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. 2019 Mar 22;10(1):1324.
doi: 10.1038/s41467-019-09256-0.

Aedes mosquitoes acquire and transmit Zika virus by breeding in contaminated aquatic environments

Senyan Du  1   2   3 , Yang Liu  1   4 , Jianying Liu  1 , Jie Zhao  1 , Clara Champagne  5 , Liangqin Tong  1 , Renli Zhang  3 , Fuchun Zhang  6 , Cheng-Feng Qin  7 , Ping Ma  8 , Chun-Hong Chen  9 , Guodong Liang  10 , Qiyong Liu  11 , Pei-Yong Shi  12 , Bernard Cazelles  5   13 , Penghua Wang  14 , Huaiyu Tian  15 , Gong Cheng  16   17   18
Affiliations

Aedes mosquitoes acquire and transmit Zika virus by breeding in contaminated aquatic environments

Senyan Du et al. Nat Commun. .

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that predominantly circulates between humans and Aedes mosquitoes. Clinical studies have shown that Zika viruria in patients persists for an extended period, and results in infectious virions being excreted. Here, we demonstrate that Aedes mosquitoes are permissive to ZIKV infection when breeding in urine or sewage containing low concentrations of ZIKV. Mosquito larvae and pupae, including from field Aedes aegypti can acquire ZIKV from contaminated aquatic systems, resulting in ZIKV infection of adult females. Adult mosquitoes can transmit infectious virions to susceptible type I/II interferon receptor-deficient (ifnagr-/-) C57BL/6 (AG6) mice. Furthermore, ZIKV viruria from infected AG6 mice can causes mosquito infection during the aquatic life stages. Our studies suggest that infectious urine could be a natural ZIKV source, which is potentially transmissible to mosquitoes when breeding in an aquatic environment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Mosquitoes are permissive to Zika virus (ZIKV) infection during breeding. a, b ZIKV survivability in human urine. ZIKV was incubated with fresh human urine or phosphate-buffered saline (PBS) (Mock). The mixtures were then maintained at either 37 °C or 28 °C over a time course and subsequently subjected to a viral titration assay. The PRVABC59 strain was used for the incubation. The initial ZIKV titer was 1 × 105 or 20 pfu/ml. Data are presented as the mean ± S.E.M. a n = 3 biologically independent samples. b n = 2 biologically independent samples. The data were repeated by two independent experiments with the similar results. F: female; M: male. c–e Acquisition of ZIKV infection by Aedes mosquitoes breeding in infectious human urine. c Experimental schematic representation. Freshly emerging mosquito pupae were used for breeding in ZIKV urine. The urine from human Donor 3 (Fig. 1a) was used in these experiments. Both ZIKV prevalence and infectivity were determined in the A. aegypti Rockefeller strain (d) and the A. albopictus Jiangsu strain (e). The mosquitoes were breeding from human urine with serial ZIKV titration. The emerging adults were reared for an additional 8 days for ZIKV detection by TaqMan quantitative PCR (qPCR). The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. One dot represents one mosquito. The percentages are represented as the ratios of mosquito infection. a–e Source data are provided in as Source Data file
Fig. 2
Fig. 2
Zika virus (ZIKV) is transmissible between AG6 mice and mosquitoes breeding in ZIKV urine. a Schematic representation of the ZIKV transmission between AG6 mice and mosquitoes breeding in the ZIKV urine. The A. aegypti pupae were breeding in the ZIKV urine (final ZIKV titer was 20 pfu/ml). The urine from human Donor 3 (Fig. 1a) was used in these experiments. After rearing for 8 days after adults emerged, 20 emerged female mosquitoes were allowed to feed together on an AG6 mouse. The mouse viremia was determined over a time course. b, c ZIKV viremia in the mosquito-bitten AG6 mice. The A. aegypti Rockefeller strain was used in this experiment. d Acquisition of ZIKV infection by a field A. aegypti Brazil Paraiba strain breeding in infectious human urine. With the same experimental procedure as Fig. 1c, both ZIKV prevalence and infectivity were determined in the A. aegypti Brazil Paraiba strain breeding in human urine with a serial ZIKV titration. The emerging adults were reared for 8 days for subsequent ZIKV detection by TaqMan quantitative PCR (qPCR). The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. One dot represents one mosquito. The percentages are represented as the ratios of mosquito infection. e, f ZIKV viremia in the AG6 mice bitten by the A. aegypti Brazil Paraiba mosquitoes breeding in human urine. b, e The presence of ZIKV RNA copies in whole blood was assessed using quantitative reverse transcription PCR (RT-qPCR) detection. n represents the mouse number used in the experiment. The results were pooled from three detection replicates. c, f The numbers of infectious particles at the viremic peak were detected by a plaque assay. c n = 3 detection replicates. f n = 2 detection replicates. Data are presented as the mean ± SEM. b–f Source data are provided as a Source Data file
Fig. 3
Fig. 3
The field A. aegypti mosquitoes breeding in the infectious sewage transmit Zika virus (ZIKV) to hosts. a, b ZIKV survivability in the sewage samples. The supernatant from ZIKV-infected Vero cells was incubated at 28 °C with the sewage samples or phosphate-buffered saline (PBS) (Mock), with either 1 × 105 (a) or 20 pfu/ml (b) of initial titer. The viral survivability was measured over a time course by a plaque assay. a n = 2 biologically independent samples for sewages and n = 3 for the mock. b n = 2 biologically independent samples. c, d Continuous supplementation of ZIKV in the sewage. c Schematic representation. Either the sewage (Sample #5) or PBS (Mock) was used. d ZIKV survivability in the infectious sewage. The mixtures were maintained at 28 °C for a time course and subsequently subjected to a plaque assay. n = 3 biologically independent samples. a–d The data were repeated by two independent biological replicates with the similar results. e Acquisition of ZIKV infection by the A. aegypti Brazil Paraiba mosquitoes breeding in the infectious sewage. Both ZIKV prevalence and infectivity in A. aegypti. The initial ZIKV urine concentration was 20 pfu/ml. f–i ZIKV is transmissible between AG6 mice and mosquitoes breeding in the infectious sewage. f Schematic representation. g, h ZIKV viremia in the mosquito-bitten AG6 mice. g The presence of ZIKV genome in whole blood was assessed using quantitative PCR (qPCR) detection. n represents the mouse number. h Detection by a plaque assay. n = 2 detection replicates. i Prevalence of ZIKV infection in the mosquitoes fed on a viremic AG6 mouse. e, i The mosquitoes for ZIKV detection by TaqMan qPCR. The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. One dot represents one mosquito. The percentages are represented as the ratios of mosquito infection. a–h Data are presented as the mean ± SEM. a–i The PRVABC59 strain was used for the incubation. Source data are provided as a Source Data file
Fig. 4
Fig. 4
Correlation between Zika virus (ZIKV) survivability and the pH value of human urine. a Adjustment of pH value regulated ZIKV survivability in human urine. The pH values of human urine samples were adjusted by either NaOH or HCl. The pH value is showed at the top of each column. n = 3 biologically independent samples. Data are presented as the mean ± SEM. b ZIKV survivability in human urine correlates with the pH value of urine. One dot represents one human urine sample. Data were analyzed by linear regression with correlation coefficients (r) and significance (p). n represents the number of human urine samples. The information for the urine samples is summarized in Supplementary Table 4. c Determination of the correlation between ZIKV survivability and the pH value in human urine. The urine samples from Donor 3 with gradient pH values were incubated with ZIKV supernatant. The percentage of ZIKV survivability was defined as the ratio of the pfu value at 4 h incubation to the value at 0 h incubation. n = 3 biologically independent samples. Data are presented as the mean ± SEM. a–c The urine samples were incubated with ZIKV supernatant from infected Vero cells (the initial titer was 1 × 105 pfu/ml) for 4 h at 37 °C. Subsequently, the ZIKV titer was measured by a plaque assay. Source data are provided as a Source Data file
Fig. 5
Fig. 5
Urination by infected animals leads to the infection of mosquitoes during breeding. a Schematic representation of mosquito infection by breeding in diluted infected mouse urine. We infected the AG6 mice by Zika virus (ZIKV) intrapleural injection (100 pfu per mouse) and subsequently subjected the infected mice to daily NaHCO3 treatment. The infected AG6 mice without NaHCO3 treatment served as untreated controls. The urine was collected and subsequently diluted 5-fold for the A. aegypti (Rockefeller strain) pupal eclosion. The emerging mosquitoes were reared for an additional 8 days for quantitative reverse transcription PCR (RT-qPCR) detection. b ZIKV viremia in the infected AG6 mice. The viral load was detected by a plaque assay over a time course after infection. c Regulation of the pH value in the urine of NaHCO3-treated infected AG6 mice. d Assessing the ZIKV infectious particles in the urine of infected AG6 mice. The urine samples collected daily were measured by a plaque assay. b–d n represents 12 independent mice in each group. The data were pooled from three independent experiments. Data are presented as the mean ± SEM. e Mosquitoes acquired ZIKV infection by breeding in diluted urine from NaHCO3-treated infected AG6 mice. The urine samples from four individual mice were mixed and then diluted 5-fold for A. aegypti pupal eclosion. The emerging adults were reared for 8 days for ZIKV detection by RT-qPCR. The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. One dot represents one mosquito. The percentages are represented as the ratios of mosquito infection. The data were pooled from three independent biological replicates. f Schematic representation of the host urination-mosquito breeding ZIKV transmission route. A Zika patient may release urine daily, resulting in various levels of infectious ZIKV being excreted by patient urination in natural circumstances. The mosquitos breeding from the water systems contaminated by ZIKV may acquire the infection. Therefore, viruria of infected hosts may facilitate ZIKV prevalence and transmission by mosquitoes. b–e Source data are provided as a Source Data file
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