Zika virus transmission in Aedes aegypti: A systematic study on the ability of mosquitoes to transmit the virus horizontally and vertically

Horizontal transmission of ZIKV from female Ae. aegypti mosquitoes to male or female mosquitoes

To investigate the potential for ZIKV-infected female Ae. aegypti mosquitoes to transmit the virus to other female or male Ae. aegypti mosquitoes, the female mosquitoes were initially fed with blood containing ZIKV using a Hemotek membrane feeding system in vitro. TaqMan qPCR was employed to determine the viral load in Ae. aegypti, revealing a high infective ratio (Fig. 1A–C). Between 0 and 8 days post blood meal, 48%–83% of female Ae. aegypti were progressively found to be infected with ZIKV, carrying 10³–10⁸ RNA copies per mosquito (Fig. 1B and C). Additionally, infectious virus was detected at 8 and 12 days post blood meal with 10³–10⁷ ​PFU per mosquito (Fig. 1D). These findings suggest that female Ae. aegypti can acquire ZIKV through the Hemotek membrane feeding system.

To visually distinguish uninfected female Ae. aegypti from their infected counterparts, we marked naïve female Ae. aegypti with a nontoxic dosage of fluorescent powder (0.2 ​mg per mosquito) (Fig. 1E and F). Subsequently, we cohabitated these marked mosquitoes with ZIKV-infected female Ae. aegypti at 8 days post blood meal in the same cage for 6 ​h to facilitate contact transmission. The viral loads in the fluorescent powder-labeled female Ae. aegypti mosquitoes at 0, 4, 8, 12 and 16 days post-contact were assessed using TaqMan qPCR. Between 58% and 96% of these mosquitoes were found to be infected with ZIKV, with RNA copies ranging from 10³ to 10⁸ per mosquito (Fig. 1G and H). An increasing abundance of viral RNA was detected from day 0 to day 8, followed by a decline from day 12 to day 16 (Fig. 1G and H). These results indicate that ZIKV-infected female Ae. aegypti mosquitoes can transmit ZIKV to other female mosquitoes via horizontal contact.

Similarly, male Ae. aegypti mosquitoes were observed to be infected with ZIKV via horizontal contact with female mosquitoes (Fig. 1I–K). We cohabitated the ZIKV-infected female Ae. aegypti at 8 days post infection with male Ae. aegypti mosquitoes in the same cage for 6 ​h (Fig. 1A). The viral loads in male Ae. aegypti mosquitoes at 0, 4, 8, 12, and 16 days post-contact were quantified using TaqMan qPCR. Surprisingly, 8%–88% of male mosquitoes were found to be infected with ZIKV, with RNA copies ranging from 10² to 10⁸ per mosquito (Fig. 1I and J). An increasing abundance of viral RNA was detected from day 0 to day 8, followed by a decline from day 12 to day 16 (Fig. 1I and J). Plaque assays were also conducted to detect infectious virus particles from representative mosquitoes, with viral loads in both female and male mosquitoes ranging from 10² to 10⁷ ​PFU per mosquito at 8 days post-contact with infected females (Fig. 1K). These findings suggest that ZIKV-infected female Ae. aegypti mosquitoes can transmit the virus to naïve male or female mosquitoes through horizontal contact.

Horizontal transmission of ZIKV from male Ae. aegypti mosquitoes to male or female mosquitoes

Since male Ae. aegypti cannot become infected by feeding on blood containing ZIKV, we co-raised male Ae. aegypti with ZIKV-infected female Ae. aegypti in the same cage for a duration of 6 ​h. This setup was designed to assess the potential of ZIKV-infected male Ae. aegypti mosquitoes to transmit the virus to either female or male Ae. aegypti mosquitoes (Fig. 2A). Consequently, female Ae. aegypti mosquitoes were confirmed to be infected with ZIKV via Hemotek blood feeding, and male Ae. aegypti mosquitoes were able to acquire the virus from ZIKV-infected females at 8 days post contact transmission (Fig. 2B and C). Notably, 97% of female mosquitoes were found to be infected with ZIKV, exhibiting 10³–10⁸ viral RNA copies per mosquito (Fig. 2B and C). Similarly, 81% of male mosquitoes were found to be infected, with 10²–10⁸ viral RNA copies per mosquito (Fig. 2B and C). Subsequently, the naïve female mosquitoes were co-raised with these male mosquitoes in the same cage without glucose for 6 ​h, and the viral loads in female Ae. aegypti mosquitoes at 0, 4, 8, 12 and 16 days post-contact were quantified using TaqMan qPCR. Infection rates among female mosquitoes ranged from 42% to 100%, with 10²–10⁶ viral RNA copies per mosquito (Fig. 2D and E). An increasing abundance of viral RNA was detected from day 0 to day 8, followed by a decline from day 12 to day 16 (Fig. 2D and E), suggesting that ZIKV-infected male Ae. aegypti mosquitoes can effectively transmit the virus to naïve female mosquitoes through horizontal contact.

Similarly, the infected male mosquitoes were cohabitated with fluorescent powder-labeled naïve male mosquitoes without glucose, and the viral loads in fluorescent powder-labeled male Ae. aegypti mosquitoes at 0, 4, 8, 12, and 16 days post-contact were assessed using TaqMan qPCR. Notably, 38%–96% of these fluorescent powder-labeled male mosquitoes were found to be infected with ZIKV, containing 10¹–10⁶ RNA copies per mosquito (Fig. 2F and G). Additionally, representative female and male mosquitoes collected at 8 days post-contact with infected males were analyzed to detect viable infectious viral particles through plaque assays, yielding 10² to 10⁶ ​PFU per mosquito (Fig. 2H). These findings suggest that ZIKV-infected male Ae. aegypti can horizontally transmit the virus to both female and male Ae. aegypti.

Vertical transmission of ZIKV by mosquitoes

To evaluate the potential for vertical transmission of ZIKV in mosquitoes, we examined the viral loads in the offspring of ZIKV-infected females (Fig. 3A). We employed the A129 mouse-mosquito model to enhance ZIKV infection rates and increase egg-laying in female mosquitoes. The female mosquitoes exhibited an effective ZIKV acquisition rate of 91% after feeding on ZIKV-infected A129 mice, with viral loads ranging from 10³ to 10⁹ RNA copies per mosquito as determined by TaqMan qPCR, and 10⁵ to 10⁸ ​PFU per mosquito via plaque assays (Fig. 3B–D). The virus was also identified in the eggs, albeit at a relatively low infection rate of 24%, with 10¹ to 10⁵ viral RNA copies per mosquito and 10¹ to 10³ ​PFU per egg (Fig. 3B–D).

Subsequently, the virus-infected mosquito eggs were cultivated, and viral loads were assessed during developmental stages I, II, III, IV, and pupae. The infection rate at stage I was 20%, with 10¹ to 10⁵ viral RNA copies per mosquito (Fig. 3E and F). At stages II and III, the infection rate increased to 23%, while the viral RNA copy ranges remained consistent (Fig. 3E and F). Notably, stage IV demonstrated an increased infection rate of 67%, with viral RNA copies per mosquito still within the range from 10¹ to 10⁵ (Fig. 3E and F). Similarly, the infection rate at the pupae stage was 63%, maintaining the same viral RNA copy range (Fig. 3E and F). Next, pupae were transferred to fresh water in newly sanitized mosquito cages to promote their maturation to adulthood. The newly emerged F1 female mosquitoes exhibited an infection rate of 69%, with 10² to 10⁶ viral RNA copies per mosquito as quantified by TaqMan qPCR, and 10³ to 10⁶ ​PFU per mosquito via plaque assays (Fig. 3G–I). In contrast, the newly emerged F1 male adults demonstrated a lower infection rate of 38%, with 10¹ to 10⁵ viral RNA copies per mosquito and 10¹ to 10³ ​PFU per mosquito (Fig. 3G–I). These findings indicate that ZIKV can be vertically transmitted to subsequent generations, resulting in persistent viral carriage in the offspring mosquitoes.

Distribution and proliferation of ZIKV in mosquito organs

To investigate the potential routes for horizontal and vertical transmission of ZIKV among mosquitoes, we further examined the distribution of ZIKV in various organs of infected specimens. We dissected and separated the salivary glands, midguts, heads, ovaries (females), malpighian tubules, and carcasses of infected mosquitoes, subsequently quantified the viral RNA loads using TaqMan qPCR (Fig. 4A). Our results revealed the presence and replication of ZIKV RNA in all examined organs in female and male mosquitoes (Fig. 4B–L). Notably, the salivary glands, midguts, ovaries, and carcasses in female mosquitoes exhibited significantly higher levels of ZIKV RNA, while the heads displayed the lowest viral loads (Fig. 4B–G). Specifically, ZIKV levels in mosquito organs increased gradually from day 0 to day 8 or 12, but viral loads decreased from day 8 or 12 to day 16 (Fig. 4B–G). Similarly, the ZIKV RNA levels in the organs of male mosquitoes were found to be gradually increased from day 0 to day 8 or 12, but subsequently decreased from day 8 or 12 to day 16 (Fig. 4H–L). Despite the decline in viral RNA abundance during the later stages of infection, both ZIKV-infected female and male mosquitoes were able to persistently harbor the virus for an extended duration.

Furthermore, a fluorescence in situ hybridization assay was conducted to detect viral RNA in various organs. The results showed the presence of ZIKV RNA in whole intestinal tissues, including the midgut and eggs of infected mosquitoes, in comparison to the mock-infected group (Fig. 4M). This widespread distribution of ZIKV across the organs of infected mosquitoes suggests potential routes for both horizontal and vertical transmission. The detection of ZIKV RNA in the salivary gland and malpighian tubules is critical for horizontal transmission among mosquitoes. ZIKV-positive salivary glands can lead to the secretion of infectious saliva, while ZIKV-positive malpighian tubules may result in infectious excreta, such as feces, which are likely to facilitate the acquisition of the virus by naïve mosquitoes from infected individuals. Additionally, the presence of ZIKV in the ovaries and eggs of infected mosquitoes further supports the possibility of vertical transmission.

The effects of ZIKV infection on the reproduction and development of mosquitoes

To investigate the influences of ZIKV infection on mosquito reproduction and development, adult female mosquitoes were infected with ZIKV and various parameters including egg number, egg size, larval size at various stages, hatching rate, pupation rate, feathering rate, and sex ratio were observed (Fig. 5A). Surprisingly, the results indicated that eggs from ZIKV-infected mosquitoes were larger in size but fewer in number (Fig. 5B–D). The average number of egg produced by uninfected females was approximately 45 per mosquito, whereas ZIKV-infected females produced around 35 eggs per mosquito, demonstrating a reduction in egg quantity due to ZIKV infection (Table 1; Fig. 5B). However, no significant morphological changes were observed (Fig. 5C). Specifically, the average length of eggs from uninfected females was about 580 ​μm, while those from ZIKV-infected females measured approximately 650 ​μm, suggesting an obvious increase in egg size associated with ZIKV infection (Fig. 5C and D).

No variations in larval size at stages I and IV were noted following ZIKV infection (Table 1; Fig. 5E and F). However, pupae from infected mosquitoes were smaller compared to those from uninfected counterparts (Table 1; Fig. 5G). ZIKV infection did not significantly affect the hatching rate, pupation rate, feathering rate, or sex ratio of the offspring (Table 1; Fig. 5H and I). Additionally, the normal and basic behaviors of mosquitoes remain unaltered after ZIKV infection. These findings imply that ZIKV infection may slightly impede the normal growth and development of the next generation of mosquitoes.

Regulation of hormone expression by ZIKV infection in mosquitoes

To explore the potential impacts of ZIKV infection on the reproduction and development of mosquitoes, we first analyzed the expression levels of key hormone genes associated with egg production in ZIKV-infected female mosquitoes, including E75A2, EcR, Hairy, ILP4, and Kr-h1 (Fig. 6A–E). Following ZIKV acquisition via a membrane feeding system, we collected Ae. aegypti at 12, 24, and 36 ​h post-infection (hpi) and subsequently measured hormone gene mRNA levels. The qRT-PCR results indicated a significant decrease in the mRNA levels of Hairy, ILP4, and Kr-h1 at 36 hpi (Fig. 6C–E), which may lead to reduced egg production, while the levels of E75A2 and EcR remained unchanged after ZIKV infection (Fig. 6A and B). Hairy, ILP4, and Kr-h1 are associated with juvenile hormones (Nouzova et al., 2018), and their suppression may suggest limited developmental potential in the subsequent generation. Conversely, E75A2 and EcR are more closely linked to mosquito aging (Ekoka et al., 2021). The stable expression levels of these genes after infection imply a co-evolutionary relationship between ZIKV and mosquitoes, which may account for the absence of increased mortality in female adult mosquitoes, consistent with natural phenotypes.

We subsequently investigated the impacts of ZIKV infection on the expression levels of hormone genes associated with growth and development in Ae. aegypti at various developmental stages (I, II, III, IV, and pupae), focusing on JHE, JHMAT, PTTH, and Torso (Fig. 6F–I). Initially, adult female mosquitoes were exposed to ZIKV through a membrane feeding system, after which they mated with adult males to lay eggs. Subsequently, mosquito eggs were collected from both control and ZIKV-infected females. The transcriptional changes of hormone genes were monitored from the egg to the pupae stage using qRT-PCR. During stages I, II, III, and IV, no significant changes were observed in the levels of JHE and JHMAT (Fig. 6F and G), which are involved in the biosynthesis and degradation pathways of insect juvenile hormones (Nouzova et al., 2018). However, during the pupae stage, the mRNA levels of JHE and JHMAT were significantly higher in the ZIKV-infected pupae group, which may be attributed to the smaller size of the pupae (Fig. 5G). Furthermore, our results indicated a decrease in the levels of PTTH and Torso, which regulate ecdysteroid content, in larvae during stages I and II (Fig. 6H and I). These findings provide insights into the potential mechanisms underlying the delayed larval development observed in the early stages.

Cells

African green monkey kidney cells (Vero, ATCC: CCL-81) were cultured and maintained in Dulbecco's modified Eagle's Medium (DMEM, Gibco; 12800082) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Every Green, 11011-8611), 1% Antibiotic/Antimycotic Solution (Gibco; 15240062) at 37 ​°C in a 5% CO₂ atmosphere.

Virus and infection assay

ZIKV strain H/PF/2013 (GenBank: KJ776791.2), obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences, was propagated and titrated by plaque assays on Vero cells. The ZIKV infection in mice and mosquitoes were conducted in a BSL-2 laboratory at the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, following standard procedures.

Mosquitoes

Ae. aegypti (PeC02 strain) mosquitoes were kindly provided by Prof. Qiuying Huang (Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, Huazhong Agricultural University, Wuhan, China). All mosquitoes were laboratory-reared strain and were maintained within a limit of 200 generations. Ae. aegypti were maintained under standard laboratory conditions at 28 ​°C, with 70%–80% relative humidity and a 12:12 light/dark photoperiod. The adult mosquito colonies were housed in cages and provided with a 5% glucose solution, while the larvae were fed minced commercial fish food.

Mice

Adult BALB/c mice (female, 4 weeks old) were obtained from the Scientific Ethic Committee of Huazhong Agriculture University, Wuhan, China. A129 (Ifnar1^−/−) mice (female, 4 weeks old) were kindly provided by Dr. Bin Wei (Fudan University, Shanghai, China). Mice were bred and maintained in accordance with the guidelines established by The Scientific Ethic Committee of Huazhong Agriculture University.

In-vitro membrane feeding

Blood was collected from healthy female BALB/c mice and subjected to centrifugation at 1,000 ×g for 10 ​min at 4 ​°C. This procedure was performed in a heparin-coated tube to separate the plasma from the blood cells. The plasma was then inactivated at 56 ​°C for 60 ​min. The separated blood cells were washed three times with phosphate-buffered saline (PBS) to eliminate the anticoagulant. Subsequently, the cells were re-suspended in heat-inactivated plasma. The Hemotek® film feeding system was then utilized to combine the material with mouse blood for mosquito oral feeding. To infect mosquitoes, a mixture of mouse blood and ZIKV was fed to Ae. aegypti mosquitoes. The infected Ae. aegypti mosquitoes were subsequently used for further experiments.

Fluorescent powder labeling of Ae. aegypti

Following the anesthetization of mosquitoes on ice, both female and male mosquitoes were transferred to a plate containing fluorescent powder (Hercules Radiant, R-103 G115). The plate was gently shaken to ensure uniform adherence of the fluorescent powder to the bodies of the mosquitoes. Each mosquito, regardless of sex, was labeled with a fluorescent dye at a dosage of 0.2 ​mg per mosquito, using a total of 20 ​mg of fluorescent powder to mark 100 mosquitoes in one cage.

Viral genome quantitation by TaqMan qPCR

RNAs from mosquitoes and mouse blood were extracted using the MagZol® Regent (Magen, R4801-02) and were reversed to cDNAs using a commercial ABscript II cDNA First Strand Synthesis Kit (ABclonal, RK20400). Subsequently, cDNAs were used as template DNAs to detect nucleic acid copy numbers of ZIKV by TaqMan qPCR with a pair of primers (F: 5′-CCGCTGCCCAACACAAG-3'; R: 5′-CCACTAACGTTCTTTTGCAGACAT-3′) and a synthesized probe (P: FAM-AGCCTACCTTGACAAGCARTCAGACACTCAA-TAMRA). PCR was performed in a 10 ​μL reaction system containing 5 ​μL of Entrans 2​× ​qPCR Probe Master Mix V2 (ABclonal, RK21212), 10 ​μM of each forward and reverse primers, 10 ​μM of probe, 0.2 ​μL of 50× ​ROX dye, 3 ​μM template cDNA, and 1.1 ​μM nucleic acid free water, via ViiA 7 Real-Time PCR System (Applied Biosystems). The cycling conditions were 95 ​°C for 3 ​min, followed by 40 cycles of 95 ​°C for 15 ​s, and 55 ​°C for 30 ​s.

The amount of viral genomic RNA per mosquito was normalized by establishing a standard curve targeting standard sample. In details, the standard sample was generated using a plasmid encoding the ZIKV E gene (pMD®19-T-E). The plasmid concentration was quantified with a Nanodrop 2000 (E112352, Thermo Fisher Scientific), and the copy number of the E gene was calculated. A standard curve was established through TaqMan qPCR with a series of standard plasmid diluted tenfold, converting cycle threshold (Ct) values into copy numbers for each diluted standard sample. The viral RNA was quantified via TaqMan qPCR amplification of the viral genes, normalized against the standard curve. The limit of detection was established at 9.212 copies of ZIKV E gene, as defined by the uninfected mosquitoes.

Horizontal transmission of ZIKV in mosquitoes

Female and male mosquitoes that were one week old post-eclosion were utilized in the infection experiments for horizontal transmission. A minimum of 50 mosquitoes per group were employed for each experiment.

Vertical transmission of ZIKV in mosquitoes

Three 4-week-old A129 mice were inoculated with 1000 ​PFU ZIKV in 100 ​μL of DMEM via intraperitoneal injection. A separate group of three mice received 100 ​μL of DMEM as a mock-infection control. After three days, female mosquitoes that had been pre-starved for 24 ​h were allowed to feed on the infected mice for 6 ​h without glucose. In each mosquito cage used for independent biological replicates, at least 100 mosquitoes were allowed to bite each mouse, which was secured with wire. Subsequently, the infected female mosquitoes were transferred to new hygienic mosquito cages containing 5% glucose, maintained at 28 ​°C and 80% humidity for 8 days. Following this period, the infected females were paired with naïve male mosquitoes for 6 ​h without glucose. Two males were matched with one female mosquito to facilitate egg-laying. The female mosquitoes and their eggs were then collected for subsequent analyses.

Fluorescence in situ hybridization (FISH) of mosquitoe slices

Mosquitoes were placed in 4% paraformaldehyde containing 0.25% Triton X-100 and fixed at 4 ​°C for 24 ​h. Following dehydration through a gradient of low to high concentrations of alcohol, the mosquitoes were rendered transparent in xylene and subsequently embedded in dipping wax. The trimmed wax block was then positioned on a microtome for sectioning, producing slices with a thickness of 3–4 ​μm. The sections were floated on water at 40 ​°C using a spreader to flatten the tissue, after which the glass slide was used to collect the tissue slices. The slices were baked in an oven at 62 ​°C for 2 ​h, and then subjected to a series of treatments: dewaxing in transparent solution I for 15 ​min, dewaxing in transparent solution II for 15 ​min, followed by anhydrous ethanol I for 5 ​min, anhydrous ethanol II for 5 ​min, 85% alcohol for 5 ​min, and 75% alcohol for 5 ​min. The samples were soaked in DEPC water and subsequently repaired with a repairing solution. After cooling to room temperature, a grouping pen was used to draw a circle around the slices, which were then digested by adding Proteinase K (20 ​μg/mL) at 40 ​°C. After rinsing with distilled water, the samples were washed three times with PBS, with each wash lasting 5 ​min. The samples were then incubated for 1 ​h at 40 ​°C with a drop of pre-hybridization solution. Following this, the pre-hybridization solution was removed, and a drop of hybridization solution containing the probes of ZIKV E gene (synthesized in Servicebio, Wuhan, China) was added, allowing for overnight incubation in a thermostat. Subsequently, the samples were washed with hybridization solution: 10 ​min at 40 ​°C with 2× ​SSC, followed by 5 ​min at 40 ​°C with 1× ​SSC, and 5 ​min at 40 ​°C with 0.5× ​SSC twice, concluding with 8 ​min at room temperature and 8 ​min in the incubation solution. The slices were then incubated with DAPI staining solution for 8 ​min. After rinsing, the slices were sealed with a drop of anti-fluorescence quenching sealer. Finally, images were captured using a Nikon Ti–U microscope, with red (CY3) indicating the presence of ZIKV and DAPI staining representing the nucleus.

Detection of hormone mRNA by using real-time reverse transcription PCR (qRT-PCR)

The RNA extraction was performed using MagZol® Regent (Magen, R4801-02) on homogenized mosquitoes, and the RNA was reverse transcribed into cDNA using ABScript® II cDNA First-Strand Synthesis Kit (ABclonal, RK20400) according to the manufacturer's instructions. Quantitative real-time reverse transcription PCR was carried out using 2 ​× ​Universal SYBR Green Fast qPCR Mix (ABclonal, RK21203) and a ViiA 7 Real-Time PCR System (Applied Biosystems). The primers used are listed in Supplementary Table S1. The relative mRNA levels were normalized to the expression of Aedes actin gene (AAEL011197).

Observation and analysis of mosquito reproduction and development

The mosquito eggs are placed in a basin and allowed to hatch into larvae. There are four stages of mosquito larval development, designated as I, II, III, and IV, which are associated with larval molting. Stage IV larvae undergo a transformation into pupae and eventually mature into adult mosquitoes.

Throughout the growth and development of mosquitoes, we observed their appearance and measured various parameters, including size, viral genome abundance, and hormone expression. For the morphological analysis of eggs and larvae, we utilized a Nikon Ti–U microscope to observe and photograph the specimens. The lengths of the eggs and larvae were measured based on images captured with the microscope, which included scale bars for reference. To assess viral load, TaqMan qPCR was employed to detect ZIKV gene copies in egg or larva. For hormone related gene expression analysis, qRT-PCR was conducted, and the relative mRNA levels of indicated genes were normalized to the Aedes actin gene.

Determination of viral titers by plaque assay

To determine viral titers, mosquito samples were homogenized in 500 ​μL of DMEM and centrifuged at 1000×g for 5 ​min at 4 ​°C to isolate the supernatant. The supernatant samples were then serially diluted and inoculated onto monolayers of Vero-E6 cells at 37 ​°C for 1 ​h. Following this, the supernatants were removed, and the cells were washed three times with serum-free DMEM. Vero-E6 cells were subsequently incubated for 4 days in DMEM supplemented with 2.5% FBS and 1.5% sodium carboxymethyl cellulose (Sigma, 419273). Finally, viral titers were calculated by counting visible plaques following staining with crystal violet.

Statistical analysis

Animals were randomly assigned to different groups. Mosquitoes that died prior to counting and measurement were excluded from the analysis. The investigators were not blinded to the allocation during the experiments or the outcome assessment. Each experiment was conducted at least twice. Quantitative data are presented as the mean ​± ​SEM using GraphPad Prism 9. A t-test was used to assess the significant differences in egg size and number, larval size, feathering rate, and the male-to-female ratio of adult mosquitoes. Two-way analysis of variance (ANOVA) was employed to compare hormonal differences in larval and adult mosquitoes. Fisher's exact test was utilized to compare survival rates. Statistical significance was established at P ​< ​0.05.