Introduction
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus in the family Flaviviridae, which includes human pathogens such as yellow fever, dengue viruses (DENVs), and West Nile virus (WNV). ZIKV was originally isolated from the blood of a sentinel rhesus macaque placed in a canopy platform in the Zika forest of Uganda in 1947.1 The virus circulates in several regions of Africa between arboreal mosquitoes such as Aedes africanus (Theobald) and Aedes furcifer (Edwards) and nonhuman primates in what is called an enzootic or sylvatic cycle.2 However, “spillover” infections in Africa are rarely detected, with only 14 human cases reported in the medical literature until contemporary outbreaks beginning in 2007.3 In 2007, independent ZIKV epidemics occurred in Gabon4 and Yap Island in the Federated States of Micronesia,5 the latter estimated to have affected 73% of the island's population of 7,391 persons. Following the path of another arthropod-borne virus (arbovirus), chikungunya virus (CHIKV), Zika began spreading in the South Pacific in 2013, affecting several islands, including French Polynesia, New Caledonia, Easter Island, and the Cook Islands.6–8 In early 2015, transmission was first detected in northeastern Brazil,9,10 followed by explosive spread throughout the Americas, with transmission reported in 48 countries and territories, including Florida and Texas in the United States.11,12
Infection with ZIKV is inapparent or subclinical in a majority (up to 80%) of people13 and symptomatic cases are generally characterized by mild signs and symptoms such as fever, headache, malaise, conjunctivitis, myalgia, arthralgia, and maculopapular rash.13–15 This syndrome can closely resemble that caused by other arboviruses such as CHIKV and DENV, which co-circulate with ZIKV in many regions. However, a spike in reports of serious outcomes of ZIKV infection such as microcephaly,13,16 neurological, ocular and muscular complications,17–19 and Guillain–Barré syndrome that have been identified in the Americas, and retrospectively during in the French Polynesian outbreak,20,21 caused the World Health Organization to declare ZIKV an international public health concern.22
In the absence of therapeutics or vaccines, control of ZIKV outbreaks is limited to mosquito abatement and prevention of mosquito–human contact. Since its discovery in 1947, ZIKV has been isolated from several species of mosquitoes within the genus Aedes, including several arboreal species such as Ae. furcifer, Ae. luteocephalus (Newsteed), and Ae. africanus.1,23,24 ZIKV was first isolated from a peridomestic mosquito, Ae. aegypti (Linnaeus) in 1969 in Malaysia,25 and later implicated in outbreaks in the Pacific due to the presence of ZIKV RNA in a single Ae. aegypti pool.26 However, the role of Ae. aegypti in outbreaks was not directly confirmed until this species was implicated in a 2015 outbreak of ZIKV in Chiapas State, Mexico,27 and later in Rio de Janeiro, Brazil.28 Other vectors have also been evaluated for their role in transmission; for example, Ae. (Stegomyia) hensilli Farner was retrospectively identified as the likely vector on Yap Island in 2007.3
Another mosquito suspected of urban ZIKV transmission is Ae. albopictus, the Asian tiger mosquito. Originating in the forests of southeast Asia, this invasive species recently spread throughout tropical and temperate regions of Asia, Europe, the Americas, and Africa.29 When compared with its close relative Ae. aegypti, Ae. albopictus is regarded as a secondary vector for several arboviruses, including DENV. In recent years, this prevailing view has changed, primarily due to the role of Ae. albopictus in Indian Ocean Basin and Asian CHIKV outbreaks. This role was facilitated by the convergent evolution in these regions of substitutions at position 226 of the envelope glycoprotein one involving Ala→Val (E1-A226V), which confers an approximately 40-fold fitness gain for transmission by Ae. albopictus, followed by additional adaptive substitutions in the E2 protein in Asia.30–32 In locations where Ae. albopictus is the predominant (or only) vector of CHIKV, such as in La Réunion, Italy, Cameroon, France, Gabon, and parts of Thailand, these Ae. albopictus-adapted strains have displaced others.31
Due to its ecological plasticity, which in part has facilitated its invasive spread, Ae. albopictus exhibits a wide geographic range extending into the northeastern United States.33 Its ability to transmit DENV and CHIKV coupled with its aggressive biting behavior, suggest a potential role in ZIKV transmission, a hypothesis supported by the identification of ZIKV-positive Ae. albopictus pools during the 2007 Gabon outbreak.4
Previous studies of the vector competence of Ae. albopictus for ZIKV provided disparate results, with some finding populations of this species to be relatively poor vectors,34 whereas others reported high susceptibility and transmission potential.35,36 However, all of these previous studies used artificial bloodmeals, known to be less infectious than viremic animals for several arboviruses including ZIKV.37 To better evaluate the potential role of Ae. albopictus in ongoing and future ZIKV outbreaks, we investigated its susceptibility to infection, dissemination, and potential for transmission following exposure to five strains of ZIKV. Because previous vector competency analyses indicate that mosquitoes from different geographic locations can differ in their susceptibility to arboviruses,34 we used populations from ZIKV-epidemic locations, including two high-risk locations in the Americas, based on histories of DENV circulation38: the Rio Grande Valley, TX; Houston, TX; and Salvador, Brazil.
Materials and Methods
Cells.
Vero cells were purchased from American Type Tissue Culture Collection (Bethesda, MD) and maintained in Dulbecco's modification of Eagle's Medium (DMEM) (Invitrogen, Carlsbad, San Diego) supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norwalk, GA) and Penicillin/Streptomycin (P/S; 100 units/mL and 100 μg/mL, respectively) (Invitrogen) in a water-jacketed incubator at 37°C with 5% CO2.
Viruses.
The following ZIKV strains were used: FSS 130125 (GenBank accession no. KU955593), a human isolate from Cambodia (2010); DakAR 41525 (KU955591), an Ae. africanus isolate from Senegal (1984); MEX 1-7 (KX247632), isolated from an Ae. aegypti pool from Chiapas State, Mexico (2015); PRVABC 59 (KX377337), a human isolate derived from Puerto Rico (2015); and PB 81 (KU365780), an isolate derived from human blood from Brazil (2015). All viruses were acquired from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch (UTMB) and passage histories are listed in Table 1. Stocks were titered by focus-forming assay as described below and frozen at −80°C in 30% FBS prior to mouse infections.
Zika virus strain Information
| Strain | Genbank accession | Origin | Location isolated | Year isolated | Passage history* |
|---|---|---|---|---|---|
| DakAR 41525 | KU955591 | Aedes africanus | Senegal | 1984 | AP61, C6/36 (2), Vero (3) |
| FSS 130125 | KU955593 | Human | Cambodia | 2010 | Vero, C6/36 (2), Vero (3) |
| MEX 1-7 | KX247632 | Aedes aegypti | Chiapas State, Mexico | 2015 | Vero (4), C6/36, Vero (3) |
| PB 81 | KU365780 | Human | Paraiba, Brazil | 2015 | Vero, C6/36, Vero |
| PRVABC 59 | KX377337 | Human | Puerto Rico | 2015 | Vero (5) |
Cells lines include AP61: Aedes pseudoscutellaris larvae; C6/36: Aedes albopictus larvae; Vero: Chlorocebus aethiops kidney epithelium.
Mosquitoes.
Adult female Ae. albopictus from the Rio Grande Valley (F5), Houston (F2), and Salvador, Brazil (F3), were housed in a 27 ± 1°C incubator (a typical mean temperature in tropical climates) with 80 ± 10% relative humidity, fed 10% sucrose ad libitum, and maintained at 16:8 light:dark photoperiod. Female mosquitoes in all experiments were fed 5 days posteclosion. Twenty-four hours prior to bloodmeals, sucrose was replaced with water, which was withdrawn 6 hours before feeding.
Murine infections.
Four-week-old interferon type I receptor-knockout (A129) mice were infected intraperitoneally with 1 × 105 focus-forming units (FFUs) of each ZIKV strain, diluted in sterile phosphate buffered saline. This model generates predictable viremia at 1, 2, and 3 days postinfection to produce varied oral doses for mosquitoes.39 One animal per day was randomly selected and anesthetized with 100 mg/kg of ketamine and placed on the screened lid of 0.5-L cardboard cartons containing sucrose-starved Ae. albopictus. Mosquitoes were allowed to feed for 30 minutes, then cold-anesthetized and fully engorged mosquitoes were incubated as described below. Following blood feeding, mice were killed and blood was collected for viremia assays as described below. All animal procedures and manipulations were approved by the UTMB Institutional Animal Care and Use Committee (IACUC).
Titrations.
Mouse sera and positive saliva samples underwent 10-fold serial dilution in DMEM with 2% FBS and 1% P/S on 96 well plates; 100 μL of each dilution were then transferred to Vero cell monolayers on 24 well plates. After 1 hour at 37°C wells were overlaid with 0.8% methylcellulose in DMEM. Following 3 days incubation at 37°C, the overlay was removed and monolayers were rinsed twice with sterile Dulbecco's phosphate buffered saline (DPBS), and fixed for 1 hour at room temperature in ice-cold methanol:acetone (1:1). Detection of virus was conducted via focus-forming assay, as detailed below.
Mosquito infection, dissemination, and transmission potential.
On days 3, 7, and 14 of incubation, 9–15 mosquitoes per group were cold-anesthetized, and legs removed and placed into microfuge tubes containing a steel ball bearing and 500 μL of DMEM, supplemented with 2% FBS, 1% P/S, and 2.5 μg/mL amphotericin B (Gibco, Waltham, MA). Mosquitoes were then restrained on a glass slide with mineral oil and their proboscis inserted for 30 minutes of salivation into a sterile 10 μL micropipette tip containing 8 μL of FBS, after which the expectorated saliva/FBS was added to 100 μL of DMEM supplemented with 1% P/S and 2.5 μg/mL amphotericin B. Mosquito bodies and legs were then triturated for 5 minutes at 26 Hz in a TissueLyser II (Qiagen, Venlo, the Netherlands). Following homogenization, samples were clarified by centrifugation at 200 × g for 5 minutes. Infections and detections were performed via the focus-forming assay described below.
Focus-forming assay.
Focus-forming assays were performed as previously described,40 with modifications. Briefly, viremic mouse sera or clarified mosquito samples were inoculated onto Vero cell monolayers on 24 or 96 well plates, respectively. After incubation for 3 days, media was removed and wells were washed. Plates were fixed using ice-cold methanol:acetone (1:1). Following complete air drying, plates were washed with PBS and then blocked with 3% FBS in PBS, followed by an overnight incubation with mouse hyperimmune serum against-ZIKV strain MR-766 (1:2,000 in blocking solution) (WRCEVA, UTMB). Plates were then washed with PBS followed by incubation with a goat antimouse secondary antibody conjugated to horseradish peroxidase (KPL, Gaithersburg, MD) diluted at 1:2,000 in blocking solution. Plates were washed with PBS, after which an aminoethylcarbazole solution (Enzo Diagnostics, Farmingdale, NY) prepared according to manufacturer's protocol, was added and plates were incubated in the dark. Development was halted by washing in tap water and plates were allowed to air dry at room temperature before scoring. For samples in 96 well plates, detection of any intracytoplasmic staining by light microscopy was defined as positive. For titrations of positive saliva samples and mouse serum on 24 well plates foci were counted either by eye or by light microscopy.
50% oral infectious dose calculation.
The oral dose of a given ZIKV strain required to infect, disseminate, and transmit in a given sample of mosquitoes was calculated where possible, utilizing the method of Reed and Muench.41
Statistical analysis.
The impact of mosquito strain, virus strain, and virus titer in the mouse and day of extrinsic incubation on infection in the mosquito body (a dichotomous yes/no variable) was analyzed using nominal logistic regression. Next, the impact of the factors listed earlier on dissemination into the legs from those mosquitoes with infected bodies, as well as secretion of virus into the saliva from those mosquitoes with a disseminated infection, was analyzed using nominal logistic regression. Saliva samples found positive in the 96 well format were titrated on 24 well plates and subjected to focus forming assay as described earlier. For samples that were positive in the initial screen but negative in the titration due to a titer below the limit of detection (10 FFU), a titer of 9 FFU was assigned to ensure conservative comparisons. Resultant titers were log-transformed, but since this failed to result in normal distribution titers were analyzed via a Wilcoxon test.
Results
Comparison of ZIKV strains.
Rio Grande Ae. albopictus.
With respect to our analyses, it should be noted that ideally, the virus strains tested in all three populations of mosquitoes would be matched, so as to facilitate easier comparisons. However, on availability, we replaced DakAR 41525 and FSS13025 with modern epidemic strains PRVABC 59 and PB 81 due to their increased public health relevance. Therefore, because virus and mosquito strains were not completely blocked, we could not compare all virus strains among all mosquito strains. Thus we first compared the impact of virus strain, viremia titer in the mouse, and day of extrinsic incubation, as well as interactions among all three factors, on infection of Rio Grande mosquitoes, which fed on MEX 1-7-, FSS 133205-, and DakAR 41525-infected mice. Substantially higher viremia titers were generated in mice infected by the DakAR 41525 strain compared with the other two (Figure 1). We found no significant interactions among the three factors, and a significant effect only of virus strain (df = 2, χ2 = 7.07, P = 0.029), with DakAR 41525 and MEX 1-7 infecting a significantly higher proportion of mosquitoes than FSS 13025. This effect was driven primarily by the greater infectivity of DakAR 41525 and MEX 1-7 at the lower viremia titers. We then compared the effect of the three factors on ZIKV dissemination in infected mosquitoes. This analysis revealed a significant interaction between viral strain and days of extrinsic incubation (df = 2, χ2 = 10.5, P = 0.005), but no significant interactions among the remaining factors. We therefore conducted a simple effects test of the impact of viral strain and viremia titer for each day of extrinsic incubation (3, 7, 14) individually. There were very few disseminated infections on day 3, precluding comparison, but on days 7 and 14 there were significant effects of both virus strain and viremia titer (P ≤ 0.001 for all comparisons). Virus dissemination increased with increasing viremia titer, and at a comparable viremia titer, the DakAR 41525 strain of ZIKV (1.6 × 106 FFU/mL) disseminated more efficiently than the FSS 13025 (3.5 × 106 FFU/mL) or MEX 1-7 (1.0 × 106 FFU/mL) strains. Finally, we compared the efficiency of dissemination by different ZIKV strains following different viremia titers and days of extrinsic incubation. This analysis detected a significant interaction between viremia titer and days of extrinsic incubation (df = 2, χ2 = 12.8, P = 0.002). Thus we conducted a simple effects test of the impact of virus strain and titer at days 7 and 14 of extrinsic incubation (no dissemination was detected at day 3 of extrinsic incubation). This analysis detected no significant differences at day 7 but a significant interaction of virus titer and strain on day 14, with virus secretion into the saliva increasing with viremia titer, and with the DakAR 41525 strain of ZIKV generating a higher percentage of infectious saliva than the other two strains. Overall, these results indicated that the DakAR 41525 disseminated more efficiently and was shed into the saliva more frequently in Rio Grande Valley Ae. albopictus compared with the MEX 1-7 and FSS 13025 ZIKV strains.
Infection, dissemination, and potential transmission of three Zika virus strains by Aedes albopictus from the Rio Grande Valley, TX, following bloodmeal from viremic A129 mice infected with (A) FSS 13025 (Cambodia, 2010), (B) MEX 1-7 (Mexico, 2015) or (C) DakAR 41525 (Senegal 41525) and assays on day 3 (N = nine mosquitoes per virus), day 7 (N = 14), and day 14 (N = 14).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Infection, dissemination, and potential transmission of three Zika virus strains by Aedes albopictus from the Rio Grande Valley, TX, following bloodmeal from viremic A129 mice infected with (A) FSS 13025 (Cambodia, 2010), (B) MEX 1-7 (Mexico, 2015) or (C) DakAR 41525 (Senegal 41525) and assays on day 3 (N = nine mosquitoes per virus), day 7 (N = 14), and day 14 (N = 14).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Infection, dissemination, and potential transmission of three Zika virus strains by Aedes albopictus from the Rio Grande Valley, TX, following bloodmeal from viremic A129 mice infected with (A) FSS 13025 (Cambodia, 2010), (B) MEX 1-7 (Mexico, 2015) or (C) DakAR 41525 (Senegal 41525) and assays on day 3 (N = nine mosquitoes per virus), day 7 (N = 14), and day 14 (N = 14).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Houston Ae. albopictus.
A similar analysis to the one described earlier detected no significant interactions or main effects of virus strain, virus titer, or days of extrinsic incubation on infection of Houston mosquitos exposed to PB 81, MEX 1-7, and PRVABC 59 ZIKV strains (Figure 2). This analysis detected no significant interactions or main effects of virus strain, virus titer, or days of extrinsic incubation on infection. Analysis of dissemination from infected bodies revealed a three-way interaction among the independent variables. A simple effects test of virus strain at viremia titer = 7.0 ± 0.2 log10 FFU/mL and day 14 of extrinsic incubation showed no differences among the three ZIKV strains. There were no interactions among the three factors on secretion of virus into saliva and only virus strain influenced this outcome, with PRVABC 59 ZIKV shed into saliva more efficiently than the other two strains (df = 2, χ2 = 17.2, P = 0.0002), which failed to produce detectable virus in saliva. Overall, ZIKV strain PRVABC 59 was more efficiently shed into the saliva of Houston Ae. albopictus compared with PB 81 and MEX 1-7 (Figure 2).
Infection, dissemination, and potential transmission of three Zika virus strains by Aedes albopictus from Houston, TX, following bloodmeal from viremic A129 mice infected with (A) PB 81 (Brazil, 2015), (B) MEX 1-7 (Mexico, 2015) or (C) PRVABC 59 (Puerto Rico 2015) and assays on day 3 (N = 12 mosquitoes per virus), day 7 (N = 15), and day 14 (N = 15).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Infection, dissemination, and potential transmission of three Zika virus strains by Aedes albopictus from Houston, TX, following bloodmeal from viremic A129 mice infected with (A) PB 81 (Brazil, 2015), (B) MEX 1-7 (Mexico, 2015) or (C) PRVABC 59 (Puerto Rico 2015) and assays on day 3 (N = 12 mosquitoes per virus), day 7 (N = 15), and day 14 (N = 15).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Infection, dissemination, and potential transmission of three Zika virus strains by Aedes albopictus from Houston, TX, following bloodmeal from viremic A129 mice infected with (A) PB 81 (Brazil, 2015), (B) MEX 1-7 (Mexico, 2015) or (C) PRVABC 59 (Puerto Rico 2015) and assays on day 3 (N = 12 mosquitoes per virus), day 7 (N = 15), and day 14 (N = 15).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Salvador Ae. albopictus.
Only days of extrinsic incubation significantly influenced infection or dissemination of PB 81 and MEX 1-7 in Salvador mosquitoes (P < 0.003 for both comparisons). Neither virus strain was detected in mosquito saliva (Figure 3).
Infection, dissemination, and transmission of two Zika virus strains by Aedes albopictus from Salvador, Brazil, following bloodmeal from viremic A129 mice infected with (A) PB 81 (Brazil, 2015) or (B) MEX 1-7 (Mexico, 2015) and assays on day 3 (N = 15 mosquitoes per virus), day 7 (N = 15), and day 14(N = 15).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Infection, dissemination, and transmission of two Zika virus strains by Aedes albopictus from Salvador, Brazil, following bloodmeal from viremic A129 mice infected with (A) PB 81 (Brazil, 2015) or (B) MEX 1-7 (Mexico, 2015) and assays on day 3 (N = 15 mosquitoes per virus), day 7 (N = 15), and day 14(N = 15).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Infection, dissemination, and transmission of two Zika virus strains by Aedes albopictus from Salvador, Brazil, following bloodmeal from viremic A129 mice infected with (A) PB 81 (Brazil, 2015) or (B) MEX 1-7 (Mexico, 2015) and assays on day 3 (N = 15 mosquitoes per virus), day 7 (N = 15), and day 14(N = 15).
Citation: The American Society of Tropical Medicine and Hygiene 97, 2; 10.4269/ajtmh.16-0969
Comparison of mosquito strains.
MEX 1-7 ZIKV.
Using the same data set that we used to compare virus strains, we next compared the effect of mosquito strain, virus titer, and days of extrinsic incubation on infection by MEX 1-7, the only ZIKV strain fed to all three Ae. albopictus populations. We detected a significant interaction between virus titer and mosquito strain (df = 2, χ2 = 6.48, P = 0.039). We therefore proceeded to conduct a simple effects test of the impact of mosquito strain and days of extrinsic incubation with a viremia titer of 7.0 ± 0.2 log10 FFU/mL. This comparison revealed no significant interaction between the two factors and a significant effect only of mosquito strain (df = 2, χ2 = 43.7, P < 0.0001), with Houston and Rio Grande mosquitoes showing significantly higher susceptibility than Salvador mosquitoes. For dissemination from infected mosquitoes, we detected no significant interactions among factors, with a significant impact of both mosquito strain (df = 2, χ2 = 16.7, P < 0.0002) and days of extrinsic incubation (df = 1, χ2 = 59.6, P < 0.0001). As expected, dissemination increased as extrinsic incubation increased. Additionally, dissemination was significantly lower in the Salvador strain of Ae. albopictus than in the two U.S. strains. MEX 1-7 produced detectable virus in the saliva of only the Rio Grande mosquitoes. Overall, only Ae. albopictus from the Rio Grande Valley proved competent for transmission potential of MEX 1-7. However, Ae. albopictus from Houston proved more susceptible to disseminated infections of ZIKV strain MEX 1-7 when compared with mosquitoes from Salvador, Brazil.
PB 81 ZIKV.
We then compared the infectivity of ZIKV strain PB 81 in the two mosquito strains tested: Houston and Salvador. We detected a significant interaction between mosquito strain and days of extrinsic incubation (df = 2, χ2 = 4.8, P = 0.03), so we conducted a simple effects test of the impact of mosquito strain and virus titer for each day of extrinsic incubation, individually. This analysis revealed a significant effect of mosquito strain on days 7 and 14 (P < 0.03 for both comparisons), with Houston mosquitoes showing significantly greater susceptibility than Salvador mosquitoes. Dissemination among infected mosquitoes was shaped by a significant three-way interaction among mosquito strain, virus titer, and days of extrinsic incubation, with the general pattern that dissemination increased with virus titer and days of extrinsic incubation, but dissemination was generally higher in Houston mosquitoes. Virus was detected in the saliva of only the Houston mosquitoes. Overall, Ae. albopictus from Houston were more competent for transmission of ZIKV strain PB 81 compared with the Salvador, Brazil, population.
Saliva titers.
Because Ae. albopictus from the Rio Grande Valley and Houston were fed on different sets of ZIKV strains, it was not possible to compare the two mosquito populations. Instead, we compared the saliva titers of three viruses, DakAR 41525, FSS 13025, and MEX 1-7, in Rio Grande mosquitoes that were fed on viremia titers of 8.5, 7.3, and 7.0 log10 FFU/mL, respectively, and sampled on day 14 of extrinsic incubation. All three viruses reached median saliva titers of 1.0 log10 FFU per collection sample and mean titers of 2.1, 1.5, and 1.6 log10 FFU per collection sample in saliva, respectively, which did not differ significantly (Wilcoxon, df = 2, N = 18, P = 0.78). We then compared the saliva titers in Rio Grande mosquitoes fed on the DakAR 41525 strain of ZIKV at three different viremia titers (6.2, 8.5, 8.8 log10 FFU/mL, respectively) and sampled at day 14 of extrinsic incubation; these titers (3.3, 3.7, and 3.6 log10 FFU per collection sample) also did not differ significantly (Wilcoxon test, df = 2, N = 29, P = 0.74). Titers generally increased between days 7 and 14 of extrinsic incubation, but the large number of negative day 7 samples precluded analysis of titer as a continuous variable.
50% oral infectious doses.
The 50% oral infectious dose (OID50) values were interpolated utilizing the method of Reed and Muench when infection encompassed 50% at the doses used in the study. For a majority of Ae. albopictus populations fed on ZIKV strains the OID50 could not be calculated due to infection rates exceeding 50%, even at the lowest tested viremia titers (e.g., DakAR 41525). The FSS 13025 strain demonstrated an OID50 of 6.7 log10 FFU/mL for assay on day 7 of extrinsic incubation which decreased to 5.9 log10 FFU/mL by day 14 of extrinsic incubation in Rio Grande population of Ae. albopictus. PB 81 exhibited an OID50 of 6.8 log10 FFU/mL for assay on day 3 of extrinsic incubation in Salvador Ae. albopictus (Supplemental Table 1).
Discussion
These results demonstrate the vector competence of Ae. albopictus from various geographic locations in the Americas for multiple strains of ZIKV, an important component of establishing risk and designing control strategies. To determine the role that Ae. albopictus may play in outbreak settings (vectorial capacity), additional factors such as range, longevity and feeding behaviors, especially as compared with the domestic and highly anthropophilic Ae. aegypti must be considered. Aedes albopictus is widespread throughout many temperate regions of the United Ststes where Ae. aegypti is not typically found, such as the upper Midwest and the northeast.11,29,31 Anthropophilic and endophilic feeding behaviors of Ae. aegypti, however, make the species more apt at transmitting human arboviruses when compared with the more opportunistic and exophilic bloodfeeding behavior of Ae. albopictus.29,42 Additionally, the tendency of Ae. aegypti to take multiple bloodmeals per gonotrophic cycle means these mosquitoes are more likely to become infected and more likely to transmit to multiple people once infected compared with Ae. albopictus mosquitoes.42 ZIKV has been detected in both species during outbreaks, Ae. albopictus in Gabon in 20074 and Ae. aegypti in Mexico in 201527 and Brazil in 2016,28 indicating that each species can play a role in ZIKV epidemics.
As demonstrated in previous studies with both Ae. aegypti and Ae. albopictus, we found that vector competence of Ae. albopictus varies with the geographic origin of both vector and virus strain. To more accurately reflect natural infection, we used viremic A129 mice, previously shown to be more infectious for mosquitoes than artificial bloodmeals. For example, when Ae. aegypti from Salvador, Brazil, were orally infected with 6 log10 FFU/mL of ZIKV (FSS 13025) by artificial bloodmeal, by day 14 of extrinsic incubation, 75% of mosquitoes were infected, with 67% of those infections disseminating, but never with virus detected in the saliva. When the same population of mosquitoes was infected via a viremic A129 mouse circulating 6 log10 FFU/mL, on day 14 of extrinsic incubation all tested mosquitoes were infected, with 92% of infections disseminating and 61% of the disseminated infections reaching the saliva.37 This pronounced difference has been observed with other arboviruses such as western equine encephalitis virus, and is at least partly explained by clotting of blood ingested from an animal resulting in greater viral concentration directly adjacent to the mosquito midgut epithelium.43 The flaviviruses, St. Louis encephalitis virus and DENV also exhibit reduced infectivity for Culex quinquefasciatus Say and Ae. aegypti, respectively, when freeze-thawed virus is compared with virus freshly harvested from cell cultures, even when matched for final infectious titer.44 Recently, the reduction of infectivity as a result of freeze-thaws was also demonstrated for ZIKV infection of Ae. aegypti.45 All of these studies support the use of viremic animals for accurately assessing vector competence.
Strikingly, our findings with Ae. albopictus from the Rio Grande Valley of Texas corroborate field findings in Gabon during the 2007 outbreak.4 When Rio Grande Valley mosquitoes were exposed to the African lineage ZIKV strain DakAR 41525 at 6 and 8 log10 FFU/mL, they were uniformly infected and developed disseminated infections, such that by day 14 of extrinsic incubation, 60% of mosquitoes had shed virus into their saliva (Figure 1C). Phylogenetically, the Gabon 2007 strain clusters closely with the 1984 Senegal ZIKV strain (DakAR 41525) based on envelope and NS3 genes.4 Additionally, recent studies45 as well as our own data37 have also demonstrated efficient transmission of African lineage ZIKV strains by American Ae. aegypti. In sum, these data suggest that the African lineage of ZIKV is well adapted for urban transmission by both Ae. aegypti and Ae. albopictus.
A limitation of our understanding of vector competence for ZIKV is the dearth of data for infectious human viremia profiles, with titers ranging from 0.49 to 3.39 log10 infectious particles/mL or 900 to 729, 000 RNA copies/mL,24,46,47 orders of magnitude below the viremia titers to which mosquitoes in our analyses were exposed, and well below our estimated OID50 of both FSS 13025 and PB 81 in Ae. albopictus from the Rio Grande and Salvador, Brazil, respectively. Another limitation is that previous studies found that colonization of Ae. aegypti and Ae. albopictus alters their competence for DENV.48,49 However, our use of low-generation colonies should have minimized these potential artifacts. Also, ideally our mosquito and virus strains would have been matched, and all possible combinations would have been tested. This was not possible because of logistical constraints and the acquisition of virus and mosquito strains after the study was initiated. For example, on availability, we replaced DakAR 41525 and FSS13025 with recent epidemic strains PRVABC 59 and PB 81 due to their increased public health relevance.
Our data strongly demonstrated that, while all tested populations of Ae. albopictus proved susceptible to midgut and disseminated infections at varying efficiencies, Houston and Salvador mosquitoes were relatively incapable of transmitting ZIKV. In order for an arbovirus to be transmitted by a competent vector, it must 1) be ingested via a bloodmeal from an infected host; 2) infect epithelial cells of the mosquito midgut; 3) disseminate from the midgut into the hemocoel and infect further tissues; 4) infect the salivary glands; and 5) be shed into acinar cavities for innoculation into a new host upon subsequent feedings.50,51 The relative inability of Houston and Salvador Ae. albopictus to transmit ZIKV strains, despite the presence of disseminated virus in hemocoel, suggests the possibility of a salivary gland infection or salivary egress barrier.52 In the case of the former, ZIKV may be failing to infect secondary amplification tissues such as the fat bodies, hemocytes, nerve, or muscle tissues following midgut escape, preventing sufficient replication to efficiently infect the salivary glands. Anatomic analyses utilizing ZIKV reporter systems53 in transmission-competent versus transmission-incompetent Ae. albopictus populations are needed to address these hypotheses.
The explosive spread of ZIKV throughout tropical and subtropical regions of the Americas has raised concerns that mosquitoes other than Ae. aegypti may be transmitting ZIKV.54 In our study, Ae. albopictus from Salvador, Brazil, orally exposed to two American strains of ZIKV (MEX 1-7 and PB 81) at high titers (6 or 7 log10 FFU/mL) shed no virus into saliva, even by day 14 of extrinsic incubation. Although we tested only one Brazilian mosquito population, the lack of transmission competence in the population tested with high viremia titers, coupled with a lack of field data from Mexico and Brazil reporting ZIKV positive Ae. albopictus pools, calls a significant role for this species into question, especially when taken together with previous reports on the competency of Ae. albopictus from Jurjuba, Rio de Janeiro. Of the seven populations of mosquitoes (all of which were populations from the Americas, five Ae. aegypti populations and two Ae. albopictus populations) tested in that study, the Jurjuba Ae. albopictus proved to be the least susceptible, although the transmission potential of this population was not tested.45
A critical component of viral pathogenesis is the infectious dose, or the saliva titer for arboviruses.55 We found a range of saliva titers, with a maximum titer of 3.72 log10 FFU/collection of DakAR 41525 in a Rio Grande Valley mosquito sample. Analyses in other arboviruses have reported a large variation in saliva titers dependent on the virus and vector in question. For example, when the alphavirus Venezuelan equine encephalitis virus (VEEV) was used to infect Ae. albopictus and Aedes taeniorhynchus (Wiedemann) and saliva was collected in vitro, the resultant titers ranged from 0.2 to 1.1 log10 and 0.2 to 3.2 log10 plaque forming units (PFU) per collection, respectively.56 Further analyses comparing in vitro saliva collection of VEEV to in vivo inoculation revealed that artificial salivation (mean 74 PFU) overestimates the in vivo inoculum by nearly 10-fold (mean 11 PFU).57 Similar analyses of in vivo saliva deposition of WNV from four different species of mosquitoes (Culex tarsalis Coquillet, Culex pipiens Linnaeus, Aedes japonicus [Theobald], and Aedes triseriatus [Say]) into a murine host yielded a range of saliva titers (3.4–6.1 log10 PFU),58 whereas a separate analysis of WNV utilizing saliva collected from Cx. tarsalis in vitro yielded a titer of 1.41 log10 PFU.59 Further complicating in vitro collection of mosquito saliva is the finding that mosquitoes demonstrate host-seeking behavior until imbibing 2.5–3.5 μL of blood.60 In vitro saliva collections (our own as well as previous reports37,57) comprise minimal volumes (≤ potential for mosquitoes to imbibe a significant percentage of expectorated saliva). To account for potential volume loss, we report our results as FFU per collection, as opposed to FFU/mL. In summary, disparate methodologies of saliva collection from different mosquito populations infected with different viruses limit what conclusions can be drawn from salivary viral titers and underscore the necessity for standardization of methodologies.
Like other vector competence studies,34 our data suggest significant variation as a function of mosquito origin and viral strain. Laboratory competency studies have produced disparate findings, with Ae. albopictus populations being shown to be both poor33 and relatively competent vectors.35,36 These disparities underscore the need for further studies in both the laboratory and the field to determine the potential role that Ae. albopictus may play in future ZIKV outbreaks, especially in temperate climates where Ae. aegypti cannot survive cold winters. Variables among this and other vector competence studies reported in the literature, such as colonization, mosquito microbiome composition, and genetics differences, should be further explored to determine their impact on ZIKV transmission.
REFERENCES
- 1.↑
Dick GWA, Kitchen SF, Haddow AJ, 1952. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg 46: 509–520.
- 2.↑
Fauci AS, Morens DM, 2016. Zika virus in the Americas: yet another arbovirus threat. N Engl J Med 374: 601–604.
- 3.↑
Weaver SC, Costa F, Garcia-Blanco MA, Ko AI, Ribeiro GS, Saade G, Shi P-Y, Vasilakis N, 2016. Zika virus: history, emergence, biology, and prospects for control. Antiviral Res 130: 69–80.
- 4.↑
Grard G, Caron M, Mombo IM, Nkoghe D, Mboui Ondo S, Jiolle D, Fontenille D, Paupy C, Leroy EM, 2014. Zika virus in Gabon (Central Africa)–2007: a new threat from Aedes albopictus?PLoS Negl Trop Dis 8: e2681.
- 5.↑
Duffy MR, Chen T-H, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB, 2009. Zika virus outbreak on yap island, federated states of Micronesia. N Engl J Med 360: 2536–2543.
- 6.↑
Musso D, Cao-Lormeau VM, Gubler DJ, 2015. Zika virus: following the path of dengue and chikungunya? Lancet 386: 243–244.
- 7.
Cao-Lormeau V-M, Roche C, Teissier A, Robin E, Berry A-L, Mallet H-P, Sall AA, Musso D, 2013. Zika virus, French Polynesia, South Pacific, 2013. Emerg Infect Dis 20: 1084–1086.
- 8.↑
Jouannic J-M, Friszer S, Leparc-Goffart I, Garel C, Eyrolle-Guignot D, 2016. Zika virus infection in French Polynesia. Lancet 387: 1051–1052.
- 9.↑
Campos GS, Bandeira AC, Sardi SI, 2015. Zika virus outbreak, Bahia, Brazil. Emerg Infect Dis 21: 1885–1886.
- 10.↑
Zanluca C, de Melo VCA, Mosimann ALP, dos Santos GIV, dos Santos CND, Luz K, 2015. First report of autochthonous transmission of Zika virus in Brazil. Mem Inst Oswaldo Cruz 110: 569–572.
- 11.↑
Centers for Disease Control and Prevention, 2016. Countries & Territories with Active Local Zika Virus Transmission. Available at: http://www.cdc.gov/zika/geo/active-countries.html. Accessed November 14, 2016.
- 12.↑
Texas Department of State Health Services, 2016. Texas Announces Local Zika Virus Case in Rio Grande Valley. Available at: http://dshs.texas.gov/news/releases/2016/20161128.aspx. Accessed December 2, 2016.
- 14.
Mlakar J, Korva M, Tul N, Popović M, Poljšak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodušek V, Vizjak A, Pižem J, Petrovec M, Avšič Županc T, 2016. Zika virus associated with microcephaly. N Engl J Med 374: 951–958.
- 16.↑
Brasil P, Pereira JP Jr, Raja Gabaglia C, Damasceno L, Wakimoto M, Ribeiro Nogueira RM, Carvalho de Sequeira P, Machado Siqueira A, Abreu de Carvalho LM, Cotrim da Cunha D, Calvet GA, Neves ES, Moreira ME, Rodrigues Baião AE, Nassar de Carvalho PR, Janzen C, Valderramos SG, Cherry JD, Bispo de Filippis AM, Nielsen-Saines K, 2016. Zika virus infection in pregnant women in Rio de Janeiro: preliminary report. N Engl J Med 375: 2321–2334.
- 17.↑
Carteaux G, Maquart M, Bedet A, Contou D, Brugières P, Fourati S, Cleret de Langavant L, de Broucker T, Brun-Buisson C, Leparc-Goffart I, Mekontso Dessap A, 2016. Zika virus associated with meningoencephalitis. N Engl J Med 374: 1595–1596.
- 18.
De Paula Freitas B, de Oliveira Dias JR, Prazeres J, Sacramento GA, Ko AI, Maia M, Belfort R, 2016. Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil. JAMA Ophthalmol 134: 529–535.
- 19.↑
Mécharles S, Herrmann C, Poullain P, Tran T-H, Deschamps N, Mathon G, Landais A, Breurec S, Lannuzel A, 2016. Acute myelitis due to Zika virus infection. Lancet 387: 148.
- 20.↑
Cao-Lormeau V-M, Blake A, Mons S, Lastère S, Roche C, Vanhomwegen J, Dub T, Baudouin L, Teissier A, Larre P, Vial A-L, Decam C, Choumet V, Halstead SK, Willison HJ, Musset L, Manuguerra J-C, Despres P, Fournier E, Mallet H-P, Musso D, Fontanet A, Neil J, Ghawché F, 2016. Guillain-Barré syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 387: 1531–1539.
- 21.↑
Paploski IAD, Prates APPB, Cardoso CW, Kikuti M, Silva MMO, Waller LA, Reis MG, Kitron U, Ribeiro GS, 2016. Time lags between exanthematous illness attributed to Zika virus, Guillain-Barré syndrome, and microcephaly, Salvador, Brazil. Emerg Infect Dis 22: 1438–1444.
- 22.↑
World Health Organization, 2016. WHO Statement on the First Meeting of the International Health Regulations (2005) (IHR 2005) Emergency Committee on Zika Virus and Observed Increase in Neurological Disorders and Neonatal Malformations. Available at: http://www.who.int/mediacentre/news/statements/2016/1st-emergency-committee-zika/en/. Accessed December 2, 2016.
- 23.↑
Berthet N, Nakouné E, Kamgang B, Selekon B, Descorps-Declère S, Gessain A, Manuguerra J-C, Kazanji M, 2014. Molecular characterization of three Zika flaviviruses obtained from sylvatic mosquitoes in the Central African Republic. Vector Borne Zoonotic Dis 14: 862–865.
- 24.↑
Vasilakis N, Weaver SC, 2017. Flavivirus transmission focusing on Zika. Curr Opin Virol 22: 30–35.
- 25.↑
Marchette NJ, Garcia R, Rudnick A, 1969. Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. Am J Trop Med Hyg 18: 411–415.
- 27.↑
Guerbois M, Fernandez-Salas I, Azar SR, Danis-Lozano R, Alpuche-Aranda CM, Leal G, Garcia-Malo IR, Diaz-Gonzalez EE, Casas-Martinez M, Rossi SL, Del Río-Galván SL, Sanchez-Casas RM, Roundy CM, Wood TG, Widen SG, Vasilakis N, Weaver SC, 2016. Outbreak of Zika virus infection, Chiapas State, Mexico, 2015, and first confirmed transmission by Aedes aegypti mosquitoes in the Americas. J Infect Dis 214: 1349–1356.
- 28.↑
Ferreira-de-Brito A, Ribeiro IP, de Miranda RM, Fernandes RS, Campos SS, da Silva KAB, de Castro MG, Bonaldo MC, Brasil P, Lourenço-de-Oliveira R, 2016. First detection of natural infection of Aedes aegypti with Zika virus in Brazil and throughout South America. Mem Inst Oswaldo Cruz 111: 655–658.
- 29.↑
Paupy C, Delatte H, Bagny L, Corbel V, Fontenille D, 2009. Aedes albopictus, an arbovirus vector: from the darkness to the light. Microbes Infect 11: 1177–1185.
- 30.↑
Tsetsarkin KA, Chen R, Yun R, Rossi SL, Plante KS, Guerbois M, Forrester N, Perng GC, Sreekumar E, Leal G, Huang J, Mukhopadhyay S, Weaver SC, 2014. Multi-peaked adaptive landscape for chikungunya virus evolution predicts continued fitness optimization in Aedes albopictus mosquitoes. Nat Commun 5: 4084.
- 31.↑
Bonizzoni M, Gasperi G, Chen X, James AA, 2013. The invasive mosquito species Aedes albopictus: current knowledge and future perspectives. Trends Parasitol 29: 460–468.
- 32.↑
Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S, 2007. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 3: e201.
- 33.↑
Centers for Disease Control and Prevention, 2016. Estimated Range of Aedes albopictus and Aedes aegypti in the United States. Available at: http://www.cdc.gov/zika/vector/range.html. Accessed November 27, 2016.
- 34.↑
Chouin-Carneiro T, Vega-Rua A, Vazeille M, Yebakima A, Girod R, Goindin D, Dupont-Rouzeyrol M, Lourenço-de-Oliveira R, Failloux A-B, 2016. Differential susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika virus. PLoS Negl Trop Dis 10: e0004543.
- 35.↑
Di Luca M, Severini F, Toma L, Boccolini D, Romi R, Remoli ME, Sabbatucci M, Rizzo C, Venturi G, Rezza G, Fortuna C, 2016. Experimental studies of susceptibility of Italian Aedes albopictus to Zika virus. Euro Surveill 21: 30223.
- 36.↑
Wong P-SJ, Li MI, Chong C-S, Ng L-C, Tan C-H, 2013. Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore. PLoS Negl Trop Dis 7: e2348.
- 37.↑
Roundy CM, Azar SR, Rossi SL, Huang JH, Leal G, Yun R, Fernadez-Salas I, Vitek CJ, Paploski IAD, Kitron U, Ribeiro GS, Hanley KA, Weaver SC, Vasilakis N, 2017. Variation in Aedes aegypti competence for Zika virus transmission as a function of viral strain blood meal type and mosquito geographic origin. Emerg Infect Dis 23: 625–632.
- 38.↑
Centers for Disease Control and Prevention (CDC), 2007. Dengue hemorrhagic fever: U.S.-Mexico border, 2005. MMWR Morb Mortal Wkly Rep 56: 785–789.
- 39.↑
Rossi SL, Tesh RB, Azar SR, Muruato AE, Hanley KA, Auguste AJ, Langsjoen RM, Paessler S, Vasilakis N, Weaver SC, 2016. Characterization of a novel murine model to study Zika virus. Am J Trop Med Hyg 94: 1362–1369.
- 40.↑
Williams M, Mayer SV, Johnson WL, Chen R, Volkova E, Vilcarromero S, Widen SG, Wood TG, Suarez-Ognio L, Long KC, Hanley KA, Morrison AC, Vasilakis N, Halsey ES, 2014. Lineage II of southeast Asian/American DENV-2 is associated with a severe dengue outbreak in the Peruvian Amazon. Am J Trop Med Hyg 91: 611–620.
- 41.↑
Reed LJ, Muench H, 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27: 493–497.
- 42.↑
Yang C-F, Hou J-N, Chen T-H, Chen W-J, 2014. Discriminable roles of Aedes aegypti and Aedes albopictus in establishment of dengue outbreaks in Taiwan. Acta Trop 130: 17–23.
- 43.↑
Weaver SC, Lorenz LH, Scott TW, 1993. Distribution of western equine encephalomyelitis virus in the alimentary tract of Culex tarsalis (Diptera: Culicidae) following natural and artificial blood meals. J Med Entomol 30: 391–397.
- 44.↑
Richards SL, Pesko K, Alto BW, Mores CN, 2007. Reduced infection in mosquitoes exposed to blood meals containing previously frozen flaviviruses. Virus Res 129: 224–227.
- 45.↑
Weger-Lucarelli J, Rückert C, Chotiwan N, Nguyen C, Garcia Luna SM, Fauver JR, Foy BD, Perera R, Black WC, Kading RC, Ebel GD, 2016. Vector competence of American mosquitoes for three strains of Zika virus. PLoS Negl Trop Dis 10: e0005101.
- 46.↑
Fourcade C, Mansuy J-M, Dutertre M, Delpech M, Marchou B, Delobel P, Izopet J, Martin-Blondel G, 2016. Viral load kinetics of Zika virus in plasma, urine and saliva in a couple returning from Martinique, French West Indies. J Clin Virol 82: 1–4.
- 47.↑
Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR, 2008. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 14: 1232–1239.
- 48.↑
Armstrong PM, Rico-Hesse R, 2001. Differential susceptibility of Aedes aegypti to infection by the American and southeast Asian genotypes of dengue type 2 virus. Vector Borne Zoonotic Dis 1: 159–168.
- 49.↑
Lambrechts L, Scott TW, Gubler DJ, 2010. Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission. PLoS Negl Trop Dis 4: e646.
- 50.↑
Forrester N, Coffey L, Weaver S, 2014. Arboviral bottlenecks and challenges to maintaining diversity and fitness during mosquito transmission. Viruses 6: 3991–4004.
- 51.↑
Lounibos LP, Kramer LD, 2016. Invasiveness of Aedes aegypti and Aedes albopictus and vectorial capacity for chikungunya virus. J Infect Dis 214 (Suppl 5): S453–S458.
- 52.↑
Franz A, Kantor A, Passarelli A, Clem R, 2015. Tissue barriers to arbovirus infection in mosquitoes. Viruses 7: 3741–3767.
- 53.↑
Shan C, Xie X, Muruato AE, Rossi SL, Roundy CM, Azar SR, Yang Y, Tesh RB, Bourne N, Barrett AD, Vasilakis N, Weaver SC, Shi P-Y, 2016. An infectious cDNA clone of Zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors. Cell Host Microbe 19: 891–900.
- 54.↑
Gardner LM, Chen N, Sarkar S, 2016. Global risk of Zika virus depends critically on vector status of Aedes albopictus. Lancet Infect Dis 16: 522–523.
- 55.↑
Althouse BM, Hanley KA, 2015. The tortoise or the hare? Impacts of within-host dynamics on transmission success of arthropod-borne viruses. Phil Trans R Soc B 370: 20140299.
- 56.↑
Smith DR, Carrara A-S, Aguilar PV, Weaver SC, 2005. Evaluation of methods to assess transmission potential of Venezuelan equine encephalitis virus by mosquitoes and estimation of mosquito saliva titers. Am J Trop Med Hyg 73: 33–39.
- 57.↑
Smith DR, Aguilar PV, Coffey LL, Gromowski GD, Wang E, Weaver SC, 2006. Venezuelan equine encephalitis virus transmission and effect on pathogenesis. Emerg Infect Dis 12: 1190–1196.
- 58.↑
Styer LM, Kent KA, Albright RG, Bennett CJ, Kramer LD, Bernard KA, 2007. Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts. PLoS Pathog 3: e132.
- 59.↑
Hanley KA, Goddard LB, Gilmore LE, Scott TW, Speicher J, Murphy BR, Pletnev AG, 2005. Infectivity of West Nile/dengue chimeric viruses for West Nile and dengue mosquito vectors. Vector Borne Zoonotic Dis 5: 1–10.
- 60.↑
Klowden MJ, Lea AO, 1978. Blood meal size as a factor affecting continued host-seeking by Aedes aegypti (L.). Am J Trop Med Hyg 27: 827–831.