The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated West Nile virus
Introduction
West Nile (WN) virus is an arthropod-borne virus of the genus Flavivirus in the family Flaviviridae. It is a member of the Japanese encephalitis virus (JEV) serocomplex, which includes JEV, St. Louis encephalitis virus, and Murray Valley encephalitis (MVE) virus. Since the outbreak of WN encephalitis in humans and horses in New York City in late August 1999, WN virus has spread throughout North and Central America (Beasley et al., 2001, Garmendia et al., 2001). Currently, no effective therapies or vaccines against human WN virus infection exist.
The WN virus strain isolated in New York City (NY strain) causes large-scale mortality in wild birds; however, this phenomenon was not observed with previously isolated WN virus strains (Garmendia et al., 2001). Therefore, the pathogenicity of the NY strain appears to differ from that of previously isolated strains, and the study of the pathogenesis of this strain may be useful for developing new vaccines and therapies.
Recently, variants with different amino acid sequences at an N-linked glycosylation site in the envelope (E) protein region were isolated from NY strain stocks (Beasley et al., 2004, Beasley et al., 2005, Shirato et al., 2004b). When 6-week-old BALB/c mice were infected subcutaneously with these variants, those infected with viruses carrying the glycosylated E protein developed lethal infections, whereas those infected with viruses carrying non-glycosylated E protein showed low mortality. In contrast, intracerebral infection was lethal, with no difference between E protein-glycosylated and non-glycosylated viruses. Therefore, the glycosylation of the E protein is a molecular determinant of the neuroinvasiveness of the NY strain of WN virus.
The initial target of infection for dengue virus, a mosquito-borne flavivirus, is dendritic cells (Wu et al., 2000); these cells migrate to the lymph nodes after arbovirus infection (Johnston et al., 2000). However, subsequent events leading to viral invasion of the central nervous system (CNS) remain unclear.
It has been reported that macrophage-depleted mice exhibited more severe WN virus-induced disease (Ben-Nathan et al., 1996), and maximum antigen-presenting activity was exhibited by activated macrophages in WN virus-infected mouse lymph nodes (Pisarev et al., 2003). Therefore, we focused on the interaction between WN virus and macrophages. In this study, we investigated virus replication and cytokine induction in macrophages infected with E protein-glycosylated and non-glycosylated WN viruses.
Section snippets
Viruses
Two variants, 6-LP (AB185914) and 6-SP (AB185915), of West Nile virus strain NY99-6922 were used in this study. These viruses were isolated from the NY99-6922 strain by plaque purification (Shirato et al., 2004b) and have a single point mutation at an N-linked glycosylation site in the envelope (E) protein region sequence. The 6-LP virus has N-linked glycosylated E protein; the 6-SP virus does not. Viruses were propagated once in the brains of suckling mice as previously described (Shirato et
Virus replication in macrophages
WN virus replication in murine peritoneal macrophages was determined (Fig. 1). The E protein-glycosylated WN virus, 6-LP, and non-glycosylated virus, 6-SP, were infected into macrophages at an m.o.i. of 1 or 0.01. Previous data (Shirato et al., 2004b) and Fig. 1C both show that the E-protein-glycosylated virus replicated at a higher titer than did the non-glycosylated virus in tissue culture and murine organs. In macrophages, the two viruses exhibited similar kinetics at both m.o.i., with a
Discussion
The aim of this study was to examine the relationship between E protein glycosylation and macrophage infection with WN virus. The results showed that virus replication in macrophages did not differ between E protein-glycosylated and non-glycosylated viruses. Previous data in BHK cells and the results of this study in Vero E6 cells both show that the final concentration of E protein-glycosylated WN virus is 10 times that of non-glycosylated virus in cell culture (Shirato et al., 2004b). In
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research and the Program of Excellence for Zoonosis Control, the 21st Century COE program, from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.
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