West Nile virus (WNV) RNA was demonstrated in 5 of 25 (20%) urine samples collected from convalescent patients 573 to 2,452 days (1.6 to 6.7 years) after WNV infection. Four of the 5 amplicons sequenced showed >99% homology to the WNV NY99 strain. These findings show that individuals with chronic symptoms following WNV infection may have persistent renal infection over several years.

Designing proteins that reflect the natural variability of a pathogen is essential for developing novel vaccines and drugs. Flaviviruses, including Dengue (DENV) and West Nile (WNV), evolve rapidly and can “escape” neutralizing monoclonal antibodies by mutation. Designing antigens that represent many distinct strains is important for DENV, where infection with a strain from one of the four serotypes may lead to severe hemorrhagic disease on subsequent infection with a strain from another serotype. Here, a DENV physicochemical property (PCP)-consensus sequence was derived from 671 unique sequences from the Flavitrack database. PCP-consensus proteins for domain 3 of the envelope protein (EdomIII) were expressed from synthetic genes in E. coli. The ability of the purified consensus proteins to bind polyclonal antibodies generated in response to infection with strains from each of the four DENV serotypes was determined. The initial consensus protein bound antibodies from DENV-1-3 in ELISA and Western blot assays. This sequence was altered in 3 steps to incorporate regions of maximum variability, identified as significant changes in the PCPs, characteristic of DENV-4 strains. The final protein was recognized by antibodies against all four serotypes. Two amino acids essential for efficient binding to all DENV antibodies are part of a discontinuous epitope previously defined for a neutralizing monoclonal antibody. The PCP-consensus method can significantly reduce the number of experiments required to define a multivalent antigen, which is particularly important when dealing with pathogens that must be tested at higher biosafety levels.

BackgroundAnalysis of large sets of biological sequence data from related strains or organisms is complicated by superficial redundancy in the set, which may contain many members that are identical except at one or two positions. Thus a new method, based on deriving physicochemical property (PCP)-consensus sequences, was tested for its ability to generate reference sequences and distinguish functionally significant changes from background variability.MethodsThe PCP consensus program was used to automatically derive consensus sequences starting from sequence alignments of proteins from Flaviviruses (from the Flavitrack database) and human enteroviruses, using a five dimensional set of Eigenvectors that summarize over 200 different scalar values for the PCPs of the amino acids. A PCP-consensus protein of a Dengue virus envelope protein was produced recombinantly and tested for its ability to bind antibodies to strains using ELISA.ResultsPCP-consensus sequences of the flavivirus family could be used to classify them into five discrete groups and distinguish areas of the envelope proteins that correlate with host specificity and disease type. A multivalent Dengue virus antigen was designed and shown to bind antibodies against all four DENV types. A consensus enteroviral VPg protein had the same distinctive high pKa as wild type proteins and was recognized by two different polymerases.ConclusionsThe process for deriving PCP-consensus sequences for any group of aligned similar sequences, has been validated for sequences with up to 50% diversity. Ongoing projects have shown that the method identifies residues that significantly alter PCPs at a given position, and might thus cause changes in function or immunogenicity. Other potential applications include deriving target proteins for drug design and diagnostic kits.