Comparison of commercially available and novel West Nile virus immunoassays for detection of seroconversion in pig-tailed macaques (Macaca nemestrina)

We report the assessment and validation of an NS1 epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to West Nile virus (WNV) in macaques. Sera from naturally infected Macaca nemestrina were tested by ELISA and plaque reduction neutralization test (PRNT). Results were correlated with hemagglutination inhibition (HAI) data. Our results demonstrate that the blocking ELISA rapidly and specifically detects WNV infection in M. nemestrina. In addition, the diagnostic value of 7 commercially available immunoassays (PanBio immunoglobulin [Ig] M ELISA, PanBio IgG ELISA, PanBio immunofluorescence assay (IFA), InBios IgG ELISA, InBios IgM ELISA, Focus Diagnostics IgG ELISA, and Focus Diagnostics IgM ELISA) in M. nemestrina was evaluated and compared with that of the epitope-blocking ELISA. The PanBio IgG ELISA was found to effectively diagnose WNV exposure in M. nemestrina. Further, PanBio IFA slides are fast and reliable screening tools for diagnosing flaviviral exposure in M. nemestrina.

Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS213-34A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

West Nile and St. Louis encephalitis virus antibody seroconversion, prevalence, and persistence in naturally infected pig-tailed macaques (Macaca nemestrina)

Pig-tailed macaques (Macaca nemestrina) naturally infected with West Nile virus were monitored from 1999 to 2005 to determine virus-specific antibody seroconversion, prevalence, and persistence. Antibodies persisted for up to 36 months, as detected by epitope-blocking enzyme-linked immunosorbent and hemagglutination inhibition assays. Exposure to cocirculating St. Louis encephalitis virus was evaluated by Western blotting and immunofluorescence assays.

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3,site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States

In Late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Genetic analysis of West Nile New York 1999 encephalitis virus

Analysis of the genome of the flavivirus responsible for the 1999 New York City encephalitis epidemic cloned from human brain by reverse-transcription polymerase chain reaction indicates its identity as a lineage I West Nile virus (WNV; WNV-NY1999) closely related to WNVs previously isolated in the Middle East.