Molecular characterization of dengue virus 1 from autochthonous dengue fever cases in Croatia

In the summer of 2010, two autochthonous dengue fever cases were detected in Croatia. Here we report the retrospective detection of an additional case of dengue fever, representing the first sustained autochthonous transmission in Europe since 1928. In addition, we present the phylogenetic analyses based on two sequences from the Pelje?ac peninsula, southern Croatia. The sequences were identified as dengue virus genotype 1 and recovered from two out of the three Pelje?ac patients in whom infection occurred.     

Autoimmune pathogenesis in dengue virus infection

The pathogenic mechanisms of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) caused by dengue virus (DV) infection remain unresolved. Patients with DHF/DSS are characterized by several manifestations, including severe thrombocytopenia, vascular leakage, and hepatomegaly. In addition to the effect of virus load and virus variation, abnormal immune responses of the host after DV infection may also account for the progression of DHF/DSS. Actually, viral autoimmunity is involved in the pathogenesis of numerous viral infections, such as human immunodeficiency virus, human hepatitis C virus, human cytomegalovirus, herpes simplex virus, Epstein- Barr virus, and DV. In this review, we discuss the implications of autoimmunity in dengue pathogenesis. Antibodies directed against DV nonstructural protein 1 (NS1) showed cross-reactivity with human platelets and endothelial cells, which lead to platelet and endothelial cell damage and inflammatory activation. Based on these findings, we hypothesize that anti-DV NS1 is involved in the pathogenesis of DF and DHF/DSS, and this may provide important information in dengue vaccine development.     

Antibody-dependent enhancement of dengue virus infection in vitro by undiluted sera from monkeys infected with heterotypic dengue virus

Two monkeys (Macaca fascicularis) each were infected with dengue virus type 1 (DENV-1) and type 2 (DENV-2). High levels of neutralizing antibody to homotypic serotype were detected from day 10 to week 58 after infection. Levels of cross-reactive neutralizing antibody to other serotypes were at lower levels or undetectable. Serum samples collected from day 10 to week 58 enhanced infection by homotypic and heterotypic serotypes of DENV when diluted, demonstrating antibody-dependent enhancement (ADE). The ADE activities to heterotypic and homotypic dengue virus infections peaked at dilutions of 1:10–1:100 and 1:100–1:1,000, respectively. Serum samples collected enhanced heterotypic dengue virus infection without any dilution. The results indicate that sera from infected monkeys have an ability to enhance heterotypic dengue virus infection in vitro without dilution, although some of these sera also possess neutralizing activity.     

Culture-amplified detection of dengue virus from serum in an outbreak of dengue fever

An outbreak of dengue type 2 occurred in North Queensland, Australia, between December 1996 and April 1997. Culture of serum in the Aedes albopictus C6/36 cell line with detection using immunofluorescent staining was compared with a culture-amplified detection system using an immunoperoxidase staining method in a microtiter plate format. A total of 374 serum specimens from individuals during the outbreak were tested. Ninety-five specimens were positive using immunofluorescence and ninety-two were positive using the immunoperoxidase method (sensitivity 91.6%; specificity 98.2%). The immunoperoxidase method is quicker, easier to perform, and does not require the use of an immunofluorescent microscope. The method is more suited to the processing of large numbers of specimens in an outbreak and could be used in endemic areas with limited virological resources. J. Med. Virol. 57:212–215, 1999. © 1999 Wiley-Liss, Inc.     

Imported cases of dengue virus infection: Emilia-Romagna,Italy, 2010

Dengue is a significant mosquito-borne infection in humans, and its worldwide prevalence is rapidly increasing. In 2010, 83 serum samples from febrile travellers returning from dengue-endemic countries to a region in north-eastern Italy, densely infested with Aedes albopictus, were analysed for dengue virus (DENV). DENV RNA was detected in 20.5% of patients. By RT-PCR, DENV serotypes 1 and 3 were the most common. DENV must be identified early in symptomatic travellers returning from high-risk countries, to prevent outbreaks where potential vectors exist.     

Genetic diversity of respiratory syncytial virus isolated during an epidemic period from children of northeastern Brazil

Outbreaks of human respiratory syncytial virus (HRSV) are the leading cause of serious acute lower respiratory viral disease in many countries in different continents. Data on clinical and epidemiological aspects of HRSV infections in this country have been reported, but there is lack of data regarding the molecular epidemiology of this virus in Salvador. The genetic variability of HRSV isolated during an outbreak in Salvador, Brazil (1999) has been analysed. Partial sequences of the G protein gene of 13 isolates from antigenic group A and 4 isolates from antigenic group B of HRSV were determined. Nucleotide sequences of C-terminal G gene were compared to sequences of HRSV isolates from countries of South America and from the rest of the world available at the GenBank. Brazilian group A and B isolates were clustered into previously characterised genotypes: GA5, GA2, GA7, and GB3, SAB3, respectively. This is the first study of GA7 and SAB3 genotypes circulation in South American countries. It is interesting to point out that viruses isolated in Salvador appear to be closer related with those from Montevideo-Uruguay and Buenos Aires, Argentina strains, suggesting circulation of similar strains among different South American countries in different seasons. Moreover, viruses closely related genetically circulated in the same year in Salvador and distant places such as Mozambique, supporting the previous suggestion on the complexity of HRSV strain circulation patterns, and the high capability of HRSV spreading world-wide.     

Three fatal cases of pandemic 2009 influenza A virus infection in Shenzhen are associated with cytokine storm

China had taken strict measures for pandemic 2009 H1N1 infection with enhanced surveillance and hospital isolation since April 2009. In Shenzhen, over 1200 confirmed cases of H1N1 infection were identified. Three young patients died of severe pneumonia. Among them, two boys developed neurological complications. Cytokine storm seemed an important cause.     

Pathogenesis of vascular leak in dengue virus infection

Endothelial dysfunction leading to vascular leak is the hallmark of severe dengue. Vascular leak typically becomes clinically evident 3–6 days after the onset of illness, which is known as the critical phase. This critical phase follows the period of peak viraemia, and lasts for 24–48 hr and usually shows rapid and complete reversal, suggesting that it is likely to occur as a result of inflammatory mediators, rather than infection of the endothelium. Cytokines such as tumour necrosis factor‐α, which are known to be elevated in the critical phase of dengue, are likely to be contributing factors. Dengue NS1, a soluble viral protein, has also been shown to disrupt the endothelial glycocalyx and thus contribute to vascular leak, although there appears to be a discordance between the timing of NS1 antigenaemia and occurrence of vascular leak. In addition, many inflammatory lipid mediators are elevated in acute dengue viral infection such as platelet activating factor (PAF) and leukotrienes. Furthermore, many other inflammatory mediators such as vascular endothelial growth factor and angiopoietin‐2 have been shown to be elevated in patients with dengue haemorrhagic fever, exerting their action in part by inducing the activity of phospholipases, which have diverse inflammatory effects including generation of PAF. Platelets have also been shown to significantly contribute to endothelial dysfunction by production of interleukin‐1β through activation of the NLRP3 inflammasome and also by inducing production of inflammatory cytokines by monocytes. Drugs that block down‐stream immunological mediator pathways such as PAF may also be beneficial in the treatment of severe disease.     

Role of antibodies in controlling dengue virus infection

The incidence and disease burden of arthropod-borne flavivirus infections have dramatically increased during the last decades due to major societal and economic changes, including massive urbanization, lack of vector control, travel, and international trade. Specifically, in the case of dengue virus (DENV), the geographical spread of all four serotypes throughout the subtropical regions of the world has led to larger and more severe outbreaks. Many studies have established that recovery from infection by one DENV serotype provides immunity against that serotype, whereas reinfection with another serotype may result in severe disease. Pre-existing antibodies thus play a critical role in controlling viral infection. Both neutralization and enhancement of DENV infection by antibodies are thought to be related to the natural route of viral entry into cells. In this review, we will describe the current knowlegde on the mechanisms involved in flavivirus cell entry and discuss how antibodies may influence the course of infection towards neutralization or enhancement of viral disease.     

Apoptosis in tissues from fatal dengue shock syndrome.

BACKGROUND: Apoptosis, or programmed cell death, has been implicated in dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) pathogenesis. OBJECTIVES: To determine the in vivo apoptosis contribution to the pathogenesis of fatal DHF/DSS during a Cuban dengue epidemic. STUDY DESIGN: We detected apoptosis by the TdT-mediated dUTP Nick-End Labeling (TUNEL) technique and dengue virus (DENV) antigens by an immunohistochemical assay in different tissues from six individuals who died of DHF/DSS during the Santiago de Cuba DENV-2 epidemic in 1997. RESULTS: DENV antigens were immunolocalized mainly in hepatocytes. Apoptotic cells were found in five of the six cases studied. Apoptosis was demonstrated in liver, brain, intestinal and lung tissues. Severe brain hypoxia and ischemia in the studied subjects during DHF/DSS probably might induce apoptosis in cerebral cells. Apoptotic microvascular endothelial cells (ECs) in pulmonary and intestinal tissues, a finding only previously reported in vitro, are likely related to vascular plasma leakage manifested by the individuals. CONCLUSIONS: Apoptosis was demonstrated in cerebral cells, white blood cells, intestinal and pulmonary microvascular ECs from Cuban fatal cases of DHF/DSS. As far as we know, these findings have not been previously reported in DHF/DSS. Our results indicate there is very likely an in vivo contribution of apoptosis to the pathophysiological mechanisms of DHF/DSS.     

Immunoglobulin A antibody responses in dengue patients: a useful marker for serodiagnosis of dengue virus infection

We determined the usefulness of an immunoglobulin A (IgA) antibody-capture enzyme-linked immunosorbent assay for serodiagnosis of dengue virus infections. The results indicate that the presence of IgA and IgM in serum samples assures recent primary dengue virus infection even with a single serum sample.     

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.     

Effect of immunosuppression on dengue virus infection in mice

Mean survival time following intracerebral inoculation of dengue virus was reduced and the titre of the virus in the brain of immunosuppressed mice was markedly increased. A single dose of cyclophosphamide given 24 h after dengue virus i.c. or i.p. substantially reduced the number of antibody forming cells in the spleen. Three doses of dengue virus, each followed by cyclophosphamide 24 h later, produced specific hyporesponsiveness to the dengue virus but not to a heterologous virus (Coxsackie B4), with a reduction in antibody forming cells in the spleen of such animals against dengue virus but not against Coxsackie B4 virus. Adoptive immunity by antiserum was abolished along with increased titres of the virus in the brain of immunosuppressed mice but the protection could be restored by a second dose of antiserum. Pre-treatment of mice with immune or normal spleen cells i.v. or reconstitution of immunosuppressed mice by such cells had no effect. Thus, humoral antibodies play a crucially important role in host defence mechanism in recovery of mice from primary dengue virus infection.