recovered as contamination in a sample of a virulent strain of dengue-2 virus (strain 44/2), which was recovered from a patient in the state of Espirito Santo, Brazil, and amplified in insect cells. ESV was found to be dependent upon coinfection with a virulent strain of dengue-2 virus and to replicate in C6/36 insect cells but not in mammalian Vero cells. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. An incomplete match to the nucleotide sequence of the RNA-dependent RNA polymerase from Drosophila X virus (DXV), another birnavirus, could be detected. Mass spectrometry analysis of ESV proteins found no matches in the protein data banks. However, peptides recovered by mass spectrometry corresponded to the de novo-assembled sequence by BLAST analysis. The composition and three-dimensional structure of ESV are presented, and its sequence is compared to those of other members of the birnavirus family. Although the virus was found to belong to the family Birnaviridae, biochemical and sequence information for ESV differed from that of DXV, the representative species of the genus Entomobirnavirus. Thus, significant differences underscore the uniqueness of this infectious agent, and its relationship to the coinfecting virus is discussed.

An alternative pathway for alphavirus entry.

The study of alphavirus entry has been complicated by an inability to clearly identify a receptor and by experiments which only tangentially and indirectly examine the process, producing results that are difficult to interpret. The mechanism of entry has been widely accepted to be by endocytosis followed by acidification of the endosome resulting in virus membrane-endosome membrane fusion. This mechanism has come under scrutiny as better purification protocols and improved methods of analysis have been brought to the study. Results have been obtained that suggest alphaviruses infect cells directly at the plasma membrane without the involvement of endocytosis, exposure to acid pH, or membrane fusion. In this review we compare the data which support the two models and make the case for an alternative pathway of entry by alphaviruses.

Alphavirus adsorption to mosquito cells as viewed by freeze fracture immunolabeling.

Sindbis Virus (SV), the prototype alphavirus in the family togaviridae, infects both mammalian and insect cells. The ability of SV to infect cells possessing significantly different biochemical environments suggests that there may be a common mode of entry into each cell type. Previous studies show that up to 4h post infection cells are permeable to small ions and alpha sarcin suggesting that the plasma membrane is compromised as infection takes place. Thin-section electron microscopy has also shown SV to bind to the plasma membrane and lose its electron dense core through a pore like structure developed upon interaction of the virus with the cell surface. Using freeze-fracture replicas, thin-sections and antibody labeling the data presented herein show virus associated with intramembrane particles on mosquito cells. These data suggest that the intramembrane particles associated with SV may be part of the pore structure consisting of virus proteins and cell receptor.

Helical virus particles formed from morphological subunits of a membrane containing icosahedral virus.

The classic publication by Caspar and Klug in 1962 [Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27:1-24.] has formed the basis of much research on virus assembly. Caspar and Klug predicted that a single virus morphological unit could form a two dimensional lattice composed of 6-fold arrays (primitive plane), a family of icosahedra of increasing triangulation numbers (T) and helical arrays of varying length. We have shown that icosahedral viruses of varying T numbers can be produced using Sindbis virus [Ferreira, D. F. et al. 2003. Morphological variants of Sindbis virus produced by a mutation in the capsid protein. Virology 307:54-66]. Other studies have shown that Sindbis glycoproteins can also form a 2-dimensional lattice confirming Caspar and Klug's prediction of the primitive plane as a biologically relevant structure [VonBonsdorff, C. H., and S. C. Harrison. 1978. Sindbis virus glycoproteins form a regular icosahedral surface lattice. J. Virol. 28:578]. In this study we have used mutations in the glycoproteins of membrane containing Sindbis virus to create helical-virus-like particles from the morphological subunits of a virus of icosahedral geometry. The resulting virus particles were examined for subunit organization and were determined to be constructed of only 6-fold rotational arrays of the virus glycoproteins. A model of the tubular virus particles created from the 6-fold rotational arrays of Sindbis virus confirmed the observed structure. These experiments show that a common morphological unit (the Sindbis E1-E2 heterodimer) can produce three different morphological entities of varying dimensions in a membrane-containing virus system.