Penelope Koraka

Dengue Virus Pathogenesis: an Integrated View

SUMMARY Much remains to be learned about the pathogenesis of the different manifestations of dengue virus (DENV) infections in humans. They may range from subclinical infection to dengue fever, dengue hemorrhagic fever (DHF), and eventually dengue shock syndrome (DSS). As both cell tropism and tissue tropism of DENV are considered major determinants in the pathogenesis of dengue, there is a critical need for adequate tropism assays, animal models, and human autopsy data. More than 50 years of research on dengue has resulted in a host of literature, which strongly suggests that the pathogenesis of DHF and DSS involves viral virulence factors and detrimental host responses, collectively resulting in abnormal hemostasis and increased vascular permeability. Differential targeting of specific vascular beds is likely to trigger the localized vascular hyperpermeability underlying DSS. A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk for development of severe disease.

Human picobirnaviruses identified by molecular screening of diarrhea samples.

ABSTRACT The global threat of (re)emerging infectious viruses requires a more effective approach regarding virus surveillance and diagnostic assays, as current diagnostics are often virus species specific and not able to detect highly divergent or unknown viruses. A systematic exploration of viruses that infect humans is the key to effectively counter the potential public health threat caused by new and emerging infectious diseases. The human gut is a known reservoir of a wide variety of microorganisms, including viruses. In this study, Dutch clinical diarrhea samples for which no etiological agent could be identified by available cell culture, serological, or nucleic acid-based tests were gathered. Large-scale molecular RNA virus screening based on host nucleic acid depletion, sequence-independent amplification, and sequencing of partially purified viral RNA from a limited number of clinical diarrhea samples revealed four eukaryotic virus species. Among the detected viruses were a rhinovirus and a new picobirnavirus variant. In total, ∼20% of clinical diarrhea samples contained human picobirnavirus sequences. The Dutch picobirnaviruses belonged to different phylogenetic clades and did not group with other picobirnaviruses according to year of isolation or host species. Interestingly, the average age of patients infected with picobirnavirus was significantly higher than that of uninfected patients. Our data show that sequence-independent amplification of partially purified viral RNA is an efficient procedure for identification of known and highly divergent new RNA viruses in clinical diarrhea samples.

The challenge of West Nile virus in Europe: knowledge gaps and research priorities.

West Nile virus (WNV) is continuously spreading across Europe, and other continents, i.e. North and South America and many other regions of the world. Despite the overall sporadic nature of outbreaks with cases of West Nile neuroinvasive disease (WNND) in Europe, the spillover events have increased and the virus has been introduced into new areas. The high genetic diversity of the virus, with remarkable phenotypic variation, and its endemic circulation in several countries, require an intensification of the integrated and multidisciplinary research efforts built under the 7th Framework Programme of the European Union (FP7). It is important to better clarify several aspects of WNV circulation in Europe, including its ecology, genomic diversity, pathogenicity, transmissibility, diagnosis and control options, under different environmental and socio-economic scenarios. Identifying WNV endemic as well as infection-free areas is becoming a need for the development of human vaccines and therapeutics and the application of blood and organs safety regulations. This review, produced as a joint initiative among European experts and based on analysis of 118 scientific papers published between 2004 and 2014, provides the state of knowledge on WNV and highlights the existing knowledge and research gaps that need to be addressed with high priority in Europe and neighbouring countries.

West Nile Virus: Immunity and Pathogenesis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans. WNV is endemic in parts of Africa, Europe, the Middle East, and Asia, and since 1999 has spread to North America, Mexico, South America, and the Caribbean. WNV infects the central nervous system (CNS) and can cause severe disease in a small minority of infected humans, mostly immunocompromised or the elderly. This review discusses some of the mechanisms by which the immune system can limit dissemination of WNV infection and elaborates on the mechanisms involved in pathogenesis. Reasons for susceptibility to WNV-associated neuroinvasive disease in less than 1% of cases remain unexplained, but one favored hypothesis is that the involvement of the CNS is associated with a weak immune response allowing robust WNV replication in the periphery and spread of the virus to the CNS.

Serologic evidence of ehrlichiosis among humans and wild animals in The Netherlands.

Abstract.The seroprevalence of antibodies directed against granulocytic and monocytic Ehrlichia was determined by use of human granulocytic ehrlichiosis agent and Ehrlichia chaffeensis as surrogate antigens. Seven hundred twenty-one serum samples were collected between 1992 and 1999 from febrile patients with unresolved aetiology (n=108), patients suspected of having Lyme disease (n=174), forestry workers (n=154) and healthy controls (n=54) as well as from wild deer (n=96), hares (n=60), wild boar (n=15) and red foxes (n=60). Reactive antibodies against granulocytic Ehrlichia were detected in 4% of febrile patients with unresolved aetiology and in 4% of patients suspected of having Lyme disease. Among the forestry workers, 1% tested positive for antibodies against granulocytic Ehrlichia, whereas all the healthy controls were negative. Antibody reaction against monocytic Ehrlichia was detected in only 2% of the febrile patients. Granulocytic Ehrlichia and monocytic Ehrlichia-reactive serum antibodies were detected in 22% and 3% of the deer samples, respectively, and in 2% of the hares. In wild boars and in red foxes, only serum antibodies reactive against monocytic Ehrlichia were detected in 13% and 7%, respectively. The demonstration of the presence of both granulocytic and monocytic Ehrlichia-reactive serum antibodies among humans and wild animals in The Netherlands indicates that patients suspected of having Lyme disease and febrile patients with unresolved aetiology should be tested for the presence of granulocytic and monocytic Ehrlichia antibodies or by polymerase chain reaction. Furthermore, granulocytic Ehrlichia are most prevalent in humans and animals in The Netherlands.

Lipopolysaccharide levels are elevated in dengue virus infected patients and correlate with disease severity

BACKGROUND Although in the majority of cases dengue virus (DENV) infection results in a self-limiting febrile disease, it can cause severe plasma leakage in a minority of patients. The appearance of plasma leakage indicates an increased permeability of the vascular wall. In this study we investigated if DENV infection can lead to leakage of lipopolysaccharide (LPS) from the intestine into the blood of the patient, indicative of an increased permeability of the intestinal mucosal barrier. OBJECTIVES The aim of this study was to investigate if LPS levels were elevated in DENV infected patients and if these levels correlated with disease severity. STUDY DESIGN LPS levels in the blood of DENV infected children were determined using the Limulus Amebocyte Lysate assay. To determine disease severity we used the 1997-WHO criteria, the expert physicians judgement and a score that focused on plasma leakage in particular. Furthermore, the modulatory factors LPS binding protein (LBP) and sCD14, as well as the immune activation marker neopterin were determined. RESULTS We showed significantly elevated LPS levels in plasma of DENV infected children compared to healthy controls. The plasma leakage severity score had the strongest correlation with levels of LPS. LBP, sCD14 and neopterin were elevated compared to healthy controls. CONCLUSION In this study we show evidence of elevated LPS levels during DENV infection. Moreover, a correlation between LPS levels and disease severity was found, especially when disease severity was determined in terms of plasma leakage.

A recombinant influenza a virus expressing domain III of west nile virus induces protective immune responses against influenza and west nile virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 105 TCID50 Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.

Characterization of the Mouse Neuroinvasiveness of Selected European Strains of West Nile Virus

West Nile virus (WNV) has caused outbreaks and sporadic infections in Central, Eastern and Mediterranean Europe for over 45 years. Most strains responsible for the European and Mediterranean basin outbreaks are classified as lineage 1. In recent years, WNV strains belonging to lineage 1 and 2 have been causing outbreaks of neuroinvasive disease in humans in countries such as Italy, Hungary and Greece, while mass mortality among birds was not reported. This study characterizes three European strains of WNV isolated in Italy (FIN and Ita09) and Hungary (578/10) in terms of in vitro replication kinetics on neuroblastoma cells, LD50 values in C57BL/6 mice, median day mortality, cumulative mortality, concentration of virus in the brain and spinal cord, and the response to infection in the brain. Overall, the results indicate that strains circulating in Europe belonging to both lineage 1 and 2 are highly virulent and that Ita09 and 578/10 are more neurovirulent compared to the FIN strain.

Kinetic Analysis of Mouse Brain Proteome Alterations Following Chikungunya Virus Infection before and after Appearance of Clinical Symptoms

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV infection in the nervous system, a kinetic analysis on the host proteome modifications in the brain of CHIKV-infected mice sampled before and after the onset of clinical symptoms was performed. The combination of 2D-DIGE and iTRAQ proteomic approaches, followed by mass spectrometry protein identification revealed 177 significantly differentially expressed proteins. This kinetic analysis revealed a dramatic down-regulation of proteins before the appearance of the clinical symptoms followed by the increased expression of most of these proteins in the acute symptomatic phase. Bioinformatic analyses of the protein datasets enabled the identification of the major biological processes that were altered during the time course of CHIKV infection, such as integrin signaling and cytoskeleton dynamics, endosome machinery and receptor recycling related to virus transport and synapse function, regulation of gene expression, and the ubiquitin-proteasome pathway. These results reveal the putative mechanisms associated with severe CHIKV infection-mediated neurological disease and highlight the potential markers or targets that can be used to develop diagnostic and/or antiviral tools.

Susceptibility of European jackdaws (Corvus monedula) to experimental infection with lineage 1 and 2 West Nile viruses

Mass bird mortality has been observed in North America after the introduction of West Nile virus (WNV), most notably massive die-offs of American crows (Corvus brachyrhynchos). In contrast, WNV epidemic activity in Europe has been characterized by very low incidences of bird mortality. As the general susceptibility of European corvids to strains of WNV remains in question, European jackdaws (Corvus monedula) were inoculated with WNV strains circulating currently in Greece (Greece-10), Italy (FIN and Ita09) and Hungary (578/10), as well as a North American (NY99) genotype with a demonstrated corvid virulence phenotype. Infection with all strains except WNV-FIN resulted in mortality. Viraemia was observed for birds inoculated with all strains and virus was detected in a series of organs upon necropsy. These results suggested that jackdaws could potentially function as a sentinel for following WNV transmission in Europe; however, elicited viraemia levels might be too low to allow for efficient transmission of virus to mosquitoes.