In a short time, West Nile virus has developed into a nationwide health and veterinary problem. The high virulence of the circulating virus and related lineage 1 WN strains hinders development of an attenuated live vaccine. We describe an attenuated WN isolate, WN1415, which is a molecularly cloned descendant of the WN prototype B956 strain. The parent virus belongs to lineage 2, members of which have not been associated with epidemic or epizootic outbreaks. A set of non-conservative mutations, mostly in non-structural protein genes, distinguishes the WN1415 isolate from the parent B956 prototype strain. Immunization with WN1415 (55-550,000 pfu) established a potent immunity, which protected the majority of mice against lethal challenge with WN NY99. The attenuated nature of the isolate and its excellent growth characteristics combined with the availability of a highly stable infectious clone make the isolate an attractive candidate for live WN vaccine development.

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The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.

Recently, we have described a lineage 2 attenuated WN virus suitable for the development of a live WN vaccine. To design vaccine candidates with an improved immunogenicity, we assembled an infectious clone of the NY99 strain and created several chimeric constructs with reciprocal exchanges of structural protein genes between attenuated W956 and virulent NY99 and investigated their biological properties. Our data indicated that, while the growth rates of NY99 and chimeric viruses in tissue culture are determined primarily by properties of the structural proteins, determinants responsible for a highly cytopathic phenotype of NY99 or lack thereof for W956 are located within the nonstructural protein region of the WN genome. The high virulence of NY99 and the attenuated phenotype of W956 were found to be associated with determinants in the nonstructural region. Chimeric viruses carrying the NY99 structural proteins were attenuated in neuroinvasiveness and demonstrated an immunogenicity superior to W956.

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