West Nile Virus Proteins Research Articles

Immunochemical Properties of Recombinant Polypeptides Mimicking Domains I and II of West Nile Virus Glycoprotein E

Complementary DNA fragments (nucleotides 935–1475, 1091–1310, and 935–1193) encoding the N-terminal portion of glycoprotein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were cloned. Recombinant polypeptides of glycoprotein E (E1–180, E53–126, and E1–86) of the WNV having amino acid sequences corresponding to the cloned cDNA fragments and mimicking the main functional regions of domains I and II of surface glycoprotein E were purified by affinity chromatography. According to ELISA and Western blotting, 12 types of monoclonal antibodies (MAbs) raised in our laboratory against recombinant polypeptide E1–180 recognized the WNV glycoprotein E. This is indicative of similarity between the antigenic structures of the short recombinant polypeptides and corresponding regions of the glycoprotein. Analysis of interactions of the MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus revealed at least six epitopes within domains I and II of the WNV protein E. We found at least seven MAb types against the region between amino acid residues (aa) 86 and 126 of domain II, which contains the peptide responsible for fusion of the virus and cell membranes (residues 98–110). The epitope for antireceptor MAbs 10H10 was mapped within the 53–86 aa region of domain II of WNV protein E, which is evidence for the spatial proximity of the fusion peptide and the coreceptor of protein E (residues 53–86) for cellular laminin-binding protein (LBP). The X-ray pattern of protein E suggests that the bc loop (residues 73–89) of domain II interacts with LBP and, together with the cd loop (fusion peptide), determines the initial stages of flavivirus penetration into the cell.

Neutralizing Monoclonal Antibodies Against Russian Strain of the West Nile Virus

We have developed a panel of 16 hybridomas secreting neutralizing monoclonal antibodies (Nt- MAbs) to Russian isolate (LEIV-Vlg99-27889-human) of the West Nile virus (WNV). Most of the Nt-Mabs were either IgG1 or IgG3 subtypes. Nine of the 16 neutralizing MAbs detected WNV protein E in Western blot. According to their Nt-activities, Western blot results and cross-reactivity, the MAbs were divided into four groups. Monoclonal antibodies from group I were able to neutralize WNV strains Vlg99-27889, Vlg00-27924, Hp-94, A-1640, A-72, Tur-2914, and Eg101. The Nt-activity of MAbs from groups II-IV towards these WNV strains was variable. Recombinant fragments E(1-180), E(1-321), and E(260-466) of protein E were used for preliminary mapping of domains recognized by Nt-MAbs. Only five Nt-MAbs were able to react with the recombinant polypeptides. The MAbs 9E2, 7G9, 11G3, and 7E6 from group Ia recognized Nt-epitope(s) between amino acids 321 and 466 of protein E and Nt-MAb 4F11 (group III) reacted with residues 1-180. This demonstrates that two discrete regions of protein E are involved in neutralization of WNV. Our data on immunochemical, biological activities of Nt-MAbs and mapping of Nt-epitopes using recombinant polypeptides suggest at least 13 different Nt-epitopes for WNV.

Characterization of West Nile viral replication and maturation in peripheral neurons in culture

The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of > or = 10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a beta-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.

Assays for detecting West Nile Virus antibodies in human serum, plasma, and cerebrospinal fluid

The rapid spread of West Nile Virus (WNV) across the North American continent has led to a need to understand what assays for WNV antibodies are available and how they are used as diagnostic and epidemiologic tools. In this article, we review six methods for measuring WNV antibodies in human serum, plasma, and cerebrospinal fluid. The complement fixation and hemagglutination inhibition assays were historically important; however, due to their low sensitivity, low specificity, and complex technical and reagent production issues, they are no longer in common use. The plaque reduction neutralization test is the gold standard for WNV antibody detection; due to its complexity and long turnaround time, however, it is increasingly reserved for establishing the presence of WNV infection in a geographic area and characterizing problematic samples. The immunofluorescence assay measures both IgG and IgM antibodies to WNV. Although historically considered insensitive, recent studies using commercially available slides have shown acceptable performance; the immunofluorescence assay is thus a cost-effective way to measure WNV antibodies in laboratories that routinely test small numbers of samples. The enzyme-linked immunosorbent assay (ELISA) format is the most popular method currently used to detect WNV IgG and IgM. Both indirect and monoclonal antibody-mediated antigen capture formats of IgG ELISAs have been described, whereas nearly all IgM ELISAs utilize the IgM capture format. Before 2000, WNV antibody ELISAs employed native WNV antigens; since then, there has been a dramatic shift toward using recombinant WNV antigens, particularly subviral particles containing the envelope protein. Like in the other assays mentioned, however, antibodies induced by other flavivirus infections may crossreact with both native and recombinant WNV antigens, necessitating concurrent measurement of antibodies to flaviviruses endemic in a given geographic area. The new microsphere immunoassay shows great promise as a sensitive, specific, and cost-effective method for simultaneously measuring antibodies to multiple flaviviruses. This method has also been used to characterize antibodies to nonstructural WNV proteins; these antibodies appear to be highly specific for WNV, and their measurement may soon be the test of choice for diagnosing WNV infection.

Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway.

West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid (Cp) by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade.

Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses.

BackgroundIn Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.ResultsConvalescent sera from 69 individuals who were known to have had dengue or Japanese encephalitis virus infection were tested by western blotting against dengue, Japanese encephalitis and West Nile virus antigens. We determined that individuals who had been infected with dengue viruses had IgG responses against the premembrane protein of dengue viruses but not Japanese encephalitis, whereas individuals who had been infected with Japanese encephalitis had IgG specific for the premembrane protein of Japanese encephalitis virus but not the dengue viruses. None reacted with the premembrane protein of West Nile virus. Using the Pearson Chi Square test, it was determined that the difference between the two groups was highly significant with a p value of <0.001.ConclusionThe use of flavivirus premembrane protein in seroepidemiological studies will be useful in determining what flaviviruses have circulated in a community.

An Analysis of the Antibody Response against West Nile Virus E Protein Purified by SDS-PAGE Indicates that This Protein Does Not Contain Sequential Epitopes for Efficient Induction of Neutralizing Antibodies

The large envelope (E) protein of flaviruses is the viral surface protein that contains neutralizing epitopes. We have analysed the E protein of the West Nile (WN) flavivirus for neutralizing epitopes generated from linear segments of the protein; if effective, these might allow the synthesis of peptides suitable for vaccination. For this study we used the E protein and defined fragments thereof as antigens in rabbits. The sera thus obtained, containing antibodies to E protein as shown by Western blot analyses, were tested for neutralizing activity by the plaque reduction neutralization test. If the E protein used as antigen was reduced (the native E protein contains six disulphide bridges) and denatured the resulting antibodies did not consistently demonstrate neutralizing activity. These results show that the E protein of WN virus does not contain a linear segment that is able to induce neutralizing antibodies efficiently. Studies using denatured E protein fragments containing subsets of the intact disulphide bridges showed that the local covalent primary structure of the protein involved in each of the six bridges was also insufficient for inducing the synthesis of neutralizing antibodies. The complete E protein, with all six disulphide bridges intact, purified by preparative SDS-PAGE under the conditions used in our experiments could, however, induce the synthesis of neutralizing antibodies in rabbits. These data indicate that at least one complex epitope which is able to induce neutralizing antibodies is not completely denatured or can be reformed to some extent if the complete E protein has been subjected to SDS-PAGE without prior destruction of the disulphide bridges.

Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure

The glycosylation pattern of several flavivirus E proteins as well as the role of carbohydrate in biological functions and the antigenic structure of tick-borne encephalitis (TBE) virus were investigated by the use of specific endoglycosidases. Endoglycosidase F digestion revealed the presence of a single asparagine-linked oligosaccharide side chain in TBE virus (Western and Far Eastern subtype), Louping III virus, Murray Valley encephalitis virus, and Rocio virus. Consistent with published sequence data, the E protein of West Nile virus apparently is not glycosylated at all. Evidence derived from digestion experiments using endoglycosidase H indicates that the tick-borne viruses contain high-mannose type N-linked oligosaccharide side chains, whereas that of the mosquito-borne Murray Valley encephalitis virus and Rocio virus is endoglycosidase H resistant. Complete deglycosylation of TBE virus by endoglycosidase F did not impair infectivity and HA activity. Carbohydrate does not seem to play a role in the antigenic structure of the TBE virus glycoprotein since the reactivity of the native virus and the deglycosylated virus was identical when analyzed with monoclonal as well as polyclonal immune sera.

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype

The nucleotide sequence of the 5′-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5′-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5′-C-prM(M)-E-3′ and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.