Detection and isolation of H5N1 influenza virus from large volumes of natural water.

Various species of aquatic or wetlands birds can be the natural reservoir of avian influenza A viruses of all hemagglutinin (HA) subtypes. Shedding of the virus into water leads to transmission between waterfowl and is a major threat for epidemics in poultry and pandemics in humans. Concentrations of the influenza virus in natural water reservoirs are often too low to be detected by most methods. The procedure was designed to detect and isolate low concentrations of the influenza virus in large volumes of water without the need for costly installations and reagents. The virus was adsorbed onto formalin-fixed erythrocytes and subsequently isolated in chicken embryos. Sensitivity of the method was determined using a reverse-genetic H5N1 virus. A concentration as low as 0.03 of the 50% egg infection dose per milliliter (EID50/ml) of the initial volume of water was effectively detected. The probability of detection was approximately 13%, which is comparable to that of detecting the influenza virus M-gene by PCR amplification. The method can be used by field workers, ecologists, ornithologists, and researchers who need a simple method to isolate H5N1 influenza virus from natural reservoirs. The detection and isolation of virus in embryonated chicken eggs may help epidemiologic, genetic, and vaccine studies.

Structure-based characterization and optimization of novel hydrophobic binding interactions in a series of pyrrolidine influenza neuraminidase inhibitors.

The structure-activity relationship (SAR) of a novel hydrophobic binding interaction within a subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified at the 4-position. Previously, potent inhibitors have targeted this subsite with hydrophilic substituents such as amines and guanidines. Inhibitor-bound crystal structures revealed that hydrophobic substituents with sp(2) hybridization could achieve optimal interactions by virtue of a low-energy binding conformation and favorable pi-stacking interactions with the residue Glu119. From a lead methyl ester, investigation of five-membered heteroaromatic substituents at C-4 produced a 3-pyrazolyl analogue that improved activity by making a targeted hydrogen bond with Trp178. The SAR of substituted vinyl substituents at C-4 produced a Z-propenyl analogue with improved activity over the lead methyl ester. The C-1 ethyl ester prodrugs of the substituted C-4 vinyl analogues gave compounds with excellent oral bioavailability (F > 60%) when dosed in rat.

Design, synthesis, and neuraminidase inhibitory activity of GS-4071 analogues that utilize a novel hydrophobic paradigm.

Structure-based design has led to the synthesis of a novel analogue of GS-4071, an influenza neuraminidase inhibitor, in which the basic amino group has been replaced by a hydrophobic vinyl group. An X-ray co-crystal structure of the new inhibitor (K(i)=45 nM) bound to the active site shows that the vinyl group occupies the same subsite as the amino group in GS-4071.

Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98).

We have characterized monoclonal antibodies raised against the neuraminidase (NA) of a Sydney-like influenza virus (A/Memphis/31/98, H3N2) in a reassortant virus A/NWS/33(HA)-A/Mem/31/98(NA) (H1N2) and nine escape mutants selected by these monoclonal antibodies. Five of the antibodies use the same heavy chain VDJ genes and may not be independent. Another antibody, Mem5, uses the same V(H) and J genes with a different D gene and different isotype. Sequence changes in escape mutants selected by these antibodies occur in two loops of the NA, at amino acid 198, 199, 220, or 221. These amino acids are located on the opposite side of the NA monomer to the major epitopes found in N9 and early N2 NAs. Escape mutants with a change at 198 have reduced NA activity compared to the wild-type virus. Asp198 points toward the substrate binding pocket, and we had previously found that a site-directed mutation of this amino acid resulted in a loss of enzyme activity (M. R. Lentz, R. G. Webster, and G. M. Air, Biochemistry 26:5351-5358, 1987). Mutations at residue 199, 220, or 221 did not alter the NA activity significantly compared to that of wild-type NA. A 3.5-A structure of Mem5 Fab complexed with the Mem/98 NA shows that the Mem5 antibody binds at the sites of escape mutation selected by the other antibodies.