Characterization of a small plaque variant of West Nile virus isolated in New York in 2000

Abstract

A small-plaque variant (SP) of West Nile virus (WNV) was isolated in Vero cell culture from kidney tissue of an American crow collected in New York in 2000. The in vitro growth of the SP and parental (WT) strains was characterized in mammalian (Vero), avian (DF-1 and PDE), and mosquito (C6/36) cells. The SP variant replicated less efficiently than did the WT in Vero cells. In avian cells, SP growth was severely restricted at high temperatures, suggesting that the variant is temperature sensitive. In mosquito cells, growth of SP and WT was similar, but in vivo in Culex pipiens (L.) there were substantial differences. Relative to WT, SP exhibited reduced replication following intrathoracic inoculation and lower infection, dissemination, and transmission rates following oral infection. Analysis of the full length sequence of the SP variant identified sequence differences which led to only two amino acid substitutions relative to WT, prM P54S and NS2A V61A.

Figure 1

Plaque morphology of wild type (WT) and a small plaque (SP) variant WNV. Vero cells in 6-well plates were infected with Vero cell amplified WT or SP stock virus, and plaques were visualized 3 days post-infection after staining with neutral red. (A) Plaques were examined with a Zeiss Stemi 2000-C stereo microscope, and images were captured with a Zeiss Axiocam MRC digital camera. (B) Plaque sizes were measured using AxioVision 3.0 software (Zeiss, Germany). For each virus, 200 individual plaques were measured and analyzed by Graph Pad Prism (v.4.0). Bar within the box represents the median value, boxes extend from 25^th to 75^th percentile, and error bars represent highest and lowest values. Statistical analysis by t-test, P<0.0001.

Figure 2

Comparison of growth of wild type (WT) and a small plaque (SP) variant WNV in three vertebrate cell lines. Vero, DF-1, and PDE cells in 6-well plates were infected with Vero cell amplified WT or SP stock virus in triplicate at an MOI of 0.01 pfu/cell, based on Vero cell titers. Both infection and incubation were carried out at the indicated temperatures. Virus was harvested from the medium at various time points after infection, and titers were determined by plaque assay on Vero cells. Symbols represent the mean titer and standard deviation for each time point. The limit of detection (LOD) was 0.7 log₁₀pfu/0.1ml of supernatant of infected cell cultures; in cases where the sample had no detectable virus, a titer of 0.65 log₁₀pfu/0.1ml was used for calculations.

Figure 3

Comparison of growth of wild type (WT) and a small plaque (SP) variant WNV in vitro in a mosquito cell line and in vivo in mosquitoes. (A) C6/36 cells in 6-well plates were infected with Vero cell amplified WT or SP stock virus in triplicate at an MOI of 0.01 pfu/cell. Virus was harvested from the medium at the indicated time points and titers determined by plaque assay on Vero cells. Symbols represent the mean titer and standard deviation for each time point. The limit of detection (LOD) was 0.7 log₁₀pfu/0.1ml of supernatant of infected cell cultures; in cases where the sample had no detectable virus, a titer of 0.65 log₁₀pfu/0.1ml was used for calculations. (B) Cx. pipiens mosquitoes were inoculated intrathoracically at 10× the ID₅₀ of WT (5 pfu per mosquito) or SP (21 pfu per mosquito) with Vero cell amplified WT or SP stock virus, and were maintained at the indicated temperatures. Five mosquitoes per virus were harvested immediately following inoculation, and 10 mosquitoes were harvested at each time point thereafter. Viral titers in the mosquito bodies were determine by plaque titration on Vero cells. Symbols represent the mean viral titer and standard deviation for each time point. The limit of detection (LOD) was 0.7 log₁₀pfu/mosquito, and in cases where the sample had no detectable virus, a titer of 0.65 log₁₀pfu/mosquito was used for calculations.

Figure 4

Virus titers of wild type (WT) and small plaque (SP) variant WNV in Cx. pipiens bodies at 7 and 14 days after oral infection with the C6/36 cell amplified virus. Mosquitoes were fed on defibrinated goose blood containing 10^8.4 pfu/ml of either WT or SP virus, incubated at 27°C, and harvested at 7 or 14 days post-feeding (PF). Virus recovered from each mosquito body was titered on Vero cells; each symbol represents the titer from an individual mosquito. The mean titer of SP was significantly less than WT at both 7 and 14 days PF by t-test (P₇ = 0.001; P₁₄=0.001). For both SP and WT the mean titer at 7 day PF was significantly lower than at 14 day PF by t-test (P_SP=0.012; P_WT=0.001).