Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Dengue type 2 outbreak in the south of the State of Bahia, Brazil: laboratorial and epidemiological studies

During March 1994 cases of a exanthematic acute disease were reported in the municipalities of Itagemirim, Eunápolis and Belmonte, state of Bahia. Dengue fever was confirmed by serology (MAC-ELISA) and by dengue virus type 2 isolation, genotype Jamaica. Signs and symptoms of classic dengue fever were observed with a high percentual of rash (73.8%) and pruritus (50.5%). Major haemorrhagic manifestations were unfrequent and only bleeding gum was reported. Dengue virus activity spreaded rapidly to important tourism counties like Porto Seguro, Ilhéus, Santa Cruz de Cabrália, Prado, Alcobaça and others, representing a risk for the spreading of dengue virus into the country and abroad.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

VERTICAL TRANSMISSION OF DENGUE VIRUS IN Aedes aegypti COLLECTED IN PUERTO IGUAZÚ, MISIONES, ARGENTINA

A finding of vertical transmission of the DEN 3 virus in male specimens of Aedes aegypti, collected in the 2009 fall-winter period, in Puerto Iguazú city, Misiones, Argentina, using the RT-PCR technique in a 15-specimen pool is reported. This result is analyzed within the context of the epidemiological situation of Argentina's northeast border.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98% similarity to sequences from Southeast Asia that were obtained from GenBank.

An outbreak of dengue virus at Rio de Janeiro - 1986

Dengue virus type 1 has been isolated in Aedes albopictus cell strain, from sera of patients living in the Nova Iguaçu county, by Rio de Janeiro. The clinical picture was characterized by fever, headache, retrobulbar pain, backache, pains in the muscles and the joints and prostration. Studies in paired sera confirmed the presence of recent infection by dengue virus type 1. The outbreak reached adjacent areas, including Rio de Janeiro city (May, 1986).

Dengue virus type 3 isolation from Aedes aegypti in the municipality of Nova Iguaçu, State of Rio de Janeiro

In a prospective field study conducted from July 2000 to June 2001, adult Aedes aegypti and Ae. albopictus mosquitoes were caught from the municipality of Nova Iguaçu, State of Rio de Janeiro, Brazil. Virus isolation in Ae. albopictus clone C6/36 cell line and a semi-nested reverse transcription-polymerase chain reaction detected only dengue virus type 3 in three pools of Ae. aegypti, despite the co-circulation of DEN-1, DEN-2 and DEN-3 serotypes in that area. No viruses were detected in Ae. albopictus mosquitoes. This virological surveillance consists in a sentinel system alerting for dengue outbreaks.

Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7%) and transmission (83.3%) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.

Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas

The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1%) were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.

Temporal distribution of dengue virus serotypes in Colombian endemic area and dengue incidence: re-introduction of dengue-3 associated to mild febrile illness and primary infection

We have investigated the temporal distribution of dengue (DEN) virus serotypes in the department (state) of Santander, Colombia, in relation to dengue incidence, infection pattern, and severity of disease. Viral isolation was attended on a total of 1452 acute serum samples collected each week from 1998 to 2004. The infection pattern was evaluated in 596 laboratory-positive dengue cases using an IgG ELISA, and PRNT test. The dengue incidence was documented by the local health authority. Predominance of DEN-1 in 1998 and DEN-3 re-introduction and predominance in 2001-2003 coincided with outbreaks. Predominance of DEN-2 in 2000-2001 coincided with more dengue hemorrhagic fever (DHF). DEN-4 was isolated in 2000-2001 and 2004 but was not predominant. There was an annual increase of primary dengue infections (from 13.7 to 81.4%) that correlated with frequency of DEN-3 (r = 0.83; P = 0.038). From the total number of primary dengue infections DEN-3 (81.3%) was the most frequent serotype. DHF was more frequent in DEN-2 infected patients than in DEN-3 infected patients: 27.5 vs 10.9% (P < 0.05). DEN-3 viruses belonged to subtype C (restriction site-specific-polymerase chain reaction) like viruses isolated in Sri-Lanka and other countries in the Americas. Our findings show the importance of continuous virological surveillance to identify the risk factors of dengue epidemics and severity.

The dynamics of dengue virus serotype 3 introduction and dispersion in the state of Bahia, Brazil

By 2002, dengue virus serotype 1 (DENV-1) and DENV-2 had circulated for more than a decade in Brazil. In 2002, the introduction of DENV-3 in the state of Bahia produced a massive epidemic and the first cases of dengue hemorrhagic fever. Based on the standardized frequency, timing and location of viral isolations by the state's Central Laboratory, DENV-3 probably entered Bahia through its capital, Salvador, and then rapidly disseminated to other cities, following the main roads. A linear regression model that included traffic flow, distance from the capital and DENV-1 circulation (r² = 0.24, p = 0.001) supported this hypothesis. This pattern was not seen for serotypes already in circulation and was not seen for DENV-3 in the following year. Human population density was another important factor in the intensity of viral circulation. Neither DENV-1 nor DENV-2 fit this model for 2001 or 2003. Since the vector has limited flight range and vector densities fail to correlate with intensity of viral circulation, this distribution represents the movement of infected people and to some extent mosquitoes. This pattern may mimic person-to-person spread of a new infection.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 μg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 μg/mL and between 1.9-33.7 μg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Complete genome of a dengue virus serotype 4 strain from Amazonas, Brazil

Dengue virus (DENV) infections represent a significant concern for public health worldwide, being considered as the most prevalent arthropod-borne virus regarding the number of reported cases. In this study, we report the complete genome sequencing of a DENV serotype 4 isolate, genotype II, obtained in the city of Manaus, directly from the serum sample, applying Ion Torrent sequencing technology. The use of a massive sequencing technology allowed the detection of two variable sites, one in the coding region for the viral envelope protein and the other in the nonstructural 1 coding region within viral populations.

Primary dengue virus infections induce differential cytokine production in Mexican patients

Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient217;s clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score ≥ ±2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.