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Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico Barry W. Alto1*, Chelsea T. Smartt1, Dongyoung Shin1, David Bettinardi1, Jolene Malicoate1, Sheri L. Anderson1, and Stephanie L. Richards2 University of Florida, IFAS, Department of Entomology and Nematology, Florida Medical Entomology Laboratory, Vero Beach, FL 32962, U.S.A., [email protected] 2 East Carolina University, College of Health and Human Performance, Department of Health Education and Promotion, Environmental Health Sciences Program, Greenville, NC 27858, U.S.A.
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Received 10 July 2014; Accepted 22 August 2014 ABSTRACT: Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue-1 during 2009, 2010, and 2013 in Florida and dengue-1 and -2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler-imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV-1 and -2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV-1 (6-14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV-2 than Ae. albopictus (Ae. aegypti, ~28%; Ae. albopictus, ~9%). The dissemination rate with dengue-2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV-2 isolate from Puerto Rico. These results are discussed in light of Florida’s vulnerability to DENV transmission. Journal of Vector Ecology 39 (2): 406-413. 2014. Keyword Index: Arbovirus infection, Florida, Flavivirus, Aedes. INTRODUCTION Traveler-associated dengue infection in the continental United States is a potential threat to public health (CDC 2002, CDC 2005, Mohammed et al. 2009), yet little is known about vector competence of U.S. mosquitoes for imported dengue viruses (DENV). Cases of locally acquired dengue in the continental U.S. have been rare (CDC 2010). However, outbreaks of dengue-1 during 2009, 2010, and 2013 in Florida and dengue-1 and -2 in Texas suggest vulnerability to the establishment of local transmission. The majority of dengue cases in the U.S. are imported, e.g., a U.S. resident travels to a DENV-endemic region, becomes infected, and then returns to the U.S. or a DENV-infected resident of an endemic region visits the U.S. Between 2010 and 2012 Florida ranked number one in the number of imported dengue cases (N=303), followed by New York (N=231) and California (N=97) (CDC 2014). In 2013, there were 519 imported dengue cases in the U.S. with Florida (N=41) ranked number three, behind New York (N=139) and California (N=68) (CDC 2014). However, unlike these other top-ranked states for imported cases, Florida is associated with year-round activity of both major DENV vectors. Florida may be a vulnerable region of the U.S. for local DENV transmission due to the high incidence of imported cases and the presence of DENV vectors Aedes (Stegomyia) aegypti and Ae. (Stegomyia) albopictus. Dengue is the most important vector transmitted viral disease in terms of public health and economic costs with an estimated 3.6 billion people residing in areas of risk (Gubler 2012, Bhatt et al. 2013). Dengue is caused by infection with
one of the four serotypes of DENV transmitted by Aedes species. Aedes aegypti is the primary vector of epidemic/ endemic DENV to humans, although other species may have secondary roles in viral transmission and maintenance, such as Ae. albopictus and Ae. mediovittatus (Gubler et al. 1985, Lambrechts et al. 2012). Since the 1981 dengue hemorrhagic fever (DHF) outbreak in Cuba, reports of DHF have increased in the Americas, in part attributable to reduced controlled efforts of Ae. aegypti in the 1970s, urbanization, socioeconomic factors, and human travel (Brandling-Bennett and Pinheiro 1996, Rey et al. 2010, San Martin et al. 2010). In the Americas, the economic burden resulting from DENV associated illnesses is $2.1 billion per year (Shepard et al. 2011). Dengue is endemic in the U.S. Commonwealth territory of Puerto Rico where all four DENV serotypes co-circulate. Outbreaks of dengue have occurred for nearly a century in Puerto Rico (King 1917). However, the frequency of outbreaks has increased over the last ~30 years. Outbreaks involving all four serotypes occurred in 1998 and large outbreaks occurred in 2007 and 2010, the latter being the largest to date with greater than 10,000 diagnosed cases (Rigau-Perez et al. 2002, Tomashek et al. 2009, CDC 2011). Frequent outbreaks of dengue in Puerto Rico pose a risk for local transmission in select states attributable to 1) frequent traveler-associated imported cases of dengue, 2) large populations of susceptible humans especially in urban areas, 3) environmental conditions conducive for mosquito development and virus replication, 4) abundant epidemic vectors Ae. aegypti and Ae. albopictus associated with human populations, and 5) recent
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DENV outbreaks such as Florida (CDC 2010) and Texas (Murray et al. 2013). As the incidence of dengue increases around the world, so may travel-associated dengue infection in the U.S. Between 1996 and 2005 there were 1,196 travel-associated dengue cases in the U.S. with the Caribbean (23%), Mexico and Central America (20%), and Southeast Asia (17%) being the most commonly visited regions (Mohammed et al. 2009). Although definitive evidence is lacking, outbreaks of dengue during 2009, 2010, and 2013 in Florida were probably attributable to travel-associated infection and suggests vulnerability to transmission in areas where dengue vectors occur (CDC 2010). Furthermore, the Florida dengue outbreaks in 2009 and 2010 were caused by genetically related DENV-1 (Shin et al. 2013), an indication of endemic transmission (Graham et al. 2011, Muñoz-Jordan 2013). The current population decline on Puerto Rico is associated with increased migration to mainland U.S. (Cohn et al. 2014). The population of Puerto Ricans living in Florida is second in abundance only to New York (U.S. Census Bureau, 2010). Further, migration to the U.S. is facilitated by Puerto Rico being a commonwealth and so individuals are citizens. High numbers of dengue cases in Puerto Rico (Sharp et al. 2014), together with ease of migration, may pose a potential risk for importation of dengue to the continental U.S. Here, we assess the relative susceptibility of Florida Ae. aegypti and Ae. albopictus for infection and dissemination of DENV originating from Puerto Rico. MATERIALS AND METHODS Mosquitoes and viruses Larval Ae. aegypti and Ae. albopictus were collected from water-filled containers in Key West (Old Town historic district) and Vero Beach, FL, respectively. We used a single population for Ae. aegypti and Ae. albopictus since there is little evidence for genetic differentiation due to the absence of barriers to gene flow in Florida (Damal et al. 2013). We chose to use Ae. aegypti from Key West, FL, because of its involvement in dengue outbreaks in 2009 and 2010 attributable to DENV1 originating from Nicaragua (Shin et al. 2013). Mosquitoes were reared to adulthood on a diet of brewer’s yeast and lactalbumin at 26-28° C. Adults were provided with 20% sucrose (food grade quality) solution and allowed to blood feed on chickens once per week in order to propagate egg production. Aedes aegypti (generation=F2) and Ae. albopictus (generation=F4) were used for DENV infection studies. DENV-1 (GenBank: EU482591) and DENV-2 (GenBank: EU482553) were used for the infection studies. We focused on dengue serotypes 1 and 2 because of their recent involvement in outbreaks of locally transmitted DENV in the U.S. (2001-02, Hawaii; 2003-05, Texas; 2009, 2010, and 2013, Florida) (CDC 2010, Effler et al. 2005, Murray et al. 2013, Florida Health 2014). Low-passage isolates of DENV were provided by the Centers for Disease Control and Prevention. Dengue-1 virus was isolated in 2006 from a human and passaged once in C6/36 cells. Dengue-2 virus was isolated in 2007 from a human and passaged twice in
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C6/36 cells. Dengue viruses were passaged once in African green monkey (Vero) cells to produce stocks used to initiate the infection studies. Infected blood meals were prepared by inoculating monolayers of Vero cells with 400 μl of virus stocks (multiplicity of infection of 0.01 viruses per cell) using methods described in Alto and Bettinardi (2013). After 1 h of incubation at 35° C and under 5% carbon dioxide, 25 ml media (199 media, 10% fetal bovine serum, 2% penicillinstreptomycin, and 0.2% antimycotic) were added to each monolayer. Depending on virus growth rate, monolayers were inoculated with DENV on different days so that virus isolates would have similar population sizes on the day infectious blood meals were provided to mosquitoes (Shin et al. 2013). Blood meals were prepared by combining defibrinated bovine blood (Hemostat, Dixon, CA) with media and virus from infected monolayers of Vero cells (1:1 ratio). Mosquito infection Adult female and male mosquitoes were housed together in 0.3 m3 cages at 25° C and 14:10 light: dark cycle and provided with 20% sucrose solution. During feeding trials, mosquitoes were transferred to 1 liter cardboard cages including a 30 ml cup with a paper towel as an oviposition substrate. Females were deprived of sucrose but not water 48 h before blood-feeding trials. Mosquitoes were allowed to feed on DENV infectious blood for 60 min from an artificial membrane feeding system (Hemotek, Discovery workshops, Accrington, UK). Feeding trials were performed in incubators at 30° C, a temperature previously demonstrated to achieve high feeding rates (Alto and Bettinardi 2013). Fully engorged females were separated from unfed and partially fed mosquitoes using microscopy and held at 28° C along with access to 20% sucrose solution. Immediately following the feeding trial, a sample of freshly blood-fed mosquitoes (six to seven mosquitoes per DENV serotype) were stored at -80˚ C and then later plaque assays determined virus titers of the ingested blood. The remaining fully engorged females were returned in groups to 1.0 liter cardboard cages and held for the designated incubation period. After seven and 14 days of incubation, surviving mosquitoes (N=709) were stored at -80˚ C until later dissection and assayed for the presence of virus RNA. Mosquito processing Blood that is ingested by mosquitoes is deposited in the posterior midgut. Typically, infectious blood meals first result in infection of midgut cells followed by dissemination of virus from the midgut to secondary tissues such as the salivary glands. Separate assays of mosquito bodies and legs are a convenient and reliable approach to determine virus infection and dissemination, the latter being necessary for transmission (Turell et al. 1984). Individual mosquitoes were dissected to remove legs from the bodies, and triturated separately in 2.0 ml microcentrifuge tubes with two 4.5-mm stainless steel beads and 0.9 ml BA-1 (10x medium 199, 1% bovine serum albumin, 0.05 M TRIS, pH 7.5, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 1 μg/ml of mycostatin) at 25 Hz for 3 min (TissueLyser, Qiagen, Inc., Valencia, CA)
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and subsequently centrifuged at 3,148 x g for 4 min at 4° C. Nucleic acid was extracted from 250-μl samples of bodies and legs and eluted in 50 μl buffer using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Indianapolis, IN). The presence of viral RNA in samples was determined using the Superscript III One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA) with a Light Cycler 480 system (Roche, Mannheim, Germany) (Callahan et al. 2001). The probe and primer sequences for DENV serotype specific assays are described in another paper (Callahan et al. 2001). The virus titer present in mosquito bodies and legs were expressed as plaque forming unit equivalents (pfue)/ml that relates cDNA synthesis by PCR to plaque forming units (Alto et al. 2008, Alto and Bettinardi 2013). Midgut infection refers to infected mosquitoes used for infection rate and leg infection refers to disseminated infection used for dissemination rate. The infection rate is the percent of all blood fed mosquitoes with DENV RNA present in their bodies. The dissemination rate is the percent of mosquitoes with infected bodies that have dengue RNA present in their legs. Statistical analysis Treatment effects of DENV serotype, species (Ae. aegypti, Ae. albopictus), incubation period (seven and 14 days), and interactions were analyzed using maximum likelihood (ML) categorical analyses of contingency tables based on the number of individual samples categorized for the presence or absence of DENV. Separate ML ANOVA tests were used for body and leg samples to identify viral barriers (e.g., midgut infection and escape barriers) that may vary between treatments. Thus, each mosquito had a binomial infection status, as opposed to percentage for each replicate. Analysis of variance (ANOVA) was used to evaluate differences in DENV titers of individual mosquito bodies between DENV serotype, mosquito species, incubation periods, and interactions. If significant differences were detected, we used pair-wise contrasts of means adjusting p-values for multiple comparisons using the Bonferroni method (Sokal and Rohlf 1995). RESULTS
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Virus titer Plaque assays determined virus titers of freshly blood-fed mosquitoes were 6.8 ± 0.5 and 7.1 ± 1.2 log10 plaque-forming unit equivalents (pfue) DENV/ml for DENV-1 and DENV-2, respectively (t-test with t = 0.53, df = 11, p = 0.61). Infection rates Between 10-14% of Ae. aegypti and Ae. albopictus were susceptible to infection with DENV-1 virus seven days after blood feeding and 6-10% were susceptible to infection 14 days after blood-feeding (Figure 1A). Aedes aegypti were more susceptible to infection with DENV-2 virus than Ae. albopictus (Ae. aegypti, ~28%; Ae. albopictus, ~9%) for both seven days and 14 days incubation periods (Figure 1B). The ML ANOVA on susceptibility to DENV infection of mosquitoes showed marginally significant effects of dengue serotype (c2=3.79, df=1, p=0.051, Table 1) and species (c2=3.83, df=1, p=0.050, Table 1). There was a significant interaction of mosquito species by serotype (c2=13.2, df = 1, p=0.0003, Table 1). All other treatment effects on susceptibility to DENV infection were not significant (Table 1). To investigate the significant interaction further, we performed all possible pairwise comparisons of each species (Ae. aegypti vs Ae. albopictus) and DENV serotype (1 or 2), resulting in six comparisons. Aedes aegypti were significantly more susceptible to infection than Ae. albopictus for DENV-2 (c2=20.14, df=1, p 14 days), hence, further investigation is needed to investigate temporal patterns in virus dissemination (Chan and Johansson 2012). Another study demonstrated that although dissemination of DENV-2 to the salivary glands was observable for both Ae. aegypti and Ae. albopictus by 11 days post-blood feeding, there was delayed dissemination of virus to the salivary glands observed in Ae. albopictus compared to Ae. aegypti (Whitehead et al. 1971). Previous studies comparing the susceptibility of Aedes mosquitoes to DENV have demonstrated variable results. Several studies have demonstrated genetic variability in oral susceptibility, or variation attributable to geographic origin, to DENV for both Ae. aegypti and Ae. albopictus (Rosen et al. 1985, Borimosa et al. 1987, Tardieuz et al. 1991, VazeilleFalcoz et al. 1999). In some instances, rates of infection were higher in Ae. albopictus than Ae. aegypti for multiple serotypes of DENV, whereas other studies have shown the opposite result (Chen et al. 1993, Whitehead 1971, Rosen et al. 1985, Vazeille et al. 2001, 2003). A meta-analysis study demonstrated that Ae. albopictus is more susceptible to DENV midgut infection, but less capable of viral dissemination than Ae. aegypti (Lambrechts et al. 2012). Although increasing generations during laboratory colonization may increase the infection rate in mosquitoes (e.g., Ae. albopictus) (Lambrechts et al. 2012, Vazeille et al. 2003), this mechanism is not wholly responsible for discrepancies in relative competence between Ae. albopictus and Ae. aegypti as variable results have been observed for both high and low generation laboratory passaged mosquito strains (references above). Our assessment of susceptibility to DENV infection and dissemination enabled us to determine the relative vulnerability of Florida to potential transmission of DENV from Puerto Rico. Here we show that both Aedes mosquitoes were susceptible to midgut infection of DENV-1 and -2. Further, Ae. albopictus is not likely to be an important transmitter of the DENV-2 isolate from Puerto Rico given low susceptibility to infection and lack of evidence for viral dissemination. Recent outbreaks of dengue-1 virus in Florida (Monroe Co. 2009, 2010; Martin Co. 2013, St. Lucie Co. 2013) may place residents at greater risk for more severe secondary infection with other dengue serotypes (Halstead 1982). Specifically, human hosts in south and central Florida previously infected with DENV-1 are at most risk for secondary infection with DENV-2 and other serotypes of DENV. However, caution is advised in interpreting these results since they do not take into account variable temperature regimes in nature known to influence DENV infection in Aedes (Lambrechts et al. 2011) as well as other environmental factors that may influence the risk of disease agent transmission. Acknowledgments Dengue viruses from Puerto Rico were provided by Jorge Muñoz-Jordán and Candimar Colón at the Centers for Disease Control and Prevention. This study is supported by Department of Agriculture and Consumer Services (DACS) 2010 grant 00090369.
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Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico Barry W. Alto1*, Chelsea T. Smartt1, Dongyoung Shin1, David Bettinardi1, Jolene Malicoate1, Sheri L. Anderson1, and Stephanie L. Richards2 University of Florida, IFAS, Department of Entomology and Nematology, Florida Medical Entomology Laboratory, Vero Beach, FL 32962, U.S.A., [email protected] 2 East Carolina University, College of Health and Human Performance, Department of Health Education and Promotion, Environmental Health Sciences Program, Greenville, NC 27858, U.S.A.
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Received 10 July 2014; Accepted 22 August 2014 ABSTRACT: Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue-1 during 2009, 2010, and 2013 in Florida and dengue-1 and -2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler-imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV-1 and -2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV-1 (6-14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV-2 than Ae. albopictus (Ae. aegypti, ~28%; Ae. albopictus, ~9%). The dissemination rate with dengue-2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV-2 isolate from Puerto Rico. These results are discussed in light of Florida’s vulnerability to DENV transmission. Journal of Vector Ecology 39 (2): 406-413. 2014. Keyword Index: Arbovirus infection, Florida, Flavivirus, Aedes. INTRODUCTION Traveler-associated dengue infection in the continental United States is a potential threat to public health (CDC 2002, CDC 2005, Mohammed et al. 2009), yet little is known about vector competence of U.S. mosquitoes for imported dengue viruses (DENV). Cases of locally acquired dengue in the continental U.S. have been rare (CDC 2010). However, outbreaks of dengue-1 during 2009, 2010, and 2013 in Florida and dengue-1 and -2 in Texas suggest vulnerability to the establishment of local transmission. The majority of dengue cases in the U.S. are imported, e.g., a U.S. resident travels to a DENV-endemic region, becomes infected, and then returns to the U.S. or a DENV-infected resident of an endemic region visits the U.S. Between 2010 and 2012 Florida ranked number one in the number of imported dengue cases (N=303), followed by New York (N=231) and California (N=97) (CDC 2014). In 2013, there were 519 imported dengue cases in the U.S. with Florida (N=41) ranked number three, behind New York (N=139) and California (N=68) (CDC 2014). However, unlike these other top-ranked states for imported cases, Florida is associated with year-round activity of both major DENV vectors. Florida may be a vulnerable region of the U.S. for local DENV transmission due to the high incidence of imported cases and the presence of DENV vectors Aedes (Stegomyia) aegypti and Ae. (Stegomyia) albopictus. Dengue is the most important vector transmitted viral disease in terms of public health and economic costs with an estimated 3.6 billion people residing in areas of risk (Gubler 2012, Bhatt et al. 2013). Dengue is caused by infection with
one of the four serotypes of DENV transmitted by Aedes species. Aedes aegypti is the primary vector of epidemic/ endemic DENV to humans, although other species may have secondary roles in viral transmission and maintenance, such as Ae. albopictus and Ae. mediovittatus (Gubler et al. 1985, Lambrechts et al. 2012). Since the 1981 dengue hemorrhagic fever (DHF) outbreak in Cuba, reports of DHF have increased in the Americas, in part attributable to reduced controlled efforts of Ae. aegypti in the 1970s, urbanization, socioeconomic factors, and human travel (Brandling-Bennett and Pinheiro 1996, Rey et al. 2010, San Martin et al. 2010). In the Americas, the economic burden resulting from DENV associated illnesses is $2.1 billion per year (Shepard et al. 2011). Dengue is endemic in the U.S. Commonwealth territory of Puerto Rico where all four DENV serotypes co-circulate. Outbreaks of dengue have occurred for nearly a century in Puerto Rico (King 1917). However, the frequency of outbreaks has increased over the last ~30 years. Outbreaks involving all four serotypes occurred in 1998 and large outbreaks occurred in 2007 and 2010, the latter being the largest to date with greater than 10,000 diagnosed cases (Rigau-Perez et al. 2002, Tomashek et al. 2009, CDC 2011). Frequent outbreaks of dengue in Puerto Rico pose a risk for local transmission in select states attributable to 1) frequent traveler-associated imported cases of dengue, 2) large populations of susceptible humans especially in urban areas, 3) environmental conditions conducive for mosquito development and virus replication, 4) abundant epidemic vectors Ae. aegypti and Ae. albopictus associated with human populations, and 5) recent
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DENV outbreaks such as Florida (CDC 2010) and Texas (Murray et al. 2013). As the incidence of dengue increases around the world, so may travel-associated dengue infection in the U.S. Between 1996 and 2005 there were 1,196 travel-associated dengue cases in the U.S. with the Caribbean (23%), Mexico and Central America (20%), and Southeast Asia (17%) being the most commonly visited regions (Mohammed et al. 2009). Although definitive evidence is lacking, outbreaks of dengue during 2009, 2010, and 2013 in Florida were probably attributable to travel-associated infection and suggests vulnerability to transmission in areas where dengue vectors occur (CDC 2010). Furthermore, the Florida dengue outbreaks in 2009 and 2010 were caused by genetically related DENV-1 (Shin et al. 2013), an indication of endemic transmission (Graham et al. 2011, Muñoz-Jordan 2013). The current population decline on Puerto Rico is associated with increased migration to mainland U.S. (Cohn et al. 2014). The population of Puerto Ricans living in Florida is second in abundance only to New York (U.S. Census Bureau, 2010). Further, migration to the U.S. is facilitated by Puerto Rico being a commonwealth and so individuals are citizens. High numbers of dengue cases in Puerto Rico (Sharp et al. 2014), together with ease of migration, may pose a potential risk for importation of dengue to the continental U.S. Here, we assess the relative susceptibility of Florida Ae. aegypti and Ae. albopictus for infection and dissemination of DENV originating from Puerto Rico. MATERIALS AND METHODS Mosquitoes and viruses Larval Ae. aegypti and Ae. albopictus were collected from water-filled containers in Key West (Old Town historic district) and Vero Beach, FL, respectively. We used a single population for Ae. aegypti and Ae. albopictus since there is little evidence for genetic differentiation due to the absence of barriers to gene flow in Florida (Damal et al. 2013). We chose to use Ae. aegypti from Key West, FL, because of its involvement in dengue outbreaks in 2009 and 2010 attributable to DENV1 originating from Nicaragua (Shin et al. 2013). Mosquitoes were reared to adulthood on a diet of brewer’s yeast and lactalbumin at 26-28° C. Adults were provided with 20% sucrose (food grade quality) solution and allowed to blood feed on chickens once per week in order to propagate egg production. Aedes aegypti (generation=F2) and Ae. albopictus (generation=F4) were used for DENV infection studies. DENV-1 (GenBank: EU482591) and DENV-2 (GenBank: EU482553) were used for the infection studies. We focused on dengue serotypes 1 and 2 because of their recent involvement in outbreaks of locally transmitted DENV in the U.S. (2001-02, Hawaii; 2003-05, Texas; 2009, 2010, and 2013, Florida) (CDC 2010, Effler et al. 2005, Murray et al. 2013, Florida Health 2014). Low-passage isolates of DENV were provided by the Centers for Disease Control and Prevention. Dengue-1 virus was isolated in 2006 from a human and passaged once in C6/36 cells. Dengue-2 virus was isolated in 2007 from a human and passaged twice in
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C6/36 cells. Dengue viruses were passaged once in African green monkey (Vero) cells to produce stocks used to initiate the infection studies. Infected blood meals were prepared by inoculating monolayers of Vero cells with 400 μl of virus stocks (multiplicity of infection of 0.01 viruses per cell) using methods described in Alto and Bettinardi (2013). After 1 h of incubation at 35° C and under 5% carbon dioxide, 25 ml media (199 media, 10% fetal bovine serum, 2% penicillinstreptomycin, and 0.2% antimycotic) were added to each monolayer. Depending on virus growth rate, monolayers were inoculated with DENV on different days so that virus isolates would have similar population sizes on the day infectious blood meals were provided to mosquitoes (Shin et al. 2013). Blood meals were prepared by combining defibrinated bovine blood (Hemostat, Dixon, CA) with media and virus from infected monolayers of Vero cells (1:1 ratio). Mosquito infection Adult female and male mosquitoes were housed together in 0.3 m3 cages at 25° C and 14:10 light: dark cycle and provided with 20% sucrose solution. During feeding trials, mosquitoes were transferred to 1 liter cardboard cages including a 30 ml cup with a paper towel as an oviposition substrate. Females were deprived of sucrose but not water 48 h before blood-feeding trials. Mosquitoes were allowed to feed on DENV infectious blood for 60 min from an artificial membrane feeding system (Hemotek, Discovery workshops, Accrington, UK). Feeding trials were performed in incubators at 30° C, a temperature previously demonstrated to achieve high feeding rates (Alto and Bettinardi 2013). Fully engorged females were separated from unfed and partially fed mosquitoes using microscopy and held at 28° C along with access to 20% sucrose solution. Immediately following the feeding trial, a sample of freshly blood-fed mosquitoes (six to seven mosquitoes per DENV serotype) were stored at -80˚ C and then later plaque assays determined virus titers of the ingested blood. The remaining fully engorged females were returned in groups to 1.0 liter cardboard cages and held for the designated incubation period. After seven and 14 days of incubation, surviving mosquitoes (N=709) were stored at -80˚ C until later dissection and assayed for the presence of virus RNA. Mosquito processing Blood that is ingested by mosquitoes is deposited in the posterior midgut. Typically, infectious blood meals first result in infection of midgut cells followed by dissemination of virus from the midgut to secondary tissues such as the salivary glands. Separate assays of mosquito bodies and legs are a convenient and reliable approach to determine virus infection and dissemination, the latter being necessary for transmission (Turell et al. 1984). Individual mosquitoes were dissected to remove legs from the bodies, and triturated separately in 2.0 ml microcentrifuge tubes with two 4.5-mm stainless steel beads and 0.9 ml BA-1 (10x medium 199, 1% bovine serum albumin, 0.05 M TRIS, pH 7.5, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 1 μg/ml of mycostatin) at 25 Hz for 3 min (TissueLyser, Qiagen, Inc., Valencia, CA)
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and subsequently centrifuged at 3,148 x g for 4 min at 4° C. Nucleic acid was extracted from 250-μl samples of bodies and legs and eluted in 50 μl buffer using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Indianapolis, IN). The presence of viral RNA in samples was determined using the Superscript III One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA) with a Light Cycler 480 system (Roche, Mannheim, Germany) (Callahan et al. 2001). The probe and primer sequences for DENV serotype specific assays are described in another paper (Callahan et al. 2001). The virus titer present in mosquito bodies and legs were expressed as plaque forming unit equivalents (pfue)/ml that relates cDNA synthesis by PCR to plaque forming units (Alto et al. 2008, Alto and Bettinardi 2013). Midgut infection refers to infected mosquitoes used for infection rate and leg infection refers to disseminated infection used for dissemination rate. The infection rate is the percent of all blood fed mosquitoes with DENV RNA present in their bodies. The dissemination rate is the percent of mosquitoes with infected bodies that have dengue RNA present in their legs. Statistical analysis Treatment effects of DENV serotype, species (Ae. aegypti, Ae. albopictus), incubation period (seven and 14 days), and interactions were analyzed using maximum likelihood (ML) categorical analyses of contingency tables based on the number of individual samples categorized for the presence or absence of DENV. Separate ML ANOVA tests were used for body and leg samples to identify viral barriers (e.g., midgut infection and escape barriers) that may vary between treatments. Thus, each mosquito had a binomial infection status, as opposed to percentage for each replicate. Analysis of variance (ANOVA) was used to evaluate differences in DENV titers of individual mosquito bodies between DENV serotype, mosquito species, incubation periods, and interactions. If significant differences were detected, we used pair-wise contrasts of means adjusting p-values for multiple comparisons using the Bonferroni method (Sokal and Rohlf 1995). RESULTS
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Virus titer Plaque assays determined virus titers of freshly blood-fed mosquitoes were 6.8 ± 0.5 and 7.1 ± 1.2 log10 plaque-forming unit equivalents (pfue) DENV/ml for DENV-1 and DENV-2, respectively (t-test with t = 0.53, df = 11, p = 0.61). Infection rates Between 10-14% of Ae. aegypti and Ae. albopictus were susceptible to infection with DENV-1 virus seven days after blood feeding and 6-10% were susceptible to infection 14 days after blood-feeding (Figure 1A). Aedes aegypti were more susceptible to infection with DENV-2 virus than Ae. albopictus (Ae. aegypti, ~28%; Ae. albopictus, ~9%) for both seven days and 14 days incubation periods (Figure 1B). The ML ANOVA on susceptibility to DENV infection of mosquitoes showed marginally significant effects of dengue serotype (c2=3.79, df=1, p=0.051, Table 1) and species (c2=3.83, df=1, p=0.050, Table 1). There was a significant interaction of mosquito species by serotype (c2=13.2, df = 1, p=0.0003, Table 1). All other treatment effects on susceptibility to DENV infection were not significant (Table 1). To investigate the significant interaction further, we performed all possible pairwise comparisons of each species (Ae. aegypti vs Ae. albopictus) and DENV serotype (1 or 2), resulting in six comparisons. Aedes aegypti were significantly more susceptible to infection than Ae. albopictus for DENV-2 (c2=20.14, df=1, p 14 days), hence, further investigation is needed to investigate temporal patterns in virus dissemination (Chan and Johansson 2012). Another study demonstrated that although dissemination of DENV-2 to the salivary glands was observable for both Ae. aegypti and Ae. albopictus by 11 days post-blood feeding, there was delayed dissemination of virus to the salivary glands observed in Ae. albopictus compared to Ae. aegypti (Whitehead et al. 1971). Previous studies comparing the susceptibility of Aedes mosquitoes to DENV have demonstrated variable results. Several studies have demonstrated genetic variability in oral susceptibility, or variation attributable to geographic origin, to DENV for both Ae. aegypti and Ae. albopictus (Rosen et al. 1985, Borimosa et al. 1987, Tardieuz et al. 1991, VazeilleFalcoz et al. 1999). In some instances, rates of infection were higher in Ae. albopictus than Ae. aegypti for multiple serotypes of DENV, whereas other studies have shown the opposite result (Chen et al. 1993, Whitehead 1971, Rosen et al. 1985, Vazeille et al. 2001, 2003). A meta-analysis study demonstrated that Ae. albopictus is more susceptible to DENV midgut infection, but less capable of viral dissemination than Ae. aegypti (Lambrechts et al. 2012). Although increasing generations during laboratory colonization may increase the infection rate in mosquitoes (e.g., Ae. albopictus) (Lambrechts et al. 2012, Vazeille et al. 2003), this mechanism is not wholly responsible for discrepancies in relative competence between Ae. albopictus and Ae. aegypti as variable results have been observed for both high and low generation laboratory passaged mosquito strains (references above). Our assessment of susceptibility to DENV infection and dissemination enabled us to determine the relative vulnerability of Florida to potential transmission of DENV from Puerto Rico. Here we show that both Aedes mosquitoes were susceptible to midgut infection of DENV-1 and -2. Further, Ae. albopictus is not likely to be an important transmitter of the DENV-2 isolate from Puerto Rico given low susceptibility to infection and lack of evidence for viral dissemination. Recent outbreaks of dengue-1 virus in Florida (Monroe Co. 2009, 2010; Martin Co. 2013, St. Lucie Co. 2013) may place residents at greater risk for more severe secondary infection with other dengue serotypes (Halstead 1982). Specifically, human hosts in south and central Florida previously infected with DENV-1 are at most risk for secondary infection with DENV-2 and other serotypes of DENV. However, caution is advised in interpreting these results since they do not take into account variable temperature regimes in nature known to influence DENV infection in Aedes (Lambrechts et al. 2011) as well as other environmental factors that may influence the risk of disease agent transmission. Acknowledgments Dengue viruses from Puerto Rico were provided by Jorge Muñoz-Jordán and Candimar Colón at the Centers for Disease Control and Prevention. This study is supported by Department of Agriculture and Consumer Services (DACS) 2010 grant 00090369.
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