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- 1. Virulencia del Virus Dengue en la evolución del Dengue 2 en Nicaragua de 2005-2007. Andrea Núñez, MSc.
- 3. Virus Dengue Genoma del ARN Poliproteína precursora ANCH C - PrM- E- NS1-NS2a-NS2b-NS3- NS4a-NS4b- NS5 C M E NS1 NS2a NS2b NS3 NS4a NS5 Estructurales No estructurales NS4b
- 7. FIS Estudio Hospitalario Muestras Longitudinales 3 meses 6 meses 12 meses 18 meses Muestra aguda Muestra convaleciente 2 semanas Enrolados al presentarse HIMJR
- 8. Estudio Cohorte Muestra anual Año 1 Año 2 Año 3 Año 4 Muestra convaleciente FIS Muestra Aguda
- 9. 2005 2006 2007 0 10 20 30 40 50 60 70 80 90 % Total Cases FD FDH/SCD Incremento en la severidad del Dengue en 2006/2007 en ambos estudios Estudio Hospitalario sintomática:inaparente 1:4.3 1:2.6 Estudio Cohorte 0% 0% 8% 15% 0% 2% 4% 6% 8% 10% 12% 14% 16% 2004 2005 2006 2007 % DHF/DSS p<0.001 4:1 0.7:1 1:1
- 10. Incremento en la severidad no se explica por la introducción de un nuevo serotipo Estudio Hospitalario 2005 DENV1 DENV2 DENV3 DENV4 2006 2007
- 13. Que habrá ocurrido ? Qué diferencia existe entre los años?
- 15. Secuencia del genoma completo del virus ( Broad NIAID Microbial Sequencing Center ) 163 cepas del serotipo 2 del dengue Hubo cambios en la secuencia viral entre estos años? Nicaragua Broad STRUCTURAL NON-STRUCTURAL
- 17. Nicaragua “ Asia Americano” “ Asiático” “ Americano” Cepas Nicaraguense más relacionados al genotipo asiático americano DENV2
- 18. DENV-2 Nicaragua: Rápido reemplazo de los clades Asiático Americano/Asiático NI-1a NI-2a NI-2b Nicaraguense Año de aislamiento y Clade significantemente relacionados (Pearson’s Chi-Square p<0.0001) % Genomes Year
- 19. Correlación con enfermedad severa Asiático Americano/Asiático NI-1 NI-2a NI-2b Correlación con enfermedad severa (DF/DHF/DSS) Pearson’s Chi-Square p=0.046 Nicaragua 2 1 DHF/DSS DF Cohorte Hospital
- 20. NI-2a NI-2b/c NI-1a 9 Amino Acidos propuestos para el reemplazamiento de Clade Cap:K97R NS1:R869K NS3:T1720P NS4:N2488S E:M772V NS1:L1054F NS5:K2691Q NS5:T2781I NS5:R2892K
- 21. Env Mukhopadhyay/Rossman Nature Reviews 2005 La mutación E es localizada en C-terminal E M772V
- 22. Tres mutaciones en NS5 están expuesta a la superficie NS5
- 23. Clade 1 Clade 2 Son los Clade 2 virales mas virulentos?? (MDDC)? X 2p 4 1° human MDDCs Nicaragua 1 2b,c Clade 1 2 3 5 6 7 8 9 1 10 100 1000 10000 0 pfu/ml Clade 1 Clade 2 p = 0.0298 (Wilcoxon)
- 24. Clade 1 Clade 2 U937 /DC-SIGN Son los virus Clade 2 “inherently” more fit (U937 #1)? X 2p Nicaragua 1 2a 2b 2c Clade 0 10 20 30 40 50 33 34 37 67 4010 187 174 191 306 694 128 % Infection p = 0.056 (Mann-Whitney)
- 25. Clade 2 virus pueden replicar mejor que los Clade 1 virales. qRT-PCR 1 2a 2b/c Plaque assay 1 2a 2b/c
- 26. Ensayo Particulas Virales Reporteras Structural Proteins GFP Replicon T.C. Pierson, NIH co-transfect Single-round RVP (Particulas de Virus reportero) infect GFP+ C prM/M E C prM/M E Clade 2 Clade 1 + suero DenV2 0 50 100 150 200 250 300 350 1 2 PRNT50
- 29. Agradecimientos Centro de Salud Socrates Flores Vivas *Guillermina Kuan Oscar Ortega Miguel Reyes Leyla Saenz Nery Sanchez Sergio Ojeda Magaly Amador Zoila Orozco Carolina Flores Hospital La Mascota *Crisanta Rocha Maria de los Angeles Pérez *Sheyla Silva Javier Silva Federico Narváez Rafael Centeno Cintia Saborío Gerardo Mejia Departamento de Virolog í a, CNDR *Angel Balmaseda *Andrea Nuñez *Douglas Elizondo *Yolanda Tellez *Tangni Gomez *Yara Saboria *Magelda Montoya Leonel Pérez Juan Carlos Mercado Juan Carlos Matute Celia Machado Maria Jos é Vargas Sonia Arguello Sustainable Sciences Institute William Avilés Kate Standish Mirtha Monterrey University of CA, Berkeley Eva Harris Aubree Gordon Molly OhAinle MINSA Nicaragua Guillermo Gonzalez SILAIS Managua Maritza Cuan Dir. de Epidemiolog í a Edmundo Sánchez CNDR, MINSA Alcides González Broad Institute Matthew Henn Niall Lennon Bruce Birren
- 30. Thanks! Gracias! Funding: NIAID/NIH Pediatric Dengue Vaccine Initiative, TDR
Notas del editor
- El genoma de los virus del dengue sufre mutaciones frecuentes que explican la existencia en cada serotipo de un gran número de cepas antigénicamente diferentes circulando en áreas distintas del mundo.
- Generalidades virus dengue Explicar la estructura virus dengue
- Hospital Study - sick kids are enrolled when they show up at hospital. followed daily while in hospital. + convalescent samples. Cohort Study - healthy kids are followed by annual visit plus any febrile illness. one acute phase sample, but come in earlier.
- Hospital Study - sick kids are enrolled when they show up at hospital. followed daily while in hospital. + convalescent samples. Cohort Study - healthy kids are followed by annual visit plus any febrile illness. one acute phase sample, but come in earlier.
- Hospital Study - sick kids are enrolled when they show up at hospital. followed daily while in hospital. + convalescent samples. Cohort Study - healthy kids are followed by annual visit plus any febrile illness. one acute phase sample, but come in earlier.
- Hospital Study - sick kids are enrolled when they show up at hospital. followed daily while in hospital. + convalescent samples. Cohort Study - healthy kids are followed by annual visit plus any febrile illness. one acute phase sample, but come in earlier.
- ADD images for Balma and kulp
- Captures complete ORF and ~95% of UTRs @ 8X coverage
- Nicaraguan sequences similarly reveal a clade replacement dynamic. In Nicaragua, 3 well defined clades have circulated in the past 3 years. In contrast to Vietnam clade replacement is happening very rapidly. In 2005 clade NI-1a dominated and witin 3 years NI-2b came to dominance. Interestingly in Nicaragua, based on our current sampling, sequences appear to be genetically distinct from any previously described genotypes of DENV-2 In the next slide, I am going to zoom in on the Nicaraguan part of this tree. Note: 9-13 yrs when 1a & 2a/b split 6-9 yrs when 2a&2b split 96 strains in analysis
- Nicaraguan sequences similarly reveal a clade replacement dynamic. In Nicaragua, 3 well defined clades have circulated in the past 3 years. In contrast to Vietnam clade replacement is happening very rapidly. In 2005 clade NI-1a dominated and witin 3 years NI-2b came to dominance. Interestingly in Nicaragua, based on our current sampling, sequences appear to be genetically distinct from any previously described genotypes of DENV-2 In the next slide, I am going to zoom in on the Nicaraguan part of this tree. Note: 9-13 yrs when 1a & 2a/b split 6-9 yrs when 2a&2b split 96 strains in analysis
- Despite the large amount of nucleotide diversity in the Nicaraguan dengue population, the emergence of the three clades just described are driven by mutations and reversions in a set of 8 specific amino acids. These changes span the genome, and happen in the Capsid, Env, NS1, NS3, NS4b, and NS5 genes 5 mutations distinguish all Nicaraguan sequences from the previously described clades 4 Reversions lead to the split between group-1a and groups-2a/2b (9-13 years ago) And 3 novel mutations lead to the split between groups 2a and 2b (6-9 years ago) These three mutations are quite interesting; if you look across all available DENV genetic sequence we find that: The ENV mutation is novel to DENV-2, and has only previously been seen in DENV-3 Both of the NS5 mutations are completely novel and have never seen before in any DENV serotype. V=Valine Q=Glutamine I=Isoleucine K=Lysine R=Arginine L=Leucine T=Threonine N=asparagine P=proline S=serine
- Determination of hotspots within dengue viral genome Investigation of relation between QS/diversity and severity Gen etic diversity Over-represented sequences (“signatures”) Investigati on of QS differences (diversity and sequence) between DEN in serum vs intracellular DEN (PBMC) vs isolate QS in asymptomatic and symptomatic DEN infection