1. DVBD Survey for Arthropod-Borne Viruses in Uganda
B. R. Miller
We are conducting adult mosquito trapping in pristine habitats, at the human-animal interface, farms and ranchland in Uganda. Captured mosquitoes are frozen and returned to the Uganda Virus Research Institute where they will be shipped to CDC Fort Collins to be sorted by species and tested for the presence of arboviruses in Vero cell cultures. Viral isolates will be identified using NIH grouping fluids, RT-PCR and sequencing in CDC-Ft. Collins. Unknowns will be further characterized in Fort Collins, Colorado in a BLS-3 enhanced laboratory with HEPA air filtration with investigators wearing personal protective suits and respirators. Low passage virus isolates will be made available to other investigators by placing isolates in WHO Collaborating Centers. This information will be useful in determining arboviral activity in different ecological zones in Uganda and because viral isolation will be done, new viral threats will also be discovered.
Crabtree, M.B., Sang, R. and Miller, B.R. (2009). Kupe virus: Description of a new virus in the genus Nairovirus,family Bunyaviridae. Emerging Infectious Diseases 15: 147-154.
Rosemary Sang, Elizabeth Kioko, Joel Lutomiah, Marion Warigia, Caroline Ochieng, Monica O’Guinn, John S. Lee,Philemon Cheruiyot, Hellen Koka, Marvin Godsey, Robin Lindsay, Barry Miller, David, Schnabel, Robert F. Brieman, and Jason Richardson. Rift Valley fever epidemic in Kenya, 2006/2007: the entomologic investigations. Am. J. Trop.Med. Hyg: 83 (Suppl 2), 2010, pp. 28 37doi:10.4269/ajtmh.2010.09-0319.
Crabtree, M.B., Nga, P.T. and Miller, B.R. 2009. Isolation and characterization of a new mosquito flavivirus, QuangBinh virus, from Vietnam. Arch. Virol. 154:857–860.
Crabtree, M.B., Sang, R., Lutomiah, J., Richardson, J. and Miller B.R. 2009. Arbovirus surveillance of mosquitoescollected at sites of active Rift Valley fever virus transmission: Kenya, 2006-2007. J. Med Entomol. 46:961-964.
2. DVBD Systematic Studies on Mosquito Vectors of Arboviruses
H. M. Savage
Arboviruses that cause human disease, e.g. St. Louis encephalitis, West Nile, Japanese encephalitis, and Rift Valley fever viruses, are transmitted in natural cycles that involve various vertebrate hosts and mosquito vectors of the genera Culex and Aedes s.l. Each virus is transmitted by a limited number of vector species. Precision in species identification is essential to evaluate vector status, and to elucidate transmission cycles. Research projects that integrate morphological and molecular studies to address species identification and evolution are encouraged. Sequence data obtained on a variety of nuclear and mitochondrial genes may be used to develop species-specific assays, and to investigate phylogenetic relationships among vectors and related species.
Crabtree, MB, HM Savage, and BR Miller. 1995. Development of a species-diagnostic polymerase chain reaction assay for the identification of Culex vectors of St. Louis encephalitis virus based on interspecies sequence variation in ribosomal DNA spacers. Am. J. Trop. Med. Hyg. 53:105-109.
Miller, BR, MB Crabtree, and HM Savage. 1996. Phylogeny of fourteen Culex mosquito species, including the Culex pipiens complex, inferred from the internal transcribed spacers of ribosomal DNA. Insect Mol. Biol. 5:1-15.
Mukwaya, LG, JK Kayondo, MB Crabtree, HM Savage, BJ Biggerstaff and BR Miller. 2000. Genetic differentiation in the yellow fever virus vector, Aedes simpsoni complex in Africa: Sequence variation in the ribosomal DNA internal transcribed spacers of anthropophilic and non-anthropophilic populations. Insect Mol. Biol. 9:85-91.
Aspen, S, MB Crabtree, and HM Savage. 2003. Polymerase chain reaction assay identifies Culex nigripalpus: Part of an assay for molecular identification of the common Culex (Culex) mosquitoes of the eastern United States. J. Am. Mosquito Contr. Assoc. 19:115-120.
Aspen, S, and HM Savage. 2003. Polymerase chain reaction assay identifies North American members of the Culex pipiens complex based on nucleotide sequence differences in the acetylcholinesterase gene Ace.2. J. Am. Mosquito Contr. Assoc. 19:323-328.
3. DVBD Ecology of Mosquito Vectors and Transmission Dynamics of Arboviruses
H. M. Savage
Knowledge of the basic biology and ecology of mosquito vectors is essential to understanding the transmission dynamics of arboviral diseases and the development of control strategies. Research projects that integrate field, laboratory and molecular studies to address unknown or poorly understood aspects of mosquito biology and arbovirus transmission are encouraged.
Apperson, C.S., H.K. Hassan, B.A. Harrison, H.M. Savage, S.E. Aspen, A. Farajollahi, W. Crans, T. J. Daniels, R.C. Falco, M. Benedict, M. Anderson, L. McMillen and T.R. Unnasch. 2004. Host feeding patterns of established and potential mosquito vectors of West Nile virus in the Eastern United States. Journal of
Vector-Borne and Zoonotic Diseases 4:71-82.
Fyodorova, M.V., H.M. Savage, J.V. Lopatina, T.A. Bulgakova, A.V. Ivanitsky, O.V. Platonova, and A.E. Platonov. 2006. Evaluation of potential West Nile virus vectors in Volgograd Region, Russia, 2003 (Diptera: Culicidae): Species composition, bloodmeal host utilization, and virus infection rates of mosquitoes. Journal of Medical Entomology 43:552-563.
Savage, H.M., M. Anderson, E. Gordon, L. McMillen, L. Colton, D. Charnetzky, M. Delorey, S. Aspen, K. Burkhalter, B.J. Biggerstaff and M.Godsey. 2006. Oviposition activity patterns and West Nile virus infection rates for members of the Culex pipiens complex at different habitat types within the hybrid zone, Shelby County, TN, 2002 (Diptera: Culicidae). Journal of Medical Entomology 43:1227-1238.
Savage, H.M., D. Aggarwal, C.S. Apperson, C.R. Katholi, E. Gordon, H.K. Hassan, M. Anderson, D. Charnetzky, L. McMillen, E.A. Unnasch, and T.R. Unnasch. 2007. Host choice and West Nile virus infection rates in blood-fed mosquitoes, including members of the Culex pipiens complex, from Memphis and Shelby County, Tennessee, 2002-2003. Journal of Vector-Borne and Zoonotic Diseases 7:365-386.
4. DVBD Molecular Epidemiology, Pathogenesis, and Transmission of Alphaviruses Using Infectious Clone Technology
Numerous alphaviruses have recently been implicated as significant human pathogens during large outbreaks while others have been designated as potential bioterrorism agents. For example, Chikungunya virus has caused a massive epidemic over the past 4 years that has resulted in over 2 million infections. Further basic, clinical, and translational research is needed to further prepare for epidemics and to develop control measures. Our lab utilizes existing and novel infectious clones of alphaviruses (particularly chikungunya; o'nyong nyong; and western, eastern, and Venezuelan equine encephalitis viruses) to gain increased knowledge of alphavirus virulence determinants and epidemiologic patterns. Potential projects include identification of elements of alphaviral genomes that correlate with virulence, development of novel assays and reagents to allow detection of altered or previously unidentified genotypes, characterization of molecular approaches to distinguish virulent versus non-virulent strains of alphaviruses, studies of immune response to alphavirus infection, and studies of virus/vector interactions using the modified infectious clones.
Kariuki Njenga, M., Nderitu, L., Ledermann, J.P., Ndirangu A., Logue, C.H., Kelly, C.H.L., Sang, R., Sergon, K., Breiman, R. and Powers, A.M. 2008. Tracking Epidemic Chikungunya Virus Into The Indian Ocean From East Africa. Journal of General Virology. In Press.Powers, A.M. & Logue, C.H. 2007. The changing patterns of chikungunya virus – reemergence of a zoonotic arbovirus. Journal of General Virology. 88(9): 2363-2377.
Roberts, B.A., Teehee, M., Kelly, C.H.L., Raetz, J.L., Baker, D.L., Powers, A.M., Bowen, R.A., Fine, D.L. 2007. Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity, and efficacy in horses. Vaccine. 25:1868-1876.
Rosemary Sang, Ouledi Ahmed, Ousmane Faye, Cindy L.H. Kelly, Ali Ahaya, Kibet Sergon, Jennifer Brown, Agata, Allerangar, Muliva, Ball, Robert Breiman, Barry R. Miller, and Ann M. Powers. 2007. Epidemic Chikungunya Virus In The Union Of The Comoros, 2005. II. Entomologic Investigations. American Journal of Tropical Medicine and Hygiene. 78(1):77–82
Myles, K.M., Kelly, C.H.L., Ledermann, J.P., and Powers, A.M. 2006. Effects of an opal termination codon preceding the nsP4 gene sequence in the o'nyong nyong virus genome on Anopheles gambiae infectivity. Journal of Virology. 80(10):4992-4997.
5. DVBD: Molecular Virology, Immunology, Pathogenesis, and Vaccine Development of Flaviviruses
C.Y.H Huang (aka C.Y.H. Kinney)
This lab focuses on research and vaccine development targeted to medically important flavivirses that cause significant human diseases worldwide. These human pathogens include 4 different dengue viruses, West Nile virus, yellow fever virus, and Japanese encephalitis virus. Our lab has pioneered the engineering of numerous full-length infectious cDNA clones for various flaviviruses, and they have been widely distributed to research collaborators in and outside of DVBD. We conduct systematic genetic and phenotypic studies of recombinant mutant viruses derived by site-directed mutagenesis of these cDNA infectious clones. We have identified critical viral genetic and protein determinants that are important in virus infection/replication, virus virulence, viral-host interaction, mosquito vector competence, and immunogenicity. We also rationally designed and developed recombinant attenuated vaccine viruses for multiple flaviviruses, and some of them are currently tested in human trials with commercial partner. In addition, we are interested in antivirals, assay development for vaccine and basic researches, and methods for efficient production and purification of viral reagents (e.g. whole viral particles and proteins). Research proposals are invited in (1) identification of molecular biological determinants or functional domains encoded by viral genomes in areas described above, (2) novel research methodology/system development or improved virus reagent production, (3) improvement of vaccine efficiency by new vaccination strategy, (4) and investigation of viral genetic targets for antivirals.
Wicker JA, Whiteman MC, Beasley DWC, Davis T, McGee CE, Lee JC, Higgs S, Kinney RM, Huang CYH, Barrett ADT (2012). Mutational analysis of the West Nile virus NS4B protein. Virology 426:22-33
Butrapet S, Childers T, Moss KJ, Erb SM, Luy BE, Calvert A, Blair CD, Roehrig JT, Huang CYH (2011) Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion. Virology 413, 118-127.
Brault AC,Kinney RM, Maharaj PD, Green ENG, Reisen WK, Huang CYH (2011). Replication of the PDK-53 dengue 2 virus vaccine candidate in Aedes aegypti is modulated by a mutation in the 5’ untranslated region and amino acid substitutions in nonstructural proteins 1 and 3. Vector-Borne and Zoonotic Dis. 11, 683-689
Osorio JE, Brewoo JN, Silengo SJ, Arguello J, Moldovan IR, Tary-Lehmann M, Powell TD, Livengood JA, Kinney RM, Huang CYH, Stinchcomb DT (2011). Efficacy of a Tetravalent Chimeric Dengue Vaccine (DENVax) in Cynomolgus Macaques. Am. J. Trop. Med. Hyg 84, 978-987.
Erb SM, Butrapet S, Moss KJ, Luy BE, Childers T, Calvert AE, Silengo SJ, Roehrig JT, Huang CYH, Blair CD (2010). Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes. Virology 406, 328-335.
Huang CYH., Butrapet S, Moss KJ, Childers T, Erb SM, Calvert AE, Silengo SJ, Kinney RM, Blair CD, Roehrig JT (2010). The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion. Virology 396, 305-315.
Stein DA, Huang CYH, Silengo S, Amantana A, Crumley S, Blouch R, Iversen PL, Kinney RM (2008). Treatment of AG129 Mice with Antisense Morpholino Oligomers Increases Survival Time Following Challenge with Dengue 2 Virus. J. Antimicrob. Chemother. 62: 555-565
Butrapet S, Kinney RM, Huang CYH (2006). Determining genetic stabilities of chimeric dengue vaccine candidates based on dengue 2 PDK-53 virus by sequencing and quantitative TaqMAMA. J. Virol. Methods 131, 1-9
Kinney RM, Huang CYH, Whiteman M, Bowen RA, Langevin SA, Miller BR, Brault AC (2006). Avian Virulence and thermostable replication of the North American West Nile viral strain. J. Gen. Virol. 87: 3611-3622.
Huang CYH, Butrapet S,Tsuchiya KR, Bhamarapravati N, Gubler DJ, Kinney RM (2003). Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. J. Virol. 77,11436-11447.
Huang CYH, Butrapet S,Tsuchiya KR, Bhamarapravati N, Gubler DJ, Kinney RM (2003). Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. J. Virol. 77,11436-11447.
6. DVBD The role of NS1 during a dengue infection with regard to disease pathogenesis
Dengue is a complex of four antigenically distinct arthropod-borne viruses belonging to the Flavivirus family. More than 2.5 billion people are at risk for dengue infection, with 50 million infections occurring annually and over 100 countries involved. At least 500,000 people are hospitalized annually for dengue hemorrhagic fever, a more severe form of the disease, with fatality rates exceeding 20% in the absence of appropriate treatment. A recent increase in hyperendemicity (co-circulation of multiple dengue serotypes) has correlated with increased frequencies of dengue hemorrhagic fever (DHF) and the serious dengue shock syndrome (DSS) that may follow, presumably brought about by immune enhancement.
Dengue-associated deaths are usually linked to the more severe form of the disease known as DHF or DSS. Although no vaccines or drugs are available, severe disease can be successfully managed by careful monitoring of warning signs and early initiation of aggressive intravenous rehydration therapy. Hence, early diagnosis saves lives. Since NS1 is secreted in the blood during the early phases of infection, it is an ideal target for the development of a diagnostic tool. Additionally, previous characterization of NS1 indicated that this viral antigen may be a marker for severe disease and could be used as a predictor of viremia levels in infected individuals. The role of NS1 during a dengue infection with regard to disease pathogenesis is not well understood.
The purpose of this research is to 1) develop a serotype-specific assay for the detection of dengue virus NS1 in human serum and to 2) understand the role of NS1 in dengue pathogenesis. To achieve the first goal, peptide antisera specific for NS1 proteins produced by each of the four dengue serotypes will be generated and incorporated into an immunobased assay system for detection of dengue NS1 in clinical samples. The results obtained in this study will contribute to the development of antibodies that are specific for NS1 proteins produced by each of the four dengue virus serotypes. In order to understand the role of NS1 in pathogenesis, mechanistic assays were established to determine the cellular role of NS1 in culture and we are developing an in vitro model to determine the mechanism of exogenous NS1 on viral replication for all DEN viruses. Previous studies have determined that exogenous NS1 mediated enhancement of infection (only demonstrated with DEN-1) and that differences in NS1 glycosylation may play a role in pathogenesis. These studies provide the basic research understanding that would eventually have utility for applied prevention public health programs for dengue.
7. DVBD Dengue Branch, Molecular Diagnostics and Research Laboratory
Our laboratory is a common ground to facilitate interactions with scientists and generate national and international, collaborative projects. These interactions stimulate the development and evaluation of new technologies to improve dengue detection and surveillance, and the execution of research to better understand viral pathogenesis or dissemination in humans and mosquito vectors. Our studies encompass 3 mayor areas: 1- Dengue surveillance in Puerto Rico and Latin America: We conduct dengue surveillance in Puerto Rico, the Caribbean and Latin American countries. Through this work, we have learned that the four serotypes are endemic in the area and rapidly disseminate across the region, showing complex patterns of transmission and evolution. Research is conducted to better understand severity of disease and outbreak potential of the strains collected over a large geographical and temporal coverage at high-resolution. 2- Development and Evaluation: Our current understanding about the prevalence of dengue in the region is constrained within our ability to detect the virus. In addition, the reported dissemination of West Nile and its recent detection in Puerto Rico exemplify a rapidly evolving panorama. We are currently working to increase the level of sensitivity of our detection systems and evaluating mass spectroscopy and Microarray technologies. 3- Research in Viral Antagonism: Flavivirus infections cannot be cured with interferon therapy. Infections by these viruses stimulate a strong interferon response; but the virus can block the antiviral effects and remain one step ahead of the host ability to control infection. Our studies are aimed to better understand dengue and West Nile mechanisms involved in interferon antagonism. These studies include analysis of circulating strains of dengue which have been linked to epidemiological and clinical data. Other areas of viral antagonism include mosquito adaptation and dissemination.
Sariol CA, Munoz-Jordán JL, Rosado L, Pantoja P, Abel K, Giavedoni L, Rodriguez IV, White LJ, Martinez M, Arana T, Kraiselburd EN (2007) Transcriptional activation of Interferon stimulated genes but not of cytokine genes after primary infection of rhesus macaques with dengue virus type 1.Journal of Vaccine Immunology, Clinical and Vaccine Immunology 14: 756-766.
Ayala A, Rivera A, Johansson M, Muñoz-Jordán JL (2006) Travel-Associated Dengue - United States, 2005; MMWR 55: 700-702.
Beatty ME, Vorndam V, Hunsperger EA, Muñoz-Jordán JL (2005), Travel - Associated Dengue Infections - United States, 2001-2004 MMWR 54: 556-558.
Puig-Basagoiti F, Tilgner M, Bennett CJ, Zhou Y, Munoz-Jordán JL (2006) Garcia-Sastre A, Bernard KA, Shi PY. A mouse cell-adapted NS4B mutation attenuates West Nile virus RNA synthesis. Virology 361: 229–241.
Munoz-Jordan J, Laurent-Rolle M, Ashour J, Martinez-Sobrido L, Ashok M, Lipkin WI, Garcia-Sastre A. (2005) Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses. Journal of Virology 79: 8004-13.
Muñoz-Jordán J, Sánchez-Burgos GG, Laurent-Rolle M, García-Sastre A (2003) Inhibition of interferon signaling by dengue virus. PNAS SPELL out. 100: 14333-14338.
8. DVBD Arboviral Molecular Determinants of Pathogenesis
Aaron C. Brault
Most arboviruses have genomes comprised of RNA genomes that are highly mutable and capable of rapidly generating large population sizes, making these viruses exceedingly proficient at rapidly evolving into new ecological niches. Identifying the mechanisms by which arboviruses (alphaviruses and flaviviruses) emerge to initiate disease outbreaks will be critical for predicting future arboviral outbreaks. Our laboratory utilizes a combination of molecular systematic and reverse genetic system approaches for the specific identification of mutations associated with the epizootic emergence of arboviruses. For example, a single amino acid substitution within the helicase protein of West Nile virus (WNV; flavivirus) has been demonstrated to result in an approximate million-fold increase in replication efficiency in certain avian species. This mutation has also been identified through positive selection modeling to be under strong selective pressure, indicating the potential importance of avian replication for WNV emergence. Additional studies are currently underway to investigate vertebrate and invertebrate pathogenesis by using novel methodologies for restricting viral replication in a tissue-dependent manner. Potential projects are welcomed that seek to identify virally encoded determinants of vector competence, arboviral pathogenesis, novel methods for pathogen detection and viral attenuation mechanisms for the development of live-attenuated arboviral vaccine candidates.
Coffey, Lark L., Vasilakis, Nikos, Brault, Aaron C., Powers, Ann M., Tripet, Frédéric and Weaver, Scott C. 2008. Arbovirus evolution in vivo is constrained by host alternation. Proceedings of the National Academy of Sciences 105: 6970-6975.
Reisen, William K., Fang, Ying and Brault, Aaron C. 2008. Limited interdecadal variation in mosquito (Diptera: Culicidae) and avian host competence for western equine encephalomyelitis virus (Togaviridae, Alphavirus). The American Journal of Tropical Medicine and Hygiene 78(4) 681-686.
Brault, Aaron C., Huang, C.Y.-H., Langevin, Stan A., Kinney, Richard M., Bowen, R.A., Ramey, W.N., Panella, Nicholas A., Holmes, Edward C., Powers, Ann M. and Miller, Barry R. 2007. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nature Genetics 39 (9) 1162-1166.
Kinney, Richard M., Huang, Claire Y.–H, Whitman, Melissa, Bowen, Richard A., Langevin, Stanley A., Miller, Barry R. and Brault, Aaron C. 2006. Avian virulence and thermostable replication of the North American strain of West Nile virus. Journal of General Virology 87 (12) 3611-3622.
Brault, Aaron C., Langevin, Stanley A., Bowen, Panella, Nicholas A., Richard A., Biggerstaff, Brad J., Miller, Barry R. and Komar, Nicholas 2004. Differential virulence of West Nile viral strains for American crows (Corvus brachyrhynchos). Emerging Infectious Diseases 10(12) 2161-2168.
9. DVBD Ecological Dynamics of Dengue Vectors in Puerto Rico
Aedes aegypti is the main dengue vector worldwide because of its close association with humans in tropical and sub-tropical urbanized areas. This mosquito encounters other invasive or native container mosquitoes (mostly treehole mosquitoes) with similar requirements of aquatic habitats for its immature development (natural and artificial containers) in some parts of the world. Examples of other container mosquitoes that overlap in aquatic habitat requirements are Aedes triseriatus in eastern North America, Ae. albopictus in Asia, Africa and the Americas, Ae. polynesiensis in the South Pacific, and Aedes mediovittatus in the Caribbean. Aedes mediovittatus is a competent vector of dengue viruses, with a high rate of vertical transmission for all dengue serotypes. Also, it has been proposed that this mosquito can act as a reservoir in the maintenance of dengue viruses in Puerto Rico during inter-epidemic periods in rural areas with low human population densities. The extent to which Ae. mediovittatus overlaps with Ae. aegypti, dengue viruses, and humans in urban areas is been determined. We have found that both species extensively overlap in sub suburban and rural areas. However, the importance of biological interactions (e.g., competition) between Ae. aegypti and Ae. mediovittatus has not yet been investigated. Previous studies have indicated that biotic interactions among larvae of Ae. aegypti and Ae. albopictus may be largely responsible for the replacement of the former mosquito species by the latter in the US. The proposed study involves conducting experiments in the laboratory and the field on the effects of environmental factors (e.g., food, temperature) on the nature and intensity of the interactions between immature Ae. aegypti and Ae. mediovittatus and assessing the effects of interactions on traits that might modify vector competence (e.g., size, fecundity).
Barrera R, 1996. Competition and resistance to starvation in larvae of container-inhabiting Aedes mosquitoes. Ecological Entomology 21: 117–127.
Barrera R. 2009. Simplified Aedes aegypti’s pupal-surveys for entomological surveillance and dengue control. American Journal of Tropical Medicine and Hygiene 81: 100-107.
Barrera R, Amador M, Diaz A. Smith J, Munoz-Jordan JL, Rosario Y. 2008. Unusual productivity of Aedes aegypti in septic tanks and its implications for dengue control. Medical and Veterinary Entomology 22:62-69
Bracks MAH, Honório NA, Lounibos LP, Lourenço-de-Oliveira R, Juliano SA, 2004. Interspecific competition between two invasive species of container mosquitoes in Brazil. Annals of the Entomological Society of America 97: 130–139.
Cox J, Grillet ME, Ramos OM, Amador M, Barrera R. 2007. Habitat segregation of dengue vectors along an urban environmental gradient. American Journal of Tropical Medicine and Hygiene 76: 820-826.
Lounibos LP, Suarez S, Menéndez Z, Nishimura N, Escher RL, O’Connell SM, Rey JR, 2002. Does temperature affect the outcome of larval competition between Aedes aegypti and Aedes albopictus? Journal of Vector Ecology 27: 86–95.
Smith J, Amador M, BARRERA R. 2009. Seasonal and habitat effects on dengue and West Nile virus vectors in San Juan, Puerto Rico. Journal of of the American Mosquito Control Association 25: 38-46.
10. DVBD Global Spread of Arboviral Diseases
M. A. Johansson
Global travel is increasingly associated with the spread of infectious diseases. Predicting the spread of arboviruses is complicated by the complex dynamics of vector populations and human-vector interactions. We have constructed a novel mathematical model to assess the potential for spread of yellow fever virus. The work included statistical modeling of key infection parameters and development of dynamic models for travel, virus transmission, and vector population dynamics. Future work will develop similar models for other pathogens and assess and compare potential interventions. We are particularly interested in developing models which can be used to inform policy.
Johansson, MA, N Arana-Vizcarrondo, BJ Biggerstaff, JE Staples, N Gallagher, & N Marano. (2011) On the treatment of airline travelers in mathematical models. PLoS One. 6(7): e22151.
Johansson, MA, N Arana-Vizcarrondo, BJ Biggerstaff, & JE Staples. (2010) The incubation periods of yellow fever. American Journal of Tropical Medicine and Hygiene. 83(1):183-188.
(In review) Johansson, MA, N Arana-Vizcarrondo, BJ Biggerstaff, N Gallagher, N Marano, & JE Staples. Assessing the risk of international spread of yellow fever virus: A mathematical analysis of an urban outbreak in Asunción, 2008.
11. DVBD Dengue Virus Transmission Dynamics
M. A. Johansson
Dengue is the most wide-spread arboviral disease in the world and its prevalence is increasing. To plan more effective large-scale interventions it is essential to understand the factors underlying dengue virus transmission. We develop large-scale analyses to identify and understand global patterns of incidence. Past studies have assessed the role of climate and weather on different spatial and temporal scales. Future efforts will build upon that work and investigate other potentially important determinants such as serotype interactions at the population level, socio-economic status, human mobility, population density, and vector control efforts. Approaches may include both statistical and mathematical modeling.
Johansson MA, Dominici F, & Glass GE. (2009) Local and Global Effects of Climate on Dengue Transmission in Puerto Rico. PLoS Neglected Tropical Diseases. 3(2): e382.
Johansson MA, Cummings DAT, & Glass GE. (2009) Multiyear Climate Variability and Dengue—El Niño Southern Oscillation, Weather, and Dengue Incidence in Puerto Rico, Mexico, and Thailand: A Longitudinal Data Analysis. PLoS Medicine. 6(11): e1000168.
12. DVBD Parameters Affecting Arbovirus Transmission Dynamics
J P Mutebi
Arboviruses are not uniformly distributed throughout their distribution ranges and numerous studies have described demographic and other physical factors associated with high arboviral activity in human populations. However, there is very little information on the biotic factors associated with high and/or low arboviral activity. Our lab focuses on studying underlying biotic factors that influence arboviral distribution patterns especially over short distances where climatic factors remain relatively constant. We integrate studies on vector biology, vector genetics and population genetics, viral genetics and molecular epidemiology to understand the epidemiology of arboviral diseases. The ultimate goal is to utilize this information in planning and/or improving surveillance programs for arboviral diseases by targeting specific biotic factors associated with elevated arboviral activity.
Mutebi JP, Lubelczyk C, Eisen R, Panella N, MacMillan K, Godsey M, Swope B, Young G, Smith Jr RP, Kantar L, Robinson S, Sears S. Using Wild White-Tailed Deer to Track Eastern Equine Encephalitis virus in Maine. Vector-Borne and Zoonotic Diseases, 2011, Jul 7 (Epub ahead of print)
Gibney KB, Pelletier AR, Robinson S, Mutebi JP, Nett, RJ, Staples JE, Fischer M. Eastern Equine Encephalitis: An Emerging Arboviral Disease Threat — Maine, 2009. Vector-Borne and Zoonotic Diseases, 2011, 11(6):637-639.
Mutebi JP, Jones RC, Plate DK, Gerber SI, Gibbs K, Sun G, Cohen NJ, Delorey MJ and Paul WS. The Impact of Spraying on Mosquito Density in Chicago, 2005. Journal of the American Mosquito Control Association, 2011, 27(1): 69-76.
Jones RC, Weaver KN, Smith S, Blanco C, Flores C., Gibbs K, Markowski D., Plate D, Mutebi JP, Use of the Vector Index and Spatial Analysis to Prospectively Estimate and Delineate Areas of West Nile Virus Transmission Risk in an Urban Jurisdiction. Journal of the American Mosquito Control Association, 2011, 27(3):315-319.
Mutebi, J. -P., René C. A. Rijnbrand, Heinman, Kate D. Ryman, Eryu Wang, Lynda D. Fulop, Richard W. Titball, and Alan D. T. Barrett. Genetic relationships and evolution of genotype of yellow fever virus and other members of the yellow fever virus group within the Flavivirus genus. Journal of Virology 2004, 78(18): 9652-65.
Mutebi, J. -P., A. Gianelli, A. Travossos, R. B. Tesh, A. D. T. Barrett and S. Higgs. Infectivity of YFV for Bolivian Aedes aegypti. Emerging Infectious Diseases, 2004 10(9): 1657-1660.
Milleron, R., J. -P. Mutebi and G. C. Lanzaro. Antigenic diversity in Maxadillan, a salivary protein from the sand fly vector of American Visceral Leishmaniasis. American Journal for Tropical Medicine Hygiene. 2004 70(3): 286-93.
McAthur, M., M. Suderman, J. -P. Mutebi, and A. D. T. Barrett. Molecular characterization of ahamster viscerotropic strain of yellow fever virus. Journal of Virology 2003, 77(2) 1494-1502.
Mutebi, J. -P., H. Wang, Li Li, J. E. Bryant and A. D. T. Barrett. Nucleotide sequence variation ofthe prM/E region of yellow fever virus identifies four distinct genotypes in Africa. Journal of Virology 2001, 75(15) 699-708.
13. DVBD Dengue Branch, Molecular Diagnostics and Research Laboratory
Our laboratory conducts dengue research in three major areas: Molecular Epidemiology, Antiviral Response to Dengue virus Infection, and Improved Molecular Diagnostics.
1- Molecular Epidemiology: The four dengue virus (DENV) serotypes are endemic in Puerto Rico and rapidly disseminate across the region, showing complex patterns of transmission and evolution. Sequencing and phylogenetic studies are conducted to better understand the molecular determinants of the long-term persistence and outbreak potential of the DENV strains. Our results show that (1) each DENV serotype evolves through periods of high and low transmission in co-existence with other serotypes, (2) virus lineages spread across the island through geographic corridors in response to important epidemiological changes, and (3) viruses find long-term refuge in geographic areas after several years of high transmission. Research to further define the viral and epidemiologic reasons for these transmission patterns may aid our efforts to prevent the spread and re-emergence of dengue in endemic areas.
2- Antiviral Response: Because dengue illness presents before a full protective antibody response is developed, our ability to eliminate the virus depends to some extend on the innate immune response. However, the virus can block the antiviral effects of interferon and remains one step ahead of the host’s ability to control infection. We know that two nonstructural proteins (NS4B and NS5) block the Jak/Stat pathway; but the precise interactions between virus and host proteins are not known. In addition, microarray studies have shown that the strong interferon response seen in patients with mild dengue illness is weakened in patients with hemorrhagic dengue or dengue shock. Our studies aim to (a) further study the interactions of NS4B and NS5 with the interferon pathway, (b) analyze the ability of currently circulating dengue strains to block interferon and stimulate T-cell response, and (c) study the expression of interferon-stimulated genes in dengue patients. This knowledge would contribute to development of specific antivirals against dengue, detect less pathogenic strains for vaccine development/improvement, and detect specific markers of disease severity useful in disease prognosis.
3- Improved Molecular Diagnostics: Most dengue patients present with symptoms when they are viremic and prior to developing a full immune response; therefore, our ability to diagnose this disease relies on detection of DENV through molecular diagnostics. However, molecular diagnostic tests are not approved in the US for commercial use. We have worked on increasing the level of sensitivity of our RT-PCR assays, evaluating high throughput and point of care diagnostic options for these assays, and conducting pre-market evaluations. These assays can positively identify 85% of confirmed cases presenting with symptoms during the first days of symptoms. Research in this area would be directed towards development of new or improved diagnostic assays and studies to validate diagnostic test algorithms.
Munoz-Jordan JL: Subversion of interferon by dengue virus. Curr Top Microbiol Immunol 2010, 338:35-44.
Munoz-Jordan JL, Bosch I: Modulation of the antiviral response by dengue virus. Frontiers in Dengue Research, Caister Academic Press, Norfolk, UK 2010:121-140.
Munoz-Jordan JL, Fredericksen B: How flaviviruses activate and suppress the interferon response. Viruses 2010, 2:676-691.
McElroy K, Henn M, Santiago G, Lennon N, Munoz-Jordan JL: Long-term dominance of dengue virus Type II Puerto Rican lineages throughout Major Epidemiological Changes, Emerging Infectious Diseases, Accepted for PublicationTomashek KM, Rivera A, Munoz-Jordan JL, Hunsperger E, Santiago L, Padro O, Garcia E, Sun W: Description of a large island-wide outbreak of dengue in Puerto Rico, 2007. Am J Trop
Med Hyg 2009, 81:467 -474.
Munoz-Jordan JL, Collins CS, Vergne E, Santiago GA, Petersen L, Sun W, Linnen JM: Highly sensitive detection of dengue virus nucleic acid in samples from clinically ill patients. J Clin Microbiol 2009, 47:927-931.
Munoz-Jordan JL, Sanchez-Burgos GG, Laurent-Rolle M, Garcia-Sastre A. Inhibition of interferon signaling by dengue virus. Proc Natl Acad Sci U S A 2003;100(24):14333-8.
Chesler DA, Munoz-Jordan JL, Donelan N, Garcia-Sastre A, Reiss CS. PKR is not required for interferon-gamma inhibition of VSV replication in neurons. Viral Immunol 2003;16(1):87-96.
Vernal J, Munoz-Jordan JL, Muller M, Cazzulo JJ, Nowicki C. Sequencing and heterologous expression of a cytosolic-type malate dehydrogenase of Trypanosoma brucei. Mol Biochem Parasitol 2001;117(2):217-21.
Munoz-Jordan JL, Cross GA, de Lange T, Griffith JD. t-loops at trypanosome telomeres. Embo J 2001;20(3):579-88.
Munoz-Jordan JL, Cross GA. Telomere shortening and cell cycle arrest in Trypanosoma brucei expressing human telomeric repeat factor TRF1. Mol Biochem Parasitol 2001;114(2):169-81.
Munoz-Jordan JL, Davies KP, Cross GA. Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei. Science 1996; 272(5269):1795-7.
14. DVBD Dengue Virus NS1 Research
Dengue is a complex of four antigenically distinct arthropod-borne viruses belonging to the Flavivirus family. It is one of the most important vector-borne diseases with more than 2.5 billion people at risk for dengue infection and 50 million infections occurring annually in over 100 tropical and sub-tropical countries. At least 500,000 people are hospitalized annually for DHF, a more severe form of the disease, with fatality rates exceeding 5% in the absence of appropriate treatment. A recent increase in hyperendemicity (co-circulation of multiple dengue serotypes) has correlated with increased frequencies of DHF and the serious dengue shock syndrome (DSS). Dengue is hyperendemic in Puerto Rico where yearly transmission occurs. The global epidemiology and the dynamics of transmission of dengue viruses have changed dramatically during the past decade due to an expansion of the geographical distribution of mosquito vector, Aedes sp., and urbanization and modern transportation. The responsibility of the Dengue Branch is to understand the pathogenesis of dengue and implement improved diagnostic tools. Conventional early diagnostic techniques for DENV infection rely on assays to detect virus including viral isolation and/or RT-PCR testing. Both of these techniques require sophisticated equipment and training which poses limitations for early diagnosis in most dengue endemic countries, most of which are resource poor. Immunoassays that detect anti-DENV IgM are more widely used for dengue diagnosis than virus isolation or RT-PCR however often requires paired serum samples. IgM antibodies are not present until five days post-onset of symptoms which is often too late for clinical management and intervention purposes. Although clinical warning signs can alert a physician that a patient is progressing into severe dengue, none of the current laboratory diagnostic techniques can determine whether a patient will progress to a more severe form of dengue (DHF or DSS) early in the infection prior to clinical warning signs. Since NS1 is secreted in the blood during the early and late phases of infection, it is an ideal target for the development of a single sample diagnostic test. Additionally, previous characterization of NS1 indicates that this viral antigen may be a marker of severe dengue disease (Libraty et al., 2002). The role of NS1 during a dengue infection with regard to disease pathogenesis is not well understood (for review, see Alcon-LePoder et al., 2006).
The purpose of this study is to 1) develop a serotype-specific enzyme-linked immunoassay assay (ELISA) for the detection of dengue virus NS1 in human serum and to 2) understand the role of NS1 in dengue pathogenesis. To achieve the first goal, peptide antisera specific for NS1 proteins produced by each of the four dengue serotypes will be generated and incorporated into an ELISA for detection of dengue NS1 in clinical samples. The results obtained from this study will contribute to the development of antibodies that are specific for NS1 proteins produced by each of the four dengue virus serotypes. In order to understand the role of NS1 in pathogenesis, the fellow will establish mechanistic assays to determine the cellular role of NS1 in culture and develop an in vitro model to determine the mechanism of exogenous NS1 in the augmentation of viral replication for all four DENV serotypes. Previous studies have determined that exogenous NS1 mediated enhancement of infection (only demonstrated with DENV-1) and that differences in NS1 glycosylation may play a role in pathogenesis (Crabtree et al., 2004) however the mechanism of this observation and its implication in human disease is unknown. These studies provide the basic research understanding that would eventually have utility for applied prevention programs for dengue.