Keywords: Mosquito,Aedes aegypti, arboviruses, Zika virus, vertical transmission, blood meal, infectious blood meal

Cell lines and viruses

A. albopictus cells (C6/36; ATCC, CRL-1660) were grown in MEM supplemented with 10 % FBS, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and maintained at 28°C in 5 % CO₂. All media had 100 U penicillin ml⁻¹ and 100 μg streptomycin ml⁻¹. Viral titers were determined by plaque assay in African green monkey kidney (Vero; ATCC CCL-81) cells, which were grown in MEM (Sigma-Aldrich, M0268) supplemented with 10 % FBS, 2.2 g/L sodium bicarbonate, and maintained at 37°C in 5 % CO₂. Zika virus strain PRVABC59 (kindly provided by the Centers for Disease Control and Prevention, Fort Collins, CO, USA; GenBank accession no.KU501215), was initially obtained from serum of a patient who had traveled to Puerto Rico in 2015 and was passaged 3 times on Vero cell culture and 1 time on C6/36 cell culture. All stock viruses were generated in C6/36 cells as follows: confluent monolayers of C6/36 cells in six-well plates were infected with ZIKV at 0.1 multiplicity of infection (MOI). After one hour of adsorption, 3 ml of maintenance medium (growth medium with 2% FBS) were added to each well and incubated at 28^oC. Supernatants from each well were collected after 5 days and pooled. Freshly prepared viruses were used for all mosquito infections.

Mosquito infections and detection of ZIKV in ovaries

All mosquito rearing was done at 28°C. Five hundred 4–7 day oldAe. aegypti (from Poza Rica, Mexico, kindly provided by Greg Ebel) were orally infected with ZIKV (strain PRVABC59, Accession number:KU501215) at 1 × 10⁸ plaque forming unit (pfu)/ml with a defibrinated sheep-blood meal as previously described³⁴. Engorged mosquitoes were separated and maintained with 10% sucrose. No additional blood meal was provided for the entire study period. An oviposition substrate was provided for laying eggs. Legs, ovaries, and carcass from each mosquito were dissected at indicated time points post blood feeding. Ovaries were washed three times in phosphate-buffered saline (PBS) before collecting in 500 μl of mosquito diluent (1xPBS containing 20% heat inactivated FBS, 100 U/ml of Penicillin, 100 μg/ml Streptomycin, 10μg/ml Gentamycin and 1μg/ml of Fungizone). They were frozen at −80°C until RNA isolation. Infection and viral dissemination were determined by RT-PCR for the presence of the viral RNA in the body or leg homogenate, respectively. Viral titer was determined by plaque assay on Vero cells. Differences in the mean viral titers were determined using a Kruskal-Wallis test with Dunn’s multiple comparisons test (GraphPad Prism).

Detection of DNA forms of the viral RNA genome

ZIKV infection was carried out as described above. No additional blood meal was given for the entire study period. Ovaries were collected from infected mosquitoes, which were identified by removing one leg and then determining the presence of the viral RNA in the leg homogenate by RT-PCR³⁵. Ovaries from 8–10 infected mosquitoes were pooled in 250 μl of PBS, homogenized and then used for DNA isolation by the Qiagen DNeasy DNA isolation kit following the manufacturer’s protocol. About 70 ng DNA was used for each conventional polymerase chain reaction (PCR) assay; primer information and PCR parameters are described in Nag et al.³⁶, except that amplifications were carried out for 46 cycles.

Immunofluorescence assay

Immunofluorescence assay (IFA) was carried out as described by Dong et. al³⁷. Dissected ovaries were fixed with 10% formalin (Fisher Chemicals) in a 50 mm petri dish (~10 ovaries per dish) for 4–7 days at 4°C. After three washes with PBS, tissues were permeabilized by incubation with PBT (1 X PBS containing 1% BSA and 0.2% Triton X-100) for one hour on an orbital shaker at room temperature. Samples were then incubated overnight at 4°C with anti-flavivirus mouse monoclonal antibody {D1–4G2–4-15 (4G2) from Absolute antibody (Boston, MA, USA)} after 1:100 dilution with PBT. The tissues were then washed four times with PBST (1 X PBS containing 1% BSA and 0.1% Triton X-100). Samples were then incubated 1.5 to 2 hours at 37°C with 1:125 dilution (in PBT) of FITC-labeled goat anti-mouse IgG (H+L) (SeraCare life sciences, Milford, MA, USA) in the dark. DAPI (0.5 μl of 0.5 μg/μl per 100 μl) was added during the last 30 min of incubation. After 4 washes with PBST, ovaries were placed on slides, mounted with Vectashield mounting medium (Vector laboratories, Burlingame, CA, USA), covered with cover slips, then viewed under an inverted spectral confocal microscope. Both primary and secondary antibody treatment were carried out on slides and the washing steps in 35 mm petri dishes.

Vertical Transmission assay

Four to seven day-old females were fed with a ZIKV (1×10⁸ pfu/ml) containing infectious blood meal. Fully engorged mosquitoes were separated into a cardboard container and maintained with 10% sucrose at 28°C. After 2 days, mosquitoes were separated into individual cups each containing a 30 ml plastic cup half filled with water and the remaining half had a coffee filter paper touching the water. After 7 days, mosquitoes that had laid eggs were transferred to a cardboard container and maintained with 10% sucrose at 28°C. Thirty-three mosquitoes were also tested by RT-PCR to determine the prevalence of infection. On day 10, oviposited mosquitoes were given a second non-infectious blood meal; engorged mosquitoes were separated individually into cups with oviposition substrates as described above. On day 17, coffee filter papers with eggs and any hatched larvae present in the 30 ml cup were transferred to a 148 ml cup containing water for hatching. Third and fourth instar larvae were collected in pools of 8 in 800 μl of mosquito diluent and stored at −80°C until they were homogenized and processed for RNA extraction. Larvae pools from individual mosquitoes were kept separate. The presence of the ZIKV in the larvae was determined by RT-PCR using ZIKV-specific primer-probe combination. Also on day 17, mosquitoes with disseminated infection were identified after separating a leg and detecting the presence of viral RNA in the leg homogenate by RT-PCR. Larvae from infected mosquitoes were used for the RT-PCR assay. Ovaries from mosquitoes with disseminated infections were dissected and fixed in 10% formalin for subsequent detection of infected ovaries by IFA.

Detection of ZIKV inAe. aegypti ovaries by plaque assay

In order to determine whether ZIKV infects ovaries,Ae. aegypti (from Poza Rica, Mexico) were infected with ZIKV using an infectious blood meal containing 1×10⁸ pfu/ml of ZIKV. Engorged mosquitoes were separated and maintained with 10% sucrose. No additional blood meal was given for the entire study period. Ovaries were dissected on days 3, 6, 10, 14, and 19 following infection (Fig. 1A). Thirty-five to 50 mosquitoes were analyzed per time point. The infection status of the mosquitoes and virus dissemination were determined by RT-PCR and the viral titer was determined by plaque assay. Ninety percent of mosquitoes were infected by day 3 with 57% dissemination among the infected mosquitoes (Table 1). The mean viral titer in the ovary increased from 2.0 log₁₀ pfu/mL at 3 days post infection (dpi) to 2.8 log₁₀ pfu/mL at 6 dpi to 3.6 log10 pfu/mL at 10 dpi and 3.8 log₁₀ pfu/mL at 19 dpi (Fig. 1B). However, viral loads at 3 and 6 dpi were not significantly different. Viral titer, as determined by plaque assay, does not always reflect a true infection of the tissue, since the tissue can be contaminated with hemolymph during dissection, or the virus particles could be loosely attached to the ovary. To address these issues, the presence of the virus in the ovaries were monitored by detecting the presence of the DNA form of the viral RNA genome and the presence of viral antigen by indirect immunofluorescence assay (IFA). The presence of the DNA form was detected in the 21-day sample from the experiment described above as well as in other independent experiments, described below.

Detection of the DNA form of the ZIKV RNA genome inAe. aegypti ovaries

Previously, we showed that DNA forms of the ZIKV RNA genome are generated following infection both in mosquito cell cultures and in mosquitoes³⁶. Therefore, detecting the presence of the viral DNA (vDNA) in the genomic DNA preparation from mosquito ovaries would be an indication of viral infection. Mosquitoes were infected with the ZIKV as described above. No additional blood meals were given for this experiment. A day before dissection, the infection status of individual mosquitoes was determined by removing a leg and then determining the presence of the viral RNA in the mosquito leg extract³⁵. We collected ovaries from 8–10 mosquitoes with disseminated infection in PBS, and total genomic DNA was prepared using the Qiagen DNeasy Blood and Tissue DNA preparation kit as described before³⁶. We prepared total DNA from mosquito ovaries at 10 and 21 dpi. DNA forms were detected using ZIKV specific primers in a conventional PCR assay, and the PCR products were confirmed by DNA sequencing. Our results indicated that the vDNA can be easily detected at 21 dpi (Fig. 2). We performed this experiment (vDNA detection) several times, because vDNA on day 10 could not always be detected (1 in 8 experiments), although it could be easily detected on day 21. These results suggest that ovaries are not frequently infected at 10 dpi or vDNAs are generated late in infection. However, the latter possibility seems unlikely based on the IFA results described below.

Detection of ZIKV infection by Immunofluorescence assay

Finally, we used indirect IFA to detect the presence of the virus in the ovaries. Mosquito infection was carried out as described above. IFA was carried out with ovary samples collected at 10 and 18 dpi from mosquitoes with disseminated infections. Infected mosquitoes were identified as described above. Ovaries from mosquitoes fed with non-infectious blood meal were used as controls. We checked ovaries from 15–35 infected mosquitoes at each time point. IFA positive samples were infrequent at 10 dpi (2 in 15 ovaries tested), but could be easily detected with 18 dpi samples (Fig. 3). At least 40% (10/25) of infected mosquitoes had the virus in ovaries at 18 dpi. These results suggest that the level of infection of ZIKV in ovaries is low at 10 dpi and virus detected by plaque assay at early time points may represent loosely attached virus particles.

It has been demonstrated that progeny from the second or third gonotrophic cycle exhibit a higher VTR than those from the first gonotrophic cycle^16,22,23,38. Based on this observation, we wanted to see if a second feeding increases the prevalence of ovary infections. Mosquitoes were fed with a second non-infectious blood meal 9 days after the first infectious meal and engorged mosquitoes were separated and provided with an oviposition substrate. Ovaries were collected from infected mosquitoes at 18 dpi. Our results showed that the prevalence of ovary infections increases to 62% (21/34) after second feeding. The difference between ovary infections at 18 dpi in single-fed and double-fed mosquitoes, however, was not statistically significant (p = 0.098). The staining intensity was slightly higher in 10% of ovaries from double-fed mosquitoes compared to single-fed mosquitoes. These results suggest that the increased VT in the second gonotrophic cycle could be due to delayed infection and/or enhanced infection status of mosquito ovaries. Sanchez-Vargas et al.²³ had similar observations in a genetically diverse laboratory strain ofAe. aegypti following DENV2 infection. DENV2 was found in ovaries at 17 dpi in both single-fed and doubled-fed mosquitoes. However, they were unable to detect the DENV2 in ovaries at 10 dpi. Since we observed ZIKV infected ovaries at 10 dpi, it is possible that DENV2 and ZIKV exhibit a slightly different infection pattern or there is a difference in the number of ovaries processed in these two studies. We were unable to determine whether oocytes were infected. Since ZIKV was passaged in mosquito cells (C6/36) prior to infection, it is difficult to say whether adaptation may have played a role in the prevalence of mosquito infections.

Vertical transmission of ZIKV

Since ZIKV infects mosquito ovaries, we determined whether infected progeny derive from mosquitoes with infected ovaries. Female mosquitoes were orally fed with a 10⁸ pfu/ml of ZIKV-containing infectious blood meal. Engorged mosquitoes were kept in individual cups (see Methods for details, andFig. 4A). By day 7, 97% (32/33) of mosquitoes were infected. On day 7, mosquitoes that laid eggs were pooled and then fed with a non-infectious blood meal on day 10. Engorged mosquitoes were kept separately in individual cups, each containing an oviposition substrate. On day 17, eggs and larvae were collected and allowed to develop until the third/fourth instar stage, when they were pooled. Pooled larvae from individual mosquitoes were kept separate. On Day 18, ovaries were dissected from mosquitoes with disseminated infections and ovary infection status was determined by IFA. Ninety-two percent (36/39) of mosquitoes had disseminated infection and 52% ovaries were infected with the virus. There were 127 pools, containing 970 larvae derived from 30 mosquitoes with disseminated infection. The infection status of the progeny larvae produced in the second gonotrophic cycle were determined by RT-PCR. Two pools turned out to be ZIKV positive. Larvae in these two pools were produced by the same mosquito, which had infected ovaries (Fig. 4B). A total of 18 larvae was generated by this mosquito which were distributed into two pools. None of the other mosquitoes irrespective of their ovary infection status produced infected progeny. These results suggest that ZIKV VTR (proportion of infected mosquitoes producing at least one infected infecting progeny) is just 3.33%. We have also determined the presence of the viral RNA in pools of larvae from the first gonotrophic cycle; among 1035 larvae in 115 pools (9 larvae per pool), none were positive for ZIKV.

Sanchez-Vargas et al²³. detected a small number of oocytes infected with DENV2 among the double-fed mosquitoes. A stabilized infection may result in a small number of females when the viruses infect germline. Once established, the stabilized infection may yield high VTRs in subsequent generations via TOT. Such stabilized infections have been demonstrated for California encephalitis serogroup viruses (orthobunyaviruses) in variousAedes species and for Culex flavivirus inCulex pipiens^26,39,40. We showed here by plaque assay, IFA, and by monitoring the generation of vDNA that ZIKV also infects mosquito ovaries. Viruses could be detected frequently at 18 dpi, but seldom at 10 dpi. These results suggest that viruses observed by plaque assay at early time points do not represent true infections. It appears that like midgut escape barrier, there might be present a barrier that restricts ovary infection by arboviruses. The above results also indicated that only a minor fraction of mosquitoes with infected ovaries produce infected offspring. It is possible that oocytes are generally not infected and only those mosquitoes with infected oocytes transmit the virus vertically. It is unclear whether all progeny were infected with the virus. It is also not clear how the oocytes are protected from virus infection. In our studies, we observed that in most ovary samples, the viral antigen was present in the tracheal system (Figs. 3 and4B). Similar results were also obtained with Rift Valley fever virus (RVFV)⁴¹. Although the infection of ovarian tissues or VT through the tracheal cells is not known, it is possible that some of the progeny might get infected due to virus entering through the tracheal system. Tracheal conduits have also been suggested to facilitate virus dissemination from the midgut to hemocoel in DENV2 and RVFV infections ofAe. aegypti^41,42.

VTRs for dual-host flaviviruses are low and the reported filial infection rates vary among reports^{16,32,33,43–47}. Several possibilities can explain these variations. First, VTs are known to be dependent on mosquito species and their geographic origin^{33,43,46–50}. Second, ovary infection is not a guarantee for VT, which likely depends on the infection status of oocytes. Third, most of the previous studies on VT were carried out using pools of eggs, larvae, or adult mosquitoes that originated from several mosquitoes with an unknown infection status or from a mixture of both infected and uninfected mosquitoes. Virus-positive pools might have had progeny that derived from the same mosquito. Consequently, the rates can vary from one experiment to another depending on how many offspring are produced by the infected mosquitoes and how the infected offspring are distributed among the pools. Finally, various stages of mosquito development are tested in different laboratories, which may impact the calculated VTRs, since virus present during larval stages can be lost during metamorphosis. In addition, sensitivities of various techniques used in different laboratories may alter the VTRs.

An increased VTR was observed following second and later gonotrophic cycles for several arboviruses. When mosquitoes are provided with a second non-infectious blood meal 3 days following the first infectious blood meal, the disseminated infection rate for ZIKV and DENV was significantly higher in early time points following the second blood meal, but no significant difference was observed at later time points (beyond 7 dpi)⁵¹. It is possible that ovary infection prevalence increases in the second or later gonotrophic cycles due to a combination of multiple factors, such as timing of dissemination of the virus to ovaries and morphological alterations of ovaries following egg formation. Such morphological alterations (stretched parous ovaries) may be necessary for a productive infection of the ovaries by arboviruses. Since an oviposition substrate was provided in our studies, it is possible that eggs were laid before the viruses reached the reproductive tissue during the first gonotrophic cycle. Supporting this idea is the observation that delayed egg laying byCulex pipiens females to 11–14 dpi and 25 dpi resulted in VT of St. Louis encephalitis virus during the first ovarian cycle⁵². We also previously showed that delaying oviposition increases VT of WNV inCx. pipiens⁵³.

It was found that when measured in immature developmental stages, VTs are higher than the corresponding adults.Ae. aegypti also exhibited delayed development when infected with YFV, Kunjin virus, ZIKV, and Japanese encephalitis virus^16,54. It is possible that vertically infected larvae have a low survival rate, leading to lower transstadial transmission rates in adults. Since we have not tested the infection status of eggs or adults, transstadial transmission could not be determined. The ISFs exhibit higher VTRs than dual-host flaviviruses, as VT is the predominant mode of survival for ISVs in nature⁵⁵. It would be interesting to determine whether similar ovary-infection barrier is also present for ISVs and the timing of infection following an infectious blood meal.

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