Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis.

Neurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells.

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.

Widespread activity of multiple lineages of Usutu virus, western Europe, 2016.

In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak.

Working with Zika and Usutu Viruses In Vitro.

Usutu (USUV) and Zika (ZIKV) viruses are emerging arboviruses of significant medical and veterinary importance. These viruses have not been studied as well as other medically important arboviruses such as West Nile (WNV), dengue (DENV), or chikungunya (CHIKV) viruses. As such, information regarding the behavior of ZIKV and USUV viruses in the laboratory is dated. Usutu virus re-emerged in Austria in 2001 and has since spread throughout the European and Asian continents causing significant mortality among birds. Zika virus has recently appeared in the Western Hemisphere and has exhibited high rates of birth defects and sexual transmission. Information about the characteristics of USUV and ZIKV viruses are needed to better understand the transmission, dispersal, and adaptation of these viruses in new environments. Since their initial characterization in the middle of last century, technologies and reagents have been developed that could enhance our abilities to study these pathogens. Currently, standard laboratory methods for these viruses are limited to 2-3 cell lines and many assays take several days to generate meaningful data. The goal of this study was to characterize these viruses in cells from multiple diverse species. Cell lines from 17 species were permissive to both ZIKV and USUV. These viruses were able to replicate to significant titers in most of the cell lines tested. Moreover, cytopathic effects were observed in 8 of the cell lines tested. These data indicate that a variety of cell lines can be used to study ZIKV and USUV infection and may provide an updated foundation for the study of host-pathogen interactions, model development, and the development of therapeutics.

Sampling Design Influences the Observed Dominance of Culex tritaeniorhynchus: Considerations for Future Studies of Japanese Encephalitis Virus Transmission.

Mosquito sampling during Japanese encephalitis virus (JEV)-associated studies, particularly in India, has usually been conducted via aspirators or light traps to catch mosquitoes around cattle, which are dead-end hosts for JEV. High numbers of Culex tritaeniorhynchus, relative to other species, have often been caught during these studies. Less frequently, studies have involved sampling outdoor resting mosquitoes. We aimed to compare the relative abundance of mosquito species between these two previously used mosquito sampling methods. From September to December 2013 entomological surveys were undertaken in eight villages in a Japanese encephalitis (JE) endemic area of Bangladesh. Light traps were used to collect active mosquitoes in households, and resting boxes and a Bina Pani Das hop cage were used near oviposition sites to collect resting mosquitoes. Numbers of humans and domestic animals present in households where light traps were set were recorded. In five villages Cx. tritaeniorhynchus was more likely to be selected from light trap samples near hosts than resting collection samples near oviposition sites, according to log odds ratio tests. The opposite was true for Cx. pseudovishnui and Armigeres subalbatus, which can also transmit JEV. Culex tritaeniorhynchus constituted 59% of the mosquitoes sampled from households with cattle, 28% from households without cattle and 17% in resting collections. In contrast Cx. pseudovishnui constituted 5.4% of the sample from households with cattle, 16% from households with no cattle and 27% from resting collections, while Ar. subalbatus constituted 0.15%, 0.38%, and 8.4% of these samples respectively. These observations may be due to differences in timing of biting activity, host preference and host-seeking strategy rather than differences in population density. We suggest that future studies aiming to implicate vector species in transmission of JEV should consider focusing catches around hosts able to transmit JEV.

Memory and learning seems to be related to cholinergic dysfunction in the JE rat model.

Cognitive changes have been known in encephalitis but in Japanese encephalitis (JE) such studies are limited. This study aims at evaluating the spatial memory and learning and correlate with markers of cholinergic activity in the brain.12day old Wistar rats were inoculated with dose of 3×10(6)pfu/ml of JE virus. On 10, 33 and 48days post-inoculation (dpi), spatial memory and learning was assessed by Y maze. Brain biopsies from frontal cortex, corpus striatum, hippocampus and cerebellum were taken. Muscarinic cholinergic receptor was assayed by Quinuclidinyl benzylate (H3-QNB) binding, CHRM2 gene expression by real time PCR and choline acetyl transferase (ChAT) by Western blot. Spatial learning and memory showed significant decline in rats inoculated with JEV on 10 and 33dpi (47.5%, p<0.01; 30.2%, p<0.01). It started recovering on 48dpi. Muscarinic cholinergic receptors showed significant decrease in frontal cortex (31%, p=0.001; 26%, p=0.003), hippocampus (57%, p=0.001; 39.9%, p=0.002) and cerebellum (31.2%, p=0.008; 21.6%, p=0.007) but not in corpus striatum as compared to control. The mRNA expression of CHRM2 receptor gene showed significant decrease in the expression in frontal cortex (48%, p<0.001; 38%, p<0.01), hippocampus (43%, p<0.001; 37%, p<0.05) and cerebellum (46%, p<0.001; 42%, p<0.05) on 10 and 33dpi. ChAT showed significant fold decrease in the expression in frontal cortex (2.11, p<0.01, 1.12, p<0.05) and hippocampus (2.2, p<0.01, 1.41, p<0.05) on 10 and 33dpi. Correlation between ChAT, CHRM2 and total muscarinic receptor activity with spatial memory were found at different dpi. There was transient spatial learning and memory impairment which was associated with reduction of total muscarinic receptor binding, CHRM2 gene and ChAT expression in different brain region of rat infected with JE Virus.

Co-circulation of Usutu virus and West Nile virus in a reed bed ecosystem.

BACKGROUND: Mosquito-borne flaviviruses are a major public health threat in many countries worldwide. In Central Europe, West Nile virus (WNV) and Usutu virus (USUV), both belonging to the Japanese encephalitis virus group (Flaviviridae) have emerged in the last decennium. Surveillance of mosquito vectors for arboviruses is a sensitive tool to evaluate virus circulation and consequently to estimate the public health risk. METHODS: Mosquitoes (Culicidae) were collected at South-Moravian (Czech Republic) fishponds between 2010 and 2014. A total of 61,770 female Culex modestus Ficalbi mosquitoes, pooled to 1,243 samples, were examined for flaviviruses by RT-PCR. RESULTS: One pool proved positive for USUV RNA. Phylogenetic analysis demonstrated that this Czech USUV strain is closely related to Austrian and other Central European strains of the virus. In addition, nine strains of WNV lineage 2 were detected in Cx. modestus collected in the same reed bed ecosystem. CONCLUSIONS: This is the first detection of USUV in Cx. modestus. The results indicate that USUV and WNV may co-circulate in a sylvatic cycle in the same habitat, characterised by the presence of water birds and Cx. modestus mosquitoes, serving as hosts and vectors, respectively, for both viruses.

Epidemic Spread of Usutu Virus in Southwest Germany in 2011 to 2013 and Monitoring of Wild Birds for Usutu and West Nile Viruses.

Mosquito-borne viruses are becoming an increasing threat for Europe. One of these viruses is Usutu virus (USUV), a single-stranded RNA virus belonging to the Japanese encephalitis virus group within the family Flaviviridae. Since the occurrence of USUV among wild birds in June, 2011, infected Blackbirds (Turdus merula) have frequently been found dead in southwest Germany, cumulating in a massive die-off. Moreover, other bird species (Strigiformes) in this region have been affected. In a first study, 209 of over 600 dead birds (wild birds and birds kept in aviaries) collected from 2011 to 2013 carried USUV, more than 88% of them Blackbirds. USUV had already been detected in 2010, one year before the epizooty, in a mosquito-based surveillance program in Germany. The main epidemic area of the USUV outbreak in wild birds in southwest Germany has been similar for the last three years. In a second study during 2011 to 2013, 902 live migratory and resident birds (representing 87 bird species belonging to 14 bird orders) from four different sampling sites were bled and tested serologically and by qPCR for West Nile virus (WNV) and USUV infections. No USUV or WNV genomes were detected. Some migratory birds (mainly long-distance migrants and some partial migrants) carried neutralizing antibodies against WNV as discriminated by USUV and WNV cross-neutralization tests. Only few resident birds showed relevant USUV-specific neutralizing antibodies. The occurrence of USUV in the Upper Rhine valley area of southwest Germany is a proof of principle for the incursion and spread of other arthropod-borne (arbo)-viruses along these routes. Therefore, monitoring studies in birds and mosquitoes for the presence of arboviruses in these areas are indispensable.

Further circulation of West Nile and Usutu viruses in wild birds in Italy.

Usutu virus (USUV) and West Nile virus (WNV) are emerging pathogens that can cause neurological disease in humans. From March 2012 to June 2013, a sero-survey on wild birds was carried out to investigate the circulation of both viruses in Northwest (NW) Italy. Samples belonging to 47 different bird species have been collected using a volunteer based network and a wildlife rehabilitation center. Four of 297 serum samples had neutralizing antibodies against USUV (P=1.34%, IC 95% 0.36-3.4), while 10 of 233 samples tested positive for WNV (P=4.29%, IC 95% 2.07-7.75). Neutralizing antibodies for WNV were significantly more prevalent (p<0.001) in trans-Saharan migrants (P=21%, IC 95% 9.55-37.3) than in resident and short-distance birds, but no migratory habit-related differences were found for USUV. Antibodies in resident bird species suggest that both viruses are circulating in NW Italy.

Lack of usutu virus RNA in cerebrospinal fluid of patients with encephalitis of unknown etiology, Tuscany, Italy.

Usutu virus (USUV) is an African mosquito-borne flavivirus associated with human neurological disorders in Europe. Recently, USUV introduction in Europe has been traced back to Eurasian blackbirds deaths in the Tuscany region of Italy in 1996. Ninety-six cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown etiology diagnosed in 2010-2013 were screened to determine whether USUV circulates in humans in Tuscany. Using real-time polymerase chain reaction, no positive patient was found. USUV does not seem to cause neuroinvasive disorders in humans in Tuscany.

Usutu virus: an emerging flavivirus in Europe.

Usutu virus (USUV) is an African mosquito-borne flavivirus belonging to the Japanese encephalitis virus serocomplex. USUV is closely related to Murray Valley encephalitis virus, Japanese encephalitis virus, and West Nile virus. USUV was discovered in South Africa in 1959. In Europe, the first true demonstration of circulation of USUV was reported in Austria in 2001 with a significant die-off of Eurasian blackbirds. In the subsequent years, USUV expanded to neighboring countries, including Italy, Germany, Spain, Hungary, Switzerland, Poland, England, Czech Republic, Greece, and Belgium, where it caused unusual mortality in birds. In 2009, the first two human cases of USUV infection in Europe have been reported in Italy, causing meningoencephalitis in immunocompromised patients. This review describes USUV in terms of its life cycle, USUV surveillance from Africa to Europe, human cases, its cellular tropism and pathogenesis, its genetic relationship with other flaviviruses, genetic diversity among USUV strains, its diagnosis, and a discussion of the potential future threat to Asian countries.

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.

Culex gelidus: an emerging mosquito vector with potential to transmit multiple virus infections.

Culex gelidus Theobald has emerged as a major vector of Japanese encephalitis virus (JEV) in India, Southeast Asian countries and Australia. The species has expanded its geographic distribution from the Indian subcontinent to Japan, China, other Southeast Asian countries, Island nations in Australasian region and Australia. In recent years, a sudden increase in its population especially in the urban and sub-urban areas has been observed in several countries, thus, becoming a dominant mosquito species. The virus has been repeatedly isolated from from different geographical locations making it one of the most important vectors of JEV. Apart from JEV, other viruses of public health importance, viz. Getah, Ross River (RRV), Sindbis and Tembusu have been isolated from the mosquito. Experimental studies have shown that the mosquito Cx. qelidus is highly competent to transmit West Nile, Kunjin and Murray valley encephalitis viruses with infection and transmission rates of >80 and >50%, respectively for each virus. The species is also found competent to transmit RRV, but at a lower rate. Experimental studies have shown that the species is susceptible to chikungunya, Chandipura and Chittoor (Batai) viruses. Development of resistance to DDT and malathion has also been detected in the species recently. The invasive nature, ability to breed both in fresh and dirty waters, development of resistance to insecticides, high anthropophily and its potential to transmit important human viruses pose an increased threat of viral encephalitis in India and Oriental region especially in the light of explosive increase in its population.

First evidence of simultaneous occurrence of West Nile virus and Usutu virus neuroinvasive disease in humans in Croatia during the 2013 outbreak.

PURPOSE: We report on first evidence of simultaneous occurrence of West Nile virus (WNV) and Usutu virus (USUV) neuroinvasive infection in humans in Croatia during the transmission season 2013. METHODS: From June to December 2013, a total of 95 patients with clinically suspected WNV infection (WNV fever and neuroinvasive disease) were tested for WNV IgM/IgG antibodies using enzyme-linked immunosorbent assay. Twenty-six reactive samples were further tested by virus neutralization test for confirmation. RESULTS: WNV neuroinvasive infection was confirmed in 20 patients, while in three patients USUV neutralizing antibodies were detected. Cases occurred during the 11-week interval (from 24 July to 07 October 2013). Both WNV and USUV cases were distributed in three north-western Croatian counties. In addition to human cases, recent asymptomatic WNV infection (detection of IgM antibodies) was recorded in 9/3,460 (0.3 %) tested sentinel horses. Infected animals were recorded in two eastern and one north-western county. CONCLUSIONS: Our results indicate co-circulation of WNV and USUV in Croatia. WNV infection could be misdiagnosed with other emerging infectious diseases presenting with neurological symptoms such as USUV infection.

Flavivirus detection and differentiation by a microsphere array assay.

Flaviviruses of the Japanese encephalitis virus (JEV) serocomplex include major human and animal pathogens that have a propensity to spread and emerge in new geographic areas. Different genotypes or genetic lineages have been defined for many of these viruses, and they are distributed worldwide. Tools enabling rapid detection of new or emerging flaviviruses and differentiation of important subgroups have widespread application for arbovirus diagnosis and surveillance, and are crucial for detecting virus incursions, tracking virus emergence and for disease control. A microsphere suspension array assay was developed to identify JEV serocomplex flaviviruses of medical and veterinary importance. Assay performance was evaluated using representative virus strains as well as clinical and surveillance samples. The assay detected all JEV serocomplex viruses tested in this study with an apparent analytical sensitivity equal or better than the reference real-time or conventional RT-PCR assays and was able to identify mixed virus populations. The ability to identify mixed virus populations at a high analytical sensitivity would be pertinent in the Australian context when attempting to detect exotic JEV or West Nile virus (WNV), and differentiate from endemic Murray Valley encephalitis virus and WNV-Kunjin virus. The relatively low cost, the ability to identify mixed virus populations and the multiplex nature makes this assay valuable for a wide range of applications including diagnostic investigations, virus exclusions, and surveillance programs.

Development of a Usutu virus specific real-time reverse transcription PCR assay based on sequenced strains from Africa and Europe.

Usutu virus (USUV) has been isolated in several African and European countries mainly from mosquitoes and birds. However, previous benign and two recent severe cases of human infections point out the need of a tool for the identification of USUV in human samples. A published real-time reverse transcription (RT) PCR assay for the detection of USUV in human blood or cerebrospinal fluid does not take into account the genetic variability of USUV in different geographic regions. Therefore, this article presents a quantitative real-time RT-PCR assay based on sequences from Europe and Africa. Primers and probe were designed in conserved regions among USUV strains that differed from closely related flaviviruses. The specificity of the assay was investigated by testing 16 other flaviviruses circulating in Africa. The sensitivity was determined by testing serial dilutions of virus and RNA standard. Intra- and inter-assay coefficients of variation were evaluated by 10 reactions in a same and in different assays, respectively. The assay provides high analytical specificity for USUV and detection limits of 1.2pfu/reaction for virus dilutions in L-15 medium or human serum and 60 copies/reaction for the RNA standard. The assay needs to be evaluated in a clinical context and integrated in standard diagnosis of flaviviral diseases.

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.

Low Usutu virus seroprevalence in four zoological gardens in central Europe.

BACKGROUND: Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused bird die-offs in Austria, Hungary and Switzerland between 2001 and 2009. While the zoological gardens of Vienna and Zurich recorded USUV-associated mortality in different species of birds during this period, incidences in Budapest were limited to areas outside the zoo, and in the greater Basel area avian mortality due to USUV infection was not observed at all. The objectives of this investigation were to gain insight into USUV infection dynamics in captive birds in zoos with varying degrees of virus exposure and to study differences in susceptibility to USUV of different species of birds. RESULTS: 372 bird sera were collected between October 2006 and August 2007. The samples were tested in parallel by hemagglutination inhibition (HI) and 90% plaque reduction neutralization tests (PRNT-90). 8.75%, 5.3% and 6.59% of birds in the zoos of Vienna, Zurich and Basel, respectively, showed USUV-specific antibodies by PRNT-90. No antibodies to USUV were detected in birds of the Budapest zoo. The order Strigiformes (owls) exhibited the highest USUV-seroprevalence, compared to other orders of birds. CONCLUSIONS: USUV seems not to pose an imminent threat to zoo bird populations in central Europe at the moment. Depending on a variety of especially environmental factors, however, this may change at any time in the (near) future, as experienced with West Nile virus (WNV). It is therefore strongly suggested to continue with combined WNV and USUV surveillance activities in affected areas.