Browsing by Author "Blair, Carol D., advisor"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Open Access Aedes aegypti and dengue virus investigation of anatomic, genomic, and molecular determinants of vector competence(Colorado State University. Libraries, 2009) Bernhardt, Scott Arthur, author; Blair, Carol D., advisor; Black, William C., IV, advisorDengue (DENV) causes one of the most rapidly expanding diseases in the tropics. Vector competence (VC) in Aedes aegypti for DENV-2 is a quantitative trait and has been shown to be highly variable. Questions remain as to whether variation in VC continues to exist after the primary field observation. What genetic factors contribute to VC and do these factors evolve from arbovirus exposure remain unclear.Item Open Access Analysis of virus-derived small RNAs reveals that the RNA silencing response to flavivirus infection differs dramatically between C6/36 and Aag2 mosquito cell lines(Colorado State University. Libraries, 2010) Scott, Jaclyn Christine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Wilusz, Carol J., committee member; Peersen, Olve, committee memberThe exogenous small RNA pathway has been shown to be an important antiviral defense in mosquitoes against arboviruses such as dengue virus (DENV), but little is known about how the pathway and the virus interact in the cell. The studies described in this dissertation examine the how small RNA pathways interact with DENV and a mosquito-only flavivirus, cell-fusing agent virus (CFAV), in mosquito cell cultures. Deep sequencing of virus-specific small RNAs in Aedes aegypti Aag2 cells indicates that DENV2 is targeted by the exogenous RNA interference (RNAi) pathway in this cell line, which is consistent with the DENV2-specific small RNAs seen in DENV2-infected A. aegypti mosquitoes. When the DENV2-specific small RNAs from the Aedes albopictus C6/36 cell line were analyzed, the size and polarity of the small RNAs was not consistent with the exogenous small interfering RNA (siRNA) pathway. Further molecular analysis of the C6/36 cell line indicated that it appears to lack functional Dicer2 processing of long double-stranded RNA (dsRNA). CFAV small RNAs were also discovered in the Aag2 cell line during the deep sequencing analysis. It appears that this cell line is persistently infected with this mosquito-only flavivirus, and the virus is also targeted by the exogenous siRNA pathway in the cells. Sequence comparisons between CFAV and DENV2 RNA did not show long regions of sequence identity between the two viruses, indicating that a sequence-specific mechanism for virus-derived small RNAs from one virus to interfere with replication of the other virus during dual infections seems unlikely. The C6/36 cell line was inadvertently infected with CFAV, but the CFAV-specific small RNAs in C6/36 cells did not appear to be generated from the exogenous siRNA pathway, consistent with the DENV2-specific small RNAs in this cell line. The larger sized, mostly positive sense virus-specific small RNAs found in the C6/36 cells suggest that virus infections may be targeted by another small RNA pathway (such as the piwi-interacting pathway) in this cell line. These studies provide a better understanding of the interactions of DENV2 with the mosquito antiviral RNAi pathway in infected mosquito cells and have revealed a dysfunctional RNAi pathway in the C6/36 cell line. This work also provides a basis for further studies examining the interactions between mosquito-only flaviviruses, arboviruses and the antiviral RNAi pathway.Item Open Access Flavivirus surveillance in mosquitoes from northern Colorado, with the detection and description of two insect-specific flaviviruses(Colorado State University. Libraries, 2010) Bolling, Bethany Gayle, author; Blair, Carol D., advisor; Moore, Chester G.(Chester Gunn), advisor; Olson, Kenneth Edward, committee member; Olea-Popelka, Francisco J., committee memberVector-borne diseases remain a major public health concern worldwide. The studies described here underline the importance of combining field surveillance activities with laboratory experiments to provide a comprehensive understanding of the dynamics of vector-borne disease systems. Entomological measures of West Nile virus (WNV) risk were found to be strongly associated with human WNV disease cases in northeastern Colorado. Specifically, Culex tarsalis abundance and the Vector Index for WNV-infected Cx. Tarsalis females (weekly mean per trap night x weekly proportion of WNV-infected females) were associated with weekly numbers of WNV human disease cases with lag times of 4-7 weeks and 1-2 weeks, respectively. This provides information that can be utilized for decision-making processes concerning when to initiate mosquito control activities and how to best utilize limited resources. These studies also describe the first detection of insect-specific flaviviruses in mosquitoes collected in Colorado. Culex flavivirus (CXFV), first described in Japan in 2007, and a new insect-specific flavivirus, designated Calbertado virus, were detected in Culex spp. mosquitoes. Experiments were conducted to explore the transmission dynamics of CXFV in a naturally infected Culex pipiens laboratory colony and also the potential effects of CXFV infection on vector competence for WNV. Results indicated that vertical transmission is the primary mechanism for viral persistence in the colony, with venereal transmission perhaps playing a supplemental role. Vector competence experiments suggested possible suppression of WNV replication by persistent CXFV infection. These findings are important as insect-specific flavivirus transmission dynamics have not been described yet and studies investigating putative interactions between insect-specific flaviviruses with arboviruses, like WNV, are lacking. This work provides a better understanding of local vector-borne disease systems, providing a basis for additional studies to further characterize the dynamics of co-circulating flaviviruses in vector mosquitoes.Item Open Access Genomic characterization, detection and molecular evolution of arthropod-borne viruses of the family Bunyaviridae(Colorado State University. Libraries, 2010) Lambert, Amy Julia, author; Blair, Carol D., advisor; Chen, Chaoping, committee member; Lanciotti, Robert S., committee member; Miller, Barry R., committee member; Black, William C., committee memberThe genomic characterization, detection and evolution of arthropod-borne human pathogens and related viruses of the family Bunyaviridae are presented. This study began with the determination of primary nucleotide sequence data for a diversity of bunyaviruses of interest that were not previously characterized at the genetic level. Following molecular characterization, an RT-PCR strategy was designed, according to previously determined and newly derived nucleotide sequence data, to target S genomic segments of 47 viruses, including 29 arthropod-borne human pathogens, of the family Bunyaviridae. Following development, this strategy was used, in some instances with an expanded capacity for the detection of multiple segments of the bunyavirus genome, for the identification of arthropod-borne bunyaviruses of medical importance and novel circumstance at a global level. Phylogenetic analyses performed on nucleotide sequence data generated by these efforts facilitate an ancestral understanding of the discovered agents. Finally, advanced molecular and phylogenetic analyses of a subset of newly derived sequences are described here to facilitate an enhanced understanding of the evolution of Bunyaviridae.Item Open Access Identification of Culex tarsalis D7 salivary protein and role of salivary protein vaccine on subsequent West Nile virus infection(Colorado State University. Libraries, 2010) Reagan, Krystle Lynn, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Foy, Brian D., committee member; Chen, Chaoping, committee member; Wang, Tian, committee memberMosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of protein factors in saliva responsible for immunomodulation should lead to new strategies to prevent and protect against arboviral infection. D7 salivary proteins are among the most abundant in mosquito saliva, and they function as both vasodilators and suppressors of local inflammation. Here we identify D7 salivary proteins in Culex tarsalis, an important disease vector in the western United States. Recombinant D7 proteins were used to analyze the systemic and local immunomodulatory properties of the host. In this project, we immunized mice with recombinant D7 and tested for protection against subsequent challenge with West Nile virus (WNV) (NY99) delivered by mosquito bite. The vaccine was able to elicit a specific immune response. However, it enhanced WNV infection in the mouse model. We suggest that WNV enhancement is due to three factors. First, vaccinated mice had significant cellular infiltrates at the mosquito bite site, which included WNV permissive monocytes and dendritic cells. Increases in these cell populations at the mosquito bite site leads to an increase in initial viral infection and dissemination. Others have shown that higher peripheral viral levels lead to a worse disease outcome from infection. Secondly, mortality curves in infected mice receiving passive transfer of serum containing antibodies from vaccinated mice mimicked those from vaccinated animals. Antibody neutralization of mosquito salivary proteins that are critical in completing a successful blood meal may lead to increased probing time by the mosquito. Increased probing by the mosquito results in an increase in amount of saliva being deposited, therefore an increase in initial viral dose. Lastly, the cytokine profile observed in vaccinated mice showed an increase in the Th2 cytokine IL-4 and regulatory cytokine IL-10 and a decrease in Th1 cytokines such as IL-12p70 and IFNγ. A protective immune response to WNV includes high levels of Th1 cytokines. Production of IL-4 by mice that received the rD7 vaccine directly inhibits the Th1 cytokines necessary for protection. This work has increased our understanding of the complex nature of immunity to MSPs. Vector saliva vaccines have been successful in protecting against other blood feeding arthropods transmitted diseases. Nevertheless, differences in vector and pathogen physiology may preclude this approach from being successful for mosquito virus systems.Item Open Access Increasing dengue virus vaccine safety and immunogenicity by manipulating antigenic determinants of the flavivirus envelope protein(Colorado State University. Libraries, 2010) Hughes, Holly Ruth, author; Blair, Carol D., advisor; Chang, Gwong-Jen J., advisor; Chen, Chaoping, committee member; Bowen, Richard Arnold, committee memberDengue virus (DENV), which exists as four closely related serotypes, is a mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). Severe dengue disease is often associated with secondary DENV infection and hypothesized to frequently be induced by cross-reactive, weakly neutralizing antibodies, a process referred to as antibody-dependent enhancement of infection (ADE). Due to the complex humoral immune response to DENV infection and the complexity of severe DENV disease, currently no licensed DENV vaccine is available. The goal of this dissertation is to increase the safety and immunogenicity of DENV vaccination through a better understanding of the antigenic properties of the envelope protein. To these ends the objectives of this research were to: 1) use site-directed mutagenesis and monoclonal antibody mapping to identify possible cross-reactive epitopes of the DENV-2 E protein which could contribute to ADE (Chapter 2), 2) use B cell epitope modification to construct a serotype-specific DENV-2 vaccine with reduced potential of vaccine-induced ADE (Chapter 3), and 3) identify potential dominant T cell epitopes in West Nile virus E which could act as immunological adjuvants for DENV-2 vaccines (Chapter 4). Humoral immune responses to DENV infection are complex and can exacerbate pathogenicity, yet are essential for immune protection. DENV-2 E protein epitope-specific antigens were created and used to measure immunoglobulin responses to three distinct epitopes in serum samples from DENV-2 infected humans. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary sera from DENV-2 infected patients, confirming previous studies and identifying the fusion peptide as an immunodominant epitope. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response, yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. These results begin to tease apart complex humoral immune responses to DENV infection and thus are important for improving our understanding of dengue disease and immunological correlates of protection relevant to DENV vaccine development and testing. DENV vaccines must induce a balanced protective immunity to all four serotypes to reduce the possibility of cross-reactive antibody induced severe disease upon subsequent infection. By modification of immunodominant B cell epitopes of E, cross-reactivity reduced (CRR) DENV-2 DNA vaccine candidates were developed and tested in mice for immunogenicity and potential reductions in developing vaccine induced ADE in mice. Unlike wild-type vaccine, CRR vaccine immunized mouse sera neutralized virus and did not enhance viral infection in vitro. Thus, reducing cross-reactivity in the envelope glycoprotein of DENV may provide a solution to increase vaccine safety and resolve the long-standing obstacle of immune enhancement in dengue vaccine development. DNA vaccination is a highly pursued vaccine platform for its safety, stability, and ease of development, use and manufacturing. However, DNA vaccination is hindered by lower immunogenicity. Methods investigated to increase the immunogenicity of DNA vaccines have included the use of immunological adjuvants; however, few are approved for human use. A dominant, naturally occurring CD4 T cell epitope located in the transmembrane domain of West Nile virus was identified and its ability to increase the immunogenicity of heterologous flavivirus vaccines was investigated. The incorporation of the West Nile virus CD4 epitope into a DENV-2 DNA or protein vaccine significantly increased neutralizing antibody titers compared to parental vaccines without the CD4 epitope. Identifying differential antigenic properties of vaccines may alleviate concerns of imbalanced immunity associated with multivalent vaccinations.Item Open Access Isolation and characterization of dengue virus membrane-associated replication complexes from Aedes aegypti(Colorado State University. Libraries, 2010) Poole-Smith, Betty Katherine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Foy, Brian D., committee member; Ross, Eric D., committee memberUltrastructural studies of flavivirus replication have long observed proliferation of host membranes. Membrane-bound replication compartments have recently been isolated and characterized from flavivirus-infected mammalian cells, providing insight into the morphology, organelle of origin, and protein components of the flavivirus membrane-associated replication complex. Our laboratory has proposed that a balance exists between dengue virus (DENV) replication in Aedes aegypti and the mosquito's RNA interference (RNAi) based antiviral response. Here, we have isolated and characterized membrane-bound replication compartments from mosquito cell culture and Ae. aegypti to evaluate the role that these membranes may play in shielding DENV double-stranded RNA (dsRNA) from RNAi. Membrane isolation techniques and immunofluorescent staining techniques for dsRNA identification were developed to isolate and characterize membrane-associated replication complexes in DENV-infected mosquito cell culture and Ae. aegypti. Here we show that double-membrane vesicles arise from the endoplasmic reticulum (ER) and are associated with DENV dsRNA in mosquitoes. These data suggest that DENV dsRNA replicative intermediates may be shielded from the RNAi response in the mosquito. DENV membrane-associated replication complexes were characterized in mosquito cell culture and Ae. aegypti using immunofluorescent staining for dsRNA, confocal microscopy, sucrose gradient cellular fractionation, and electron microscopy. In addition, we compared immunofluorescent staining for dsRNA between DENV and Sindbis virus (SINV). We also evaluated replication of DENV mutants in the DENV-resistant transgenic mosquito strain known as Carb77 and whether mutations in DENV genome sequence lead to evasion of the enhanced RNAi response of Carb77 mosquitoes. This is the first isolation of membrane-associated replication complexes and first characterization of dsRNA staining from DENV-infected mosquito cell culture and Ae. aegypti, providing knowledge which can be used to develop improved RNAi-based control strategies for DENV in mosquitoes.Item Open Access Metabolomics-based diagnosis and prognosis of dengue virus infections and NS1 antigen detection for diagnosis and surveillance in humans and mosquitoes(Colorado State University. Libraries, 2013) Voge, Natalia Victoria, author; Blair, Carol D., advisor; Beaty, Barry J., advisor; Loroño-Pino, Maria A., committee member; Eisen, Lars, committee member; Belisle, John T., committee member; Prenni, Jessica E., committee memberDengue (DEN) is a mosquito-borne viral disease of significant public health importance. There are currently no commercialized vaccines or accepted pharmacological treatments for DEN disease, making mosquito surveillance critical for the prevention of outbreaks of this disease. Aedes aegypti is the principal vector of DENV, although Aedes albopictus and other Aedes species have been reported to be able to transmit it. Improved surveillance methods for DENV in mosquito populations would be of great value for public health and vector control programs and would provide better risk assessment of potential DENV infections in humans. Improved mosquito-based surveillance would also improve vector control programs by targeting areas at higher risk for ongoing or potential epidemics for vector control. Non-structural protein 1 (NS1) detection by ELISA is a commercially available test with the ability to detect DENV NS1 protein in DENV infected samples. Studies were conducted to determine the ability of the NS1 antigen test to detect DENV in Aedes aegypti mosquitoes. The NS1 antigen detection test proved to be highly sensitive and specific for DENV-antigen detection in pools of mixed infected and non-infected mosquitoes under various field-simulated conditions and in different sizes of mosquito pools. This test could facilitate mosquito-based surveillance for early warning of DEN outbreaks. The capacity of this test to diagnose human DENV infections in non-invasive clinical specimens, i.e., urine and saliva, was also investigated. NS1 protein detection in acute phase, non-invasive clinical specimens was found to be less sensitive than NS1 detection in serum samples. Most dengue virus (DENV) infections are subclinical. The clinical manifestations of apparent infections range from DEN fever (DF), typically a self-limiting illness with fever and rash, to severe DEN hemorrhagic fever and shock syndrome (DHF/DSS). Upon presentation, there is no way to predict whether or not the patient will experience DF or will progress to the severe form of disease (DHF/DSS). A sensitive and specific test that utilizes acute phase clinical specimens and that provides diagnosis of DENV infection as well prognosis of progression to severe disease is sorely needed. Such a test would permit identifying those patients destined for severe disease for appropriate patient management and therapeutic intervention. An innovative metabolomics platform was used in these studies to determine a biosignature of small molecular biomarkers (SMBs) that can potentially differentiate patients with the most severe forms of disease from DF and non-DEN patients. SMBs that could potentially be diagnostic of DENV infections and prognostic of disease outcomes were identified in acute phase serum, saliva, and urine specimens obtained from DEN and non-DEN patients from Mexico and Nicaragua. Using acute phase serum specimens, a panel of six candidate SMB compounds was identified by tandem liquid chromatography-mass spectrometry (LC-MS/MS); five of these six biomarkers differentiated DF patients from those with DHF/DSS and have potential to diagnose and predict DEN disease or DEN severity (DHF/DSS) outcomes. Two candidate SMB compounds were identified in urine and one in saliva that could potentially be used for diagnosis and prognosis of DEN infections. Tandem mass spectra of candidate compounds and commercial standards were obtained and compared to identify the SMB metabolites. Some of the SMB identities were confirmed by using the NIST (National Institute of Standards and Technology) or METLIN spectrum libraries. The biochemical nature of the identified metabolites included phospholipids, fatty acids, amino acids, nucleosides, and vitamin D. These SMBs have potentially important roles in DEN pathogenesis and in endothelial cell metabolism (these cells are among the principal target cells affected during severe DEN disease). The immediate goal of this dissertation research was to identify a biosignature panel of LC-MS/MS-identified candidate SMB metabolites that differentiate the DEN disease diagnosis groups (DHF/DSS, DF, and ND) and that have potential for diagnosis and prognosis of DENV infections using acute phase serum and non-invasive clinical specimens. However, it is unlikely that LC-MS/MS technology will be applicable in the front-line clinics where DEN patients first present. Thus a long-term goal of the research project is to select a subset of these pathogenically and physiologically relevant SMBs and then determine the potential for the metabolite analyte or a surrogate (e.g., a protein involved in the metabolic pathway conditioning the metabolic change) to be incorporated into diagnostic formats amenable to point of care tests (POC), such as ELISA based formats. A diagnostic algorithm incorporating results from such a POC test and conventional laboratory and clinical biomarkers could provide dramatically improved capability for diagnosis and prognosis of DENV infections and would be of immense value to physicians in managing patients.Item Open Access Mutagenesis of the dengue virus envelope glycoprotein gene can significantly alter virus infectivity phenotypes in cultured cells and live mosquitoes(Colorado State University. Libraries, 2011) Erb, Steven Michael, author; Blair, Carol D., advisor; Roehrig, John T., committee member; Olson, Kenneth E., committee member; Chen, Chaoping, committee memberThe dengue virus (DENV) envelope (E) glycoprotein is the primary determinant for initiation of host cell infection. To date, studies investigating the contribution of DENV genetics to mosquito infection are limited. A infectious clone cDNA of DENV type 2 strain 16681 (30P-NBX) provided the ability to introduce site-specific amino acid (AA) mutations into the E protein. The results of the studies herein analyze the effects that AA mutations in the E protein have on infectivity of cultured cells and live mosquitoes. The ability of 30P-NBX to infect Aedes aegypti RexD strain mosquitoes after oral infectious blood-meal was investigated and showed that both 30P-NBX and the parent virus 16681 have low, but equivalent midgut infection rates (MIRs). Mosquito midgut infection with 30P-NBX is not affected by the virus titer in the blood-meal as long as titers are above 6 log 10 pfu/ml or 7 log 10 TCID 50 /ml. Additionally, multiple experimental repetitions with at least 20 mosquitoes per infectious blood-feed were required to obtain an accurate average MIR for 30P-NBX. Serial passage of 30P-NBX in RexD mosquito midguts identified a single AA mutation at position 122 in domain II of the E protein from lysine to glutamic acid that correlated with increased MIRs. Introduction of this AA mutation into the infectious clone (mutant virus K122E) reproduced the results from the serial passage experiment. Compared to 30P-NBX, K122E was not only shown to infect a higher proportion of mosquitoes as early as day 2 post blood-feed, but also to produce a disseminated infection in a higher proportion of mosquitoes by day 6 post blood-feed. Also, K122E consistently produced a midgut infection that spread throughout the entire tissue while 30P-NBX stayed restricted by comparison. Virus attachment to midgut cells was compared and showed that 30P-NBX and K122E could attach with equal efficiencies via our midgut-virus attachment assay. Additionally, incorporation of a single AA mutation into the infectious clone at E protein AA 120 from arginine to threonine significantly enhanced mosquito midgut infection compared to 30P-NBX. This is the first time that mosquito infection determinants have been identified in the DENV E protein. Amino acid mutations were engineered into the E protein on the lateral ridge of domain III, the fusion peptide at the distal end of domain II, and the molecular hinge region between domains I and II. Mutant virus phenotypes were analyzed in cell culture and live mosquitoes. In contrast to previous suggestions, domain III mutant virus phenotypes showed that the FG loop structure (previously suggested as a mosquito-specific infection determinant) and not the specific AA sequence is important for infection of mammalian cells and live mosquitoes, while the structure and sequence of the FG loop is dispensable for infection of cultured C6/36 cells. Additionally, mutations that remove positively charged residues from the A strand in DIII significantly attenuate infection of mosquitoes after oral infectious blood-meal and completely abrogate infection in mammalian cells. The results of this study suggest that there may be multiple structures in the E protein that are contributing to virus-receptor interactions. Viruses with mutations in the fusion peptide and hinge region of the E protein were intrathoracically (IT) inoculated into mosquitoes and showed variable infectivity phenotypes. All of the mutants except for one virus from both the fusion peptide and hinge region viruses attenuated infection of mosquito tissues outside the midgut. Importantly, considering that almost all of these viruses were able to replicate as efficiently as wild type in C6/36 cells, the IT inoculation results provide evidence that C6/36 cells are not a complete surrogate for DENV replication in mosquitoes.Item Open Access Pathogenesis and immunity of rabies virus infection in bats(Colorado State University. Libraries, 2007) Davis, April, author; Bowen, R. A., advisor; Blair, Carol D., advisorRabies is one of the oldest known viral diseases and, with a >99% fatality rate, it is also one of the most deadly. Although a major public health concern, human deaths due to rabies in the developed world are rare. Worldwide, statistics are very different as rabies kills in excess of 50,000 humans per year, most often the result of canine rabies variants. During the 1950's, canine rabies cases began decreasing in the United States as a result of vaccination efforts, followed by a decrease in human rabies cases. Rabies in insectivorous bats was first reported in the US in the 1950's and has now been reported in most North American bat species. Over the last 20 years, 92% of the human rabies cases have been the result of a bat variant. This purpose of this work was to expand our knowledge of rabies variants associated with silver hair bats, Mexican free-tailed bats, and big brown bats. The goal of the first study was to characterize disease progression between two closely related big brown bat rabies variants. The data from this experiment indicated that changes in the rabies virus genome may have profound effects on infectivity and virulence. The second study was designed to determine if rabies virus could be transmitted to bats through the aerosol route. Outbred mice and two species of bats were exposed to three variants of rabies virus by aerosol exposure. Although all bats survived the aerosol exposure, 44% of the mice died of rabies. Following exposure, anti-rabies virus neutralizing antibodies were demonstrated in all bats. Following an intramuscular inoculation of rabies virus six months after the aerosol exposure, the number of seropositive bats that developed rabies was equal to the control bats. The third study examined the dynamics of rabies virus infection in bats from colonies in Texas and Colorado. The ability of healthy wild bats incubating rabies to transmit rabies virus was also examined. Rabies virus antigen was found in 50% of the salivary glands from rabid bats yet infectious rabies virus was isolated from less than 35% of rabid bats. Evaluation of of bats from both the field and in captive colonies demonstrated that approximately 0.5-2% of clinically healthy bats were in the incubation phase of rabies virus infection.