Germán Añez

Increment of interleukin 6, tumour necrosis factor alpha, nitric oxide, C-reactive protein and apoptosis in dengue

This study evaluated the levels of TNFalpha, IL-6, IL-1beta, nitric oxide (NO), CRP, C3 and apoptosis in 36 patients with dengue fever (DF), 34 patients with dengue haemorrhagic fever (DHF) and in virus-infected monocyte cultures. IL-6, TNFalpha, NO (nitrites) and CRP levels were increased and C3 diminished in patients with DF and DHF. IL-6, TNFalpha, CPR and C3 values were associated with disease severity (DHF). Nitrite content was incremented in DF patients. TNFalpha, NO and CRP levels were associated with secondary infection. IL-6 and CRP levels were associated with dengue virus type 4 (DENV-4) and DENV-2, respectively. Low levels of C3 were associated with DENV-2 and DENV-4 infections. Similarly, increased content of TNFalpha, IL-6 and nitrites were observed in supernatants from infected monocyte cultures. IL-6 was associated with DENV-4 infection. The different virus serotypes induced apoptosis in monocyte cultures. Dengue infection did not induce elevated IL-1beta production, either in patients or in infected cultures. These results suggest that TNFalpha, IL-6, NO and CRP are involved in dengue infection and that monocytes could be an important source of cytokine and NO production.

Circulation of Different Lineages of Dengue Virus Type 2 in Central America, Their Evolutionary Time-Scale and Selection Pressure Analysis

Dengue is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Each serotype is genetically distant from the others, and each has been subdivided into different genotypes based on phylogenetic analysis. The study of dengue evolution in endemic regions is important since the diagnosis is often made by nucleic acid amplification tests, which depends upon recognition of the viral genome target, and natural occurring mutations can affect the performance of these assays. Here we report for the first time a detailed study of the phylogenetic relationships of DENV-2 from Central America, and report the first fully sequenced DENV-2 strain from Guatemala. Our analysis of the envelope (E) protein and of the open reading frame of strains from Central American countries, between 1999 and 2009, revealed that at least two lineages of the American/Asian genotype of DENV-2 have recently circulated in that region. In occasions the co-circulation of these lineages may have occurred and that has been suggested to play a role in the observed increased severity of clinical cases. Our time-scale analysis indicated that the most recent common ancestor for Central American DENV-2 of the American/Asian genotype existed about 19 years ago. Finally, we report positive selection in DENV-2 from Central America in codons of the genes encoding for C, E, NS2A, NS3, and NS5 proteins. Some of these identified codons are novel findings, described for the first time for any of the DENV-2 genotypes.

Dengue in the United States of America: A Worsening Scenario?

Dengue is a febrile illness caused by any of the four dengue virus types (DENV-1 to -4, genus Flavivirus, family Flaviviridae) mainly transmitted by the mosquito Aedes aegypti. DENV can be transmitted by blood transfusion. Dengue has been historically present in the continental United States (US), in the state of Hawaii, and in the US insular territories in the Caribbean and the Pacific. During the second half of the 20th century, most of the cases reported in the US were imported cases brought to the country by travelers. Since 2009, cases of autochthonous dengue have been recognized in the state of Florida after 75 years of absence, followed by intensification of transmission in endemic places including the US territories of US Virgin Islands and Puerto Rico, which experienced a large dengue epidemic in 2010. The widespread distribution of dengue mosquito vectors, deficient mosquito control measures and increased frequency of DENV-infected visitors to the US coming from dengue-endemic locations or places experiencing epidemics appear to be jointly responsible for the emergence and reemergence of dengue in the US and its territories.

Ultrastructural studies on dengue virus type 2 infection of cultured human monocytes

BackgroundEarly interaction of dengue virus and monocyte/macrophages could be an important feature for virus dissemination after its initial entry via the mosquito vector. Since ultrastructural analysis of this interaction has not been reported, dengue type 2 (DEN2) virus-infected human monocyte cultures were studied at 1, 2, 4 and 6 hours after infection.ResultsTypical dengue particles and fuzzy coated viral particles were 35 to 42 nm and 74 to 85 nm respectively. Viruses were engulfed by phagocytosis and macropicnocytosis leading to huge vacuoles and phagosomes inside the monocytes. Interaction of monocytes with DEN2 virus induced apoptosis, characterized by nuclear condensation and fragmentation, cellular shrinkage, blebbing and budding phenomena and phagocytosis of apoptotic cells by neighboring monocytes. This finding was confirmed by TUNEL. Ultrastructural features associated to DEN2 virus replication were not observed.ConclusionThese data suggest that clearance of the virus by monocytes and cellular death are the main features during the initial interaction of DEN2 virus and monocytes and this could be important in the rapid elimination of the virus after infection by mosquito vector.

Evolutionary dynamics of West Nile virus in the United States, 1999-2011: phylogeny, selection pressure and evolutionary time-scale analysis.

West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10−4 substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.

Passage of Dengue Virus Type 4 Vaccine Candidates in Fetal Rhesus Lung Cells Selects Heparin-Sensitive Variants That Result in Loss of Infectivity and Immunogenicity in Rhesus Macaques

ABSTRACT Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3′ noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu327-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu327-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu327-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu327-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy.

Epidemiological Scenario of Dengue in Brazil.

Dengue is the most important reemerging mosquito-borne viral disease worldwide. It is caused by any of four Dengue virus types or serotypes (DENV-1 to DENV-4) and is transmitted by mosquitoes from the genus Aedes. Ecological changes have favored the geographic expansion of the vector and, since the dengue pandemic in the Asian and Pacific regions, the infection became widely distributed worldwide, reaching Brazil in 1845. The incidence of dengue in Brazil has been frequently high, and the number of cases in the country has at some point in time represented up to 60% of the dengue reported cases worldwide. This review addresses vector distribution, dengue outbreaks, circulating serotypes and genotypes, and prevention approaches being utilized in Brazil.

Impacto económico del dengue y del dengue hemorrágico en el Estado de Zulia, Venezuela, 1997-2003

OBJETIVOS: Determinar los costos directos e indirectos asociados con la atencion de los casos de dengue y de dengue hemorragico o sindrome de choque por dengue (DH/SCD) entre los anos 1997 y 2003 en el Estado de Zulia, Venezuela. METODOS: El numero total de pacientes con dengue y DH/SCD se obtuvo de los registros de la Direccion Regional de Epidemiologia del Estado de Zulia y de los informes de casos confirmados en la Seccion de Virologia del Instituto de Investigaciones Clinicas Dr. Americo Negrette, de la Facultad de Medicina, Universidad del Zulia, Maracaibo, entre el 1.° de enero de 1997 y el 31 de diciembre de 2003. Como costos directos se consideraron el costo de la atencion medica de urgencia de todos los casos y los costos de hospitalizacion de los casos con DH/SCD (costo por dia-cama y costos de laboratorio). Los costos asociados con la ausencia laboral de los enfermos mayores de 15 anos y de las madres acompanantes de los enfermos menores de 15 anos conformaron los costos indirectos, ajustados segun la proporcion de hombres y mujeres en la fuerza laboral activa del pais. Para el calculo se utilizo el salario minimo anual y los resultados se expresaron en dolares estadounidenses, segun la tasa de cambio promedio de cada ano. RESULTADOS: En el periodo estudiado se atendieron 33 857 casos de dengue y de DH/SCD; de ellos, 30 251 (89,35%) fueron de dengue y 3 606 (10,65%) de DH/SCD. Seis de estos fallecieron (letalidad 0,2 por 100 casos de DH/SCD). Los costos directos fueron US

Phylogenetic Analysis of Dengue Virus Types 1 and 4 Circulating in Puerto Rico and Key West, Florida, during 2010 Epidemics

474 251,70; de esa suma, US

Differential Induction of Cytokines by Human Neonatal, Adult, and Elderly Monocyte/Macrophages Infected with Dengue Virus

132 042,30 correspondieron a la atencion en los servicios de urgencia y US