Susceptibility of Carrion Crows to Experimental Infection with Lineage 1 and 2 West Nile Viruses.

West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains' virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

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.

In vitro and in vivo isolation and characterization of Duvenhage virus.

A fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization of DUVV in vitro and its passage in BALB/c mice. The virus proved to be neuroinvasive in both juvenile and adult mice, resulting in about 50% lethality upon peripheral infection. Clinical signs in infected mice were those of classical rabies. However, the distribution of viral antigen expression in the brain differed from that of classical rabies virus infection and neither inclusion bodies nor neuronal necrosis were observed. This is the first study to describe the in vitro and in vivo isolation and characterization of DUVV.

Dengue virus pathogenesis: an integrated view.

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.

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, approximately 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.

Bioinformatics in new generation flavivirus vaccines.

Flavivirus infections are the most prevalent arthropod-borne infections world wide, often causing severe disease especially among children, the elderly, and the immunocompromised. In the absence of effective antiviral treatment, prevention through vaccination would greatly reduce morbidity and mortality associated with flavivirus infections. Despite the success of the empirically developed vaccines against yellow fever virus, Japanese encephalitis virus and tick-borne encephalitis virus, there is an increasing need for a more rational design and development of safe and effective vaccines. Several bioinformatic tools are available to support such rational vaccine design. In doing so, several parameters have to be taken into account, such as safety for the target population, overall immunogenicity of the candidate vaccine, and efficacy and longevity of the immune responses triggered. Examples of how bio-informatics is applied to assist in the rational design and improvements of vaccines, particularly flavivirus vaccines, are presented and discussed.

RNA secondary structures in the proximal 3'UTR of Indonesian Dengue 1 virus strains.

The characteristics of DENV-1 viruses, isolated during the 2001-2002 outbreak in Indonesia were studied. The secondary structure of the 3'UTR of different DENV-1 strains derived from Indonesian patients was compared with the 3'UTR of previously described DENV-1 sequences. The complete 3'UTR of DENV-1 was sequenced from 13 patients suffering from the severe form of dengue virus infection (dengue hemorrhagic fever). Prediction of RNA secondary structure of the 3'UTR revealed some previously unidentified conserved structures in the proximal region of the 3'UTR, the role of which in viral replication is still unknown. In addition our data suggest that some structural elements previously described in the distal part of the 3'UTR are partly dependent on the proximal part of the UTR. Our data support the existence of previously unidentified conserved secondary structures in the proximal part of the 3'UTR and their roles need to be further investigated.

The effect of combined drugs therapy on the course of clinical rabies infection in a murine model.

Rabies is a fatal disease of all mammals causing almost 60,000 human deaths every year. To date, there is no effective treatment of clinical rabies once the symptoms appear. Here, we describe the promising effect of combination therapy composed of molecules that target replication of the rabies virus (RV) at different stages of life cycle and molecules that inhibit some pathways of the innate host immune response accompanied by a blood-brain barrier opener on the outcome of RV infection. The study reports statistically significant extension of survival of mice treated with the drug cocktail containing T-705, ribavirin, interferon α/ß, caspase-1 inhibitor, TNF-α inhibitor, MAPKs inhibitor and HRIG compared to the survival of mice in the virus control group (p = 0.0312). Furthermore, the study points to the significant impact of interferon α/ß on the survival of RV-infected mice. We have shown a significant down regulation of pro-inflammatory molecules (caspase-1 and TNF-a) in the CNS in RV-infected mice treated with a combination of drugs including interferon α/ß.

Combination drug treatment prolongs survival of experimentally infected mice with silver-haired bat rabies virus.

Rabies is a lethal disease in humans and animals, killing approximately 60,000 people every year. Currently, there is no treatment available, except post-exposure prophylaxis (PEP) that can be administered whenever exposure to a rabid animal took place. Here we describe the beneficial effects of a combination treatment initiated at day 4 post infection, containing anti-viral drugs and immune modulators in infected mice. Combination therapy resulted in significant increase in survival time (P < 0.05) and significantly lowers viral RNA in the brain and spinal cord (P < 0.05). Furthermore, treatment influenced markers of pyroptosis and apoptosis and early inflammatory response as measured by the levels of TNF-α. Morphological lesions were absent in rabies virus infected mice with few signs of inflammation. However, these were not significant between the different groups.

Re-evaluating the effect of Favipiravir treatment on rabies virus infection.

There is no antiviral treatment available once clinical disease following rabies virus infection has initiated. Considered a neglected tropical disease, >60,000 human rabies deaths are estimated each year despite the availability of pre- and post-exposure prophylaxis for pre-immunisation or administration following a potential exposure before the onset of clinical disease. Such post-exposure treatments include administration of rabies immunoglobulin (RIG) and vaccination at a distant site to prime a humoral immune response. However, current therapeutic options are limited. Regardless there is a need for molecules that target virus infection following the onset of clinical disease where the outcome of infection is invariably fatal. Numerous molecules have been assessed as potential antivirals against rabies virus (RABV) but with little promise. Favipiravir (T-705) is a broad-spectrum RNA polymerase inhibitor, which has been shown to have antiviral activity against a range of RNA viruses including some against RABV. In the present study, the utility of T-705 has been reassessed in vitro as well as in vivo in a murine model using intraperitoneal administration to investigate any immune protective effect of the molecule. In vitro T-705 effectively reduces RABV replication. However, in vivo, following assessment of various applications of the molecule in both pre- and post-exposure scenarios, the effect was limited. T-705 treatment delayed the onset of clinical signs when virus was delivered intramuscularly at a higher dose (106.8 TCID50/ml) and reduced the number of mice that developed clinical signs when virus was delivered at a lower dose (105.8 TCID50/ml) during the observation period. The day at which treatment commenced did not appear to have a statistically significant effect on the results in either experiment. The use of T-705 as a single biological entity may be limited, however, further work is required to assess the synergistic effect of T-705 as a component of a multi-drug therapy for treating human rabies infections.

Inhibition of caspase-1 prolongs survival of mice infected with rabies virus.

Rabies virus infects almost all mammals resulting in lethal disease. To date there is no treatment available for symptomatic rabies and there is an urgent need to develop treatment strategies that would prolong survival, thereby providing a window of opportunity for the host to mount a protective immune response. We hypothesized that both virus and excessive immune response contribute to disease and that interfering with both is necessary to prevent lethal disease. Here, we have inhibited the pro-inflammatory response associated with pyroptosis and showed that inhibition of CASP-1 had a beneficial effect on survival time. Our results confirm that some inflammatory responses may be involved in the pathogenesis of severe disease and the results suggest that effective intervention includes inhibition of virus and host response.

Combination therapy of rabies-infected mice with inhibitors of pro-inflammatory host response, antiviral compounds and human rabies immunoglobulin.

Recent studies demonstrated that inhibitors of pro-inflammatory molecular cascades triggered by rabies infection in the central nervous system (CNS) can enhance survival in mouse model and that certain antiviral compounds interfere with rabies virus replication in vitro. In this study different combinations of therapeutics were tested to evaluate their effect on survival in rabies-infected mice, as well as on viral load in the CNS. C57Bl/6 mice were infected with Silver-haired bat rabies virus (SHBRV)-18 at virus dose approaching LD50 and LD100. In one experimental group daily treatments were initiated 4 h before-, in other groups 48 or 96 h after challenge. In the first experiment therapeutic combination contained inhibitors of tumour necrosis factor-α (infliximab), caspase-1 (Ac-YVAD-cmk), and a multikinase inhibitor (sorafenib). In the treated groups there was a notable but not significant increase of survival compared to the virus infected, non-treated mice. The addition of human rabies immunoglobulins (HRIG) to the combination in the second experiment almost completely prevented mortality in the pre-exposure treatment group along with a significant reduction of viral titres in the CNS. Post-exposure treatments also greatly improved survival rates. As part of the combination with immunomodulatory compounds, HRIG had a higher impact on survival than alone. In the third experiment the combination was further supplemented with type-I interferons, ribavirin and favipiravir (T-705). As a blood-brain barrier opener, mannitol was also administered. This treatment was unable to prevent lethal consequences of SHBRV-18 infection; furthermore, it caused toxicity in treated mice, presumably due to interaction among the components. In all experiments, viral loads in the CNS were similar in mice that succumbed to rabies regardless of treatment. According to the findings, inhibitors of detrimental host response to rabies combined with antibodies can be considered among the possible therapeutic and post-exposure options in human rabies cases.

Mannitol treatment is not effective in therapy of rabies virus infection in mice.

Rabies is a deadly viral disease with an extremely high fatality rate in humans. Previously, it was suggested that an enhancement of the blood-brain barrier (BBB) permeability, which allows immune cells and/or antibodies to enter the central nervous system (CNS) tissue, is critical to clear the infection. In this study, we utilised mannitol to increase BBB permeability in mice infected with highly pathogenic silver-haired bat rabies virus (SHBRV). We found that intraperitoneal injection of mannitol causes a slight, transient increase of BBB permeability in the treated mice. SHBRV-infected mice were treated with intraperitoneally administered mannitol daily from day 3 or day 4 post-infection, but no effect of this treatment on the time of disease onset, clinical signs or survival was observed. This data indicates that the increase of BBB permeability by mannitol is not efficient in promoting CNS virus clearance in SHBRV-infected mice.

Analysis of Mouse Brain Transcriptome After Experimental Duvenhage Virus Infection Shows Activation of Innate Immune Response and Pyroptotic Cell Death Pathway.

Rabies is an important neglected disease, characterized by invariably fatal encephalitis. Several studies focus on understanding the pathogenic mechanisms of the prototype lyssavirus rabies virus (RABV) infection, and little is known about the pathogenesis of rabies caused by other lyssaviruses. We sought to characterize the host response to Duvenhage virus infection and compare it with responses observed during RABV infection by gene expression profiling of brains of mice with the respective infections. We found in both infections differentially expressed genes leading to increased expression of type I interferons (IFNs), chemokines, and proinflammatory cytokines. In addition several genes of the IFN signaling pathway are up-regulated, indicating a strong antiviral response and activation of the negative feedback mechanism to limit type I IFN responses. Furthermore we provide evidence that in the absence of significant neuronal apoptotic death, cell death of neurons is mediated via the pyroptotic pathway in both infections. Taken together, we have identified several genes and/or pathways for both infections that could be used to explore novel approaches for intervention strategies against rabies.

Transcriptomic Analyses Reveal Differential Gene Expression of Immune and Cell Death Pathways in the Brains of Mice Infected with West Nile Virus and Chikungunya Virus.

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases.

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model.

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans.

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.

A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice.

Rabies is a disease characterized by an invariably lethal encephalitis of viral origin that can be controlled by preventive vaccination programs of wildlife, domestic animals and humans in areas with a high risk of exposure. Currently available vaccines are expensive, cumbersome to produce and require intensive immunization and booster schemes to induce and maintain protective immunity. In the present study, we describe the development of candidate recombinant subunit rabies vaccines based on the glycoprotein G of the prototype rabies virus (RABV-G) expressed either as a monomer (RABV-mG) or in its native trimeric configuration (RABV-tG), with or without Matrix-M™ adjuvant. Immunogenicity and protective efficacy of the respective candidate vaccines were tested in outbred NIH Swiss albino mice. The RABV-tG candidate vaccine proved to be superior to the RABV-mG vaccine candidate both in terms of immunogenicity and efficacy. The relatively poor immunogenicity of the RABV-mG vaccine candidate was greatly improved by the addition of the adjuvant. A single, low dose of RABV-tG in combination with Matrix-M™ induced high levels of high avidity neutralizing antibodies and protected all mice against challenge with a lethal dose of RABV. Consequently RABV-tG used in combination with Matrix-M™ is a promising vaccine candidate that overcomes the limitations of currently used vaccines.

Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA.

Studying the tropism and replication kinetics of West Nile virus (WNV) in different cell types in vitro and in tissues in animal models is important for understanding its pathogenesis. As detection of the negative strand viral RNA is a more reliable indicator of active replication for single-stranded positive-sense RNA viruses, the specificity of qRT-PCR assays currently used for the detection of WNV positive and negative strand RNA was reassessed. It was shown that self- and falsely-primed cDNA was generated during the reverse transcription step in an assay employing unmodified primers and several reverse transcriptases. As a result, a qRT-PCR assay using the thermostable rTth in combination with tagged primers was developed, which greatly improved strand specificity by circumventing the events of self- and false-priming. The reliability of the assay was then addressed in vitro using BV-2 microglia cells as well as in C57/BL6 mice. It was possible to follow the kinetics of positive and negative-strand RNA synthesis both in vitro and in vivo; however, the sensitivity of the assay will need to be optimized in order to detect and quantify negative-strand RNA synthesis in the very early stages of infection. Overall, the strand-specific qRT-PCR assay developed in this study is an effective tool to quantify WNV RNA, reassess viral replication, and study tropism of WNV in the context of WNV pathogenesis.