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.

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.

SUMMARYA Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5'-C.GP20.E.GP44-P19-P10-P71.(?).P21.P98-3' YF 5'-C.prM-E. NSl.ns2a.ns2b.NS3-ns4a.ns4b-NS5-3'. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site VaI-X-AIa~ where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg,[Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85~ homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN > Murray Valley > Japanese encephalitis > St Louis encephalitis viruses within this closely related serological complex. A non-coding 5' sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3' sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87 % homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.

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Because Venezuelan equine encephalitis viruses (VEEVs) are infectious by aerosol, they are considered to be a biological-weapons threat. Nonhuman-primate models are needed to evaluate the efficacy of candidate vaccines. In the present study, cynomolgus macaques, after aerosol exposure to either VEEV-IE or VEEV-IIIA, developed fever, viremia, and lymphopenia; the severity of the fever response, viremia, and lymphopenia correlated with the inhaled dose of VEEV. Of the 10 macaques in our study, 7 developed clinical signs indicative of encephalitis, including loss of balance and hypothermia. In the macaque, the enzootic strains used are infectious by aerosol and lead to disease, including clinical encephalitis.

To elucidate the pathogenesis of eastern equine encephalitis (EEE) virus infections, we used histopathology, immunohistochemistry, and in situ hybridization to track the spread and early cellular targets of viral infection in mice. Young mice were inoculated with virulent EEE virus in their right rear footpad and were followed in a time-course study for 4 days. Virulent EEE virus produced a biphasic illness characterized by an early self-limiting replication phase in peripheral tissues followed by an invariably fatal central nervous system (CNS) phase. In the early extraneural phase, there was primary amplifying replication of virus within fibroblasts at the inoculation site and within osteoblasts in active growth areas of bone that resulted in a transient high-titer viremia. Pathological changes and viral infection were observed as early as 12 hours post-infection (PI) in osteoblasts, skeletal muscle myocytes, and in fibroblasts along fascial sheaths. The severity and extent of infection in peripheral tissues peaked at day 1 PI. In the neural phase of infection, virus was first detected in the brain on day 1 PI, with rapid interneuronal spread of infection leading to death by day 4 PI. EEE virus appeared to be directly cytopathic for neurons. The very rapid onset and apparently random and widely dispersed infection in the CNS, with concurrent sparing of olfactory neuroepithelium, strongly suggests that invasion of the CNS by EEE occurs by a vascular route, rather than via peripheral nerves or the olfactory neuroepithelium. Our finding that metaphyseal osteoblasts are an early site of amplifying viral replication may explain the higher-titer viremias and higher incidence of neuroinvasion and fulminant encephalitis seen in the young, and may also explain why mature animals become refractory to encephalitis after peripheral inoculation with EEE virus. Eastern equine encephalitis (EEE) virus is a singlestranded RNA virus in the genus Alphavirus (family Togaviridae) that can cause a severe mosquito-borne encephalitis in humans, horses, and game birds. 1 In North America, EEE virus is prevalent in freshwater swamps along the Atlantic and Gulf coasts of the United States where both its mosquito vector, Culiseta melanura, and its passerine bird reservoir hosts live. 2 The virus has a very wide host range, but as Culiseta mosquitoes rarely feed on mammalian hosts, other bridging mosquito species actually transmit the virus from infected birds to horses, and occasionally, to humans. Most EEE infections in humans are inapparent or produce a low-grade fever followed by malaise, arthralgia, and myalgia. However, in some cases, EEE virus crosses the blood-brain barrier and causes a severe, and often fatal, acute encephalitis, which kills 50 to 75% of infected humans and leaves many survivors with serious neurological sequelae. 1,3 It has long been recognized that EEE infection in children tends to have a more rapid onset and to be more severe. Goldfield et al reported that one in eight young children developed fulminant ence...

SUMMARYPartial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5'...GP44-P19.P10.P71.(?)-P21-P98-3', YF 5'...NSl.ns2a.ns2b.NS3.ns4a-ns4b.NS5-3'. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg~Gly, similar in form to the sequence Arg-Arg~Ser defined at the cleavage sites ofYF NS3 and NSS. A new consensus cleavage site for NS1, NS2A and NS4B in the form VaI-X-Ala~, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flav ivirus sequences (a total of 18 sites). N S 1 and N S4B, but not N S2A, were preceded by a putative signal sequence.