Southwest Virginia has recently become an emerging focus of activity for La Crosse (LAC) virus, a mosquito-transmitted arbovirus in the California serogroup of Bunyaviruses. In 2005 and 2006, ovitrap surveys were conducted to access the spatiotemporal oviposition activity of LAC virus vectors Aedes triseriatus, Ae. albopictus and Ae. japonicus across a wide region of southwest Virginia. Egg abundance and oviposition patterns of these vectors were significantly different across the three study areas. The primary LAC virus vector, Ae. triseriatus, was collected in the greatest abundance from all three areas, and favored forested habitats. Aedes albopictus was the second most abundant species collected, and was found to favor urban environments. Aedes japonicus also has a preference for urban habitats, and is actively expanding its range throughout southwest Virginia. Dogs were used to determine their efficacy as sentinels for assessing the distribution of LAC virus in southwest Virginia. Canine serum samples were tested using plaque reduction neutralization (PRNT) assays. Of the 436 collected canine serum samples, 21 (4.8%) were positive for LAC virus antibodies. LAC virus seroprevalence was evident in dogs from each study region, including areas where LAC virus human cases and LAC virus positive mosquito isolates have not been reported. As a result, this study provided documentation of horizontal transmission of LAC virus throughout southwest Virginia, demonstrating that dogs make useful sentinels for assessing the distribution of LAC virus in an area. The final objective examined the effects of coinfection with D. immitis microfilariae and LAC virus in three species of Aedes mosquitoes. No significant differences were found between mosquitoes fed dually infected bloodmeals (i.e. D. immitis microfilariae and LAC virus) and those fed bloodmeals containing LAC virus only. A follow-up study found low mosquito midgut penetration rates by D. immitis, despite using biologically significant doses of microfilariae. Failure to demonstrate enhancement of LAC virus in vector mosquitoes suggests that D. immitis does not have a significant impact on LAC virus epidemiology in areas where these organisms co-exist. / Ph. D.

Microfilarial enhancement of viral transmission is well documented, however only one previously studied model used components that occur together in nature and therefore has realistic implications. La Crosse (LAC) virus encephalitis is the most common mosquito-borne illness affecting children in the United States. LAC virus is prevalent in the Great Lake and Mid-Atlantic states and coincidently this area overlaps the region of highest infection for Dirofilaria immitis, the nematode that cause canine heartworm disease. Ae. albopictus and Oc. triseriatus are important vectors of La Crosse virus and among the numerous species able to transmit D. immitis. In this study, Aedes albopictus and Ochlerotatus triseriatus were infected with La Crosse virus and Dirofilaria immitis to determine the effects of dual infection on the dissemination and transmission of the virus. The effects of dual infection varied between the species tested. Ae. albopictus had significantly higher tolerance to D. immitis infection than Oc. triseriatus. Dissemination for dually infected Ae. albopictus were higher than the control group for all days tested, except one. Transmission rates for D. immitis infected Ae. albopictus were significantly higher than the control group on day 14 post infection. No microfilarial enhancement of viral dissemination or transmission was observed for Oc. triseriatus. The infection, dissemination, and tranmission rates were low for both species compared to rates of previous studies. Low rates could be a result of low susceptibility for the strains tested. In a second study, mosquitoes were collected from two counties in Southwestern Virginia to determine the abundance of potential La Crosse virus and D. immitis vector species. The abundance and distribution of mosquito species were examined in 2003 and 2004 using gravid traps. An unexpected finding was the significant increase in the abundance of Ochlerotatus japonicus. In 2003, collections were made over 192 trap nights from June to August yielding 5,879 mosquitoes of which only 24 were Oc. japonicus. In 2004, 12,151 mosquitoes were trapped from June to September over 160 trap nights. Oc. japonicus was the second most abundant mosquito species and the dominant Ochlerotatus species collected in gravid traps. Oc. japonicus was collected in low numbers in June, but the abundance increased significantly in July and remained consistent throughout the rest of the season. Of the other major mosquito species collected in this study, only Aedes albopictus exhibited a similar seasonal pattern as Oc. japonicus. Other biological similarities of Oc. japonicus and Ae. albopictus are discussed. / Master of Science in Life Sciences

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20211号 / 理博第4296号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 工藤 洋, 教授 髙林 純示, 教授 田村 実 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Le séquençage haut débit a été utilisé dans ce travail pour reconstruire avec des méthodes adaptées le génomeviral entier du virus de l’hépatite C (VHC) notamment pour le typer avec précision. Une étude a ainsi permisde mettre en évidence la présence d’une forme recombinante du VHC chez un patient. Une autre a permisde typer et détecter les mutations de résistance de plusieurs souches de VHC de génotypes différents. Enfin,une dernière étude basée sur cette approche a permis de découvrir une souche VHC appartenant à un nouveausous-type. Le séquençage haut débit a aussi été utilisé dans ce travail pour détecter des infections multiples etanalyser l’évolution virale en ciblant des gènes du VHC et en mettant en œuvre des méthodes non spécifiquespour 2 patients VHC sous traitement. Cette étude rétrospective a permis de définir la composition de chaqueéchantillon temporel, estimer leur diversité nucléotidique, explorer la structure génétique de la population viraleet son évolution temporelle et dater les infections secondaires. Les résultats obtenus supportent l’hypothèse d’unmécanisme d’apparition de résistance au traitement (selective sweeps). / High-throughput sequencing has been used in this work to reconstruct with adapted methods the whole genomeof the hepatitis C virus (HCV) particularly for accurately typing the virus. Thus, we managed to detect in a studya recombinant form of HCV circulating within a patient. We typed and detected in another study resistancemutations of several HCV strains of different genotypes. Finally, a last study based on this approach enabled touncover a HCV strain belonging to a new subtype. High-throughput sequencing has also been used in this workto detect multiple infections and analyze viral evolution with targeted HCV genes and non-specific methods for2 HCV patients under treatment. This retrospective study enabled to define the composition of each temporalsample, assess their nucleotide diversity, investigate viral population genetic structure and temporal evolutionand date secondary infections. Results of this analysis support the hypothesis of onset mechanism of treatmentresistance (selective sweeps).