Several neuroinvasive and non-neuroinvasive WestNile (WN) viruses were characterized by nucleotide sequencing of their envelope (E) protein regions. Prolonged passage in mosquito cells caused loss of neuroinvasiveness and acquisition of an N-linked glycosylation site, which is utilized. Limited passage in cell culture also caused glycosylation but not attenuation, suggesting that glycosylation may not be directly responsible for attenuation and that a second mutation (L 68 P) may also be involved. A monoclonal antibody-neutralization escape mutant with a substitution at residue 307, a site common to other flavivirus escape mutants, was also attenuated. A partially neuroinvasive revertant regained the parental E sequence, implying that determinants outside of the E region may also influence attenuation. Data suggest that the neuroinvasive determinants may be similar to those for other flaviviruses. Also, sequence comparison with the WN virus (Nigeria) strain revealed considerable divergence of the E protein at the nucleotide and amino acid levels.West Nile (WN) virus, a member of the Japanese encephalitis (JE) serogroup of the genus Flavivirus, family Flaviviridae, is distributed widely throughout Africa, the Middle East, parts of Europe (Camargue, France), the former Soviet Union, India and Indonesia (Hayes, 1989). The virus is classified into African and Middle Eastern subtypes based on antigenic variation of the envelope (E) protein (Hammam et al.,

The neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.

A strain of Sindbis virus (SV), recently isolated from mosquitoes in Israel, was used as a source for variants which differ in neuroinvasiveness and virulence that were generated by serial passage of SV in suckling and weanling mouse brain. At the 15th passage a neurovirulent variant was observed and designated SVN (neurovirulent). After 7 more passages in weanling mouse brains, another variant was observed and designated SVNI (neuroinvasive) and both were isolated and purified. All strains caused similar viremia after intraperitoneal (I.P.) injection of weanling mice, but whereas SV was neuroinvasive but nonvirulent, SVN was neurovirulent but noninvasive and SVNI was both virulent and invasive. SVNI is the first SV variant which is both neurovirulent and neuroinvasive in weanling mice. Co-injection I.P. of SV + SVN resulted in presence of SV alone in the mouse brain; co-injection of SVNI + SVN resulted in full-titered replication of both strains in the brain. We assume that this is achieved through a breach of the blood brain barrier effected by SVNI replication and used by SVN for co-invasion. SV probably invades the brain by a different mechanism. I.P. infection with SVNI of inbred BALB/c mice gave rise to clinical signs only in a few mice even though substantial viremia was demonstrated.

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.

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