Interferon alpha suppresses alphaherpesvirus immediate early protein levels in sensory neurons, leading to the establishment of a latent infection

Alphaherpesviruses are a subfamily of the herpesviruses containing closely related human and animal pathogens, including human herpes simplex virus (HSV-1) and porcine pseudorabies virus (PRV)

Porcine NK cells stimulate proliferation of pseudorabies virus-experienced CD8+ and CD4+ CD8+ T cells

Natural killer (NK) cells belong to the innate immune system and play a central role in the defense against viral infections and cancer development, but also contribute to shaping adaptive immune responses. NK cells are particularly important in the first line defense against herpesviruses, including alphaherpesviruses. In addition to their ability to kill target cells and produce interferon-g, porcine and human NK cell subsets have been reported to display features associated with professional antigen presenting cells (APC), although it is currently unclear whether NK cells may internalize debris of virus-infected cells and whether this APC-like activity of NK cells may stimulate proliferation of antiviral T cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we show that vaccination of pigs with a live attenuated PRV vaccine strain triggers expression of MHC class II on porcine NK cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8(+) and CD4(+) CD8(+) PRV-experienced T cells. These results highlight the potential of targeting these NK cell features in future vaccination strategies

Reduced virulence of a pseudorabies virus isolate from wild boar origin in domestic pigs correlates with hampered visceral spread and age-dependent reduced neuroinvasive capacity

Morbidity and mortality associated with pseudorabies virus (PRV) infection are dependent on the age of the pig and the virulence of the strain. PRV strains circulating in wild boar are considered to be low virulent, but no mechanistic explanation for their reduced virulence is available. Here infection of 2-and 15-week-old domestic pigs with the PRV wild boar strain BEL24043 did not induce clinical symptoms in 15-week-old pigs, but resulted in important neurological and respiratory disease in 2-week-old piglets. A detailed study of the (neuro) pathogenesis and associated cytokine mRNA expression showed that the reduced virulence of the wild boar strain, compared to what was previously reported for the virulent domestic NIA3 strain, is due to a severely hampered spread to visceral organs in pigs of both age categories and to an efficient suppression of viral replication at primary replication sites of 15-week-old pigs and to a lesser extent in those of 2-week-old piglets. The age-dependent difference in induced symptoms seems to be due to an immature development state of the immune and/or nervous system in 2-week-old pigs. An extensive viral replication associated with a robust expression of cytokine-related mRNA was found in the olfactory bulb of 2-week-old piglets, correlating with observed neurological disease. Neuroinvasion also occurred via the trigeminal route in 2-week-old pigs, but viral replication was efficiently suppressed in the trigeminal ganglion in the presence of a moderate induction of cytokine-related mRNA. Viral replication in the peripheral and central nervous system of 15-week-old pigs was limited and efficiently suppressed

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

DNase SISPA-next generation sequencing confirms Schmallenberg virus in Belgian field samples and identifies genetic variation in Europe

In 2011, a novel Orthobunyavirus was identified in cattle and sheep in Germany and the Netherlands. This virus was named Schmallenberg virus (SBV). Later, presence of the virus was confirmed using real time RT-PCR in cases of congenital malformations of bovines and ovines in several European countries, including Belgium. In the absence of specific sequencing protocols for this novel virus we confirmed its presence in RT-qPCR positive field samples using DNase SISPA-next generation sequencing (NGS), a virus discovery method based on random amplification and next generation sequencing. An in vitro transcribed RNA was used to construct a standard curve allowing the quantification of viral RNA in the field samples. Two field samples of aborted lambs containing 7.66 and 7.64 log(10) RNA copies per mu L total RNA allowed unambiguous identification of SBV. One sample yielded 192 SBV reads covering about 81% of the L segment, 56% of the M segment and 13% of the S segment. The other sample resulted in 8 reads distributed over the L and M segments. Three weak positive field samples (one from an aborted calf, two from aborted lambs) containing virus quantities equivalent to 4.27-4.89 log(10) RNA copies per mu L did not allow identification using DNase SISPA-NGS. This partial sequence information was compared to the whole genome sequence of SBV isolated from bovines in Germany, identifying several sequence differences. The applied viral discovery method allowed the confirmation of SBV in RT-qPCR positive brain samples. However, the failure to confirm SBV in weak PCR-positive samples illustrates the importance of the selection of properly targeted and fresh field samples in any virus discovery method. The partial sequences derived from the field samples showed several differences compared to the sequences from bovines in Germany, indicating sequence divergence within the epidemic

Interferon Alpha Induces Establishment of Alphaherpesvirus Latency in Sensory Neurons In Vitro

Background: Several alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), establish lifelong latency in neurons of the trigeminal ganglion (TG). Although it is thought that efficient establishment of alphaherpesvirus latency is based on a subtle interplay between virus, neurons and the immune system, it is not clear which immune components are of major importance for the establishment of latency. Methodology/Principal Findings: Here, using an in vitro model that enables a natural route of infection, we show that interferon alpha (IFNalpha) has the previously uncharacterized capacity to induce a quiescent HSV-1 and PRV infection in porcine TG neurons that shows strong similarity to in vivo latency. IFNalpha induced a stably suppressed HSV-1 and PRV infection in TG neurons in vitro. Subsequent treatment of neurons containing stably suppressed virus with forskolin resulted in reactivation of both viruses. HSV and PRV latency in vivo is often accompanied by the expression of latency associated transcripts (LATs). Infection of TG neurons with an HSV-1 mutant expressing LacZ under control of the LAT promoter showed activation of the LAT promoter and RT-PCR analysis confirmed that both HSV-1 and PRV express LATs during latency in vitro. Conclusions/Significance: These data represent a unique in vitro model of alphaherpesvirus latency and indicate tha

α-Herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites

α-Herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine α-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of α-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus

Risk assessment of SARS-CoV-2 infection in free-ranging wild animals in Belgium.

The aim of this review paper is to evaluate the putative susceptibilities of different free-ranging wild animal species in Belgium to SARS-CoV-2 and provide a risk assessment of SARS-CoV-2 infection in those animals. Since the onset of the Covid-19 pandemic, natural SARS-CoV-2 infections have mainly been confirmed in domestic and production animals, and in wild animals kept in captivity, although the numbers remain limited when compared to human cases. Recently, the first SARS-CoV-2 infections in presumably escaped minks found in the wild have been detected, further addressing the much-feared scenario of transmission of the virus to animals living in the wild and its consequences. Considering the most likely origin of the virus being a wild animal and the putative susceptibilities of free-ranging wild animal species to SARS-CoV-2, the risk of infection with possible establishment of the virus in these populations has to be investigated closely. The authors conclude that most attention should be given to surveillance and awareness raising activities for SARS-CoV-2 infection in wild mustelids, bats, wild canids and felids, particularly these collected in wildlife rescue centres. People involved in frequent and close contact with wild animals should take all necessary precautionary measures to protect wild animals against exposure to the virus. One year after the first detection of SARS-CoV-2 in humans, the time has come to increase investments in research and surveillance activities in animals, including in free-ranging wild animals, as part of a One Health control of this pandemic. This study focusing on Belgium could be helpful for other countries with similar animal densities and ecosystems

Putative role of arthropod vectors in African swine fever virus transmission in relation to their bio-ecological properties

African swine fever (ASF) is one of the most important diseases in Suidae due to its significant health and socioeconomic consequences and represents a major threat to the European pig industry, especially in the absence of any available treatment or vaccine. In fact, with its high mortality rate and the subsequent trade restrictions imposed on affected countries, ASF can dramatically disrupt the pig industry in afflicted countries. In September 2018, ASF was unexpectedly identified in wild boars from southern Belgium in the province of Luxembourg, not far from the Franco-Belgian border. The French authorities rapidly commissioned an expert opinion on the risk of ASF introduction and dissemination into metropolitan France. In Europe, the main transmission routes of the virus comprise direct contact between infected and susceptible animals and indirect transmission through contaminated material or feed. However, the seasonality of the disease in some pig farms in Baltic countries, including outbreaks in farms with high biosecurity levels, have led to questions on the possible involvement of arthropods in the transmission of the virus. This review explores the current body of knowledge on the most common arthropod families present in metropolitan France. We examine their potential role in spreading ASF—by active biological or mechanical transmission or by passive transport or ingestion—in relation to their bio-ecological properties. It also highlights the existence of significant gaps in our knowledge on vector ecology in domestic and wild boar environments and in vector competence for ASFV transmission. Filling these gaps is essential to further understanding ASF transmission in order to thus implement appropriate management measures

VectorNet Data Series 3: Culicoides Abundance Distribution Models for Europe and Surrounding Regions

This is the third in a planned series of data papers presenting modelled vector distributions produced during the ECDC and EFSA funded VectorNet project. The data package presented here includes those Culicoides vectors species first modelled in 2015 as part of the VectorNet gap analysis work namely C. imicola, C. obsoletus, C. scoticus, C. dewulfi, C. chiopterus, C. pulicaris, C. lupicaris, C. punctatus, and C. newsteadi. The known distributions of these species within the Project area (Europe, the Mediterranean Basin, North Africa, and Eurasia) are currently incomplete to a greater or lesser degree. The models are designed to fill the gaps with predicted distributions, to provide a) first indication of vector species distributions across the project geographical extent, and b) assistance in targeting surveys to collect distribution data for those areas with no field validated information. The models are based on input data from light trap surveillance of adult Culicoides across continental Europe and surrounding regions (71.8°N –33.5°S, – 11.2°W – 62°E), concentrated in Western countries, supplemented by transect samples in eastern and northern Europe. Data from central EU are relatively sparse.Peer reviewe