Vertical transmission of Rabensburg virus in three strains of <i>Culex pipiens.</i>^*

*<p>MLE, maximum likelihood estimation of infection rates; <i>CpUS</i>, a United States strain of <i>Culex pipiens</i> that contain genetic signatures of both form pipiens and form molestus; <i>CpEU,</i> a European strain of <i>Culex pipiens</i> form molestus; <i>CxM</i>, a United States strain of <i>Culex pipiens</i> form molestus; OP, oviposition; dpi, days post infection; Vertical transmission of RABV was observed in two independent experiments for <i>CpUS</i> and <i>CxM</i> and in one out of two independent replicates for <i>CpEU.</i> All surviving parental females were pooled in groups of five, screened for viral infection, and all pools were RABV positive.</p>†<p>pool size = 5.</p

Proportion of chickens infected following inoculation of 10-fold dilutions of SLEV before (P0) and after (CP) passage in chickens.

<p>*Chi-squared, p<0.05.</p><p>1Calculated using Reed-Muench formula.</p

Intrahost nucleotide diversity of bases 1315–3325 for individual lineages of SLEV during sequential passage in chickens or mosquitoes.

<p>Nt diversity is equivalent to the total # of mutations relative to the consensus/total # of bases sequenced. S_n(nt) refers to normalized Shannon entropy based on the frequency of individual genotypes.</p

SLEV viremia kinetics in 1-day old chickens before (P0) and after (CP20) passage in chickens.

<p>Data points represent means of 4–5 chickens +/− S.D. Statistically significance differences are indicated by an asterisk (t-test, p<0.05).</p

Proportion of <i>Cx. pipiens</i> mosquitoes infected following inoculation of 10-fold dilutions of SLEV before (P0) or after passage in mosquitoes (MP) or chickens (CP).

<p>1Calculated using Reed-Muench formula.</p

Consensus sequence changes of SLEV following passage in chickens (CP) or <i>Cx. pipiens</i> mosquitoes (MP).

<p>Consensus sequence changes of SLEV following passage in chickens (CP) or <i>Cx. pipiens</i> mosquitoes (MP).</p

Viral load of individual lineages of SLEV during sequential passage in chickens (A) or mosquitoes (B).

<p>Viral load of individual lineages of SLEV during sequential passage in chickens (A) or mosquitoes (B).</p

Evaluation of WNV NS5 variants with an in vitro primer extension assay reveals differences in polymerase incorporation fidelity.

<p><b>A.</b> Schematic of primer extension assay for evaluating WNV NS5 polymerase activity. <b>B.</b> Evaluation of elongation reactions using either correct or incorrect nucleotide substrates. Shown is the experimental design and denaturing PAGE gel of both correct and incorrect nucleotide addition. Experimental design: WNV NS5 is assembled for 30 min to produce elongation-competent complexes at which point a trap (heparin) for free enzyme is added. The reaction is then rapidly mixed with NTP substrate and quenched at various times. Denaturing PAGE gel: The 15-mer RNA is rapidly extended to 16-mer RNA product in the presence of correct nucleotide substrate, ATP. The presence of both ATP and GTP together promote full extension to 20-mer RNA as the terminal templating bases are competent for correct nucleotide addition. The presence of GTP alone allows for the kinetics of incorrect nucleotide incorporation to be observed (G:U mispair). <b>C.</b> Comparison of the kinetics of misincorporation between WNV-IC, V793I/G806R and T248I NS5. There is no observable difference in the kinetics of GMP misincorporation (G:U mispair). Shown is the percentage of RNA product plotted as a function of time and fit to a single exponential. <b>D.</b> By using an alternative substrate that allows for A:C mispairs to be evaluated, a difference in the efficiency of AMP misincorporation is observed between the three NS5 proteins.</p

Host-specific effects of WNV replication complex mutations on replicative fitness <i>in vitro</i>.

<p>Growth kinetics of WNV strains in vertebrate (BHK) and mosquito (CxT) cell culture are shown for both 1-step (<b>A</b>; MOI 10 and 8, respectively) and multi-step (<b>B</b>; MOI 0.01) infections. Viral growth kinetics were equivalent in BHK cells and significantly different in CxT cells (repeated measures ANOVA, p<0.001). Specifically, WNV-IC titers were significantly higher in CxT cells at both MOIs relative to all mutant strains (ANOVA, Tukey post-test, p<0.05).</p

Specific infectivity of WNV replication complex mutants.

<p>WNV RNA (viral particles) and infectious virus (pfu) were quantified following 1-step growth on mosquito (CxT) or vertebrate (BHK) cell culture and specific infectivity (particle/pfu) was compared among WNV-IC, WNV V793I/G806R and WNV T248I. Graphs represent means +/- standard deviation. Significantly decreased infectivity (*) was measured for WNV T248I relative to WNV-IC and WNV V793I/G806R (t-test, df = 4, p = 0.0028).</p