Forced Zika Virus Infection of Culex pipiens Leads to Limited Virus Accumulation in Mosquito Saliva

Zika virus (ZIKV) is a mosquito-borne pathogen that caused a large outbreak in the Americas in 2015 and 2016. The virus is currently present in tropical areas around the globe and can cause severe disease in humans, including Guillain-Barré syndrome and congenital microcephaly. The tropical yellow fever mosquito, Aedes aegypti, is the main vector in the urban transmission cycles of ZIKV. The discovery of ZIKV in wild-caught Culex mosquitoes and the ability of Culex quinquefasciatus mosquitoes to transmit ZIKV in the laboratory raised the question of whether the common house mosquito Culex pipiens, which is abundantly present in temperate regions in North America, Asia and Europe, could also be involved in ZIKV transmission. In this study, we investigated the vector competence of Cx. pipiens (biotypes molestus and pipiens) from the Netherlands for ZIKV, using Usutu virus as a control. After an infectious blood meal containing ZIKV, none of the tested mosquitoes accumulated ZIKV in the saliva, although 2% of the Cx. pipiens pipiens mosquitoes showed ZIKV–positive bodies. To test the barrier function of the mosquito midgut on virus transmission, ZIKV was forced into Cx. pipiens mosquitoes by intrathoracic injection, resulting in 74% (molestus) and 78% (pipiens) ZIKV–positive bodies. Strikingly, 14% (molestus) and 7% (pipiens) of the tested mosquitoes accumulated ZIKV in the saliva after injection. This is the first demonstration of ZIKV accumulation in the saliva of Cx. pipiens upon forced infection. Nevertheless, a strong midgut barrier restricted virus dissemination in the mosquito after oral exposure and we, therefore, consider Cx. pipiens as a highly inefficient vector for ZIKV.


Introduction
Mosquito-borne viruses are a severe threat to human health [1,2]. Climate change, increased global trade and travel, and the ability of viruses to adapt to new vectors and hosts contribute to the geographic expansion of these mosquito-borne pathogens [1,2]. Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) was first isolated from a caged, sentinel rhesus monkey in the canopy of the Zika forest in Uganda in 1947 [2,3]. In 1948, the virus was discovered in Aedes africanus mosquitoes in the same forest [2,3], and in 1954 the first human ZIKV isolate was obtained from a Nigerian female [2,4]. Much later, the virus re-emerged in Asia and the Pacific islands, and started a large outbreak in humans the Vero cells were seeded in HEPES-buffered DMEM medium (Gibco) supplemented with 10% FBS and P/S. When mosquito body lysate or saliva was added to the cells, the HEPES-buffered DMEM medium was additionally supplemented with gentamycin (50 µg/mL; Gibco) and fungizone (2.5 µg/mL of amphotericin B and 2.1 µg/mL of sodium deoxycholate; Gibco). This medium will hereafter be named DMEM HEPES complete.
All experiments involving infectious ZIKV and USUV were executed in the biosafety level

Infectious Blood Meal
Prior to the infectious blood meal, female mosquitoes were starved for one day. Mosquitoes were then orally exposed to ZIKV or USUV by providing them with infectious blood from a Hemotek PS5 feeder (Discovery Workshops, Lancashire, UK) in a dark room for 1 h. Infectious blood meals were prepared by mixing virus stock with human blood (Sanquin Blood Supply Foundation, Nijmegen, the Netherlands) to obtain a final virus titer of 1.0 × 10 7 TCID 50 /mL. Cx. pipiens molestus and Cx. pipiens pipiens received ZIKV during four and three independent experiments, respectively. As positive controls, Ae. aegypti was infected with ZIKV to test the quality of the virus stock, and Cx. pipiens molestus and Cx. pipiens pipiens were exposed to USUV to test the competence of the mosquitoes. After the blood meal, the mosquitoes were anesthetized using CO 2 , and fully engorged females were selected. A small number of engorged females was collected in individual SafeSeal micro tubes (Sarstedt, Nümbrecht, Germany) containing 0.5 mm zirconium oxide beads (Next Advance, Averill Park, NY, USA) to determine the virus titer in the mosquito body directly after engorgement. All other females were incubated at 28 • C with access to 6% glucose.

Salivation Assay
Fourteen days post infection, the mosquitoes were immobilized with CO 2 and the legs and wings of each mosquito were removed. Next, mosquito saliva was collected by inserting the mosquito proboscis into a 200 µL pipet tip containing 5 µL of a 50% FBS and 25% sugar solution in sterilized tap water. After 45 min, the mosquito bodies were stored at −80 • C in individual SafeSeal micro tubes (Sarstedt) containing 0.5 mm zirconium oxide beads (Next Advance). Individual mosquito saliva samples were mixed with 55 µL DMEM HEPES complete and stored at −80 • C.

Infectivity Assay
Frozen mosquito body samples were homogenized in a Bullet Blender Storm (Next Advance) at maximum speed for 2 min. The body homogenates were centrifuged in an Eppendorf 5424 centrifuge at 14,500 rpm for 1 min. Next, 100 µL of DMEM HEPES complete was added to each body homogenate. The homogenates in medium were blended again at maximum speed for 2 min, and centrifuged at Viruses 2020, 12, 659 4 of 12 14,500 rpm for 2 min. Thirty µL of each mosquito body or saliva sample was then added to one well of a 96-well plate containing a monolayer of Vero cells in DMEM HEPES complete. After 2 h at 37 • C, the medium of the cells was replaced by 100 µL fresh DMEM HEPES complete. Six days post infection, the wells were scored virus-positive or -negative based on cytopathic effect (CPE). The number of virus-positive mosquito bodies or salivas was expressed as a percentage of the total number of mosquitoes tested. Viral titers in TCID 50 /mL were measured for mosquito bodies and salivas using EPDAs on Vero cells. After 6 days, the wells were scored virus-positive or -negative based on CPE.

Mosquito Wing Length Measurement
The right wings of 20 female Cx. pipiens molestus, Cx. pipiens pipiens and Ae. aegypti were removed and mounted on sticky tape on a slide. The wing length was measured from the end of the alula to the top of the wing, excluding the fringe scales, using ImageFocus software (Euromex Microscopes, Arnhem, the Netherlands) calibrated with a slide graticule of 0.01 mm. The wing length measurements were used as an estimate of body size, as wing length is known to be correlated with body mass [38].

Statistical Analysis
The Kolmogorov-Smirnov test was used to determine whether mosquito wing lengths and viral titers of engorged mosquitoes were normally distributed. Differences in wing lengths and differences in viral titers were then tested for significance using an unpaired, two-tailed t-test. All statistical tests were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

No ZIKV Transmission by Cx. pipiens after an Infectious Blood Meal
To assess the vector competence of Cx. pipiens molestus and Cx. pipiens pipiens for ZIKV, the mosquitoes were offered an infectious blood meal containing 1.0 × 10 7 TCID 50 /mL of ZIKV. As positive controls, Ae. aegypti and both Cx. pipiens biotypes were infected with 1.0 × 10 7 TCID 50 /mL of ZIKV or USUV, respectively. To investigate the variability in engorgement among individual mosquitoes, viral body titers were determined for a selection of mosquitoes directly after ingestion of an infectious blood meal ( Figure 1A,B). Cx. pipiens mosquitoes, blood fed with ZIKV or USUV, showed very similar median viral titers ranging from 4.6 × 10 5 to 6.3 × 10 5 TCID 50 /mL. Ae. aegypti mosquitoes blood fed with ZIKV showed significantly lower viral titers compared to the Cx. pipiens biotypes blood fed with ZIKV (p < 0.05). This can be explained by the fact that the Ae. aegypti mosquitoes were smaller in size than the Cx. pipiens mosquitoes. The Ae. aegypti mosquitoes showed an average wing length (±standard deviation) of 2.60 mm (±0.18 mm), whereas average wing lengths of 3.34 mm (±0.32 mm) and 3.65 mm (±0.24 mm) were measured for molestus and pipiens, respectively. The measured wing lengths of Ae. aegypti were significantly lower compared to the wing lengths of each Cx. pipiens biotype  After the infectious blood meal, all other engorged mosquitoes were incubated at 28 °C for 14 days, and afterwards the mosquito bodies and salivas were tested for the presence of virus using infectivity assays on Vero cells. The presence of virus was scored based on CPE, and the presence of viral RNA was also confirmed using RT-PCR for a subset of the results. None of the 55 tested Cx. pipiens molestus mosquitoes showed a ZIKV-positive body or saliva (Figure 2A,B). Out of the 133 Cx. pipiens pipiens tested, two mosquitoes showed a ZIKV-positive body but no positive saliva ( Figure  2A,B). For ZIKV-blood fed Ae. aegypti mosquitoes, which served as positive controls, 100% of the tested mosquitoes were infected and 65% showed virus-positive saliva ( Figure 2A,B), which corresponds with our previous work [37]. In addition, the USUV-blood fed Cx. pipiens showed 67% (molestus) and 88% (pipiens) virus-positive bodies (Figure 2A), and 31% (molestus) and 21% (pipiens) virus-positive salivas ( Figure 2B), demonstrating that both Cx. pipiens biotypes were competent vectors for USUV. Given that none of the Cx. pipiens mosquitoes accumulated ZIKV in the saliva after oral infection, we conclude that Cx. pipiens is a highly inefficient vector for ZIKV. After the infectious blood meal, all other engorged mosquitoes were incubated at 28 • C for 14 days, and afterwards the mosquito bodies and salivas were tested for the presence of virus using infectivity assays on Vero cells. The presence of virus was scored based on CPE, and the presence of viral RNA was also confirmed using RT-PCR for a subset of the results. None of the 55 tested Cx. pipiens molestus mosquitoes showed a ZIKV-positive body or saliva (Figure 2A,B). Out of the 133 Cx. pipiens pipiens tested, two mosquitoes showed a ZIKV-positive body but no positive saliva (Figure 2A,B). For ZIKV-blood fed Ae. aegypti mosquitoes, which served as positive controls, 100% of the tested mosquitoes were infected and 65% showed virus-positive saliva (Figure 2A,B), which corresponds with our previous work [37]. In addition, the USUV-blood fed Cx. pipiens showed 67% (molestus) and 88% (pipiens) virus-positive bodies (Figure 2A), and 31% (molestus) and 21% (pipiens) virus-positive salivas ( Figure 2B), demonstrating that both Cx. pipiens biotypes were competent vectors for USUV. Given that none of the Cx. pipiens mosquitoes accumulated ZIKV in the saliva after oral infection, we conclude that Cx. pipiens is a highly inefficient vector for ZIKV.

Intrathoracic ZIKV Injection Leads to Virus Replication in Cx. pipiens with Limited Dissemination to the Mosquito Saliva
To investigate whether ZIKV was unable to pass the mosquito midgut barrier in Cx. pipiens, mosquitoes were injected in the thorax with 1.0 × 10 4 TCID50 of ZIKV. As positive controls, Ae. aegypti was injected with ZIKV and both Cx. pipiens biotypes were injected with USUV. After 14 days at 28 °C, mosquito bodies and salivas were analyzed for the presence of virus. During infectivity assays, the presence of virus was scored based on CPE and, for a subset of the results, these scores were also confirmed by RT-PCR. High percentages (74% for molestus and 78% for pipiens) of the injected mosquitoes had ZIKV-positive bodies ( Figure 4A). Interestingly, 14% (molestus) and 7% (pipiens) of the injected mosquitoes also showed infectious ZIKV in their saliva ( Figure 4B). After injection of Ae. aegypti with ZIKV, which served as a positive control experiment, 100% of the injected mosquitoes showed virus-positive bodies ( Figure 4A), whereas 72% of the injected mosquitoes showed viruspositive saliva ( Figure 4B), which is in line with our earlier results [37]. As another positive control, both biotypes of Cx. pipiens were injected with USUV, which showed that 100% of the tested Cx.

Intrathoracic ZIKV Injection Leads to Virus Replication in Cx. pipiens with Limited Dissemination to the Mosquito Saliva
To investigate whether ZIKV was unable to pass the mosquito midgut barrier in Cx. pipiens, mosquitoes were injected in the thorax with 1.0 × 10 4 TCID 50 of ZIKV. As positive controls, Ae. aegypti was injected with ZIKV and both Cx. pipiens biotypes were injected with USUV. After 14 days at 28 • C, mosquito bodies and salivas were analyzed for the presence of virus. During infectivity assays, the presence of virus was scored based on CPE and, for a subset of the results, these scores were also confirmed by RT-PCR. High percentages (74% for molestus and 78% for pipiens) of the injected mosquitoes had ZIKV-positive bodies ( Figure 4A). Interestingly, 14% (molestus) and 7% (pipiens) of the injected mosquitoes also showed infectious ZIKV in their saliva ( Figure 4B). After injection of Ae. aegypti with ZIKV, which served as a positive control experiment, 100% of the injected mosquitoes showed virus-positive bodies ( Figure 4A), whereas 72% of the injected mosquitoes showed virus-positive saliva ( Figure 4B), which is in line with our earlier results [37]. As another positive control, both biotypes of Cx. pipiens were injected with USUV, which showed that 100% of the tested Cx. pipiens molestus and Cx. pipiens pipiens had USUV-positive bodies ( Figure 4A), and 94% (molestus) and 88% (pipiens) of the injected mosquitoes had USUV-positive saliva ( Figure 4B). This indicates that virus dissemination to the saliva in Cx. pipiens is more efficient with USUV than with ZIKV.

Effect of Injected Viral Dose on ZIKV Infection of Cx. pipiens
To investigate whether the percentage of ZIKV-positive mosquitoes after intrathoracic injection was affected by the viral dose provided, Cx. pipiens pipiens mosquitoes were also injected with lower doses of ZIKV. After 14 days, 40% of the Cx. pipiens pipiens mosquitoes injected with 1.0 × 10 2 TCID50 of ZIKV showed virus-positive bodies, whereas 2% showed virus-positive salivas ( Figure 4A,B). Additionally, when a viral dose of 3.0 × 10 1 TCID50 was supplied, 13% of the tested Cx. pipiens pipiens mosquitoes showed virus-positive bodies, whereas none of the mosquitoes showed virus-positive saliva ( Figure 4A,B). This indicated that the infection and transmission potential of ZIKV-injected Cx. pipiens pipiens was dependent on the viral dose provided, but also that a low ZIKV dose of 3.0 ×10 1 TCID50 can infect a mosquito.

Variability of Viral Titers in ZIKV-Injected Cx. pipiens
To obtain better insight into the ZIKV replication dynamics in injected Cx. pipiens mosquitoes, viral body and saliva titers were measured by EPDAs. To validate ZIKV replication in the primary ZIKV vector Ae. aegypti (positive control), the bodies and salivas of ZIKV-injected Ae. aegypti were also titrated. ZIKV body titers in Cx. pipiens injected with a dose of 1.0 × 10 4 TCID50 were highly variable with maximum titers of 1.1 × 10 6 TCID50/mL (molestus) and 6.3 × 10 5 TCID50/mL (pipiens) (Figure 5A,B). This showed that ZIKV is intrinsically capable of replication to high viral titers in Cx. pipiens. The median viral body titers of ZIKV-injected Cx. pipiens were 9.6 × 10 3 TCID50/mL for pipiens and below the detection limit of 1.0 × 10 3 TCID50/mL for molestus. For Ae. aegypti, a high median viral body titer of 6.32 × 10 6 TCID50/mL was found ( Figure 5C).

Effect of Injected Viral Dose on ZIKV Infection of Cx. pipiens
To investigate whether the percentage of ZIKV-positive mosquitoes after intrathoracic injection was affected by the viral dose provided, Cx. pipiens pipiens mosquitoes were also injected with lower doses of ZIKV. After 14 days, 40% of the Cx. pipiens pipiens mosquitoes injected with 1.0 × 10 2 TCID 50 of ZIKV showed virus-positive bodies, whereas 2% showed virus-positive salivas ( Figure 4A,B). Additionally, when a viral dose of 3.0 × 10 1 TCID 50 was supplied, 13% of the tested Cx. pipiens pipiens mosquitoes showed virus-positive bodies, whereas none of the mosquitoes showed virus-positive saliva ( Figure 4A,B). This indicated that the infection and transmission potential of ZIKV-injected Cx. pipiens pipiens was dependent on the viral dose provided, but also that a low ZIKV dose of 3.0 × 10 1 TCID 50 can infect a mosquito.

Variability of Viral Titers in ZIKV-Injected Cx. pipiens
To obtain better insight into the ZIKV replication dynamics in injected Cx. pipiens mosquitoes, viral body and saliva titers were measured by EPDAs. To validate ZIKV replication in the primary ZIKV vector Ae. aegypti (positive control), the bodies and salivas of ZIKV-injected Ae. aegypti were also titrated. ZIKV body titers in Cx. pipiens injected with a dose of 1.0 × 10 4 TCID 50 were highly variable with maximum titers of 1.1 × 10 6 TCID 50 /mL (molestus) and 6.3 × 10 5 TCID 50 /mL (pipiens) (Figure 5A,B). This showed that ZIKV is intrinsically capable of replication to high viral titers in Cx. pipiens. The median viral body titers of ZIKV-injected Cx. pipiens were 9.6 × 10 3 TCID 50 /mL for pipiens and below the detection limit of 1.0 × 10 3 TCID 50 /mL for molestus. For Ae. aegypti, a high median viral body titer of 6.32 × 10 6 TCID 50 /mL was found ( Figure 5C).
injected Cx. pipiens pipiens mosquitoes with ZIKV-positive saliva, two mosquitoes had relatively high viral body titers of 3.6 × 10 5 TCID50/mL and 5.0 × 10 5 TCID50/mL, whereas the third mosquito had a relatively low viral body titer of 8.0 × 10 3 TCID50/mL ( Figure 5B). This indicates that viral dissemination into the saliva of Cx. pipiens does not always correlate with a high viral titer in the mosquito body.

Discussion
In this study, we set out to determine the vector competence of Dutch Cx. pipiens (biotypes molestus and pipiens) for ZIKV and we found that Cx. pipiens was unable to experimentally transmit ZIKV after an infectious blood meal. However, to the best of our knowledge, our study is the first study to demonstrate ZIKV dissemination to the saliva of Cx. pipiens mosquitoes after intrathoracic injection. Previous studies reported the presence of ZIKV in Cx. pipiens bodies [30,31] and heads [30] after injection but no virus accumulation in the saliva [30,31]. This suggests that ZIKV is incapable of infecting the salivary glands and/or of entering the saliva of Cx. pipiens [31]. We showed, however, that ZIKV can accumulate in the mosquito saliva after an intrathoracic injection with a viral dose as low as 1.0 × 10 2 TCID50. This viral dose for injection is similar or lower compared to other studies that did not report ZIKV presence in the saliva [30,31]. Based on our results, we conclude that ZIKV is intrinsically capable of dissemination to the saliva of Cx. pipiens upon forced infection.
Nonetheless, even after forced infection, ZIKV replication in Cx. pipiens appeared to be suboptimal, as 74% (molestus) and 78% (pipiens) of the Cx. pipiens injected with a viral dose of 1.0 × 10 4 TCID50 of ZIKV showed virus-positive bodies compared to 100% of the ZIKV-injected Ae. aegypti and USUV-injected Cx. pipiens. These results suggest a general replication deficiency of ZIKV in Culex cells, which has also been observed by others [31]. The underlying mechanisms responsible for the specific restriction of ZIKV in Cx. pipiens are currently unknown and need further investigation. Important factors that should be considered are physical barriers at the mosquito midgut and salivary glands, mosquito host factors required for virus replication, mosquito immune responses and the mosquito midgut microbiome [39]. Flaviviruses, such as ZIKV, but also yellow fever virus and dengue virus, are primarily associated with Aedes vectors [40,41]. Other flaviviruses, such as WNV and USUV, are mainly associated with Culex vectors [40,41]. Genetic differences between the mosquito genera and/or between the respective flaviviruses likely constrain and maintain the observed vector specificity. However, arboviruses have previously been shown to have the potential to quickly adapt to new vectors [42,43] and, therefore, it is important to investigate the molecular Interestingly, four Cx. pipiens molestus mosquitoes with ZIKV-positive saliva showed viral body titers below the detection limit of 1.0 × 10 3 TCID 50 /mL ( Figure 5A). Moreover, out of the three ZIKV-injected Cx. pipiens pipiens mosquitoes with ZIKV-positive saliva, two mosquitoes had relatively high viral body titers of 3.6 × 10 5 TCID 50 /mL and 5.0 × 10 5 TCID 50 /mL, whereas the third mosquito had a relatively low viral body titer of 8.0 × 10 3 TCID 50 /mL ( Figure 5B). This indicates that viral dissemination into the saliva of Cx. pipiens does not always correlate with a high viral titer in the mosquito body.

Discussion
In this study, we set out to determine the vector competence of Dutch Cx. pipiens (biotypes molestus and pipiens) for ZIKV and we found that Cx. pipiens was unable to experimentally transmit ZIKV after an infectious blood meal. However, to the best of our knowledge, our study is the first study to demonstrate ZIKV dissemination to the saliva of Cx. pipiens mosquitoes after intrathoracic injection. Previous studies reported the presence of ZIKV in Cx. pipiens bodies [30,31] and heads [30] after injection but no virus accumulation in the saliva [30,31]. This suggests that ZIKV is incapable of infecting the salivary glands and/or of entering the saliva of Cx. pipiens [31]. We showed, however, that ZIKV can accumulate in the mosquito saliva after an intrathoracic injection with a viral dose as low as 1.0 × 10 2 TCID 50 . This viral dose for injection is similar or lower compared to other studies that did not report ZIKV presence in the saliva [30,31]. Based on our results, we conclude that ZIKV is intrinsically capable of dissemination to the saliva of Cx. pipiens upon forced infection.
Nonetheless, even after forced infection, ZIKV replication in Cx. pipiens appeared to be suboptimal, as 74% (molestus) and 78% (pipiens) of the Cx. pipiens injected with a viral dose of 1.0 × 10 4 TCID 50 of ZIKV showed virus-positive bodies compared to 100% of the ZIKV-injected Ae. aegypti and USUV-injected Cx. pipiens. These results suggest a general replication deficiency of ZIKV in Culex cells, which has also been observed by others [31]. The underlying mechanisms responsible for the specific restriction of ZIKV in Cx. pipiens are currently unknown and need further investigation. Important factors that should be considered are physical barriers at the mosquito midgut and salivary glands, mosquito host factors required for virus replication, mosquito immune responses and the mosquito midgut microbiome [39]. Flaviviruses, such as ZIKV, but also yellow fever virus and dengue virus, are primarily associated with Aedes vectors [40,41]. Other flaviviruses, such as WNV and USUV, are mainly associated with Culex vectors [40,41]. Genetic differences between the mosquito genera and/or between the respective flaviviruses likely constrain and maintain the observed vector specificity. However, arboviruses have previously been shown to have the potential to quickly adapt to new vectors [42,43] and, therefore, it is important to investigate the molecular basis underlying the vector specificity of ZIKV, and also to investigate whether virus evolutionary trajectories can be predicted that could potentially lead to new epidemic variants of ZIKV with altered vector specificity.
We provided evidence via injection experiments that the mosquito midgut acted as an important barrier against ZIKV dissemination in Cx. pipiens. Our finding, with regard to the inability of Cx. pipiens to transmit ZIKV, is in line with other recent studies suggesting that mosquitoes of the Culex genus are poor ZIKV vectors [30][31][32][33][34][35][36]44]. Even at an incubation temperature as high as 28 • C, Cx. pipiens did not accumulate ZIKV in the saliva after oral exposure [30,31,34]. However, when considering the massive number of mosquitoes in the field and the fact that our laboratory study only measures vector competence and does not take into account all factors contributing to the vectorial capacity of the Cx. pipiens mosquito species [39], we cannot completely rule out the possibility of ZIKV transmission by Cx. pipiens in the field. Nevertheless, the reports of others [30][31][32][33][34][35][36] and our experiments with high numbers of tested Cx. pipiens mosquitoes and positive controls to validate the competence of the tested Cx. pipiens colonies, and the used ZIKV isolate, consolidate the conclusion that Cx. pipiens is a highly inefficient vector for ZIKV.