H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase mutant explained by kinetic and structural analysis.

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

Variability of diagnostic approach, surgical technique, and medical management for children with biliary atresia in Canada - Is it time for standardization?

BACKGROUND: The Canadian 4-year native liver survival rate for biliary atresia (BA) after Kasai Portoenterostomy (KP) is 39%. The Canadian Biliary Atresia Registry (CBAR) was used to examine variability of surgical and medical management of BA. METHODS: Gastroenterologists and surgeons in all 14 Canadian pediatric tertiary centers were invited to complete an online survey of their BA management practices. RESULTS: Of gastroenterologists, diagnostic procedures included liver biopsy (92%), HIDA scan (58%), and percutaneous cholangiogram (46%). Surgeons reported Roux-en-Y lengths of 20-50cm with 78% avoiding diathermy at the portal plate; 16% performed laparoscopic exploration, but none laparoscopic KP. Postoperative corticosteroids and antibiotics were used by 24% and 85% of gastroenterologists, respectively, with similar rates for surgeons. At discharge, gastroenterologists prescribed oral antibiotics (80%), and ursodeoxycholic acid (95%), while surgeons reported lower rates (62% and 55%). Considerable variation existed in follow-up monitoring. No center had a standard protocol for evaluating suspected cholangitis. There was a lack of consensus for defining failed KP and referral criteria for transplant evaluation. CONCLUSION: In Canada, treatment of BA is not centralized, and there is variability in diagnostic approaches and management. Collaboration through CBAR will allow for implementation and evaluation of standardized surgical and medical management with a goal to improve outcomes. LEVEL OF EVIDENCE: Survey study. Level IV evidence.

Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism.

Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.