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While fatal infections caused by the Usutu virus appeared to concern only passerines (especially the blackbird) and Strigiformes (especially the great gray owl), we report herein that the virus also naturally causes a fatal disease in an anseriforme species, the common scoter (Melanitta nigra).

Type I/III interferons provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced, Mx proteins of some species appear as key components of defense against influenza A viruses. It is expected that such an antiviral protein must display a platform dedicated to the recognition of said viruses. In an attempt to identify such platform in human MxA, an evolution-guided approach capitalizing on the antagonistic arms race between MxA and its viral targets and the genomic signature it left on primate genomes revealed that the surface-exposed so-called "loop L4", which protrudes from the compact structure of the MxA stalk, is a hotspot of recurrent positive selection. Since MxA is archetypic of Mx1 proteins in general, we reasoned that the L4 loop also functions as a recognition platform for influenza viruses in the Mx1 proteins of other species that had been exposed to the virus for ever. In this study, the anti-influenza activity of 5 distinct mammalian Mx1 proteins was measured by comparing the number of viral nucleoprotein-positive cells 7 h after infection in a sample of 100,000 cells expected to contain both Mx1-positive and Mx1-negative cell subpopulations. The systematic depletion (

Schmallenberg virus emerged in 2011 in Europe. The epicentre of primordial spreading was the region straddling Germany, the Netherlands and Belgium. One of the key questions is whether the newcomer would establish a lasting presence on the continent. The apparent seroprevalence in southern Belgium wild deer populations was followed for 6 years. Two years of intense circulation were revealed, 2012 and 2016, characterized by a peak seroprevalence in the two studied populations (Capreolus capreolus and Cervus elaphus). Between the peak years and after 2016, apparent seroprevalences declined rapidly among adults and became nil among juveniles. The general pattern of apparent seroprevalence evolution observed is consistent with a cyclic circulation of Schmallenberg virus, similar to what is observed for other Orthobunyaviruses in endemic areas. These data also suggest that wild cervids play no central role in the circulation dynamics of the virus.

Orthobunyaviruses are arboviruses (Arthropod Borne Virus) and possess multipartite genomes made up of three negative RNAs corresponding to the small (S), medium (M) and large (L) segments. Reassortment and recombination are evolutionary driving forces of such segmented viruses and lead to the emergence of new strains and species. Retrospective studies based on phylogenetical analysis are able to evaluate these mechanisms at the end of the selection process but fail to address the dynamics of emergence. This issue was addressed using two Orthobunyaviruses infecting ruminants and belonging to the Simbu serogroup: the Sathuperi virus (SATV) and the Shamonda virus (SHAV). Both viruses were associated with abortion, stillbirth and congenital malformations occurring after transplacental transmission and were suspected to spread together in different ruminant and insect populations. This study showed that different viruses related to SHAV and SATV are spreading simultaneously in ruminants and equids of the Sub-Saharan region. Their reassortment and recombination potential was evaluated in mammalian and in insect contexts. A method was set up to determine the genomic background of any clonal progeny viruses isolated after in vitro coinfections assays. All the reassortment combinations were generated in both contexts while no recombinant virus was isolated. Progeny virus populations revealed a high level of reassortment in mammalian cells and a much lower level in insect cells. In vitro selection pressure that mimicked the host switching (insect-mammal) revealed that the best adapted reassortant virus was connected with an advantageous replicative fitness and with the presence of a specific segment.

We report the detection of Moku virus in invasive Asian hornets (Vespa velutina nigrithorax) in Belgium. This constitutes an unexpected report of this iflavirus outside Hawaii, USA, where it was recently described in social wasps. Although virulence of Moku virus is unknown, its potential spread raises concern for European honeybee populations.

During late summer 2016, in a northwest European region extending over Belgium, the Netherlands and the eastern border of the German state of North Rhine Westphalia, an outbreak of wild bird deaths occurred similar to those reported on the continent since 1996. Dead birds were necropsied and examined by complementary methods. Pathologic and immunohistological investigations strongly suggested an infection by Usutu virus. Subsequently, genomic segments of the said virus were detected, the virus was isolated and its complete genome was sequenced. The strain, designated Usutu-LIEGE, is a close phylogenetic relative of those isolated in Germany which form a distinct group within the USUV phylogeny, the so-called Europe_3 lineage. Should this outbreak recapitulate the characteristics of those in southwest Germany in 2011 and in/around Vienna (Austria) in 2001, it is expected that specific avian populations in the affected area will face a significant reduction in size for a few years.

Wildlife surveillance allowed the monitoring of the zoonotic mosquito-borne Usutu virus (USUV) in birds and bats (Pipistrellus pipistrellus) in southern Belgium in 2017 and 2018. USUV-RNA was detected in 69 birds (of 253) from 15 species, among which 7 species had not previously been reported to be susceptible to the infection. Similarly, 2 bats (of 10) were detected positive by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). USUV-associated lesions were mainly found in Eurasian Blackbirds (Turdus merula), in which USUV antigens were demonstrated by immunohistochemistry in the brain, heart, liver, kidney, intestine, and lung. Partial nonstructural protein 5 gene-based phylogenetic analysis showed several identical or closely related strains from 2016, 2017, and 2018 clustering together within Europe 3 or Africa 3 lineages. Further, one USUV strain detected in a common chaffinch (Fringilla coelebs) manifested a close genetic relationship with the European 1 strains circulating in Hungary and Austria. Our data provide evidence of USUV endemization in southern Belgium in local birds and bats, extension of the host range of the virus and ongoing virus introduction from abroad, likely by migratory birds. Our results highlight the need for vigilance in the forthcoming years toward new virus-associated outbreaks in birds and possible human infections in Belgium.

In a new example of pathogens hopscotching the globe, African swine fever virus hit north-western Europe's wildlife in summer 2018, marking a further spread of a disease that had invaded Central and Eastern Europe recently. The complete genomic sequence of the Belgium/Etalle/wb/2018 virus is reported, with the hope it will provide a valuable tool for tracing geographical spread and biologic evolution of the virus.

The hygiene hypothesis postulates that the recent increase in allergic diseases such as asthma and hay fever observed in Western countries is linked to reduced exposure to childhood infections. Here we investigated how infection with a gammaherpesvirus affected the subsequent development of allergic asthma. We found that murid herpesvirus 4 (MuHV-4) inhibited the development of house dust mite (HDM)-induced experimental asthma by modulating lung innate immune cells. Specifically, infection with MuHV-4 caused the replacement of resident alveolar macrophages (AMs) by monocytes with regulatory functions. Monocyte-derived AMs blocked the ability of dendritic cells to trigger a HDM-specific response by the TH2 subset of helper T cells. Our results indicate that replacement of embryonic AMs by regulatory monocytes is a major mechanism underlying the long-term training of lung immunity after infection.

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal new CD46-encoding transcripts that are predicted to encode CCP1-containing soluble forms. Further, we show that the serum of most adult cattle contains soluble CD46 (sCD46) and that a recombinant soluble isoform neutralizes BVDV infectivity in an in vitro assay. We have then established an ELISA for determination of plasma sCD46 in a large cohort of animals. Overall, serum sCD46 amounts to 8 ± 18 ng/mL (mean ± SD, n = 440), with a IC [95-105] ranging from 6,4 to 9,8 ng/mL and extreme values ​​between 0 and 178 ng/mL. We found that sCD46 is not detectable in fetal and neonatal sera and that its plasma concentration increases progressively up to adulthood. We also detected high- and low-sCD46 performers and show that this phenotype does not depend of environment. As modern rearing techniques make it possible to disseminate genetically-determined phenotypes very quickly in a population, a large-scale study examining whether high-sCD46 animals provide epidemiological protection against BVDV infection and transmission should be undertaken.

The family Parvoviridae contains diverse viruses that are capable of infecting a wide range of hosts. In this study, metagenomic sequencing of Ixodes ricinus ticks harvested in 2016 on red deer (Cervus elaphus) and European roe deer (Capreolus capreolus) in Belgium detected a new 6296-bp parvoviral genome. Phylogenetic and sequence analyses showed the new virus belongs to a new species within the Copiparvovirus genus. PCR screening of 4 pools of 10 serum samples from both deer species identified the new copiparvovirus DNA only in roe deer sera. Together, these results are the first evidence of a copiparvovirus in a deer species. Besides its potential pathogenicity to roe deers, the detection of this new virus in ticks raises questions about the possible transmission of parvoviruses by ticks. This report further increases the current knowledge on the evolution and diversity of copiparvoviruses.

West Nile Virus, Usutu virus, Bagaza virus, Israel turkey encephalitis virus and Tembusu virus currently constitute the five flaviviruses transmitted by mosquito bites with a marked pathogenicity for birds. They have been identified as the causative agents of severe neurological symptoms, drop in egg production and/or mortalities among avian hosts. They have also recently shown an expansion of their geographic distribution and/or a rise in cases of human infection. This paper is the first up-to-date review of the pathology of these flaviviruses in birds, with a special emphasis on the difference in susceptibility among avian species, in order to understand the specificity of the host spectrum of each of these viruses. Furthermore, given the lack of a clear prophylactic approach against these viruses in birds, a meta-analysis of vaccination trials conducted to date on these animals is given to constitute a solid platform from which designing future studies.

In September 2018, African swine fever in wild boars was detected in Belgium. We used African swine fever–infected spleen samples to perform a phylogenetic analysis of the virus. The causative strain belongs to genotype II, and its closest relatives are viruses previously isolated in Ukraine, Belarus, Estonia, and European Russia.

Between 30 and 50 red foxes ( Vulpes vulpes ) are submitted each year to the University of Liege’s veterinary faculty for postmortem examination. Occasionally, other wild carnivores such as European badgers ( Meles meles ) and raccoons ( Procyon lotor ) are also submitted. A PCR-based screening for canine distemper virus (CDV)1 has been performed systematically. Since 2014, all submitted cases tested negative for CDV. On November 14, 2017, a wild raccoon with severe neurological impairment was euthanased in a revalidation centre, near the Liege veterinary faculty. …

Over recent decades, metagenomic studies have expanded the number of newly described, often unclassified, viruses within the family Circoviridae. Using broad-spectrum circovirus and cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative ORFs shared very low amino acid identity with those of previously characterized circoviruses and cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defined, together with previously described mosquito- and bat faeces-derived circo-like viruses, a different genus, tentatively called Krikovirus, within the family Circoviridae. We further demonstrated that viruses of the putative genus Krikovirus all shared a genomic organization that was unique among the family Circoviridae. Further investigations are needed to determine the host range, tissue tropism and transmission route(s). This report increases the current knowledge of the genetic diversity and evolution of the members of the family Circoviridae.

Schmallenberg virus, a novel orthobunyavirus, is spreading among ruminants, especially sheep and cattle, throughout Europe. To determine the risk for domestic dog infection, we conducted a survey among cases referred to the university Companion Animal Clinic to assess possible seroconversion. No evidence of transmission to dogs was detected.

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.

In southern Belgium, a brown hare ( Lepus europaeus ) was found moribund and killed for ethical reason. The animal was transmitted for postmortem examination. Major histopathological findings consisted of multifocal subacute necrotising meningitis and multifocal subacute necrotising orchitis. Infection with Francisella tularensis was confirmed by both bacteriological isolation and detection by real-time PCR. Further, subtyping of F tularensis colonies stated that it was F tularensis subspecies holarctica biovar I . It is the first case of tularaemia detected in wildlife in Belgium since 2003. The event pushed health professionals to communicate with hunters and other groups with outdoor activities about the Francisella risk and the ways to take care of it, such as wearing gloves to handle found-dead or hunted hares and taking protective measures against tick bites.

Background: Perinatal infections with feline panleukopenia virus (FPV) have long been known to be associated with cerebellar hypoplasia in kittens due to productive infection of dividing neuroblasts. FPV, like other parvoviruses, requires dividing cells to replicate which explains the usual tropism of the virus for the digestive tract, lymphoid tissues and bone marrow in older animals. Results: In this study, the necropsy and histopathological analyses of a series of 28 cats which died from parvovirus infection in 2013 were performed. Infections were confirmed by real time PCR and immunohistochemistry in several organs. Strikingly, while none of these cats showed cerebellar atrophy or cerebellar positive immunostaining, some of them, including one adult, showed a bright positive immunostaining for viral antigens in cerebral neurons (diencephalon). Furthermore, infected neurons were negative by immunostaining for p27Kip1, a cell cycle regulatory protein, while neighboring, uninfected, neurons were positive, suggesting a possible re-entry of infected neurons into the mitotic cycle. Next-Generation Sequencing and PCR analyses showed that the virus infecting cat brains was FPV and presented a unique substitution in NS1 protein sequence. Given the role played by this protein in the control of cell cycle and apoptosis in other parvoviral species, it is tempting to hypothesize that a cause-to-effect between this NS1 mutation and the capacity of this FPV strain to infect neurons in adult cats might exist. Conclusions: This study provides the first evidence of infection of cerebral neurons by feline panleukopenia virus in cats, including an adult. A possible re-entry into the cell cycle by infected neurons has been observed. A mutation in the NS1 protein sequence of the FPV strain involved could be related to its unusual cellular tropism. Further research is needed to clarify this point., SCOPUS: ar.j, info:eu-repo/semantics/published

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal the existence of ten distinct allelic versions of the CCP1 module, varying significantly in frequency among taurine and indicine races. A complex mRNA splicing pattern was also evidenced for bovine CD46, generating three different serine-threonine-proline segments and five different cytoplasmic domains. The four most frequent allelic variants and the six splice variants were then expressed in BVDV-nonpermissive porcine cells and the quantity of progeny virions generated by each cell preparation was measured 48 h post-infection. As expected, ectopic expression of the 10 bovine CD46 isoforms rendered the PK15 cells permissive to BVDV, as attested by the 100,000-fold greater recovery of virions from these cells than from non-transfected cells. This permissivity increase was significantly lower (-33%, P

After a short winter break, bluetongue virus serotype 8 was responsible in 2007 for a large-scale epidemic among ruminant populations in Western Europe. Little is known about the mechanisms allowing the virus to survive winter conditions. A yearly mass vaccination of cattle and sheep started in spring 2008, which was recognized as successful in terms of clinical protection, but occult circulation of the bluetongue virus has not been adequately addressed. We studied the carriage of bluetongue RNA in the spleen of cattle in the vector-free period and the circulation of bluetongue virus in cattle populations in Belgium since the introduction of vaccination programmes. Overall, the results presented here show evidence for the long-term carriage of bluetongue virus RNA in the spleen of cattle and demonstrated a low but significant circulation and transplacental transmission of bluetongue virus in Belgian cattle in 2009, with apparent disappearance in 2010.

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2 , is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves ( n = 14, 87 isolates) and from a group of enterotoxaemic calves ( n = 8, 41 isolates). PCR results revealed the exclusive presence of the typical “consensus” cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.

The aim of this study was to evaluate the effect of several risk/protective factors and predictors on the prevalence of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infections in 302 stray cats captured during a trap-neuter-release programme in a mixed urban-rural area from Belgium, from 2010 to 2012. The impact of selective removal of FIV-positive cats on the apparent prevalence in the remaining population over this three-year period was also assessed. The seroprevalences over three years were 18.8 per cent for FIV and 0.7 per cent for FeLV. For FIV, the seroprevalence decreased significantly from the first year of the programme (2010; 30.5 per cent) to the last (2012; 13.1 per cent). Sex (male) and age (adult and old cats) were risk factors, while the year of sampling (years 2011 and 2012) was a protective factor. Age, sex and location were the most relevant predictors of FIV status. The data presented in this study revealed a very high FIV seroprevalence in Belgian stray cats, while FeLV was almost absent. The selective removal of positive cats had a drastic effect on the FIV seroprevalence in the remaining cat population.

Myxoma virus (MYXV) induces a lethal disease called Myxomatosis in European rabbits. MYXV is one of the rare viruses that encodes an α2,3-sialyltransferase through its M138L gene. In this study, we showed that although the absence of the enzyme was not associated with any in vitro deficit, the M138L deficient strains are highly attenuated in vivo. Indeed, while all rabbits infected with the parental and the revertant strains died within 9 days post-infection from severe myxomatosis, all but one rabbit inoculated with the M138L deficient strains survived the infection. In primary lesions, this resistance to the infection was associated with an increased ability of innate immune cells, mostly neutrophils, to migrate to the site of virus replication at 4 days post-infection. This was followed by the development of a better specific immune response against MYXV. Indeed, at day 9 post-infection, we observed an important proliferation of lymphocytes and an intense congestion of blood vessels in lymph nodes after M138L knockouts infection. Accordingly, in these rabbits, we observed an intense mononuclear cell infiltration throughout the dermis in primary lesions and higher titers of neutralizing antibodies. Finally, this adaptive immune response provided protection to these surviving rabbits against a challenge with the MYXV WT strain. Altogether, these results show that expression of the M138L gene contributes directly or indirectly to immune evasion by MYXV. In the future, these results could help us to better understand the pathogenesis of myxomatosis but also the importance of glycans in regulation of immune responses., info:eu-repo/semantics/published

Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV-3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV-3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin., Author Summary Common carp, and its colorful ornamental variety koi, is one of the most economically valuable species in aquaculture. Since the late 1990s, the common and koi carp culture industries have suffered devastating worldwide losses due to cyprinid herpesvirus 3 (CyHV-3). In the present study, we report the development of an attenuated recombinant vaccine against CyHV-3. Two genes were deleted from the viral genome, leading to a recombinant virus that is no longer capable of causing the disease but can be propagated in cell culture (for vaccine production) and infect fish when added to the water, thereby immunizing the fish. This attenuated recombinant vaccine also had a drastic defect in spreading from vaccinated to non-vaccinated cohabitant fish. The vaccine induced a protective mucosal immune response capable of preventing the entry of virulent CyHV-3 and is compatible with the simultaneous vaccination of a large number of carp by simply immersing the fish in water containing the vaccine. This vaccine represents a promising tool for controlling the most dreadful disease ever encountered by the carp culture industries. In addition, the present study highlights the importance of the CyHV-3 - carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

Rabies is a fatal anthropozoonotic viral infection of the central nervous system that remains a serious public health problem in many countries. As several animal cases of spontaneous survival to infection were reported and because type 1 interferons were shown to protect against the virus, it was suggested that innate resistance mechanisms exist. Among the antiviral proteins that are synthesized in response to interferon-alpha/beta stimulation, Mx proteins from several species are long known to block the replication of vesicular stomatitis virus (VSV). As both VSV and rabies virus belongs to the Rhabdoviridae family, this study was started with the aim to establish whether the anti-VSV activity of a mammalian Mx protein could be extended to rabies virus. This question was addressed by inoculating the virus onto a bovine Mx1 or human MxA-expressing Vero cell clone. Plaque formation was unambiguously blocked, and viral yields were reduced 100- to 1000-fold by bovine Mx1 expression for both SAG2 and SADB19 viral strains. In opposition, only SAG2 strain could be inhibited by the expression of human MxA protein. The effect of both proteins expression was then evaluated at the viral protein expression level. Again, boMx1 was able to repress protein expression in both strain, whereas only SAG2 proteins were inhibited in human MxA-expressing cells. These results suggest that protection conferred by interferon-alpha/beta against rabies could be, at least partially, attributable to the Mx pathway. Alternatively, bovine Mx1 could be unique in its ability to repress rabies virus which, if confirmed in vivo, would open an avenue for the development of new antirabies therapeutic strategies.

Toll-like receptors (TLR) are an important component in the innate immune response to a wide variety of pathogens. Recently, a series of studies has addressed the hypothesis that TLR4 also participates in the host innate response against respiratory syncytial virus (RSV), the leading cause of lower respiratory tract infections in infants and young children. In most of the studies available, RSV, which is not a natural pathogen of mice, has been systematically used in mouse models of human bronchiolitis, with conflicting results. Pneumonia virus of mice (PVM), a member of the pneumovirus genus, shares many similarities with RSV. The serological and structural relationships that exist between them suggest that the immune response to these viruses may be similar in their respective natural hosts. To determine the role of TLR4 in host defense against PVM, TLR4-competent and TLR4-deficient mice were intranasally infected with PVM. Variation of body weight, pulmonary function values, histopathology, and pulmonary viral loads were analyzed. None of the investigated clinical, functional, histological and virological parameters was different between strains, which demonstrates that the sensitivity of the mouse to its natural pneumovirus infection is independent of the presence or absence of TLR4 sensing.

In October 2012, a 3-year-old bullfinch (Pyrrhula pyrrhula) held in captivity for its entire lifespan and a wild adult great spotted woodpecker (Dendrocopos major), both with neurological signs, were found 4 km from each other and 5 days apart in the Meuse Valley, Belgium. Non-suppurative encephalitis and mild degeneration and necrosis were identified in the brain and cerebellum, and Usutu virus antigen and RNA were detected by immunohistochemistry and real-time reverse transcriptase PCR, respectively. The two cases reported here represent the most western distribution of clinical disease in birds due to Usutu virus in Europe.

The Paramyxoviridae family includes some of the most important and ubiquitous disease-causing viruses of infants and children, most of which cause significant infections of the respiratory tract. Evidence is accumulating in humans that genetic factors are involved in the severity of clinical presentation. As a first step toward the identification of the genes involved, this study was undertaken to establish whether laboratory mouse strains differ in susceptibility to Sendai virus, the murine counterpart of human type-1 parainfluenza virus which, historically, has been used extensively in studies that have defined the basic biological properties of paramyxoviruses in general. With this purpose in mind, double-chamber plethysmography data were collected daily for 7 days after inoculation of Sendai virus in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values closely reflected the success of viral replication in the lungs and revealed a pattern of continuous variation with resistant, intermediate, and susceptible strains. The results unambiguously suggest that BALB/c (resistant) and 129Sv (susceptible) strains should be used in crossing experiments aimed at identifying the genes involved in resistance to Paramyxoviridae by the positional cloning approach.

Chronic wasting disease (CWD) has not been reported in Europe, whereas it is considered to be enzootic in free-ranging mule deer, Rocky mountain elk and white-tailed deer in the area of Colorado, Wyoming, and Nebraska, and new foci of CWD have been detected in other parts of the United States. However, no large-scale active epidemiosurveillance of European wild cervids has been installed in Europe. In accordance with the opinion of the European Scientific Steering Committee, a preliminary (active) surveillance scheme was installed, in order to improve the knowledge of the CWD status of the Belgian free-ranging cervids (roe deer and red deer). Spleen samples (n=866) of roe deer and red deer collected in the south-eastern part of Belgium, were examined for CWD using a enzyme-linked immunosorbent assay of Bio-Rad. Afterwards, the ELISA was systematically confirmed by immunohistochemistry using three antibodies, namely R524, 2G11 and 12F10. There were no indications on the occurrence of transmissible spongiform enncephalopathy (TSE) in any of the samples. A Bayesian framework was used for the estimation of the true prevalence of CWD in south-eastern part of Belgium that was estimated to have a median value of zero with a 95% percentile value of 0.00115.

Typical targets of type I interferon (IFN)-induced antiviral Mx proteins known to date have been shown to share a common profile: single-stranded negative-sense RNA viruses. Among them, human MxA is known to interfere with the replication of measles, human, and bovine parainfluenza-3 viruses (BoPi3V), that is, three members of the Paramyxoviridae family. Recently, bovine Mx1 protein (BoMx1) was included in the group of Mx proteins with authenticated antiviral potential, as it dramatically represses the replication of vesicular stomatitis virus (VSV). As replication in bovine cells of Pi3, respiratory syncytial (RS), and Sendai (Se) viruses, all members of the same family, is known to be reduced on IFN-alpha incorporation into the culture medium, it was hypothesized that the BoMx1 pathway possibly was involved, its antiviral spectrum thus probably extending to Paramyxoviridae. In this study, probing of BoMx1-inhibiting effects was carried out by infecting a transgenic Vero cell line that allows tightly regulated conditional expression of BoMx1 after doxycycline treatment with a wide array of Paramyxoviridae. Expressing and nonexpressing cells displayed similar viability, cytopathic effects (CPEs), and amounts of infectious virus yields, whatever the infecting virus or the multiplicity of infection (moi) imposed. It is, therefore, concluded that BoMx1 does not interfere with Paramyxoviridae.

Mannheimia haemolytica induced pneumonias are only observed in goats, sheep and cattle. The bacterium produces several virulence factors,whose principal ones are lipopolysaccharide and leukotoxin. The latter is cytotoxic only for ruminant leukocytes, a phenomenon that is correlated with its ability to bind and interact with the ruminant β2-integrin Lymphocyte Function-associated Antigen 1. This paper globally reviews all the information available on host-pathogen interactions underlying respiratory mannheimiosis (formerly pasteurellosis), from the stable and the Petri dish to the biochemical cascade of events triggered by the leukotoxin inside ruminant leukocytes. One conclusion can be made: the most widespread cattle respiratory disease with the most important impact on beef production worldwide, is probably due to a tiny ruminant-specific focal variation in the CD18- and/or CD11a-expressing genes. Mannheimia haemolytica / leukotoxin / β2-integrin / ruminant

In some vertebrate species, type I interferon(IFN)-induced Mx gene expression has been shown to confer resistance to some single-stranded RNA (ssRNA) viruses in vitro. Because the bovine species is subject to an exceptionally wide array of infections caused by such viruses, it is anticipated that an antiviral allele should have been retained by evolution at the bovine Mx locus. The identification of such allele may help in evaluating the real significance of the Mx genotype for disease resistance in vivo, in deciphering host-virus molecular interactions involved, or in improving innate disease resistance of livestock through marker-assisted selection. We validated a double transgenic Vero cell clone in which the bovine Mx1 reference allele is placed under control of the human cytomegalovirus (CMV) enhancer-promoter sequence containing elements from the bacterial tetracycline resistance operon to regulate transcription. In the selected clone, transgene repression was very tight, and derepression by doxycycline led to homogeneous 48-h duration expression of physiologic levels of bovine Mx1. Expression of the transgene caused a dramatic decrease in cytopathic efficiency and a 500-5000-fold yield reduction of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV). To our knowledge, the transgenic clone developed here is the first ever reported that allows conditional expression of an Mx protein, thus providing a valuable tool for studying functions of Mx proteins in general and that of bovine Mx1 in particular. This latter may henceforward be included in the group of Mx proteins with authenticated anti-VSV activity, which offers new research avenues into the field of host-virus interactions.

Double-chamber plethysmography has been recognized since 1979 as a reference technique to measure pulmonary function values in guinea pigs, but it has not gained attention for use in mice. Theoretically, however, this technique combines the advantages of single-chamber plethysmography with a quantitative assessment of flow and/or volume and a calculated resistance, the interpretation of which in terms of bronchoconstriction is not disputed. Here we show that, when appropriately preconditioned, mice are able to gradually grow accustomed to the apparatus and display extremely stable nasal and thoracoabdominal flow tracings. Overall, strain, sex, and somatic growth had a significant effect on pulmonary function values. The changes in specific airway resistance (sRaw) and enhanced pause (Penh) values were never in the same direction, indicating that they measure different things. The respiratory frequency was far higher in C57BL/6 compared with BALB/c mice. Peak flows, minute volume, specific tidal and minute volumes, and sRaw were also higher, but Penh was smaller. Males breathed at a higher frequency than females, leading to a higher minute volume. Nevertheless, the specific volumes were considerably higher among females. Penh was lower in males, whereas sRaw was identical in both sexes. Changes associated with somatic growth were rapid and important between 5 and 9 wk, then slowed down between 9 and 12–13 wk and became almost imperceptible after.

Schmallenberg virus was detected in cattle and sheep in northwestern Europe in 2011. To determine whether wild ruminants are also susceptible, we measured antibody seroprevalence in cervids (roe deer and red deer) in Belgium in 2010 and 2011. Findings indicated rapid spread among these deer since virus emergence ≈250 km away.

To determine prevalence of antibodies against Schmallenberg virus in adult cows and proportion of infection transmitted to fetuses, we tested serum samples from 519 cow/calf pairs in Belgium in spring 2012. Of cattle within 250 km of location where the virus emerged, ≈91% tested positive for IgG targeting nucleoprotein. Risk for fetal infection was ≈28%.

Cycloviruses, small ssDNA viruses of the Circoviridae family, have been identified in the cerebrospinal fluid from symptomatic human patients. One of these species, cyclovirus-Vietnam (CyCV-VN), was shown to be restricted to central and southern Vietnam. Here we report the detection of CyCV-VN species in stool samples from pigs and humans from Africa, far beyond their supposed limited geographic distribution.

In the summer-fall of 2011, a nonspecific febrile syndrome characterized by hyperthermia, drop in milk production and watery diarrhea was reported in adult dairy cows from a series of farms located in North-West Europe. Further, in November 2011, an enzootic outbreak of abortion, stillbirth and birth at term of lambs, kids and calves with neurologic signs and/or head, spine or limb malformations emerged throughout several European countries. Both syndromes were associated with the presence in the blood (adults) or in the central nervous system (newborns) of the genome of a new Shamonda-Sathuperi reassortant orthobunyavirus provisionally named Schmallenberg virus after the place where the first positive samples were collected. The clinical, pathological, virological and epidemiological facts that were made publicly available during the first 6 months after the emergence are presented here. Current knowledge of the epidemiology of the phylogenetically closest relatives of the newcomer (Shamonda, Sathuperi, Aino and Akabane viruses) is not exhaustive enough to predict whether the current outbreak of Schmallenberg virus is the prelude to endemicity or to a 2 years long outbreak before the infection burns out when serologically naive animals are no longer available. In the future, cyclic epizootic reemergences are a possibility too, either synchronized with a global decrease of herd immunity or due to antigenic variants escaping the immunity acquired against their predecessors. The latter hypothesis seems unlikely because of the wide array of biologic constraints acting on the genome of viruses whose life cycle requires transmission by a vector, which represses genetic drift. The remarkable stability of the Shamonda virus genome over the last forty years is reassuring in this regard.

IN Europe, cases of Lyme borreliosis, tickborne encephalitis and granulocytic anaplasmosis have increased in human beings, domestic animals and wildlife, expanding their geographical ranges to much of Scandinavia and eastern Europe. Proposed explanations include improved surveillance and diagnosis

Veterinary inspection in slaughterhouses allows for the detection of macroscopic lesions reminiscent of bovine tuberculosis, but the presence of Mycobacterium bovis must be confirmed by laboratory methods. This study aimed at comparing the performances of the standard diagnostic tools used to identify M. bovis in tissue specimens sampled from suspicious animals. During a two years period, 1390 cattle were inspected at the Machachi abattoir in the Mejia canton Ecuador. A total of 33 animals with granulomatous lesions were detected, representing 2.33% (16/687) and 2.42% (17/703) animals examined in 2007 and 2008, respectively. Ninety-four tissue specimens were sampled and screened for the presence of mycobacteria. Acid-fast bacilli were identified in one third of the suspicious cattle (11/33) and suggestive microscopic lesions in 27.3% (9/33) of the samples examined by direct microscopy and histopathology, respectively. Culturing on Stonebrink medium and 16S-rRNA-based polymerase chain reaction (PCR) yielded 36.4% (12/33) and 27.3% (9/33) of positives, respectively. Compared to culture, other diagnostic procedures displayed a lower sensitivity, with 56.5% for PCR, and 43.5% for direct microscopy and histopathology; however, the specificity was higher (94.4% for PCR and microscopy, and 97.2% for histopathology). We conclude that reliable post-mortem laboratory testing either requires the combination of a set of available diagnostic tools or necessitates the development of improved new-generation tools with better sensitivity and specificity characteristics.

Gammaherpesviruses are archetypal pathogenic persistent viruses. The known human gammaherpesviruses (Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus) are host-specific and therefore lack a convenient in vivo infection model. This makes related animal gammaherpesviruses an important source of information. Infection by murid herpesvirus 4 (MuHV-4), a virus originally isolated from bank voles (Myodes glareolus), was studied here. MuHV-4 infection of inbred laboratory mouse strains (Mus musculus) is commonly used as a general model of gammaherpesvirus pathogenesis. However, MuHV-4 has not been isolated from house mice, and no systematic comparison has been made between experimental MuHV-4 infections of mice and bank voles. This study therefore characterized MuHV-4 (strain MHV-68) infection of bank voles through global luciferase imaging and classical virological methods. As in mice, intranasal virus inoculation led to productive replication in bank vole lungs, accompanied by massive cellular infiltrates. However, the extent of lytic virus replication was approximately 1000-fold lower in bank voles than in mice. Peak latency titres in lymphoid tissue were also lower, although latency was still established. Finally, virus transmission was tested between animals maintained in captivity. However, as observed in mice, MuHV-4 was not transmitted between voles under these conditions. In conclusion, this study revealed that, despite quantitative differences, replication and the latency sites of MuHV-4 are comparable in bank voles and mice. Therefore, it appears that, so far, Mus musculus represents a suitable host for studying gammaherpesvirus pathogenesis with MuHV-4. Establishing transmission conditions in captivity will be a vital step for further research in this field.

International audience; The interferon-induced Mx proteins of vertebrates are dynamin-like GTPases, some isoforms of which can additionally inhibit the life cycle of certain RNA viruses. Here we show that the porcine Mx1 protein (poMx1) inhibits replication of influenza A virus and we attempt to identify the step at which the viral life cycle is blocked. In infected cells expressing poMx1, the level of transcripts encoding the viral nucleoprotein is significantly lower than normal, even when secondary transcription is prevented by exposure to cycloheximide. This reveals that a pretranscriptional block participates to the anti-influenza activity. Binding and internalization of incoming virus particles are normal in the presence of poMx1 but centripetal traffic to the late endosomes is interrupted. Surprisingly but decisively, poMx1 significantly alters binding of early endosome autoantigen 1 to early endosomes and/or early endosome size and spatial distribution. This is compatible with impairment of traffic of the endocytic vesicles to the late endosomes.

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against pathogenic RNA viruses, such as members of the orthomyxoviridae, bunyaviridae, and rhabdoviridae families. Among the diverse mammalian Mx proteins examined so far, we have recently demonstrated in vitro that the Bos taurus isoform 1 (boMx1) is endowed with exceptional anti-rabies-virus activity. This finding has prompted us to seek an appropriate in vivo model for confirming and evaluating gene therapy strategies. Using a BAC transgene, we have generated transgenic mouse lines expressing the antiviral boMx1 protein and boMx2 proteins under the control of their natural promoter and short- and long-range regulatory elements. Expressed boMx1 and boMx2 are correctly assembled, as deduced from mRNA sequencing and western blotting. Poly-I/C-subordinated expression of boMx1 was detected in various organs by immunohistochemistry, and transgenic lines were readily classified as high- or low-expression lines on the basis of tissue boMx1 concentrations measured by ELISA. Poly-I/C-induced Madin-Darby bovine kidney cells, bovine turbinate cells, and cultured cells from high-expression line of transgenic mice were found to contain about the same concentration of boMx1, suggesting that this protein is produced at near-physiological levels. Furthermore, insertion of the bovine Mx system rendered transgenic mice resistant to vesicular-stomatitis-virus-associated morbidity and mortality, and embryonic fibroblasts derived from high-expression transgenic mice were far less permissive to the virus. These results demonstrate that the Bos taurus Mx system is a powerful anti-VSV agent in vivo and suggest that the transgenic mouse lines generated here constitute a good model for studying in vivo the various antiviral functions—known and yet to be discovered—exerted by bovine Mx proteins, with priority emphasis on the antirabic function of boMx1.

In a recent study dealing with a mouse model of Trypanosoma evansi-associated disease, a remarkable synchrony between the parasitaemia peak and the white-blood-cell count nadir was noticed. The present study was designed to establish whether there is a direct causal link between the parasite load during its exponential phase of growth and the disappearance of peripheral blood leukocytes. In vitro experiments performed with trypanosomes and purified peripheral blood mononucleated cells revealed the existence of a lymphotoxin embedded in the T. evansi membrane: a protein sensitive to serine proteases, with a molecular mass of less than 30 kDa. Lymphocytes death induced by this protein was found to depend on the intervention of a lymphocytic protein tyrosine phosphatase. When lymphocytes were exposed to increasing quantities of a monoclonal antibody raised against the extracellular portion of CD45, a transmembrane protein tyrosine phosphatase covering over 10% of the lymphocyte surface, T. evansi membrane extracts showed a dose-dependent decrease in cytotoxicity. As the regulatory functions of CD45 concern not only the fate of lymphocytes but also the activation threshold of the TCR-dependent signal and the amplitude and nature of cytokinic effects, this demonstration of its involvement in T. evansi-dependent lymphotoxicity suggests that T. evansi might manipulate, via CD45, the host's cytokinic and adaptive responses.

Background Actinobacillus pleuropneumoniae, the causative bacterial agent of porcine pleuropneumonia, produces Apx toxins which belong to RTX toxin family and are recognized as the major virulence factors. So far, their target receptor(s) has not been identified and the disease cytopathogenesis remains poorly understood. Production of an active Apx toxin and characterization of its toxic activity constitute the premises necessary to the description of its interaction with a potential receptor. From this point of view, we produced an active recombinant ApxIIIA toxin in order to characterize its toxicity on peripheral blood mononucleated cells (PBMCs) isolated from several species. Findings Toxin preparation exercises a strong cytotoxic action on porcine PBMCs which is directly related to recombinant ApxIIIA since preincubation with polymyxin B does not modify the cytotoxicity rate while preincubation with a monospecific polyclonal antiserum directed against ApxIIIA does. The cell death process triggered by ApxIIIA is extremely fast, the maximum rate of toxicity being already reached after 20 minutes of incubation. Moreover, ApxIIIA cytotoxicity is species-specific because llama, human, dog, rat and mouse PBMCs are resistant. Interestingly, bovine and caprine PBMCs are slightly sensitive to ApxIIIA toxin too. Finally, ApxIIIA cytotoxicity is cell type-specific as porcine epithelial cells are resistant. Conclusion We have produced an active recombinant ApxIIIA toxin and characterized its specific cytotoxicity on porcine PBMCs which will allow us to get new insights on porcine pleuropneumonia pathogenesis in the future.

African trypanosomiasis is a severe parasitic disease affecting both man and livestock. It is crucial to expand our fundamental knowledge of the intimate interactions between trypanosomes and their vertebrate hosts in order to develop new and efficient control strategies. The mouse model of trypanosomiasis is the most popular for research purposes because of all the logistic advantages of using this species. Studies of any aspect of trypanosomiases in the mouse systematically require the quantification of some phenotypic traits which translate its degree of resistance/susceptibility to the disease, as blood cell counts. The present study presents a methodological approach combining everyday microsampling of tail blood and its analysis by flow cytometry. The technical options and conditions permitting a fast, reliable and reproducible daily quantification of erythrocyte, reticulocyte, leucocyte and trypanosome counts in the inoculated mouse were established. The protocol proposed allows the multiplication of blood samplings without being exposed to the time-consuming constraint of visual countings, without causing iatrogenic blood cell alterations in the mouse and without requiring specific anti-trypanosome antibodies.

BackgroundThe most predominantbeta2-integrin lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18,alphaLbeta2), expressed on all leukocytes, is essential for many adhesive functions of the immune system. Interestingly, RTX toxin-producing bacteria specifically target this leukocytebeta2-integrin which exacerbates lesions and disease development.ResultsThis study reports the sequencing of the wild boarbeta2-integrin CD11a and CD18 cDNAs. Predicted CD11a and CD18 subunits share all the main structural characteristics of their mammalian homologues, with a larger interspecies conservation for the CD18 than the CD11a. Besides these strong overall similarities, wild boar and domestic pig LFA-1 differ by 2 (CD18) and 1 or 3 (CD11a) substitutions, of which one is located in the crucial I-domain (CD11a, E168D).ConclusionAs most wild boars are seropositive to the RTX toxin-producing bacteriumActinobacillus pleuropneumoniaeand because they have sustained continuous natural selection, future studies addressing the functional impact of these polymorphisms could bring interesting new information on the physiopathology ofActinobacillus pleuropneumoniae-associated pneumonia in domestic pigs.

Background The effects of proanthocyanidins (PACs), isolated from blackcurrant (Ribes nigrum L.) leaves, on neutrophil accumulation during inflammatory processes were investigated in vivo and in vitro. Methods In vivo studies were performed using carrageenin-induced pleurisy in rats pre-treated with PACs. Exudate volume and PMNs accumulation were measured. Leukocyte cell adhesion molecules (LFA-1, Mac-1 and VLA-4) mobilization in circulating granulocytes were analysed by flow cytometry and endothelial cell adhesion molecules (ICAM-1 and VCAM-1) were detected by immunohistochemistry on lung sections. In vitro studies were conducted on endothelial LT2 cells, stimulated with TNF-α, to evaluate ICAM-1, IL-8 and VEGF mRNA expression upon PACs treatment. Data sets were examined by one-way analysis of variance (ANOVA) followed by a Scheffe post-hoc test. Results Pretreatment of the animals with PACs (10, 30 and 60 mg/kg) inhibited dose-dependently carrageenin-induced pleurisy in rats by reducing pleural exudate formation and PMNs infliltration. Leukocyte cell adhesion molecules mobilization was not down-regulated on granulocytes by PACs. Immunohistochemistry on lung sections showed a decreased production of endothelial cell adhesion molecules. In vitro experiments demonstrated that PACs were able to significantly inhibit ICAM-1 but not IL-8 and VEGF165 mRNA expression. Moreover, VEGF121 mRNA expression was dose-dependently enhanced. Conclusion This study provides evidence to support the anti-inflammatory activity of proanthocyanidins is related to an inhibition of leukocyte infiltration which can be explained at least in part by a down-regulation of endothelial adhesion molecules, ICAM-1 and VCAM-1 and that these compounds are capable of modulating TNF-α-induced VEGF transcription.