Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ∼2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (∼six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.

BackgroundRift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors.Methodology and Principal Findings Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus.Conclusions/SignificanceIn both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.

Commercially available wood-fiber pots used to collect resting mosquitoes were modified to improve sampling efficiency. The modified traps, called the Centers for Disease Control and Prevention resting traps, collected 16.0 and 5.2 times more adult Culex pipiens and Cx. tarsalis than the conventional wood-fiber pots. The resting trap increases the mean number of resting mosquitoes collected per trap-night and is useful for collecting blood-engorged mosquitoes. Keywords Resting mosquitoes; Culex pipiens; Culex tarsalis; resting trapMost mosquito species are typically nocturnal or crepuscular, and remain relatively inactive during daylight hours. Samples of these resting mosquitoes provide effective estimates of mosquito population densities. Similarly, resting mosquito collections provide robust samples of blood-engorged mosquitoes. Tree cavities (Burkett-Cadena et al. 2008), animal burrows (Kay 1983), root masses (Mullen 1971), dense vegetation (Kay 1983), and caves are examples of natural daytime resting places for mosquitoes. Man-made structures such as barns, culverts, and basements, which provide dark, moist, and cool environments, also accommodate resting mosquitoes (Crans 1989). Researchers have taken advantage of the resting behavior of mosquitoes by sampling with artificial structures such as nail kegs (Burbutis and Jobbins 1958), wooden boxes (Gusciora 1971), walk-in red boxes (Meyer 1985), plastic trash cans (Burkett-Cadena et al. 2008), and wood-fiber pots (Komar et al. 1995).Vector control and public health surveillance programs rarely collect resting mosquitoes, for several reasons. First, resting collections capture adult mosquitoes from a broad range of physiological states (i.e., host-seeking, blood-engorged, gravid, postovipositional) and thus are not useful for calculating vector indices, which are derived from collections of hostseeking female mosquitoes only (Gujral et al. 2007). Second, resting traps predominantly attract mosquito vectors in the genera Culex, Culiseta, and Anopheles (Goodwin 1942, Meyer 1985, Komar et al. 1995, Burkett-Cadena etal. 2008), but are ineffective for sampling many genera of nuisance and vector mosquitoes such as Coquilletidia, Psorophora, and Aedes (Gusciora 1971). However, resting collections offer the most efficient source of blood-engorged mosquitoes needed for several research applications, suchasvector and arbovirus ecology studies. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptIn order to enhance the collection of resting mosquitoes, a novel trap design was devised to combine the advantages of the wood-fiber pot (e.g., low cost and portability) with those of conventional, suction-type traps (few escapees, larger samples). Traditionally, collections from these pots are made daily by aspirating resting mosquitoes from the interior walls of the pots using either a handheld aspirator or a backpack aspirator. However, slight movements can startle resting mosquitoes before they can be aspirated, and changing envi...