mosquitoes transovarially transmit (TOT) La Crosse virus (LACV) to their offspring with minimal damage to infected ovaries. al. 1972). An important part of the LACV transmission cycle in the field involves the infection of ovaries in an infected mosquito and subsequent transovarial and transtadial transmission of the virus to her adult offspring, which are then infected and capable of transmission. Transovarial transmission (TOT) is also an important part of LACV overwintering in temperate climates (Watts et al. 1973; Watts et al. 1974; Watts et al. 1975; Beaty and Thompson 1975; McGaw et al. 1998). TOT refractory and Rabbit polyclonal to PLA2G12B permissive strains of have been selected (Graham et al. 1999), and three quantitative trait loci were mapped and shown to contribute additively to a females ability to TOT LACV (Graham et al. 2003). In order for LACV to be transmitted transovarially, the virus must infect but not disrupt ovarian tissues. The LACV s-segment encodes a small nonstructural protein (NSs) similar to the pro-apoptotic protein, Reaper (Colon-Ramos et al. 2003). In mammalian cells and MMAD tissues, NSs expression or LACV infection may promote apoptosis. In contrast, LACV induced apoptosis has not been detected in LACV infected mosquito tissues. A candidate protein that may suppress apoptosis in infected tissues is the inhibitor of apoptosis protein 1 (AtIAP1) (Blitvich et al. 2002), which is an ortholog of the well-characterized inhibitor of apoptosis 1 (DIAP1). DIAP1 ubiquitinates the apical caspase Dronc to stop activation of downstream caspases that would eventually lead to apoptosis (Palaga and Osborne 2002). For apoptosis to occur, Reaper, Hid, Grim, and Sickle proteins must bind at their IAP binding motifs (IBMs) to the Baculovirus inhibitor of apoptosis repeat (BIR) domains of DIAP1 (Bergmann et al. 2003). This binding blocks the ability of DIAP1 to inactivate Dronc and the apoptotic cascade begins (Wang et al. 1999; Chai et al. 2000; Liu et al. 2000; Wu et al. 2000). AtIAP1 may act in a similar fashion to DIAP1 to counter the potential apoptotic effect of LACV in mosquitoes. Previous observations concerning have also led us to consider it a candidate gene affecting LACV TOT. LACV is known to scavenge the 5 methylated guanine cap plus the adjacent oligonucleotide from host mRNAs to prime transcription of viral mRNAs (Beaty et al. 2000). Dobie et al. (1997) found that LACV predominantly scavenged the cap from an mRNA similar to AtIAP1 in a persistently infected larval cell line and in eggs emerging from diapause (Dobie et al. 1997; Borucki et al. 2002). The biology of the LACV TOT system provides a unique opportunity to exploit association mapping to determine if specific genotypes condition efficient TOT and overwintering. eggs were collected from oviposition sites throughout southwestern Wisconsin, southeastern Minnesota, and northeastern Iowa. These were hatched, reared to adults, tested for LACV infection and then separated into TOT+ (infected) and TOT? (uninfected) groups. The gene of individual mosquitoes from both groups was amplified by polymerase chain reaction (PCR) and sequenced. The purpose of this study was to determine whether specific polymorphisms in the gene condition whether an mosquito will become transovarially infected MMAD with LACV (in eggs being laid MMAD by an infected female). An association between specific polymorphisms and increased TOT potential would allow mosquito control agencies to focus more effort on controlling populations that contain these polymorphisms in a large number of individuals. While testing this hypothesis, several additional genetic analyses were performed on the sequence. 2. Materials and Methods A. Mosquito Collection and DNA Extraction eggs were collected by the La Crosse MMAD County Health Department from LACV endemic areas in southwestern Wisconsin, southeastern Minnesota, and northeastern Iowa where La Crosse encephalitis cases were reported. The eggs were collected from June through August of 2004 in cans that were painted black, half filled with tap water, and lined with seed germination paper as an oviposition.