Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future.

Dengue: a continuing global threat

Today, dengue viruses are the most prevalent arthropod-borne viruses in the world. Since the 1960s, numerous reports have identified a second heterologous dengue virus (DENV) infection as a principal risk factor for severe dengue disease (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). Modifiers of dengue disease response include the specific sequence of two DENV infections, the interval between infections, and contributions from the human host, such as age, ethnicity, chronic illnesses and genetic background. Antibody-dependent enhancement (ADE) of dengue virus infection has been proposed as the early mechanism underlying DHF/DSS. Dengue cross-reactive antibodies raised following a first dengue infection combine with a second infecting virus to form infectious immune complexes that enter Fc-receptor-bearing cells. This results in an increased number of infected cells and increased viral output per cell. At the late illness stage, high levels of cytokines, possibly the result of T cell elimination of infected cells, result in vascular permeability, leading to shock and death. This review is focused on the etiological role of secondary infections (SI) and mechanisms of ADE.

Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection

The mosquito Aedes aegypti is more widely dispersed now than at any time in the past, placing billions of humans at risk of infection with one or more of the four dengue viruses. This review presents and discusses information on mosquito-dengue infection dynamics and describes the prominent role that temperature and rainfall play in controlling dengue viral transmission including discussions of the effect of interannual climate variations and the predicted effect of global warming. Complementary human determinants of dengue epidemiology include viremia titer, variation in viremic period, enhanced viremias, and threshold viremia. Topics covered include epidemiological phenomena such as traveling waves, the generation of genetic diversity of dengue viruses following virgin soil introductions and in hyperendemic settings, and evidence for and against viral virulence as a determinant of the severity of dengue infections. Also described is the crucial role of monotypic and heterotypic herd immunity in shaping dengue epidemic behavior.

Dengue virus-mosquito interactions.

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Molecular Evolution of Dengue Viruses: Contributions of Phylogenetics to Understanding the History and Epidemiology of the Preeminent Arboviral Disease

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.

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Antibodies to Envelope Glycoprotein of Dengue Virus during the Natural Course of Infection Are Predominantly Cross-Reactive and Recognize Epitopes Containing Highly Conserved Residues at the Fusion Loop of Domain II

A dengue epidemic caused by dengue virus 3 (DENV-3) occurred in Cuba in 2001-2002. It included cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). We report neutralizing antibody studies on sera from 54 of 78 DHF/DSS patients that provide evidence of infections occurring in the sequence DENV-1 followed by DENV-3. No sera showed infection in the sequence DENV-2 followed by DENV-3. Some sera showed a pattern of infection in the sequence DENV-1 followed by DENV-2 and then DENV-3. However definitive categorization of a tertiary infection was not possible because of broadly reactive antibodies, which could have been raised by infections in the sequence DENV-1 then DENV-3.

Dengue hemorrhagic fever caused by sequential dengue 1-3 virus infections over a long time interval: havana epidemic, 2001-2002

!lupe@ipk.sld.cu; " 53-7-2020450; Fax: 53-7-2046051 Neutralizing antibodies play a key role in the prevention of dengue infection. It is important that dengue virus plaque titration and plaque reduction neutralization tests (PRNT) be highly reproducible using standardized methods. To evaluate factors that have influence in the PRNT for dengue viruses, neutralizing antibodies were determined in 24 serum samples and 12 blood samples collected on filter paper, obtained through national dengue surveillance in Cuba. The influence of pH in the overlay medium, the percentage of ambient CO 2 , the use of two different cell lines and of rapid centrifugation on dengue plaque formation were evaluated. The efficiency of the plaquing system was optimal when the overlay medium was buffered to pH 7.5. The rapid centrifugation of the virus on confluent cells increased the virus titres. Higher virus titres were obtained on BHK21 rather than LLCMK 2 cells when the viruses were added to cell suspension. Under optimal conditions, PRNT was highly reproducible and is recommended for seroepidemiological and vaccine studies using either BHK21 or LLCMK 2 cells. This communication also highlights the infection of LLCMK 2 cell suspensions for measuring neutralizing antibodies.

Improved Dengue Virus Plaque Formation on BHK21 and LLCMK 2 Cells: Evaluation of Some Factors

It was recently reported that disease severity increased during the 1997 Cuban dengue 2 virus epidemic and it was suggested that this might be explained by the appearance of neutralization resistant escape mutants. We investigated these observations and ideas by sequencing 20 dengue 2 virus isolates obtained during the early (low case fatality rate) and the late (high case fatality rate) phases of the outbreak. Our results showed total conservation of the E gene sequence for these isolates suggesting that the selection of envelope gene escape mutants was not the determinant of increased disease severity. Alignment of these sequences with those available in GenBank, followed by Maximum likelihood phylogenetic analysis generated a tree, which indicated that our isolates are closely related to the virus that circulated in Venezuela in 1997/98 and subsequently in Martinique in 1998. This "American/Asian" genotype has therefore gradually dispersed across the Caribbean region during the past 5 years.

Dengue virus type 2 in Cuba, 1997: conservation of E gene sequence in isolates obtained at different times during the epidemic

A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non-human primates using this tetravalent formulation in a prime-boost strategy with attenuated viruses are strongly recommended.

Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus.

Background: Zika Virus (ZIKV) is an emerging mosquito-transmitted flavivirus currently causing large epidemics and represents a global public health. Most ZIKV infections in humans are asymptomatic or mild with self-limiting clinical manifestations. Certainty, an available and sustainable surveillance for risk individual groups to ZIKV infection it is necessary and serological methods offer a good alternative.

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Mayling Alvarez

Papers

Dengue hemorrhagic fever caused by sequential dengue 1-3 virus infections over a long time interval: havana epidemic, 2001-2002

Improved Dengue Virus Plaque Formation on BHK21 and LLCMK 2 Cells: Evaluation of Some Factors

Dengue virus type 2 in Cuba, 1997: conservation of E gene sequence in isolates obtained at different times during the epidemic

Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus.

Clinical performance evaluation of five commercial IgM tests for diagnostic of Zika virus infection