Gain without pain: adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector

Abstract

Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice.

Keywords: Zika virus; arbovirus; evolution; pathogenesis; transmission.

Fig 1

Vector competence of serially passaged Zika virus lineages. Female Ae. aegypti mosquitoes were exposed to passaged ZIKV strains via an artificial infectious bloodmeal and collected 7, 14, and 21 dpf. Infection (A), dissemination (B), and transmission (C) rates over the three collection timepoints are shown. Infection rate is the percentage of ZIKV-positive bodies, dissemination rate is the percentage of positive legs, and transmission rate is the percentage of positive saliva samples of total mosquitoes that took a bloodmeal (determined by plaque assay). Data points represent the empirically measured percentages (n = 39–80 per data point). The lines represent the logistic regression results and the shaded areas represent the 95% confidence intervals of the logistic regression fits. Infection, dissemination, and transmission rates of VP-A and VP-C were compared to ZIKV-BC at each timepoint (two-tailed Fisher’s exact test). *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001; ****, P  <  0.0001; ns, not significant. Infectious virus was quantified via plaque assay from bodies (D), legs (E), and saliva (F) from all positive samples. Mean titers were not significantly different between virus groups in any tissue at any timepoint (one-way ANOVA with Tukey’s multiple comparisons test). ns, not significant. (G) In vitro growth kinetics of passaged ZIKV strains in C6/36 cells. Data points represent the mean of three replicates at each timepoint. Error bars represent standard deviation. Differences in viral titer at each timepoint were compared to ZIKV-BC (two-way ANOVA with Holm-Sidak’s multiple comparisons test). *, P  <  0.05; **, P  <  0.01. The dotted line indicates the assay limit of detection.

Fig 2

Deep sequencing of viral populations in mosquito tissues. Paired legs and saliva samples from individual mosquitoes exposed to VP-A (A) and VP-C (B) 21 dpf were deep sequenced. Lines represent single nucleotide variant (SNV) frequency percentages between the stock or bloodmeal, legs, and saliva.

Fig 3

In vitro and in vivo characterization of ZIKV mutant clones. In vitro growth kinetics of ZIKV clones in Vero (A) and C6/36 (B) cells. Data points represent the mean of three replicates at each timepoint. Error bars represent standard deviation. (C) Serum viremia 2 and 4 days post infection (dpi) of Ifnar1 ^-/- mice inoculated with 10³ PFU of different ZIKV clones [n = 8 for virus groups, n = 4 for phosphate buffered saline (PBS) control]. Serum viremia for mutant viruses was compared to previously published viremia data from VP-C infection. Differences in mean serum viremia between virus groups was compared by one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant. The dotted line indicates the assay limit of detection. (D) Survival curves of Ifnar1 ^-/- mice inoculated with 10³ PFU of virus, or a PBS control. HD-E19G and HD-WTic groups were inoculated with 10⁴ PFU. Survival curves were compared to WTic by Fisher’s exact test. *, P < 0.05. (E) Chromatograms from Sanger sequencing of a subset of E19G 7d and 9d and VP-C 7d brains showing confirmation of the maintained NS4A position 19 substitution (nucleotide substitution at nt 6519 of the polyprotein: GAG → GGG). Sequenced amplicons were aligned to ZIKV-PRVABC59.

Fig 4

Differential brain viral loads and innate immune gene responses to ZIKV mutants. Viral RNA (A) and infectious virus (B) were quantified from brain tissue collected 3, 7, and 9–10 days after 10³ PFU inoculation of Ifnar1^-/- mice with ZIKV-IC, NS4A-E19G, VP-C or, double-mutant clones by qRT-PCR and plaque assay, respectively. The dotted line indicates the assay limit of detection (B). Transcript abundance of RIG-I (C), MDA-5 (D), MAVS (E), and TLR3 (F) was analyzed from brains collected 3, 7, and 9–10 dpi by qPCR. Expression levels were normalized to ActB and Hprt and the ddC_T value was calculated as log2 fold change expression relative to mock-inoculated mice. Data points represent individual samples. Means with standard deviation are shown. Viral loads, infectious viral titers, and relative fold change of transcript abundance were compared between virus groups at each timepoint by one-way ANOVA with Tukey’s multiple comparisons test. *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001; ****, P  <  0.0001; ns, not significant.

Fig 5

Vector competence for ZIKV mutants. Female Ae. aegypti mosquitoes were exposed to passaged ZIKV strains via an artificial infectious bloodmeal and collected 7, 14, and 21 dpf. Infection (A), dissemination (B), and transmission (C) rates over the three collection timepoints are shown. Infection rate is the percentage of ZIKV-positive bodies, dissemination rate is the percentage of positive legs, and transmission rate is the percentage of positive saliva samples (determined by plaque assay screen). Data points represent the empirically measured percentages (n = 20–80 per data point, point size is proportional to group size). The lines represent the logistic regression results and the shaded areas represent the 95% confidence intervals of the logistic regression fits.