Abstract
The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of ≥10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a β-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.
Similar content being viewed by others
References
Centers for Disease Control and Prevention (1999). Update: West Nile Virus encephalitis—New York, 1999. MMWR Morb Mortal Wkly Rep 48: 944–946, 955.
Anderson JF, Andreadis TG, Vossbrinck CR, Tirrell S, Wakem EM, French RA, Garmendia AE, Van Kruiningen HJ (1999). Isolation of West Nile virus from mosquitoes, crows, and a Cooper’s hawk in Connecticut. Science 286: 2331–2333.
Beasley DW, Li L, Suderman MT, Barrett AD (2002). Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 296: 17–23.
Biggerstaff BJ, Petersen LR (2002). Estimated risk of West Nile virus transmission through blood transfusion during an epidemic in Queens, New York City. Transfusion 42: 1019–1026.
Briese T, Jia XY, Huang C, Grady LJ, Lipkin WI (1999). Identification of a Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis. Lancet 354: 1261–1262.
Brinton MA (1981). Isolation of a replication-efficient mutant of West Nile virus from a persistently infected genetically resistant mouse cell culture. J Virol 39: 413–421.
Brinton MA (2002). The molecular biology of West Nile Virus: a new invader of the western hemisphere. Annu Rev Microbiol 56: 371–402.
Chu JJ, Ng ML (2002). Infection of polarized epithelial cells with flavivirus West Nile: polarized entry and egress of virus occur through the apical surface. J Gen Virol 83: 2427–2435.
Dinter A, Berger EG (1998). Golgi-disturbing agents. Histochem Cell Biol 109: 571–590.
Elshuber S, Allison SL, Heinz FX, Mandl CW (2003). Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J Gen Virol 84: 183–191.
Greene LA, Tischler AS (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73: 2424–2428.
Gromeier M, Wimmer E (1998). Mechanism of injury-provoked poliomyelitis. J Virol 72: 5056–5060.
He RT, Innis BL, Nisalak A, Usawattanakul W, Wang S, Kalayanarooj S, Anderson R (1995). Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. J Med Virol 45: 451–461.
Jordan MA, Thrower D, Wilson L (1992). Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. J Cell Sci 102(Pt 3): 401–416.
Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, Bowen M, McKinney N, Morrill WE, Crabtree MB, Kramer LD, Roehrig JT (2002). Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology 298: 96–105.
Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Banet C, Weissman J, Komar N, Savage HM, Stone W, McNamara T, Gubler DJ (1999). Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286: 2333–2337.
Mabit H, Nakano MY, Prank U, Saam B, Dohner K, Sodeik B, Greber UF (2002). Intact microtubules support adenovirus and herpes simplex virus infections. J Virol 76: 9962–9971.
Mackenzie JM, Jones MK, Westaway EG (1999). Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells. J Virol 73: 9555–9567.
Mackenzie JM, Westaway EG (2001). Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 75: 10787–10799.
Monath TP, Cropp CB, Harrison AK (1983). Mode of entry of a neurotropic arbovirus into the central nervous system. Reinvestigation of an old controversy. Lab Invest 48: 399–410.
Moss B, Ward BM (2001). High-speed mass transit for poxviruses on microtubules. Nat Cell Biol 3: E245-E246.
Nash D, Mostashari F, Fine A, Miller J, O’Leary D, Murray K, Huang A, Rosenberg A, Greenberg A, Sherman M, Wong S, Layton M (2001). The outbreak of West Nile virus infection in the New York City area in 1999. N Engl J Med 344: 1807–1814.
O’Leary DR, Marfin AA, Montgomery SP, Kipp AM, Lehman JA, Biggerstaff BJ, Elko VL, Collins PD, Jones JE, Campbell GL (2004). The epidemic of West Nile virus in the United States, 2002. Vector-Borne Zoonot Dis 4: 61–70.
Pogodina VV, Frolova MP, Malenko GV, Fokina GI, Koreshkova GV, Kiseleva LL, Bochkova NG, Ralph NM (1983). Study on West Nile virus persistence in monkeys. Arch Virol 75: 71–86.
Randolph VB, Hardy JL (1988). Establishment and characterization of St Louis encephalitis virus persistent infections in Aedes and Culex mosquito cell lines. J Gen Virol 69(Pt 9): 2189–2198.
Raux H, Flamand A, Blondel D (2000). Interaction of the rabies virus P protein with the LC8 dynein light chain. J Virol 74: 10212–10216.
Sampson BA, Armbrustmacher V (2001). West Nile encephalitis: the neuropathology of four fatalities. Ann N Y Acad Sci 951: 172–178.
Sejvar JJ, Haddad MB, Tierney BC, Campbell GL, Marfin AA, Van Gerpen JA, Fleischauer A, Leis AA, Stokic DS, Petersen LR (2003). Neurologic manifestations and outcome of West Nile virus infection. JAMA 290: 511–515.
Shrestha B, Gottlieb D, Diamond MS (2003). Infection and injury of neurons by West Nile encephalitis virus. J Virol 77: 13203–13213.
Smith R, Wilcox C (1996). Studies of herpes simplex virus 1 latency using primary neuronal cultures of dorsal root ganglion neurons. In: Protocols for gene transfer in neuroscience: Towards gene therapy of neurological disorders. Lowenstein PR, Enquist LW (eds). West Sussex, UK: John Wiley & Sons, pp 221–231.
Smithburn K, Hughes T, Burke A (1940). A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg 20: 471–492.
Solomon T, Willison H (2003). Infectious causes of acute flaccid paralysis. Curr Opin Infect Dis 16: 375–381.
Sreenivasan V, Ng KL, Ng ML (1993). Brefeldin A affects West Nile virus replication in Vero cells but not C6/36 cells. J Virol Methods 45: 1–17.
Stadler K, Allison SL, Schalich J, Heinz FX (1997). Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71: 8475–8481.
Vasquez RJ, Howell B, Yvon AM, Wadsworth P, Cassimeris L (1997). Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro. Mol Biol Cell 8: 973–985.
Vetterlein M, Niapir M, Ellinger A, Neumuller J, Pavelka M (2003). Brefeldin A-regulated retrograde transport into the endoplasmic reticulum of internalised wheat germ agglutinin. Histochem Cell Biol 120: 121–128.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hunsperger, E., Roehrig, J.T. Characterization of West Nile viral replication and maturation in peripheral neurons in culture. Journal of NeuroVirology 11, 11–22 (2005). https://doi.org/10.1080/13550280590900454
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1080/13550280590900454