Foot and mouth disease virus: transmission, pathogenesis, diagnosis and surveillance / Le virus de la fièvre aphteuse : transmission, pathogenèse, diagnostic et surveillance

Foot-and-mouth disease (FMD) is one of the most contagious viral animal diseases. It is an old disease which still poses a permanent threat of re-emergence for free zones. Foot-and-Mouth Disease Virus (FMDV), a Picornavirus belonging to genus Aphthovirus affects domestic and wild artiodactyls. FMD has a considerable socio-economic impact on agricultural production and trade in endemic regions, but also when incursions occur into FMD free areas, as in Europe in 2001. FMDV is historically one of the most studied viruses. Due to its high genetic and antigenic variability, the absence of cross-immunity between its seven serotypes, its ability to survive in the environment, its high contagiousness, its wide range of hosts and its particular biology, FMDV remains of major interest in animal health and the subject of many research projects. This review presents different aspects of FMDV infection, ranging from basic biology to diagnosis, surveillance and control.

La fièvre aphteuse (FA) est l'une des maladies virales animales les plus contagieuses. Bien que très ancienne, la FA reste toujours d'actualité et représente une menace permanente de réémergence pour les pays indemnes. Le virus de la FA ou FMDV (pour foot-and-mouth disease virus), de la famille Picornaviridae, genre Aphthovirus, affecte les artiodactyles domestiques comme sauvages (principalement bovins, ovins, caprins, porcins, camélidés et cervidés). La fièvre aphteuse a un impact socio-économique considérable sur la production et le commerce agricoles en zone d'enzootie mais également en cas d'incursion dans une zone précédemment indemne comme ce fut le cas en 2001 en Europe. Le virus de la FA est historiquement l'un des virus les plus étudiés. Par sa grande variabilité génétique et antigénique, l'absence d'immunité croisée entre ses sept sérotypes, sa capacité de survie dans l'environnement, sa grande contagiosité, son large spectre d'hôtes ainsi que sa biologie particulière, ce virus reste d'intérêt majeur en santé animale et l'objet de nombreux travaux de recherche. Cette revue vise à présenter différents aspects de l'infection par le virus de la fièvre aphteuse et ses problématiques actuelles, de la biologie fondamentale au diagnostic en passant par la surveillance et les moyens de lutte.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce. METHODS: Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 105 TCID50 of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS. RESULTS: Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed. CONCLUSIONS: These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression.

The severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related to the age of the pig. During this study, 2- and 15-week-old pigs were inoculated with PRV strain NIA3. This resulted in important clinical disease, although the associated morbidity and mortality were lower in older pigs. Quantitative PCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence, however, were determined from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was detected in the nasal mucosa, suggesting that other sites may serve as the primary replication site. Second, PRV reached the trigeminal ganglion (TG) and brain stem rapidly upon infection but, compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs, and the decrease in viral mRNA expression was not preceded by or associated with an increased cytokine expression. Third, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulbs of pigs from both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV-induced clinical signs are probably due to enhanced viral replication and associated immunopathology in immature TG and the central nervous system neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb. IMPORTANCE: It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

Genetically stable infectious Schmallenberg virus persists in foetal envelopes of pregnant ewes.

Schmallenberg virus (SBV) is a recently emerged vector-borne virus, inducing congenital defects in bovines, ovines and caprines. Here we have shown that infectious SBV is capable of persisting until the moment of birth in the foetal envelopes of ewes infected with SBV-infectious serum at day 45 (1/5 positive) and 60 (4/6 positive) of gestation. This persistence of at least 100 days is a new aspect of the SBV pathogenesis that could help to explain how SBV overwinters the cold season in temperate climate zones. Furthermore, sequencing of the M segment shows that the persisting virus in the foetal envelopes is genetically stable since only a few mutations compared to the inoculum were found. This supports the hypothesis that persisting virus could start the infection of new hosts. Finally, neutralization tests showed that infectious SBV present in the foetal envelopes at birth can be neutralized by the humoral immunity present in the infected ewes.

Pseudorabies virus isolates from domestic pigs and wild boars show no apparent in vitro differences in replication kinetics and sensitivity to interferon-induced antiviral status.

Pseudorabies virus is the causative agent of Aujeszky's disease. Domestic pigs and wild boars are its natural hosts, and strains circulating within both populations differ in their capacity to induce clinical disease. Cell biological and molecular explanations for the observed differences in virulence are, however, lacking. Different virulence determinants that can be assessed in vitro were determined for five domestic swine strains, four wild boar strains and the NIA3 reference strain. Replication kinetics and plaque formation capacity in continuous swine testicular cells and different primary porcine cell lines were highly similar for isolates from both populations. Treatment of these cell lines with IFNα, IFNγ or a combination of both provoked similar plaque-reducing effects for all strains. In conclusion, our results indicate that isolates from domestic swine and wild boar differ neither in intrinsic replication and dissemination capacity nor in sensitivity to antiviral effects of IFNs.

Age- and strain-dependent differences in the outcome of experimental infections of domestic pigs with wild boar pseudorabies virus isolates.

Although pseudorabies virus (PRV) has been eradicated in domestic swine in many countries, its presence in wild boars remains a threat for a reintroduction into the currently unprotected swine population. To assess the possible impact of such a reintroduction in a naive herd, an in vivo infection study using two genetically characterized wild boar PRV isolates (BEL24043 and BEL20075) representative for wild boar strains circulating in south-western and central Europe and the virulent NIA3 reference strain was performed in 2- and 15-week-old domestic pigs. Our study revealed an attenuated nature of both wild boar strains in 15-week-old pigs. In contrast, it showed the capacity of strain BEL24043 to induce severe clinical symptoms and mortality in young piglets, thereby confirming that the known age dependency of disease outcome after PRV infection also holds for wild boar isolates. Despite the absence of clinical disease in 15-week-old sows, both wild boar PRV strains were able to induce seroconversion, but to a different extent. Importantly, differences in infection and transmission capacity of both strains were observed in 15-week-old sows. Strain BEL24043 induced a more prolonged and disseminated infection than strain BEL20075 and was able to spread efficiently to contact animals, indicative of its capacity to induce a sustained infection. In conclusion, it was shown that a reintroduction of a wild boar isolate into the domestic swine population could have serious economic consequences due to the induction of clinical symptoms in piglets and by jeopardizing the PRV-negative status.

Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep.

Schmallenberg virus (SBV) is an Orthobunyavirus that induces abortion, stillbirths and congenital malformations in ruminants. SBV infection induces a long lasting seroconversion under natural conditions. The persistence of the protective immunity and the isotype specific antibody response upon SBV infection of sheep has however not been studied in detail. Five sheep were kept in BSL3 facilities for more than 16 months and subjected to repeated SBV infections. Blood was regularly sampled and organs were collected at euthanasia. The presence of SBV RNA in serum and organs was measured with quantitative real-time PCR. The appearance and persistence of neutralizing and SBV nucleoprotein (N) isotype specific antibodies was determined with virus neutralization tests (VNT) and ELISAs. The primo SBV infection protected ewes against clinical signs, viraemia and virus replication in organs upon challenge infections more than 15 months later. Production of neutralizing SBV specific antibodies was first detected around 6 days post primo-inoculation with VNT and correlated with the appearance of SBV-N specific IgM antibodies. These IgM antibodies remained present for 2 weeks. SBV-N specific IgG antibodies were first detected between 10 and 21 dpi and reached a plateau at 28 dpi. This plateau remained consistently high and no significant decrease in titre was found over a period of more than 1 year. Similar results were found for the neutralising antibody response. In conclusion, the SBV specific IgM response probably eliminates SBV from the blood and the protective immunity induced by SBV infection protects sheep against reinfection for at least 16 months.

Detection of total and PRRSV-specific antibodies in oral fluids collected with different rope types from PRRSV-vaccinated and experimentally infected pigs.

BACKGROUND: Oral fluid collected by means of ropes has the potential to replace serum for monitoring and surveillance of important swine pathogens. Until now, the most commonly used method to collect oral fluid is by hanging a cotton rope in a pen. However, concerns about the influence of rope material on subsequent immunological assays have been raised. In this study, we evaluated six different rope materials for the collection of oral fluid and the subsequent detection of total and PRRSV-specific antibodies of different isotypes in oral fluid collected from PRRSV-vaccinated and infected pigs. RESULTS: An initial experiment showed that IgA is the predominant antibody isotype in porcine saliva. Moreover, it was found that synthetic ropes may yield higher amounts of IgA, whereas all rope types seemed to be equally suitable for IgG collection. Although IgA is the predominant antibody isotype in porcine oral fluid, the PRRSV-specific IgA-based IPMA and ELISA tests were clearly not ideal for sensitive detection of PRRSV-specific IgA antibodies. In contrast, PRRSV-specific IgG in oral fluids was readily detected in PRRSV-specific IgG-based IPMA and ELISA tests, indicating that IgG is a more reliable isotype for monitoring PRRSV-specific antibody immunity in vaccinated/infected animals via oral fluids with the currently available tests. CONCLUSIONS: Since PRRSV-specific IgG detection seems more reliable than PRRSV-specific IgA detection for monitoring PRRSV-specific antibody immunity via oral fluids, and since all rope types yield equal amounts of IgG, it seems that the currently used cotton ropes are an appropriate choice for sample collection in PRRSV monitoring.

Bluetongue, Schmallenberg - what is next? Culicoides-borne viral diseases in the 21st Century.

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

Development and Validation of a New DIVA Real-Time PCR Allowing to Differentiate Wild-Type Lumpy Skin Disease Virus Strains, Including the Asian Recombinant Strains, from Neethling-Based Vaccine Strains.

The current epidemic in Asia, driven by LSDV recombinants, poses difficulties to existing DIVA PCR tests, as these do not differentiate between homologous vaccine strains and the recombinant strains. We, therefore, developed and validated a new duplex real-time PCR capable of differentiating Neethling-based vaccine strains from classical and recombinant wild-type strains that are currently circulating in Asia. The DIVA potential of this new assay, seen in the in silico evaluation, was confirmed on samples from LSDV infected and vaccinated animals and on isolates of LSDV recombinants (n = 12), vaccine (n = 5), and classic wild-type strains (n = 6). No cross-reactivity or a-specificity with other capripox viruses was observed under field conditions in non-capripox viral stocks and negative animals. The high analytical sensitivity is translated into a high diagnostic specificity as more than 70 samples were all correctly detected with Ct values very similar to those of a published first-line pan capripox real-time PCR. Finally, the low inter- and intra-run variability observed shows that the new DIVA PCR is very robust which facilitates its implementation in the lab. All validation parameters that are mentioned above indicate the potential of the newly developed test as a promising diagnostic tool which could help to control the current LSDV epidemic in Asia.

Belgian Anopheles plumbeus Mosquitoes Are Competent for Japanese Encephalitis Virus and Readily Feed on Pigs, Suggesting a High Vectorial Capacity.

Anopheles plumbeus, a day-active mosquito known to feed aggressively on humans, was reported as a nuisance species near an abandoned pigsty in Belgium. Since Japanese encephalitis virus (JEV) is an emerging zoonotic flavivirus which uses pigs as amplification hosts, we investigated (1) whether An. plumbeus would feed on pigs and (2) its vector competence for JEV, to investigate whether this species could be a potential vector. Three- to seven-day-old F0-generation adult mosquitoes, emerged from field-collected larvae, were fed on a JEV genotype 3 Nakayama strain spiked blood meal. Blood-fed mosquitoes were subsequently incubated for 14 days at two temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient. Our results show that An. plumbeus is a competent vector for JEV at the 25 °C condition and this with an infection rate of 34.1%, a dissemination rate of 67.7% and a transmission rate of 14.3%. The vector competence showed to be influenced by temperature, with a significantly lower dissemination rate (16.7%) and no transmission when implementing the temperature gradient. Moreover, we demonstrated that An. plumbeus readily feeds on pigs when the opportunity occurs. Therefore, our results suggest that Belgian An. plumbeus mosquitoes may play an important role in the transmission of JEV upon an introduction into our region if temperatures increase with climate change.

Relevant Day/Night Temperatures Simulating Belgian Summer Conditions Reduce Japanese Encephalitis Virus Dissemination and Transmission in Belgian Field-Collected Culex pipiens Mosquitoes.

Japanese encephalitis virus (JEV), a zoonotic mosquito-borne Flavivirus, can be considered an emerging infectious disease. Therefore, vector competence studies with indigenous mosquitoes from regions where JEV is not yet endemic are of great importance. In our study, we compared the vector competence of Culex pipiens mosquitoes emerged from Belgian field-caught larvae under two different temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient representing typical summer temperatures in Belgium. Three- to seven-day-old F0-generation mosquitoes were fed on a JEV genotype 3 Nakayama strain spiked blood-meal and incubated for 14 days at the two aforementioned temperature conditions. Similar infection rates of 36.8% and 35.2% were found in both conditions. The observed dissemination rate in the gradient condition was, however, significantly lower compared to the constant temperature condition (8% versus 53.6%, respectively). JEV was detected by RT-qPCR in the saliva of 13.3% of dissemination positive mosquitoes in the 25 °C condition, and this transmission was confirmed by virus isolation in 1 out of 2 RT-qPCR positive samples. No JEV transmission to saliva was detected in the gradient condition. These results suggest that JEV transmission by Culex pipiens mosquitoes upon an accidental introduction in our region is unlikely under current climatic conditions. This could change in the future when temperatures increase due to climate change.

Lumpy Skin Disease Virus Genome Sequence Analysis: Putative Spatio-Temporal Epidemiology, Single Gene versus Whole Genome Phylogeny and Genomic Evolution.

Lumpy Skin Disease virus is a poxvirus from the genus Capripox that mainly affects bovines and it causes severe economic losses to livestock holders. The Lumpy Skin Disease virus is currently dispersing in Asia, but little is known about detailed phylogenetic relations between the strains and genome evolution. We reconstructed a whole-genome-sequence (WGS)-based phylogeny and compared it with single-gene-based phylogenies. To study population and spatiotemporal patterns in greater detail, we reconstructed networks. We determined that there are strains from multiple clades within the previously defined cluster 1.2 that correspond with recorded outbreaks across Eurasia and South Asia (Indian subcontinent), while strains from cluster 2.5 spread in Southeast Asia. We concluded that using only a single gene (cheap, fast and easy to routinely use) for sequencing lacks phylogenetic and spatiotemporal resolution and we recommend to create at least one WGS whenever possible. We also found that there are three gene regions, highly variable, across the genome of LSDV. These gene regions are located in the 5' and 3' flanking regions of the LSDV genome and they encode genes that are involved in immune evasion strategies of the virus. These may provide a starting point to further investigate the evolution of the virus.

Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence.

Tick-borne encephalitis virus (TBEV) is a flavivirus transmitted by ticks. Serological screenings in animals are performed to estimate the prevalence and distribution of TBEV. Most screenings consist of a primary screening by ELISA, followed by confirmation of positive samples by plaque reduction neutralization tests (PRNTs). In this study, 406 wild boar sera were tested with 2 regularly used commercial ELISAs for flavivirus screening in animals (Immunozym FSME (TBEV) IgG All Species (Progen) and ID Screen West Nile Competition (Innovative Diagnostics)) and PRNTs for TBEV and USUTU virus. The results showed that the Immunozym and IDScreen ELISAs had low relative sensitivities of 23% and 20%, respectively, compared to the PRNT results. The relative specificities were 88% and 84% due to cross reactions with USUTU virus-specific antibodies. The minimal TBEV prevalence in our sample set was 8.6% when determined by PRNT. When the screening approach of ELISA testing followed by PRNT confirmation was applied, a TBEV seroprevalence of only 2.0% and 1.7% was found. The suboptimal performance of the ELISAs was confirmed by testing sera collected from experimentally TBEV-infected sheep. While the PRNT detected TBEV specific antibodies in 94% of samples collected between 7 and 18 days post-infection, the ELISAs classified only 50% and 31% of the samples as positive. Both routinely used ELISAs for TBEV antibody screening in animal sera were shown to have a low sensitivity, potentially leading to an underestimation of the true prevalence, and furthermore cross-react with other flavivirus antibodies.

Duration of Immunity Induced after Vaccination of Cattle with a Live Attenuated or Inactivated Lumpy Skin Disease Virus Vaccine.

Vaccines have proven themselves as an efficient way to control and eradicate lumpy skin disease (LSD). In addition to the safety and efficacy aspects, it is important to know the duration for which the vaccines confer protective immunity, as this impacts the design of an efficient control and eradication program. We evaluated the duration of immunity induced by a live attenuated vaccine (LSDV LAV) and an inactivated vaccine (LSDV Inac), both based on LSDV. Cattle were vaccinated and challenged after 6, 12 and 18 months for LSDV LAV or after 6 and 12 months for the LSDV Inac. The LSDV LAV elicited a strong immune response and protection for up to 18 months, as no clinical signs or viremia could be observed after a viral LSDV challenge in any of the vaccinated animals. A good immune response and protection were similarly seen for the LSDV Inac after 6 months. However, two animals developed clinical signs and viremia when challenged after 12 months. In conclusion, our data support the annual booster vaccination when using the live attenuated vaccine, as recommended by the manufacturer, which could potentially even be prolonged. In contrast, a bi-annual vaccination seems necessary when using the inactivated vaccine.

Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans.

Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. Stomoxys calcitrans flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby S. calcitrans flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after Stomoxys calcitrans flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Japanese Encephalitis Virus Interaction with Mosquitoes: A Review of Vector Competence, Vector Capacity and Mosquito Immunity.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus and a major cause of human viral encephalitis in Asia. We provide an overview of the knowledge on vector competence, vector capacity, and immunity of mosquitoes in relation to JEV. JEV has so far been detected in more than 30 mosquito species. This does not necessarily mean that these species contribute to JEV transmission under field conditions. Therefore, vector capacity, which considers vector competence, as well as environmental, behavioral, cellular, and biochemical variables, needs to be taken into account. Currently, 17 species can be considered as confirmed vectors for JEV and 10 other species as potential vectors. Culex tritaeniorhynchus and Culex annulirostris are considered primary JEV vectors in endemic regions. Culex pipiens and Aedes japonicus could be considered as potentially important vectors in the case of JEV introduction in new regions. Vector competence is determined by various factors, including vector immunity. The available knowledge on physical and physiological barriers, molecular pathways, antimicrobial peptides, and microbiome is discussed in detail. This review highlights that much remains to be studied about vector immunity against JEV in order to identify novel strategies to reduce JEV transmission by mosquitoes.

Tick-Borne Encephalitis Virus Prevalence in Sheep, Wild Boar and Ticks in Belgium.

Tick-borne encephalitis virus (TBEV) is the most important tick-borne zoonotic virus in Europe. In Belgium, antibodies to TBEV have already been detected in wildlife and domestic animals, but up-to-date prevalence data for TBEV are lacking, and no studies have assessed its seroprevalence in sheep. Serum samples of 480 sheep from all over Belgium and 831 wild boar hunted in Flanders (northern Belgium) were therefore screened for TBEV antibodies by ELISA and plaque reduction neutralization test (PRNT), respectively. The specificity of positive samples was assessed by PRNTs for TBEV and the Louping Ill, West Nile, and Usutu viruses. TBEV seroprevalence was 0.42% (2/480, CI 95%: 0.11-1.51) in sheep and 9.27% (77/831, CI 95%: 7.48-11.43) in wild boar. TBEV seroprevalence in wild boar from the province of Flemish Brabant was significantly higher (22.38%, 15/67) compared to Limburg (7.74%, 34/439) and Antwerp (8.61%, 28/325). Oud-Heverlee was the hunting area harboring the highest TBEV seroprevalence (33.33%, 11/33). In an attempt to obtain a Belgian TBEV isolate, 1983 ticks collected in areas showing the highest TBEV seroprevalence in wild boars were tested by real-time qPCR. No TBEV-RNA-positive tick was detected. The results of this study suggest an increase in TBEV prevalence over the last decade and highlight the need for One-Health surveillance in Belgium.