A brief summary of the epidemiology and genetic relatedness of avian influenza H9N2 virus in birds and mammals in the Middle East and North Africa.

H9N2 is the most widespread avian influenza virus subtype in poultry worldwide. It infects a broad spectrum of host species including birds and mammals. Infections in poultry and humans vary from silent to fatal. Importantly, all AIV, which are fatal in humans (e.g. H5N1, H7N9) acquired their 'internal' gene segments from H9N2 viruses. Although H9N2 is endemic in the Middle East (ME) and North Africa since the late 1990s, little is known about its epidemiology and genetics on a regional level. In this review, we summarised the epidemiological situation of H9N2 in poultry and mammals in Iran, Iraq, Kuwait, Qatar, United Arab Emirates, Oman, Bahrain, Yemen, Saudi Arabia, Jordan, Palestine, Israel, Syria, Lebanon, Turkey, Egypt, Sudan, Libya, Tunisia, Algeria and Morocco. The virus has been isolated from humans in Egypt and serosurveys indicated widespread infection particularly among poultry workers and pigs in some countries. Some isolates replicated well in experimentally inoculated dogs, mice, hamsters and ferrets. Insufficient protection of immunised poultry was frequently reported most likely due to concurrent viral or bacterial infections and antigenic drift of the field viruses from outdated vaccine strains. Genetic analysis indicated several distinct phylogroups including a panzootic genotype in the Asian and African parts of the ME, which may be useful for the development of vaccines. The extensive circulation of H9N2 for about 20 years in this region where the H5N1 virus is also endemic in some countries, poses a serious public health threat. Regional surveillance and control strategy are highly recommended.

Metagenomic approaches to identifying infectious agents.

Since the advent of next-generation sequencing (NGS) technologies, the untargeted screening of samples from outbreaks for pathogen identification using metagenomics has become technically and economically feasible. However, various aspects need to be considered in order to exploit the full potential of NGS for virus discovery. Here, the authors summarise those aspects of the main steps that have a significant impact, from sample selection through sample handling and processing, as well as sequencing and finally data analysis, with a special emphasis on existing pitfalls.

Depuis l'avènement des technologies de séquençage de nouvelle génération, le criblage non ciblé d'échantillons prélevés au cours d'un foyer de maladie afin d'identifier l'agent pathogène en recourant à la métagénomique est devenu accessible aux plans technique et économique. Néanmoins, un certain nombre d'aspects restent à élucider afin d'exploiter pleinement les possibilités offertes par le séquençage de nouvelle génération pour déceler des virus précédemment inconnus. Les auteurs résument ces aspects pour chaque étape déterminante, depuis le choix des échantillons jusqu'à leur manipulation et traitement, et du séquençage à l'analyse des données, en mettant l'accent sur les difficultés éventuelles.

Desde el advenimiento de las técnicas de secuenciación de próxima generación, el cribado no selectivo de muestras tomadas durante un brote para identificar al patógeno empleando la metagenómica ha pasado a ser técnica y económicamente viable. Sin embargo, hay una serie de aspectos que conviene tener en cuenta a fin de poder aprovechar plenamente las posibilidades que ofrecen esas técnicas para descubrir virus. Los autores resumen esos aspectos en relación con las principales etapas que tienen una influencia importante, desde la selección hasta la manipulación y el procesamiento de las muestras, pasando por la secuenciación y el análisis de datos, haciendo especial hincapié en sus posibles inconvenientes.

Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt, 2014/15.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

Prevalence and control of H7 avian influenza viruses in birds and humans.

The H7 subtype HA gene has been found in combination with all nine NA subtype genes. Most exhibit low pathogenicity and only rarely high pathogenicity in poultry (and humans). During the past few years infections of poultry and humans with H7 subtypes have increased markedly. This review summarizes the emergence of avian influenza virus H7 subtypes in birds and humans, and the possibilities of its control in poultry. All H7Nx combinations were reported from wild birds, the natural reservoir of the virus. Geographically, the most prevalent subtype is H7N7, which is endemic in wild birds in Europe and was frequently reported in domestic poultry, whereas subtype H7N3 is mostly isolated from the Americas. In humans, mild to fatal infections were caused by subtypes H7N2, H7N3, H7N7 and H7N9. While infections of humans have been associated mostly with exposure to domestic poultry, infections of poultry have been linked to wild birds or live-bird markets. Generally, depopulation of infected poultry was the main control tool; however, inactivated vaccines were also used. In contrast to recent cases caused by subtype H7N9, human infections were usually self-limiting and rarely required antiviral medication. Close genetic and antigenic relatedness of H7 viruses of different origins may be helpful in development of universal vaccines and diagnostics for both animals and humans. Due to the wide spread of H7 viruses and their zoonotic importance more research is required to better understand the epidemiology, pathobiology and virulence determinants of these viruses and to develop improved control tools.

Twenty years of active bat rabies surveillance in Germany: a detailed analysis and future perspectives.

SUMMARY: In Germany, active bat rabies surveillance was conducted between 1993 and 2012. A total of 4546 oropharyngeal swab samples from 18 bat species were screened for the presence of EBLV-1- , EBLV-2- and BBLV-specific RNA. Overall, 0·15% of oropharyngeal swab samples tested EBLV-1 positive, with the majority originating from Eptesicus serotinus. Interestingly, out of seven RT-PCR-positive oropharyngeal swabs subjected to virus isolation, viable virus was isolated from a single serotine bat (E. serotinus). Additionally, about 1226 blood samples were tested serologically, and varying virus neutralizing antibody titres were found in at least eight different bat species. The detection of viral RNA and seroconversion in repeatedly sampled serotine bats indicates long-term circulation of the virus in a particular bat colony. The limitations of random-based active bat rabies surveillance over passive bat rabies surveillance and its possible application of targeted approaches for future research activities on bat lyssavirus dynamics and maintenance are discussed.

Isolation and partial characterization of a novel virus from different carp species suffering gill necrosis - ultrastructure and morphogenesis.

Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100-350 nm after negative staining and 100-300 nm in ultrathin sections, covered by short projections of 8-10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family.

Pseudorabies virus in wild swine: a global perspective.

Suid herpesvirus 1 (SuHV1, syn. Aujeszky's disease virus [ADV] or pseudorabies virus [PrV]), which belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus is the causative agent of Aujeszky's disease (AD, pseudorabies), a notifiable disease, that causes substantial economic losses to the swine industry in countries, where AD is present. Members of the family Suidae (true pigs) are the only natural hosts for PrV, although the virus can infect numerous other mammals including ruminants, carnivores and rodents. Despite the tremendous progress that has been made in controlling and eliminating PrV in domestic pigs, there is mounting evidence that PrV infections are more widespread in wild swine across the world than originally thought. Unfortunately, our understanding of the extent of PrV infections in these wild populations and of the threat to domestic swine is still fragmentary. This review aims at giving a global perspective on PrV infections in wild swine by scrutinizing the current state of knowledge concerning (i) the global occurrence of PrV infections in free-living populations of wild swine, e.g., wild boar and feral swine, (ii) the molecular characterization of wild swine PrV, (iii) infection characteristics of PrV in populations of wild swine, (iv) the risk of spillover infections to domestic pigs, (v) potential risk-mitigating measures, focusing on further research needs.

Characterization of pseudorabies virus of wild boar origin from Europe.

Pseudorabies virus (PrV) infections appear to be more widely distributed in the European wild boar (Sus scrofa) population than assumed. In Europe, attempts to isolate and characterize the causative agents have been limited so far. We therefore collected and examined a total of 35 PrV isolates obtained from wild boar or hunting dogs in Germany, France, Spain, Italy, Slovakia and Hungary between 1993 and 2008. Restriction enzyme analysis of genomic DNA using BamHI showed that all isolates, except one, belonged to genogroup I but different subtypes were evident. For further investigations of the phylogenetic relationships, a 732-bp fragment of the glycoprotein C (gC) gene was amplified by PCR. Sequence analysis revealed about 40 variant positions within this fragment. Comparison of the nucleotide sequences supported the separation into a clade containing isolates from North-Rhine Westphalia, Rhineland-Palatinate (Germany), France and Spain (clade B) and an apparently more variable clade comprising isolates from Brandenburg, Baden-Wurttemberg, Saxony, Saxony-Anhalt (Germany), Slovakia, Hungary, Italy and France (clade A).

The prefusion structure of herpes simplex virus glycoprotein B.

Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.

Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response.

During stress, the activity of the sympathetic nervous system is changed in a global fashion, leading to an increase in cardiovascular function and a release of adrenal catecholamines. This response is thought to be regulated by a common set of brain neurons that provide a dual input to the sympathetic preganglionic neurons regulating cardiac and adrenal medullary functions. By using a double-virus transneuronal labeling technique, the existence of such a set of central autonomic neurons in the hypothalamus and brainstem was demonstrated. These neurons innervate both of the sympathetic outflow systems and likely function in circumstances where parallel sympathetic processing occurs, such as in the fight-or-flight response.

Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria.

To eradicate rabies in foxes, almost 97 million oral rabies vaccine baits have been distributed in Germany and Austria since 1983 and 1986, respectively. Since 2007, no terrestrial cases have been reported in either country. The most widely used oral rabies vaccine viruses in these countries were SAD (Street Alabama Dufferin) strains, e.g. SAD B19 (53.2%) and SAD P5/88 (44.5%). In this paper, we describe six possible vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) detected during post-vaccination surveillance from 2001 to 2006, involving two different vaccines and different batches. Compared to prototypic vaccine strains, full-genome sequencing revealed between 1 and 5 single nucleotide alterations in the L gene in 5 of 6 SAD isolates, resulting in up to two amino acid substitutions. However, experimental infection of juvenile foxes showed that those mutations had no influence on pathogenicity. The cases described here, coming from geographically widely separated regions, do not represent a spatial cluster. More importantly, enhanced surveillance showed that the vaccine viruses involved did not become established in the red fox population. It seems that the number of reported vaccine virus-associated rabies cases is determined predominantly by the intensity of surveillance after the oral rabies vaccination campaign and not by the selection of strains.

Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses.

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C(t) 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C(t) values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.

Novel avian influenza virus vaccines.

Current vaccines against avian influenza (AI) virus infections are primarily based on classical inactivated whole-virus preparations. Although administration of these vaccines can protect poultry from clinical disease, sterile immunity is not achieved under field conditions, allowing for undetected virus spread and evolution under immune cover. Therefore, there is an urgent need for a robust and reliable system of differentiation between infected and vaccinated animals. Moreover, current AI vaccines must be administered individually, requiring the handling of excessively large numbers of animals, which makes it difficult to obtain high vaccine coverage. Consequently, AI vaccines conferring solid immunity that could be used for mass application would be advantageous. Several approaches are being pursued to improve existing vaccines and develop novel vaccines, all of which will be covered in this overview.

Imaging Mass Spectrometry and Proteome Analysis of Marek's Disease Virus-Induced Tumors.

The highly oncogenic alphaherpesvirus Marek's disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected chickens, including neurological disorders and immunosuppression. Most notably, MDV induces transformation of lymphocytes, leading to T cell lymphomas in visceral organs with a mortality of up to 100%. While several factors involved in MDV tumorigenesis have been identified, the transformation process and tumor composition remain poorly understood. Here we developed an imaging mass spectrometry (IMS) approach that allows sensitive visualization of MDV-induced lymphoma with a specific mass profile and precise differentiation from the surrounding tissue. To identify potential tumor markers in tumors derived from a very virulent wild-type virus and a telomerase RNA-deficient mutant, we performed laser capture microdissection (LCM) and thereby obtained tumor samples with no or minimal contamination from surrounding nontumor tissue. The proteomes of the LCM samples were subsequently analyzed by quantitative mass spectrometry based on stable isotope labeling. Several proteins, like interferon gamma-inducible protein 30 and a 70-kDa heat shock protein, were identified that are differentially expressed in tumor tissue compared to surrounding tissue and naive T cells. Taken together, our results demonstrate for the first time that MDV-induced tumors can be visualized using IMS, and we identified potential MDV tumor markers by analyzing the proteomes of virus-induced tumors.IMPORTANCE Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy.

Encephalitis in a stone marten (Martes foina) after natural infection with highly pathogenic avian influenza virus subtype H5N1.

Recent outbreaks of disease in different avian species, caused by the highly pathogenic avian influenza virus (HPAIV), have involved infection by subtype H5N1 of the virus. This virus has also crossed species barriers and infected felines and humans. Here, we report the natural infection of a stone marten (Martes foina) from an area with numerous confirmed cases of H5N1 HPAIV infection in wild birds. Histopathological examination of tissues from this animal revealed a diffuse nonsuppurative panencephalitis with perivascular cuffing, multifocal gliosis and neuronal necrosis. Additionally, focal necrosis of pancreatic acinar cells was observed. Immunohistochemically, lesions in these organs were associated with avian influenza virus antigen in neurons, glial cells and pancreatic acinar cells. Thus, the microscopical lesions and viral antigen distribution in this stone marten differs from that recently described for cats naturally and experimentally infected with the same virus subtype. This is the first report of natural infection of a mustelid with HPAIV H5N1.

Vaccines against pseudorabies virus (PrV).

Aujeszkýs disease (AD, pseudorabies) is a notifiable herpesvirus infection of pigs causing substantial economic losses to swine producers. AD in pigs is controlled by the use of vaccination with inactivated and attenuated live vaccines. Starting with classically attenuated live vaccines derived from low virulent field isolates, AD vaccination has pioneered novel strategies in animal disease control by the first use of genetically engineered live virus vaccines lacking virulence-determining genes, and the concept of DIVA, i.e. the serological differentiation of vaccinated from field-virus infected animals by the use of marker vaccines and respective companion diagnostic tests. The basis for this concept has been the molecular characterization of PrV and the identification of so-called nonessential envelope glycoproteins, e.g. glycoprotein E, which could be eliminated from the virus without harming viral replication or immunogenicity. Eradication of AD using the strategy of vaccination-DIVA testing has successfully been performed in several countries including Germany and the United States. Furthermore, by targeted genetic modification PrV has been developed into a powerful vector system for expression of foreign genes to vaccinate against several infectious diseases of swine, while heterologous vector systems have been used for expression of major immunogens of PrV. This small concise review summarizes the state-of-the-art information on PrV vaccines and provides an outlook for the future.

Fish rhabdoviruses: molecular epidemiology and evolution.

Rhabdoviruses may cause serious diseases in wild and farmed fish. Within the Rhabdoviridae six genera have been established: Ephemerovirus, Cytorhabdovirus, Nucleorhabdovirus, Lyssavirus, Vesiculovirus, and Novirhabdovirus. Viruses that infect fish are official or tentative members of the genera Vesiculovirus and Novirhabdovirus, or are listed as unassigned rhabdoviruses. In this report, we summarize and discuss published and our own unpublished data on the molecular epidemiology and phylogeography of fish rhabdoviruses including intrapopulational differences and subgrouping of fish rhabdoviruses, in particular the species spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV).

Poultry food products--a source of avian influenza virus transmission to humans?

Global human mobility and intercontinental connectivity, expansion of livestock production and encroachment of wildlife habitats by invasive agricultural land use contribute to shape the complexity of influenza epidemiology. The OneHealth approach integrates these and further elements into considerations to improve disease control and prevention. Food of animal origin for human consumption is another integral aspect; if produced from infected livestock such items may act as vehicles of spread of animal pathogens, and, in case of zoonotic agents, as a potential human health hazard. Notifiable zoonotic avian influenza viruses (AIV) have become entrenched in poultry populations in several Asian and northern African countries since 2003. Highly pathogenic (HP) AIV (e.g. H5N1) cause extensive poultry mortality and severe economic losses. HPAIV and low pathogenic AIV (e.g. H7N9) with zoonotic propensities pose risks for human health. More than 1500 human cases of AIV infection have been reported, mainly from regions with endemically infected poultry. Intense human exposure to AIV-infected poultry, e.g. during rearing, slaughtering or processing of poultry, is a major risk factor for acquiring AIV infection. In contrast, human infections through consumption of AIV-contaminated food have not been substantiated. Heating poultry products according to kitchen standards (core temperatures ≥70°C, ≥10 s) rapidly inactivates AIV infectivity and renders fully cooked products safe. Nevertheless, concerted efforts must ensure that poultry products potentially contaminated with zoonotic AIV do not reach the food chain. Stringent and sustained OneHealth measures are required to better control and eventually eradicate, HPAIV from endemic regions.

Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

CNS inputs to the suprachiasmatic nucleus of the rat.

The neural circuits that modulate the suprachiasmatic nucleus (SCN) of the rat were studied with the retrograde transneuronal tracer--pseudorabies virus. First-order afferents were also identified using cholera toxin beta subunit. Olfactory processing regions (viz., main olfactory bulb, anterior olfactory nucleus, taenia tecta, endopiriform nucleus, medial amygdaloid nucleus, piriform cortex, and posteriomedial cortical amygdaloid nucleus) were virally labeled. The subfornical organ directly innervates SCN; two other circumventricular organs: organum vasculosum of the lamina terminalis and area postrema provide multisynaptic inputs. Direct limbic afferents arise from lateral septum, bed nucleus of the stria terminalis, amygdalohippocampal zone, and ventral subiculum; multineuronal connections come from the basolateral and basomedial amygdaloid nuclei, ventral hippocampus, amygdalopiriform area, as well as lateral entorhinal, perirhinal, and ectorhinal cortices. Most preoptic regions project directly to SCN. Multisynaptic inputs come from the lateral preoptic region. Hypothalamic inputs originate from the anterior, arcuate, dorsal, dorsomedial, lateral, paraventricular, posterior, periventricular posterior, retrochiasmatic, subparaventricular, ventromedial and tuberomammillary nuclei. Paraventricular thalamic nucleus, intergeniculate leaflet and zona incerta directly innervate SCN. Polyneuronal inputs arise from the subparafascicular parvicellular thalamic nucleus. Brainstem afferents originate from the pretectum, superior colliculus, periaqueductal gray matter, parabrachial nucleus, pedunculopontine nucleus, raphe system, locus coeruleus, nucleus incertus and reticular formation. Nucleus tractus solitarius, C3 catecholamine region, rostral ventrolateral medulla and spinal trigeminal nucleus provide indirect inputs. We propose that the SCN receives feedback primarily from interoceptive systems such as the circumventricular, autonomic, and neuroendocrine systems that are important in the central regulation of glucose metabolism (e.g., insulin and glucocorticoids).