Acute cerebellar ataxia: a rare Toscana Virus (TOSV) meningoencephalitis complication.

Purpose: Arbovirosis, viral infection transmitted by arthropods, is a widespread health problem. In Italy, as well for all Mediterranean basin, from late spring to the end of summer, Toscana Virus (TOSV), a sandfly borne virus, accounts for the majority of aseptic meningitis/meningoencephalitis cases. TOSV meningitis/meningoencephalitis has usually a self-extinguishing benign course. Our aim is to report a case of a young healthy women diagnosed with Toscana Virus meningoencephalitis with a complicated clinical course.Materials and methods/results: Case report of a 33-years old woman, admitted to the Infectious Diseases Unit at Careggi General Hospital (Florence-Italy), with a diagnosis of Toscana Virus meningoencephalitis. Seventy-two hours after the admission, she developed typical symptoms, as impaired legs coordination, slurred speech, stumbling and dysmetria, of acute cerebellar ataxia (ACA). Urgent neurological assessment was provided performing an electroencephalography study followed by a brain and brainstem magnetic resonance imaging. In the meanwhile, bilateral nystagmus arised. Through neurologist consultation ACA clinical diagnosis was then made and intravenous steroid therapy was administered with prompt symptoms resolution. The patient was finally discharged at day 10 since the ACA onset in good clinical conditions.Conclusions: To raise awareness among physicians about possible neurological complications during Toscana Virus meningoencephalitis.

Analysis of the Zika and Japanese Encephalitis Virus NS5 Interactomes.

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

The Involvement of Neuroinflammation in Dengue Viral Disease: Importance of Innate and Adaptive Immunity.

Neuroinflammation (inflammation in brain) has been known to play an important role in the development of dengue virus disease. Recently, studies from both clinical and experimental models suggest the involvement of neuroinflammation in dengue viral disease. Studies in clinical setup demonstrated that, microglial cells are actively involved in the patients having dengue virus infection, showing involvement of innate immune response in neuroinflammation. It was further proved that, clinical isolates of dengue-2 virus were able to initiate the pathologic response when injected in the mice brain. Natural killer cells were also found to play a crucial role to activate adaptive immune response. Notably, CXCL10/IFN-inducible protein 10 and CXCR3 are involved in dengue virus-mediated pathogenesis and play an important role in the development of dengue virus-mediated paralysis. In a latest report, it was seen that intracranial injection of dengue virus increases the CD8+ T-cell infiltration in brain, showing an important mechanism of neuroinflammation during the dengue virus infection. A similar study has described that, when DENV-3 is injected into the mice, it enhances the infiltration of CD8+ and CD4+ T cells as well as neutrophils. Cells immune-reactive against NS3 antigen were found throughout the brain. In conclusion, we focus on the various molecular mechanisms which contribute to the basic understanding about the role of neuroinflammation in dengue fever. These mechanisms will help in better understanding dengue pathophysiology and thus help in the development of possible therapeutics.

Fatal Infection with Murray Valley Encephalitis Virus Imported from Australia to Canada, 2011.

Murray Valley encephalitis virus (MVEV), a flavivirus belonging to the Japanese encephalitis serogroup, can cause severe clinical manifestations in humans. We report a fatal case of MVEV infection in a young woman who returned from Australia to Canada. The differential diagnosis for travel-associated encephalitis should include MVEV, particularly during outbreak years.

Evidence of exposure of laughing doves (Spilopelia senegalensis) to West Nile and Usutu viruses in southern Tunisian oases.

It has previously been suggested that southern Tunisian oases may be suitable areas for the circulation of flaviviruses. In order to anticipate and prevent possible epidemiological spread of flaviviruses in humans and domestic animals, the ecology of their transmission in the oasis system needs to be better understood. Thus, the aim of this study was to assess the seroprevalence of anti-flavivirus antibodies in the laughing dove (Spilopelia senegalensis), an abundant resident bird in Tunisian oases. Anti-flavivirus antibodies were detected in 17% of sampled doves. Ten per cent of the total tested doves were West Nile virus (WNV) seropositive and 4% were Usutu virus (USUV) seropositive, which provides the first evidence of USUV circulation in Tunisian birds. We also found that the occurrence probability of anti-flavivirus antibodies in dove plasma increased with decreasing distance to coast, suggesting that doves inhabiting coastal oases were more exposed to flaviviruses compared with those inhabiting inland oases. We also found significantly higher antibody occurrence probability in adult doves compared with young doves, which underlines the effect of exposure time. Overall, our results suggest that the laughing dove may be used for WNV and USUV surveillance in southern Tunisia. They also stress the need for investigations combining data on birds and mosquitoes to better understand the ecological factors governing the circulation of flaviviruses in this area.

The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.

UNLABELLED: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II. IMPORTANCE: In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

First cases of human Usutu virus neuroinvasive infection in Croatia, August-September 2013: clinical and laboratory features.

Few reports of human Usutu virus (USUV) infection have been reported to date. We describe the first three patients with USUV neuroinvasive infection in Zagreb and its surroundings from 30 August to 7 September 2013 during a West Nile virus (WNV) outbreak. Patients were aged 29, 56, and 61 years. The two older patients had several comorbidities (arterial hypertension, hyperlipidemia, and diabetes mellitus). All patients presented with meningitis and meningoencephalitis closely resembling WNV neuroinvasive disease. The main clinical features in all patients were headache, fever, nuchal rigidity, hand tremor, and hyperreflexia. Neuroimaging studies were normal and electroencephalography (EEG) revealed diffusely slow activity. The 29 years old, a previously healthy female patient, was deeply somnolent and disoriented for 4 days. Her recovery was slow and even 10 weeks after disease onset, she had memory and speech-fluency difficulties. The other two patients recovered promptly. USUV IgG antibodies were detected in all patients by ELISA with seroconversion documented in two of them. Titers of USUV-neutralizing antibodies were 10, 80, and 10, respectively. Because USUV and WNV share many clinical characteristics, USUV infection could be misdiagnosed as WNV. Testing for USUV should be considered in all suspected cases of meningoencephalitis, especially in areas where both viruses cocirculate.

Usutu virus, Italy, 1996.

Retrospective analysis of archived tissue samples from bird deaths in the Tuscany region of Italy in 1996 identified Usutu virus. Partial sequencing confirmed identity with the 2001 Vienna strain and provided evidence for a much earlier introduction of this virus into Europe than previously assumed.

Lack of identification of Flaviviruses in oral and cloacal swabs from long- and short-distance migratory birds in Trentino-Alto Adige (North-eastern Italy).

BACKGROUND: West Nile virus (WNV) and Usutu virus (USUV), both belonging to the genus Flavivirus, are emerging in Italy as important human and animal pathogens. Migratory birds are involved in the spread of Flaviviruses over long distances, particularly from Africa to Europe. Once introduced, these viruses can be further be dispersed by short-distance migratory and resident bird species. Thus far, there is still a considerable knowledge gap on the role played by different bird species in the ecology and transmission mechanisms of these viruses. The Region of Trentino-Alto Adige (north-eastern Italy) is located on the migratory route of many of the short- and long-distance migratory birds that cross the Alps, connecting northern Europe and western Asia with southern Europe and Africa. Until now, only a silent circulation of WNV and USUV within the territory of the Province of Trento has been confirmed by serological screening, whilst no cases of infected humans or animals have so far been reported. However, continuous spillover events of both viruses have been reported in neighbouring Regions. The aim of this study was to monitor the circulation of WNV and USUV in Trentino-Alto Adige, in order to detect if active virus shedding occurs in migratory birds captured during their seasonal movements and to evaluate the role that different bird species could play in the spreading of these viruses. METHODS: We carried out a biomolecular survey on oral and cloacal swabs collected from migratory birds during seasonal migrations. Birds belonging to 18 transaharian and 21 intrapaleartic species were examined during spring (n = 176) and autumn (n = 146), and were tested using a generic nested-PCR. RESULTS: All samples tested negative for Flaviviruses. The possible causes of unapparent shedding, along with ecological and epidemiological implications are discussed. CONCLUSIONS: The lack of detection of active virus shedding in these bird species does not exclude the circulation of these viruses within the Trentino-Alto Adige region, as reported in previous studies. The possible ecological implications are discussed.

Dengue in patients with central nervous system manifestations, Brazil.

We investigated the prevalence of dengue in patients with suspected viral meningitis/meningoencephalitis in a dengue-endemic area. Cerebrospinal fluid analysis showed positive results and a 6.74× greater likelihood of identifying positive fluid in patients who died. Our findings support testing patients with neurologic manifestations for the virus in dengue-endemic areas.

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy.

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.

Update in Diagnostics of Toscana Virus Infection in a Hyperendemic Region (Southern Spain).

The sandfly fever Toscana virus (TOSV, genus Phlebovirus, family Phenuiviridae) is endemic in Mediterranean countries. In Spain, phylogenetic studies of TOSV strains demonstrated that a genotype, different from the Italian, was circulating. This update reports 107 cases of TOSV neurological infection detected in Andalusia from 1988 to 2020, by viral culture, serology and/or RT-PCR. Most cases were located in Granada province, a hyperendemic region. TOSV neurological infection may be underdiagnosed since few laboratories include this virus in their portfolio. This work presents a reliable automated method, validated for the detection of the main viruses involved in acute meningitis and encephalitis, including the arboviruses TOSV and West Nile virus. This assay solves the need for multiple molecular platforms for different viruses and thus, improves the time to results for these syndromes, which require a rapid and efficient diagnostic approach.

Encephalitic Arboviruses of Africa: Emergence, Clinical Presentation and Neuropathogenesis.

Many mosquito-borne viruses (arboviruses) are endemic in Africa, contributing to systemic and neurological infections in various geographical locations on the continent. While most arboviral infections do not lead to neuroinvasive diseases of the central nervous system, neurologic diseases caused by arboviruses include flaccid paralysis, meningitis, encephalitis, myelitis, encephalomyelitis, neuritis, and post-infectious autoimmune or memory disorders. Here we review endemic members of the Flaviviridae and Togaviridae families that cause neurologic infections, their neuropathogenesis and host neuroimmunological responses in Africa. We also discuss the potential for neuroimmune responses to aide in the development of new diagnostics and therapeutics, and current knowledge gaps to be addressed by arbovirus research.

Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus.

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Serologic evidence of West Nile virus and Saint Louis encephalitis virus in horses from Southern Brazil.

Flaviviruses as West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilhéus virus (ILHV), and Rocio virus (ROCV) are previously reported in different Brazilian regions, but studies in Southern Brazil are still scarce. To improve the information regarding flaviviruses in Southern Brazil, horse serum samples were analyzed using RT-qPCR and a commercial ELISA-Ab against WNV followed by PRNT75. All 1000 samples analyzed by real-time RT-PCR resulted negative. The 465 subsampled samples were analyzed by a commercial ELISA-Ab against WNV, and the 18.5% (86/465) positive samples were further analyzed by PRNT75. In the PRNT75, 13/86 and 2/86 horses were positive for SLEV and WNV, respectively. It was observed that 5.8% (13/226) of the farms presented at least one positive animal for SLEV in PRNT75, whereas 0.9% (2/226) for WNV. Apart from the lower seroprevalences identified when compared to data previously reported in other Brazilian regions, our results suggest that public health professionals must be aware of the presence of these potential zoonotic pathogens.

False-positive transcription-mediated amplification assay detection of West Nile virus in blood from a patient with viremia caused by an Usutu virus infection.

Detection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus.

Vaccination for tropical mosquito borne encephalitis.

Tropical mosquito borne encephalitis is an important condition in neurology. This bring public health burden for many countries. An important way to face up these infections is the vaccination. In this article, the author will detail and discuss on vaccination for two important tropical mosquito borne encephalitis, Japanese encephalitis and West Nile virus infection.

Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication.

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates.

Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are important mosquito-borne agents that pose public health and bioterrorism threats. Despite considerable advances in understanding alphavirus replication, there are currently no available effective vaccines or antiviral treatments against these highly lethal pathogens. To develop a potential countermeasure for viral encephalitis, we generated a trivalent, or three-component, EEV vaccine composed of virus-like particles (VLPs). Monovalent VLPs elicited neutralizing antibody responses and protected mice and nonhuman primates (NHPs) against homologous challenges, but they were not cross-protective. In contrast, NHPs immunized with trivalent VLPs were completely protected against aerosol challenge by each of these three EEVs. Passive transfer of IgG from immunized NHPs protected mice against aerosolized EEV challenge, demonstrating that the mechanism of protection was humoral. Because they are replication incompetent, these trivalent VLPs represent a potentially safe and effective vaccine that can protect against diverse encephalitis viruses.