INTRODUCTION RESULTS

1. Vector competence of local Aedesalbopictusmosquitoes to dengue virus 1 in Hawaii Estelle Martin1, Korina Hernandez1, Chaz Kuniyoshi1,Ryuei Sato1,Deon Canyon2, Shannon Bennett1 and Vivek R. Nerurkar1 1Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Mānoa, Honolulu HI, USA 2 Department of Public Health Sciences, University of Hawaii at at Mānoa, Honolulu HI, USA INTRODUCTION RESULTS • Replication of DENV-1 strains in vitro • Oahu 1943 strain replicates at higher levels in (A) Vero cells and (C) C6/36 cells • No difference in the virus kinetics was observed at early and late time point in (B) HepG2 cells Dengue, an arboviral disease, is caused by four distinct serotypes of dengue virus (DENV 1-4). While most of the cases are asymptomatic, some develop a febrile illness (dengue fever) or progress towards the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue is caused by an enveloped positive sense RNA virus of approximatiely 11-kb in the genus Flavivirus of the Flaviviridae family and is transmitted by mosquitoes of the genus Aedes. Aedesaegypti (Ae. aegypti) is the primary vector of dengue viruses followed by Aedesalbopictus (Ae. albopictus). The mosquito becomes infected when it feeds on an infected human during the 5 days period of viremia. The ability of a mosquito to ingest, replicate and transmit a virus is know as vector competence or vector susceptibility to the virus. For a virus to succeed in infecting the mosquito it must cross multiple barriers: the midgut infection barrier, the midgut escape barrier and the salivary glands infection barrier. Differences in susceptibility have been observed amongst Ae. albopictuspopulations which has a significant impact on the course of the dengue outbreak. • DENV-1 infection in Ae. Albopictus. • Virus loads averaged at (A) 3.1 x 104 PFU/μgRNA at day 7 and at 9.2 x 104 PFU/μgRNA at day 14 for the Maui 2001 and (B) 5.1 x 103 PFU/μgRNA at day 7 and at 7.3 x 103 PFU/μgRNA at day 14 for the Oahu 1943 strain. • Proportion of infection was (C) 75% at day 7 and 94% at day 14 for the Maui 2001 strain, and (D) 33% and 67%, at day 7 and day 14 for the Oahu 1945 strain, respectively A) B) C) A) B) C) D) DENGUE IN HAWAII Figure 1. Three different cell lines (A) Vero cells, (B) HepG2, and (C) C6/36 were infected with DENV-1 Oahu 1943 and DENV-1 Maui 2001 strains at MOI 0.01. Supernatant was collected at different times after infection (6, 12, 24, 48, 96, 144, 192 hours) and analyzed for DENV-1 copy numbers using qRT-PCR. Figure 4: Mosquito bodies were dissected 7 and 14 days after the infectious bloodmeal to assess for infection. Bodies were homogenized in lysis buffer using 1-mm glass beads and total RNA was extracted using the QIAamp Viral Mini Kit (Qiagen, Germany). Viral loads in the mosquito body were obtained by qRT-PCR using DENV-1 specific primers and are shown (A) for the Maui 2001 strain and (B) for the Oahu 1945 strain. Percentage of infected females was calculated as the number of females with a positive body amongst all females receiving the bloodmeal and are shown for (C) the Maui 2001 strain and (D) the Oahu 1945 strain Dengue outbreaks in Hawaii occurred in 1840, 1903, 1912, 1943-44, 2001, and 2011. Following the eradication campaign in 1940, Ae. agyptiis now confined to specific location on the Big Island and Ae. albopictusis responsible for the subsequent DENV outbreaks in Hawaii. In 1943, DENV-1 was responsible of over 1500 cases. Fifty years after the 1943 outbreak, the virus re-emerged in 2001 in Hawaii. One would have expected an explosive epidemic associated with severe cases of the disease but in reality the epidemic was mild with only cases of DF reported. The focus of this study is to study the interactions between dengue virus and its vector Ae. Albopictus to better understand the vector competence to develop eradication strategies • Field collection of A. albopictusacross the island of Oahu • Eleven different Ae. albopictuspopulations were collected across Oahu: three on the Windward side, one on the North shore, three in Central Oahu and four on the South shore (Fig. 2) • Dissemination of DENV-1 in Ae. albopictus. • Virus loads averaged at (A) 1.1 x 103 PFU/μgRNA at day 7 and at 5.7 x 104 PFU/μgRNA at day 14 for the DENV-1 Maui 2001 strain. Virus was absent (B) in the legs and wings at d7 and averaged at 1.0 x 102 PFU/μgRNA at day14 for the DENV-1 Oahu 1945 strain. • Proportion of infection was (C) 53% at day 7 and 100% at day 14 for the Maui 2001 strain. No dissemination was observed at (D) day 7 whereas 17% of the infected females had a disseminated infection at day 14. A) B) C) D) Figure 5: Mosquito legs were dissected 7 and 14 days after the infectious bloodmeal to assess for dissemination. Legs and wings were homogenized in lysis buffer using 1 mm glass beads and total RNA was extracted using the QIAamp Viral Mini Kit (Qiagen, Germany). DENV-1 virus loads were analyzed by qRT-PCR and are shown (A) for the Maui 2001 strain and (B) for the Oahu 1945 strain. Percentage of dissemination was calculated as the number of females with positive legs amongst all infected females and are shown for (C) the Maui 2001 strain and (D) the Oahu 1945 strain • Transmission of DENV-1 by Ae. albopictus • DENV-1 was absent (A,B) in the saliva at day 7 for both strains. • Virus loads average (A) at 6.01 x 101 PFU/μgRNA at day 14 for the Maui 2001 and (B) 5.9 x 102 PFU/μgRNA at day 14 for the Oahu 1945 strain. • Proportion of infection at day 14 was (C) 23% and 50% for the Maui 2001 and Oahu 1945, DENV-1 strains, respectively. DENV-1 was identified in viral isolates obtained from 15 patients. Molecular studies demonstrated that most of the Hawaii isolates were related to the “Pacific Subtype” and clustered with the Tahitian DENV-1 strain suggesting that Tahiti was the source of the epidemic. One isolate was closely related to a DENV-1 isolate previously obtained from an infected traveler returning from Samoa suggesting a independent event of introduction. On this phylogenetic tree is also present one isolate from the 1943 outbreak. • Figure 2: Mosquito eggs (F0) where collected during weekly ovitrap surveillance. Mosquitoes were colonized under standard insectary conditions (constant 28°C± 2°C; 80% RH, 12:12 light:dark cycle). Immature stages were fed with 3:1 mix of Brewer’s yeast and liver powder and adult stages were maintained on a water and 10% sugar solution. Non-infectious bloodmeals were given every week to ensure egg production. In order to obtain enough mosquitoes for the in vivo study, the F3 generation was used. A) B) C) During the 2001 epidemic, a total of 122 cases were reported between 2001 and 2002. Ninety-two cases occurred on Maui, twenty six on Oahu and four on Kauai. • Experimental infection • Ae. albopictusmosquitoes from Pearl City were orally infected either with DENV-1 Oahu 1943 or DENV-1 Maui 2001 strain. The bloodmeal titer was 1.2 x 103 PFU/mL and 1.1 x 105 PFU/mL for the Oahu 1943 and Maui 2001 DENV-1 strains, respectively. • The engorgement rate was 35% and 20.8% for the Oahu 1943 and Maui 2001 DENV-1 strains, respectively. OBJECTIVE AND HYPOTHESIS • The objectiveof this study is to demonstrate whether Ae. albopictusis a competent vector of DENV-1 strains isolated during the 2001 outbreak. • We hypothesizethat Ae. albopictusfrom Oahu is able to support DENV-1 infection, dissemination and transmission. • The rationale being that variation in susceptibility to dengue virus have been observed between Aedes species and also amongst Ae. albopictuspopulations. Figure 6: Mosquito saliva was collected 7 and 14 days after the infectious bloodmeal to assess for transmission. Mosquitoes were anesthetized using triethylamine and put on ice. After removing the legs and wings, mosquito proboscis was inserted into a capillary filled with a 10% FBS solution. DENV-1 virus loads were analyzed by qRT-PCR and are shown (A) for the Maui 2001 strain and (B) for the Oahu 1945 strain. Percentage of transmission was calculated as the number of females with positive saliva amongst all females presenting a disseminated infection and are shown for (C) the Maui 2001 strain and the Oahu 1945 strain. CONCLUSIONS AND FUTURE DIRECTIONS DENV-1 Oahu 1943 • We have successfully colonized local population of Ae. albopictususing standard insectary conditions • Our studies demonstrate that Ae. albopictusfrom Pearl City sustains infection at day 7 and that the viremia is maintained in mosquitoes till day 14 after infection • Dissemination occurs at both day 7 and day 14 after infection and transmission was only observed at day 14 with few mosquitoes being able to transmit the virus in their saliva. This could explain the mild 2001 epidemic • For the Oahu 1943 strain, no dissemination or transmission was observed at day 7. Dissemination was low at day 14 as well as transmission. Nevertheless, no comparison between the two viral strains can be made because of the 2 log difference in the infectious bloodmeal • In order to be able to compare both virus strains, we will repeat the Oahu 1943 experiment with a higher bloodmeal titer • In future studies, we will compare the vector competence of other Ae. albopictuspopulations. Population from the Windward, South shore, North shore and Leeward side of Oahu. SIGNIFICANCE DENV-1 Maui 2001 • There are currently no licensed vaccines or specific therapeutics against DENV and vector control efforts are the best way to control the disease. • Nevertheless, dengue epidemic remain a threat to the regions within the United States that have competent mosquito vector populations and large number of travelers from dengue-endemic regions. • Most studies focus on Ae. aegyptiwhich is considered as the main vector for dengue virus. This study will provide insights into the role of Ae. Albopictus, the only dengue virus vector in Hawaii, in local dengue virus transmission. Knowledge of the susceptibility of local mosquitoes to DENV will provide information crucial for vector control strategies in order to control and prevent future outbreaks. This could also give insight in the general role of Ae. Albopictusin areas where Ae. aegyptiis absent or where Ae. albopictuspredominates over Ae. Aegypti. • Figure 3: Five to seven day old females were transferred into containers and starved for 24 h prior to the bloodmeal. The bloodmeal consisted of 50% human blood, 45% viral supernatant harvested at day 8, 2.5% sucrose solution and 2.5% adenosine triphosphate disodium salt at a final concentration of 5 x 10-3 M. Mosquitoes were allowed to feed for 30 min and anesthetized on ice in order to select only fully engorged females. Mosquitoes were maintained under standard insectary conditions (constant 28°C± 2°C; 80% RH, 12:12 light:dark cycle) for 7 or 14 days to assess for vector competence. • Three components of vector competence were assessed: infection by dissecting body, dissemination by dissecting legs and wings and transmission by quantitation of the virus in the saliva. This work was supported by grants (3ADVC-60318) from the Hawaii Community Foundation, (P20GM103516) from the Pacific Center for Emerging Infectious Diseases Research, Centers of Biomedical Research Excellence, NIGMS, (U54AI065359) from the Pacific Southwest Research Center of Excellence, NIAID, NIH, and Institutional funds.