Limited Capacity for Aedes aegypti to Mechanically Transmit Chikungunya Virus and Dengue Virus

ABSTRACT.

Mechanical transmission is an understudied mode of arbovirus transmission that occurs when a biting insect transmits virus among hosts by the direct transfer of virus particles contaminating its mouthparts. Multiple arboviruses have been shown to be capable of utilizing this transmission route, but most studies were conducted 40 to 70 years ago using dated methodologies. To gain a better understanding of this phenomenon, we used molecular techniques to evaluate the efficiency of mechanical transmission by Aedes aegypti mosquitoes for two evolutionarily divergent arboviruses, chikungunya virus (CHIKV) and dengue virus (DENV). Viral RNA and/or infectious DENV could be detected on 13.8% of mosquito proboscises sampled immediately after an infectious bloodmeal, but positivity rates declined within hours. CHIKV RNA and/or infectious virus was detected on 38.8% of proboscises immediately after feeding but positivity rates dropped to 2.5% within 4 hours. RNA copy numbers were low for both viruses, and we were unable to demonstrate mechanical transmission of CHIKV using an established animal model, suggesting that this mode of transmission is unlikely under natural conditions.

Numerous arboviruses, such as chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV), have emerged in recent decades and pose a significant public health threat to much of the world’s population.¹ Many of these viruses remain silent for years, circulating in sylvatic cycles via horizontal transmission (vector ↔ host), vertical transmission (female → offspring), and sexual transmission (male ↔ female).^2–5 However, once introduced into a naive human population, they can quickly develop into explosive epidemics. Such scenarios are largely driven by horizontal transmission, which can occur biologically and/or mechanically. In both forms, virus is transmitted from one host to another through the bite of an infectious mosquito. The difference between the two forms is that biological transmission requires the virus to infect and replicate within the mosquito, resulting in a delay, measured in days, from virus acquisition to transmission. Conversely, mechanical transmission occurs when virus particles contaminating the mouthparts of a mosquito are transferred to a new host without the need for replication within the mosquito.^6,7 This could occur when a mosquito becomes interrupted while feeding on a viremic host and subsequently resumes feeding shortly after on a naive host. Certain species, such as Aedes aegypti and Aedes albopictus, are known to take frequent, partial blood meals that could facilitate virus spread by this mechanism.⁸ Although it is widely believed that biological transmission is the primary driver of epidemics, it has been suggested that mechanical transmission may also play a significant role during outbreaks.^6,7,9 There is evidence that mosquito-mediated mechanical transmission occurs in a diversity of arbovirus families, including Togaviridae, Flaviviridae, and Phenuiviridae^10–15; however, many of these observations were reported 40 to 70 years ago using dated methodologies. We therefore sought to reexamine this phenomenon for the Aedes-borne viruses CHIKV and DENV using modern molecular techniques.

To estimate the likelihood of mechanical transmission, we harvested the proboscises from Ae. aegypti mosquitoes at various time points after exposure to an infectious blood meal and assayed them for the presence of viral RNA and infectious virus. Briefly, Ae. aegypti (Orlando strain) were provided an infectious blood meal consisting of a 1:1 mixture of defibrinated sheep’s blood and virus: CHIKV 3.40 × 10⁶ plaque forming units (PFU)/mL (1.35 × 10¹¹ genome equivalents [GE]/mL; strain: R99659; passage history: Vero-1, BHK-1; GenBank: KX713902) and DENV Serotype 2 2.55 × 10⁶ PFU/mL (1.68 × 10¹¹ GE/mL; strain: 125270; passage history: C6/36-4; GenBank: U91870). Upon engorgement, 20 mosquitoes were sampled at 0, 4, and 24 hours post–blood meal (hpbm). Flame-sterilized razor blade and forceps were used to remove the proboscis as close to the maxillary palpi as possible. Each proboscis was immediately placed in a 2-mL microcentrifuge tube with 300 µL of phosphate-buffered saline-G, homogenized using a copper BB and mixer mill as previously described.¹⁶ Experiments were completed in quadruplicate (N = 80 mosquitoes/time point). Viral RNA was extracted from 200 µL homogenate using the MagMAX^™ Viral RNA Isolation Kit (Life Technologies, Carlsbad, CA) on a Kingfisher Flex (ThermoFisher Scientific, Waltham, MA). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed using the iTaq Universal Probes One-Step Kit (Bio-Rad, Hercules, CA) and previously developed primer/probe sets: CHIKV6856/CHIKV6981/CHIKV6919-FAM¹⁷ and DEN-2-fwd/DEN-2-rev/DEN-2-probe.¹⁸ Samples were quantified using viral RNA standards developed in-house with a cycle threshold value (Ct) < 35 considered positive. Infectious virus detection was performed by inoculating 100 μL of proboscis homogenate on Vero E6 (CHIKV) or BHK-21 (DENV) cells with daily checks noting signs of cytopathologic effects (CPE) for up to 7 days. After 7 days or the first sign of CPE, supernatant was collected, RNA extracted and tested for the presence of virus.

DENV RNA could be detected on the mouthparts of 10, three, and one mosquitoes by RT-qPCR at 0, 4, and 24 hpbm, respectively (Table 1). Recovery of infectious DENV by cell culture assay was detected from one proboscis at the 0 hpbm time point; however, the presence of DENV RNA was not confirmed by RT-qPCR. We estimated a mean of 2.30 × 10³ GE per DENV positive proboscis at 0 hpbm, which was greater than those sampled at 4 (3.08 × 10² GE) or 24 (2.47 × 10² GE) hpbm (Figure 1). On the basis of the PFU-to-GE ratio of our virus stock, we estimated that there were on average 0.035, 0.005, and 0.004 PFU on RT-qPCR positive proboscises at 0, 4, and 24 hpbm, respectively. Rates of mouthpart contamination were higher for CHIKV with 18, 2, and 1 testing positive by RT-qPCR and 26, 1, and 1 by cell culture at 0, 4, and 24 hpbm, respectively (Table 1). The total number of positive proboscises between the two assays were 31 (38.8%) at 0 hours, 2 (2.5%) at 4 hours, and 1 (1.3%) at 24 hpbm. We estimated an average of 8.12 × 10³, 8.18 × 10³, and 3.68 × 10⁴ GE per CHIKV positive proboscis at 0, 4, and 24 hpbm, respectively (Figure 1). The average PFU quantities were estimated to be 0.20, 0.21, and 0.93 at 0, 4 and 24 hpbm, respectively. Together, these data demonstrate that viruses can persist on contaminated mosquito mouthparts for up to 24 hours, albeit at low levels.

Table 1.

Results of cell culture and RT-qPCR assays performed on the proboscises of mosquitoes that were allowed to feed on infectious blood meals

No. positive proboscises/no. tested (% positive)
Hours post blood meal	DENV			CHIKV
	Cell culture assay	RT-qPCR assay	Total*	Cell culture assay	RT-qPCR assay	Total*
0	1/80 (1.3)	10/80 (12.5)	11/80 (13.8)	26/80 (32.5)	18/80 (22.5)	31/80 (38.8)
4	0/80 (0)	3/80 (3.8)	3/80 (3.8)	1/80 (1.3)	2/80 (2.5)	2/80 (2.5)
24	0/80 (0)	1/80 (1.3)	1/80 (1.3)	1/80 (1.3)	1/80 (1.3)	1/80 (1.3)

CHIKV = chikungunya virus; DENV = dengue virus; RT-qPCR = reverse transcription quantitative polymerase chain reaction.

^*Total = proboscises that were positive with either or both assays.

Figure 1.

Number of genome equivalents (GE) on reverse transcription quantitative polymerase chain reaction–positive mosquito proboscises at 0, 4, and 24 hours post-infectious blood meal. CHIKV = chikungunya virus; DENV = dengue virus.

To assess the likelihood that virus found contaminating the proboscis could be transmitted to a naive host we performed a transmission study using CHIKV and suckling mice. Studies involving animals were approved by CAES Institutional Animal Care and Use Committee (protocol P34-20) and adhered to the guidelines outlined in the Animal Welfare Act Regulations. This protocol was previously used to demonstrate biological transmission of CHIKV by Ae. aegypti to mice.¹⁶ Ae. aegypti mosquitoes were provided a CHIKV infectious blood meal and allowed to partially feed. At 0 and 4 hpbm, partially fed mosquitoes were individually offered a suckling mouse (CD-1 IGS mice, Charles River Laboratories, Wilmington, MA). Mosquito abdomens and proboscises were harvested for viral detection immediately after feeding on mice. Suckling mice were examined daily for signs of illness and euthanized on day 4. Brain tissue and one hind limb were collected from each mouse and processed in 500 µL PBS-G using a mixer mill as described earlier. CHIKV was recovered from three of 17 mosquito proboscises and 16 of 17 abdomens sampled at 0 hpbm by cell culture assay, whereas 0 of 16 proboscises but all 16 abdomens were positive for CHIKV via cell culture assay at 4 hpbm (Table 2). The abdomen results were the same between cell culture and RT-qPCR assays; however, the proboscis results differed. All the proboscises from 0 hpbm were negative, yet one from the 4 hpbm group had a Ct value < 35 by RT-qPCR. None of the 32 mosquitoes that had partially fed on an infectious blood meal successfully transmitted virus to a mouse host at 0 or 4 hpbm. None of the mice demonstrated signs of illness before euthanasia, and all brain and limb tissues were negative by RT-qPCR.

Table 2.

Results of cell culture and RT-qPCR assays performed on mosquito and mouse tissues to assess mechanical transmission of CHIKV to suckling mice

	No. samples positive for CHIKV/no. tested (% positive)
Hours post blood meal	RT-qPCR assay				Cell culture assay
	Mouse tissues		Mosquito tissues		mosquito tissues
	Brain	Limbs	Proboscises	Abdomens	Proboscises	Abdomens
0	0/17 (0)	0/17 (0)	0/17 (0)	16/17 (94.1)	3/17 (17.6)	16/17 (94.1)
4	0/16 (0)	0/16 (0)	1/16 (6.3)	16/16 (100)	0/16 (0)	16/16 (100)

CHIKV = chikungunya virus; RT-qPCR = reverse transcription quantitative polymerase chain reaction.

Our findings indicate that mechanical transmission is unlikely to play a significant role in the natural transmission cycles of DENV and CHIKV. Viral RNA and/or infectious virus could be detected on the mouthparts of 13.8% DENV-exposed and 38.8% CHIKV-exposed mosquitoes sampled immediately after an infectious bloodmeal. Positivity rates declined over time, and suckling mice did not become infected by the mechanical transmission route. In another study, we found that 62.5% to 82.4% of sucking mice became infected by CHIKV after being fed upon by Ae. aegypti with disseminated viral infections via the biological transmission route.¹⁶ The failure of mechanical transmission being demonstrated in vivo, despite the use of a highly susceptible animal model, can be explained by low levels of virus on the mouthparts—levels that do not meet the minimum dose necessary to cause infection.

It is notable that the percentage of virus-positive proboscises was significantly lower after allowing mosquitoes to probe or feed on naive mice than that found in mosquitoes sampled immediately after an infectious bloodmeal, absent an intermediary host. This may be due to the proboscises being “cleaned” while the mosquitos are probing or feeding on the mice. It is likely that some virus particles were deposited into the mouse during penetration into or removal of the proboscis from the skin, although evidently not enough to cause infection. Another possibility is that the mosquitoes “groomed” the virus off their proboscises using setea that make up the tibial combs.¹⁹ Grooming behavior has been found to increase in frequency and duration following a feeding.²⁰

Humans are the primary host for DENV and CHIKV, and one study found an average of 6.53 log₁₀ GE/mL and 6.92 log₁₀ GE/mL in the serum of patients infected with DENV and CHIKV, respectively.²¹ The viral titers of the bloodmeals used in our experimental study exceed what would normally be encountered in nature; therefore, our results indicate that mechanical transmission of CHIKV or DENV by Aedes aegypti mosquitoes is highly unlikely to occur under ordinary circumstances. However, due to the higher than natural titers often used to infect mosquitoes in laboratory experiments, there may be a low risk to laboratory workers if bitten or probed by a mosquito shortly after an infectious bloodmeal. This risk decreases rapidly within hours. Future studies might focus on whether differences among viruses and/or mosquito species or strains affect mechanical transmission potential.