Infection, dissemination, and transmission of a West Nile virus green fluorescent protein infectious clone by Culex pipiens quinquefasciatus mosquitoes

Abstract

We report the construction and comparative characterization of a full-length West Nile virus (WNV) cDNA infectious clone (ic) that contains a green fluorescent protein (GFP) expression cassette fused within the viral open reading frame. Virus derived from WNV-GFP ic stably infected Culex pipiens quinquefasciatus mosquitoes at comparable rates to virus derived from the parental (non-GFP) ic. However, insertion of this GFP cassette resulted in a temporal delay in in vivo replication kinetics and significantly decreased dissemination to head tissue. Consistent with previous reports of WNV-infected mosquito midguts, focal GFP expression was observed at 3 days post-infection (dpi), with the majority of posterior midgut epithelial cells being positive by 7 dpi. GFP foci were observed in one pair of salivary glands (1/15) dissected 14 dpi. Mice exposed to WNV-GFP-infected mosquitoes developed viremia, and GFP was detected in lymph node homogenates. These data demonstrate the effectiveness of our strategy to generate a replication competent construct with increased reporter gene stability that may be used to study early events in infection.

FIG. 1.

pBelo-WNV-GFP-RZ ic plasmid map. WNV, West Nile virus; GFP, green fluorescent protein; ic, infectious clone; RB2, rybozyme.

FIG. 2.

Average whole-body titers (expressed as log₁₀TCID₅₀/mosquito) of Culex pipiens quinquefasciatus mosquitoes infected (per os) with virus derived from either pBelo-WNV-RZ ic or pBelo-WNV-GFP ic. ▴, WNV-TX02 infectious bloodmeal titer 7.52 log₁₀TCID₅₀/mL; □, WNV-GFP infectious bloodmeal titer 6.95 log₁₀TCID₅₀/mL; •, WNV-GFP infectious bloodmeal titer 4.95 log₁₀TCID₅₀/mL. Mean titers calculated only from virus-positive mosquitoes. Error bars represent standard deviation about the mean. TCID, tissue culture infectious dose.

FIG. 3.

WNV-GFP in selected segments of the Cx. p. quinquefasciatus digestive tract. (A, B) Green foci at 3 days postinfection (dpi) distributed throughout the cardia/intussuscepted foregut, anterior, and posterior midgut. (C) WNV-GFP infectious foci in the posterior midgut 5 dpi. (D, E) GFP expression posterior in midguts 7 dpi.

FIG. 4.

Green fluorescent foci (A) in a Cx. p. quinquefasciatus salivary gland pair. (B) 14 days postingestion of a high-titer (6.95 log₁₀ TCID₅₀/mL) WNV-GFP infectious bloodmeal.