FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.     

Social Restrictions versus Testing Campaigns in the COVID-19 Crisis: A Predictive Model Based on the Spanish Case

The global COVID-19 spread has forced countries to implement non-pharmacological interventions (NPI) (i.e., mobility restrictions and testing campaigns) to preserve health systems. Spain is one of the most severely impacted countries, both clinically and economically. In an effort to support policy decision-making, we aimed to assess the impacts of different NPI on COVID-19 epidemiology, healthcare costs and Gross Domestic Product (GDP). A modified Susceptible-Exposed-Infectious-Removed epidemiological model was created to simulate the pandemic evolution. Its output was used to populate an economic model to quantify healthcare costs and GDP variation through a regression model which correlates NPI and GDP change from 42 countries. Thirteen scenarios combining different NPI were consecutively simulated in the epidemiological and economic models. Both increased testing and stringency could reduce cases, hospitalizations and deaths. While policies based on increased testing rates lead to higher healthcare costs, increased stringency is correlated with greater GDP declines, with differences of up to 4.4% points. Increased test sensitivity may lead to a reduction of cases, hospitalizations and deaths and to the implementation of pooling techniques that can increase throughput testing capacity. Alternative strategies to control COVID-19 spread entail differing economic outcomes. Decision-makers may utilize this tool to identify the most suitable strategy considering epidemiological and economic outcomes.     

Molecular Characterization of Closely Related H6N2 Avian Influenza Viruses Isolated from Turkey,Egypt, and Uganda

Genetic analysis of circulating avian influenza viruses (AIVs) in wild birds at different geographical regions during the same period could improve our knowledge about virus transmission dynamics in natural hosts, virus evolution as well as zoonotic potential. Here, we report the genetic and molecular characterization of H6N2 influenza viruses isolated from migratory birds in Turkey, Egypt, and Uganda during 2017–2018. The Egyptian and Turkish isolates were genetically closer to each other than they were to the virus isolated from Uganda. Our results also suggest that multiple reassortment events were involved in the genesis of the isolated viruses. All viruses contained molecular markers previously associated with increased replication and/or pathogenicity in mammals. The results of this study indicate that H6N2 viruses carried by migratory birds on the West Asian/East African and Mediterranean/Black Sea flyways have the potential to transmit to mammals including humans. Additionally, adaptation markers in these viruses indicate the potential risk for poultry, which also increases the possibility of human exposure to these viruses.     

Groove pancreatitis has a spectrum of severity and can be managed conservatively

Background & aimsThe natural history of groove pancreatitis is incompletely characterized. Published literature suggests a high rate of surgery. We describe the short- and long-term outcomes in a cohort of patients with groove pancreatitis treated at our institution.MethodsMedical records of patients hospitalized in the University of Pittsburgh Medical Center system from 2000 to 2014 and diagnosed with groove pancreatitis based on imaging were retrospectively reviewed. Clinical presentation and outcomes during index admission and follow-up were recorded.ResultsForty-eight patients with groove pancreatitis were identified (mean age 53.2 years, 79% male). Seventy-one percent were alcohol abusers and an equal number were cigarette smokers. Prior histories of acute and chronic pancreatitis were noted in 30 (62.5%) and 21 (43.8%), respectively. Forty-four (91.7%) met criteria for acute pancreatitis during their index admission. Alcohol was the most common etiology (68.8%). No patient experienced organ failure. The most frequent imaging findings were fat stranding in the groove (83.3%), duodenal wall thickening (52.1%), and soft tissue mass/thickening in the groove (50%). Over a mean follow-up of 5.0 years, seven (14.6%) required a pancreas-related surgery. Patients had a high burden of pancreatitis-related readmissions (68.8%, 69.4/100 patient-years). Incident diabetes and chronic pancreatitis were diagnosed in 5 (13.9% of patients at risk) and 8 (29.6% of patients at risk) respectively.ConclusionsGroove pancreatitis has a wide spectrum of severity; most patients have mild disease. These patients have a high burden of readmissions and progression to chronic pancreatitis. A small minority requires surgical intervention.     

Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-d-glucoside

Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-d-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (K_m) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (K_i = 24 mM DON), and UDP strongly inhibited the enzyme. Cu²⁺ and Zn²⁺ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.