A total of 12 horses of different breeds and ages were infected with West Nile virus (WNV) via the bites of infected Aedes albopictus mosquitoes. Half the horses were infected with a viral isolate from the brain of a horse (BC787), and half were infected with an isolate from crow brain (NY99-6625); both were NY99 isolates. Postinfection, uninfected female Ae. albopictus fed on eight of the infected horses. In the first trial, Nt antibody titers reached >1:320, 1:20, 1:160, and 1:80 for horses 1 to 4, respectively. In the second trial, the seven horses with subclinical infections developed Nt antibody titers >1:10 between days 7 and 11 post infection. The highest viremia level in horses fed upon by the recipient mosquitoes was approximately 460 Vero cell PFU/mL. All mosquitoes that fed upon viremic horses were negative for the virus. Horses infected with the NY99 strain of WNV develop low viremia levels of short duration; therefore, infected horses are unlikely to serve as important amplifying hosts for WNV in nature.

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Cells activate the transcription factor NFB in a wide variety of situations, including responses to stress-inducing insults such as UV irradiation and virus infection or in response to cytokines such as tumor necrosis factor alpha (TNF-␣). NFB has an important role in suppression of apoptosis and regulates the expression of many important antiapoptotic functions (5,31,48,50). NFB, as a p65/p50 heterodimer, is normally sequestered in the cytoplasm in a complex with inhibitor of B (IB). IB␣ is targeted for phosphorylation at serine residues 32 and 36, and IB␤ is targeted for phosphorylation at serine residues 19 and 23 (42, 46, 51), by the multisubunit IB kinase (IKK) (11,23,36,52,58). This phosphorylation triggers its polyubiquitylation and destruction by the 26S proteosome (6,7,12), and, as a result, NFB is translocated to the nucleus (14, 57). Key roles for IKK and IB in NFB signaling were demonstrated in studies in which overexpression of a kinase-dead, trans-dominant form of IKK prevents IB phosphorylation and inhibits NFB activation (36, 52). IKK is activated by phosphorylation mediated by mitogen-activated protein (MAP) kinase kinase kinases (MAP3Ks) MEKK1, -2, or -3 or NFBinducing kinase (NIK) (23,30,37,59). Under stress conditions, the double-stranded RNA-activated protein kinase (PKR) has been shown to activate NFB through a pathway dependent on NIK and IKK (57). Distinct roles for the catalytic components of the IKK have been recognized. IKK␣ appears to play a major role in transducing signals for NFB activation during embryonic development (18, 45), while IKK␤ is essential for cytokine and other stress-induced signaling pathways (10,27,29). Besides cytoplasmic roles in the activation of NFB, recent studies have identified IKK␣ and the IKK scaffold protein IKK␥/NEMO in direct regulation of NFB-dependent transcription in the nucleus (2, 49, 53). NFB activation is also dependent on distinct signaling pathways which target p65 for phosphorylation (32).The ability of herpes simplex virus type 1 (HSV-1) to activate NFB has been well documented (1,15,40). Beginning at 3 to 5 h postinfection (hpi), HSV-1 induced a strong and persistent nuclear translocation, increased p50/p65-dependent DNA binding activity as measured by electrophoretic mobility shift assay (EMSA), and induced activation of a 3XNFB-luciferase reporter. Persistent NFB activation required virus binding and entry as well as de novo infected cell protein synthesis, including expression of functional viral immediateearly (IE) protein ICP27. Activation was also accompanied by increased IKK activity and loss of both IB␣ and IB␤. Interference with NFB activation occurred following expression of a dominant-negative IB␣ (DNIB) containing alanine substitutions for serine residues 32 and 36 normally targeted by IKK. The resulting substantial reduction in NFB EMSA activity correlated with a reduction in virus yield. The latter may be related to the reported role of NFB in preventing HSV-1-induced apoptosis (15). The foregoing results argue that the observe...

We have constructed a series of plasmids encoding premembrane (prM) and envelope (E) protein genes of dengue virus type 2 (DEN-2). These plasmids included an authentic DEN-2 prM-E construct (pCBD2-14-6), and two chimeric constructs, 90% DEN-2 E-10% Japanese encephalitis (JE) virus E (pCB9D2-1J-4-3) and 80% DEN-2 E-20% JE E (pCB8D2-2J-2-9-1). Monoclonal antibody (MAb) reactivity indicated that all three plasmids expressed authentic DEN-2 virus E protein epitopes representative of flavivirus domains 1, 2, and 3. However, only the pCB8D2-2J-2-9-1 construct secreted high levels of prM, M (membrane), and E proteins into the culture fluid of plasmid-transformed COS-1 cells. The major portion of the prM and E proteins expressed by COS-1 cells transformed by pCBD2-14-6 or pCB9D2-4-3 plasmids remained membrane-bound. The results supported the notion that an unidentified membrane retention sequence is located between E-397 and E-436 of DEN-2 virus E protein. Replacing the carboxyl-terminal 20% of DEN-2 E (397-450) with the corresponding JE sequence had no effect on anti-DEN-2 MAb reactivity, indicating that this region is antigenically inert, although it is required for antigen secretion. Plasmid pCBD2-2J-2-9-1, which expressed secreted forms of prM/M and E that have the potential to form subviral particles, was superior to other constructs in stimulating an antibody response. Ninety percent neutralization titers ranging from 1:40 to >1:1000 were observed in seven of nine serum specimens from pCB8D2-2J-2-9-1-immunized mice. Eleven of twelve 2-day-old neonatal mice, derived from a pCB8D2-2J-2-9-1 immunized female mouse, survived intraperitoneal challenge of DEN-2 New Guinea C virus.