Chintamani D. Atreya

Defective rotavirus particle assembly in lovastatin-treated MA104 cells

Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in “empty-looking” rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion.

Nucleotide Sequence Analysis of Rotavirus Gene 11 from Two Tissue Culture-adapted ATCC Strains, RRV and Wa

We report here nucleotide sequence and characterization of gene 11 from two tissue culture-adapted ATCC1 rhesus (RRV) and human (Wa) strains of rotavirus. Gene 11 sequence encodes a nonstructural protein, NSP5 and also encodes NSP6, from an out of phase open reading frame. Sequence of RRVATCC gene 11 represents the first report from a rhesus rotavirus which has more than 90% homology at the nucleotide and deduced amino acid sequence level with that of its closely related simian SA11 strain. The WaATCC gene sequence differed from that of published Wa (WaPub) at three nucleotide positions, one at 264 (GWa-Pub to AATCC-Wa), another a nucleotide insertion (A) at position 388 and the third, a deletion (A) at 416. The latter two changes in WaATCC NSP5 resulted in drastic amino acid changes within a 10-residue region (123–132) from VHVYQFQLTN in WaPub to DSCVSISTNH in WaATCC NSP5 protein. In this region, WaATCC NSP5 is closer to published sequences from other strains, suggesting the authenticity of the present sequence. The nucleotide difference between WaPub and WaATCC NSP5 sequences, however, did not affect the NSP6 deduced amino acid sequence, which is overall highly conserved among all the strains compared. Sequence-based phylogenetic analysis of gene 11 identified a high degree of conservation within the Group A rotaviruses. In addition, it also separated RRVATCC and WaATCC, suggesting rotavirus segregation by genogroup. An anti-NSP5 monoclonal antibody of SA11 recognized RRV NSP5 protein but not WaATCC NSP5 from the infected cells, further supporting the phylogenetic segregation of RRVATCC and WaATCC strains based on their NSP5 coding sequence.

FDA workshop on emerging infectious diseases: evaluating emerging infectious diseases (EIDs) for transfusion safety.

O n May 11, 2010, experts in the field of emerging infectious diseases (EIDs) and other participants from blood centers, academia, government agencies, and the industry gathered at the Hilton Hotel in Gaithersburg, Maryland, for a 2-day public workshop entitled “FDA Workshop on Emerging Infectious Diseases: Evaluating Emerging Infectious Diseases (EIDs) for Transfusion and Transplantation Safety.” The first day of the workshop focused on transfusion safety was opened with a few welcome remarks from Dr Carolyn Wilson, Associate Director for Research, CBER, FDA, followed by introductory remarks from Dr Jay Epstein, Director, Office of Blood Research and Review, CBER, FDA. Subsequently 10 speakers presented their views on the workshop topic ranging from the lessons learned from our past experiences with infectious agents to the current cutting edge technologies to detect as well as to reduce the infectious agent burden in transfusion settings. A brief commentary on each presentation followed by the presentation as submitted by the speakers and a summary of the panel discussion are reported here. A transcript of the entire public workshop is available online at http://www.fda.gov/cber/minutes/workshopmin.htm.

A New Proof of Concept in Bacterial Reduction: Antimicrobial Action of Violet-Blue Light (405 nm) in Ex Vivo Stored Plasma

Bacterial contamination of injectable stored biological fluids such as blood plasma and platelet concentrates preserved in plasma at room temperature is a major health risk. Current pathogen reduction technologies (PRT) rely on the use of chemicals and/or ultraviolet light, which affects product quality and can be associated with adverse events in recipients. 405u2009nm violet-blue light is antibacterial without the use of photosensitizers and can be applied at levels safe for human exposure, making it of potential interest for decontamination of biological fluids such as plasma. As a pilot study to test whether 405u2009nm light is capable of inactivating bacteria in biological fluids, rabbit plasma and human plasma were seeded with bacteria and treated with a 405u2009nm light emitting diode (LED) exposure system (patent pending). Inactivation was achieved in all tested samples, ranging from low volumes to prebagged plasma. 99.9% reduction of low density bacterial populations (≤103u2009CFUu2009mL−1), selected to represent typical “natural” contamination levels, was achieved using doses of 144u2009Jcm−2. The penetrability of 405u2009nm light, permitting decontamination of prebagged plasma, and the nonrequirement for photosensitizing agents provide a new proof of concept in bacterial reduction in biological fluids, especially injectable fluids relevant to transfusion medicine.

The rubella virus nonstructural protease recognizes itself via an internal sequence present upstream of the cleavage site for trans-activity

Summary.The substrate requirement for rubella virus protease trans-activity is unknown. Here, we analyzed the cleavability of RV P200-derived substrates varying in their N-terminal lengths (72–475 amino acids) from the cleavage site by the RV protease trans-activity. Only substrates with at least 309 amino acid residues N-terminal to the cleavage site were able to undergo cleavage. Further, rubella sequence was found to be necessary in the N-terminal region of the substrate, whereas a heterologous sequence C-terminal to the cleavage site was tolerated. These results demonstrated a requirement for residues located between amino acids 994–1102 of the RV P200 polyprotein, besides its cleavage site for RV protease trans-activity. This region overlaps with the starting site of the essential cis-protease activity of RV P200 polyprotein. This is a novel observation for a viral protease of the family Togaviridae.

New proof-of-concept in viral inactivation: virucidal efficacy of 405 nm light against feline calicivirus as a model for norovirus decontamination

The requirement for novel decontamination technologies for use in hospitals is ever present. One such system uses 405xa0nm visible light to inactivate microorganisms via ROS-generated oxidative damage. Although effective for bacterial and fungal inactivation, little is known about the virucidal effects of 405xa0nm light. Norovirus (NoV) gastroenteritis outbreaks often occur in the clinical setting, and this study was designed to investigate potential inactivation effects of 405xa0nm light on the NoV surrogate, feline calicivirusxa0(FCV). FCV was exposed to 405xa0nm light whilst suspended in minimal and organically-rich media to establish the virucidal efficacy and the effect biologically-relevant material may play in viral susceptibility. Antiviral activity was successfully demonstrated with a 4 Log10 (99.99%) reduction in infectivity when suspended in minimal media evident after a dose of 2.8xa0kJxa0cm−2. FCV exposed in artificial faeces, artificial saliva, blood plasma and other organically rich media exhibited an equivalent level of inactivation using between 50–85% less dose of the light, indicating enhanced inactivation when the virus is present in organically-rich biologically-relevant media. Further research in this area could aid in the development of 405xa0nm light technology for effective NoV decontamination within the hospital environment.

Preclinical safety evaluation of human platelets treated with antimicrobial peptides in severe combined immunodeficient mice

Bacterial sepsis is a complication attributed to room temperature (RT)‐stored platelets (PLTs) in transfusion medicine. Antimicrobial peptides (AMPs) are emerging as new therapeutic agents against microbes. We had previously demonstrated bactericidal activity of select synthetic AMPs against six types of bacteria in stored PLTs. In this report, we tested these AMPs for their potential antibody response and interference with the recovery and survival of human PLTs in an animal model.