Andreas Kurth

Pan-European Distribution of White-Nose Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality

Background The dramatic mass mortalities amongst hibernating bats in Northeastern America caused by “white nose-syndrome” (WNS) continue to threaten populations of different bat species. The cold-loving fungus, Geomyces destructans, is the most likely causative agent leading to extensive destruction of the skin, particularly the wing membranes. Recent investigations in Europe confirmed the presence of the fungus G. destructans without associated mass mortality in hibernating bats in six countries but its distribution remains poorly known. Methodology/Principal Findings We collected data on the presence of bats with white fungal growth in 12 countries in Europe between 2003 and 2010 and conducted morphological and genetic analysis to confirm the identity of the fungus as Geomyces destructans. Our results demonstrate the presence of the fungus in eight countries spanning over 2000 km from West to East and provide compelling photographic evidence for its presence in another four countries including Romania, and Turkey. Furthermore, matching prevalence data of a hibernaculum monitored over two consecutive years with data from across Europe show that the temporal occurrence of the fungus, which first becomes visible around February, peaks in March but can still be seen in some torpid bats in May or June, is strikingly similar throughout Europe. Finally, we isolated and cultured G. destructans from a cave wall adjacent to a bat with fungal growth. Conclusions/Significance G. destructans is widely found over large areas of the European continent without associated mass mortalities in bats, suggesting that the fungus is native to Europe. The characterisation of the temporal variation in G. destructans growth on bats provides reference data for studying the spatio-temporal dynamic of the fungus. Finally, the presence of G. destructans spores on cave walls suggests that hibernacula could act as passive vectors and/or reservoirs for G. destructans and therefore, might play an important role in the transmission process.

White-Nose Syndrome Fungus (Geomyces destructans) in Bats, Europe

Unlike bats in North America, bats in Europe are not killed by this fungus.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

BackgroundAs a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.ResultsThe selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.ConclusionThe MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

An Insect Nidovirus Emerging from a Primary Tropical Rainforest

ABSTRACT Tropical rainforests show the highest level of terrestrial biodiversity and may be an important contributor to microbial diversity. Exploitation of these ecosystems may foster the emergence of novel pathogens. We report the discovery of the first insect-associated nidovirus, tentatively named Cavally virus (CAVV). CAVV was found with a prevalence of 9.3% during a survey of mosquito-associated viruses along an anthropogenic disturbance gradient in Côte d’Ivoire. Analysis of habitat-specific virus diversity and ancestral state reconstruction demonstrated an origin of CAVV in a pristine rainforest with subsequent spread into agriculture and human settlements. Virus extension from the forest was associated with a decrease in virus diversity (P < 0.01) and an increase in virus prevalence (P < 0.00001). CAVV is an enveloped virus with large surface projections. The RNA genome comprises 20,108 nucleotides with seven major open reading frames (ORFs). ORF1a and -1b encode two large proteins that share essential features with phylogenetically higher representatives of the order Nidovirales, including the families Coronavirinae and Torovirinae, but also with families in a basal phylogenetic relationship, including the families Roniviridae and Arteriviridae. Genetic markers uniquely conserved in nidoviruses, such as an endoribonuclease- and helicase-associated zinc-binding domain, are conserved in CAVV. ORF2a and -2b are predicted to code for structural proteins S and N, respectively, while ORF3a and -3b encode proteins with membrane-spanning regions. CAVV produces three subgenomic mRNAs with 5′ leader sequences (of different lengths) derived from the 5′ end of the genome. This novel cluster of mosquito-associated nidoviruses is likely to represent a novel family within the order Nidovirales. IMPORTANCE Knowledge of microbial diversity in tropical rainforests is sparse, and factors driving the emergence of novel pathogens are poorly understood. We discovered and mapped the spread and genetic evolution of a novel mosquito nidovirus from a pristine rainforest to human settlements. Notably, virus diversity decreased and prevalence increased during the process of spreading into disturbed habitats. The novel virus, tentatively termed Cavally virus, contains genetic features common to members of the order Nidovirales (families Coronaviridae, Arteriviridae, and Roniviridae), including conservation of the replicase machinery and expression of subgenomic RNA messages, has a basal phylogenetic relationship to the family Coronaviridae, and clearly differs from the established nidovirus families. Inclusion of this putative novel family in the nidovirus phylogeny suggests that nidoviruses may have evolved from arthropods. Knowledge of microbial diversity in tropical rainforests is sparse, and factors driving the emergence of novel pathogens are poorly understood. We discovered and mapped the spread and genetic evolution of a novel mosquito nidovirus from a pristine rainforest to human settlements. Notably, virus diversity decreased and prevalence increased during the process of spreading into disturbed habitats. The novel virus, tentatively termed Cavally virus, contains genetic features common to members of the order Nidovirales (families Coronaviridae, Arteriviridae, and Roniviridae), including conservation of the replicase machinery and expression of subgenomic RNA messages, has a basal phylogenetic relationship to the family Coronaviridae, and clearly differs from the established nidovirus families. Inclusion of this putative novel family in the nidovirus phylogeny suggests that nidoviruses may have evolved from arthropods.

Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family

Significance Knowledge of the origin and evolution of viruses provides important insight into virus emergence involving the acquisition of genes necessary for the infection of new host species or the development of pathogenicity. The family Bunyaviridae contains important arthropod-borne pathogens of humans, animals, and plants. In this study, we provide a comprehensive characterization of two novel lineages of insect-specific bunyaviruses that are in basal phylogenetic relationship to the rodent-borne hantaviruses, the only genus within the Bunyaviridae that is not transmitted by arthropod vectors. These data, together with ancestral state reconstruction of bunyavirus hosts for major virus lineage bifurcations, suggest that the vertebrate-infecting viruses evolved from arthropod-specific progenitors. The evolutionary origins of arboviruses are unknown because their typical dual host tropism is paraphyletic within viral families. Here we studied one of the most diversified and medically relevant RNA virus families, the Bunyaviridae, in which four of five established genera are transmitted by arthropods. We define two cardinally novel bunyavirus groups based on live isolation of 26 viral strains from mosquitoes (Jonchet virus [JONV], eight strains; Ferak virus [FERV], 18 strains). Both viruses were incapable of replicating at vertebrate-typical temperatures but replicated efficiently in insect cells. Replication involved formation of virion-sense RNA (vRNA) and mRNA, including cap-snatching activity. SDS/PAGE, mass spectrometry, and Edman degradation identified translation products corresponding to virion-associated RNA-dependent RNA polymerase protein (RdRp), glycoprotein precursor protein, glycoproteins Gn and Gc, as well as putative nonstructural proteins NSs and NSm. Distinct virion morphologies suggested ancient evolutionary divergence, with bunyavirus-typical morphology for FERV (spheres of 60–120 nm) as opposed to an unusual bimorphology for JONV (tubular virions of 60 × 600 nm and spheres of 80 nm). Both viruses were genetically equidistant from all other bunyaviruses, showing <15% amino acid identity in the RdRp palm domain. Both had different and unique conserved genome termini, as in separate bunyavirus genera. JONV and FERV define two novel sister taxons to the superclade of orthobunyaviruses, tospoviruses, and hantaviruses. Phylogenetic ancestral state reconstruction with probabilistic hypothesis testing suggested ancestral associations with arthropods at deep nodes throughout the bunyavirus tree. Our findings suggest an arthropod origin of bunyaviruses.

Rat-to-elephant-to-human transmission of cowpox virus.

To the Editor: Despite the eradication of smallpox in the past century, other orthopoxviruses, such as monkeypox virus, vaccinia virus in Brazil, and cowpox virus (CPXV) in Europe (1), still infect humans. CPXV has been restricted to the Old World with wild rodents as its natural reservoir (2,3). Human CPXV infections are commonly described in relation to contact with diseased domestic cats, rarely directly from rats (2,4). Human infections usually remain localized and self-limiting but can become fatal in immunosuppressed patients (5). CPXV infections in captive exotic animals have been reported to be transmitted by rodents (2,6). In February 2007, a circus elephant (Elephas maximus) in northern Germany exhibited disseminated ulcerative lesions of the skin and mucosal membranes (Figure, panel A) caused by CPXV infection; the elephant was euthanized after treatment attempts failed. Electron micrographs of negative-stained biopsy specimens of tongue lesions showed orthopoxvirus particles. The presence of orthopoxvirus after routine virus isolation in Hep2 cells was confirmed in direct immunofluorescence assay with orthopox-specific antibodies. The morphologic feature of hemorrhagic pocks on the chorioallantoic membrane (CAM) of infected embryonated hen’s eggs indicated CPXV. This finding was confirmed by sequence analysis of the complete hemagglutinin (HA) open reading frame (ORF), which showed 99% homology of 921 bp to CPXV isolated in 1984 from an elephant in Hamburg, Germany (Figure, panel B). A serum sample was drawn from the elephant 2 weeks before euthanasia. An indirect fluorescent antibody test (IFAT) detected immunoglobulin (Ig) G antibodies against the new corresponding elephant virus isolate (termed CPXV GuWi) with a titer of 1,260. According to the owner, the >40-year-old female elephant had never been vaccinated with vaccinia virus. Figure Route of cowpox virus (CPXV) transmission and phylogenetic analysis of orthopoxviruses. A) Disseminated ulcerative lesions of the skin around the eye of the circus elephant. Although transmission of CPXV has been confirmed from cats and cows to humans ... Eight days after the elephant’s death, a circumscribed lesion developed on the back of the right hand of a 19-year-old immunocompetent, healthy, unvaccinated animal keeper. CPXV was isolated from lesion fluid and was found to be homologous by using the HA ORF to CPXV GuWi. A convalescent-phase serum sample from the keeper taken 3 weeks later showed a significant increase in IgM (from 40 to 2,560), IgG (from 20 to 10,240), and neutralizing antibody (from <5 to 80) titers. Further simultaneous investigations were undertaken to determine the source of infection. Because no felids were kept on the circus premises, the focus centered on wild rodents that had propagated and infested the area because of the mild winter. Six days after the elephant’s death, 4 rats (Rattus norvegicus) were caught and tested for orthopoxvirus antibodies. Although none of the rats had epidermal lesions or other pathologic changes indicative of a poxvirus infection, all were tested by IFAT and found to be serologically positive (IgG titers 40, 320, 2,560, and >10,240; IgM titers <5, <5, 160, and 2,560), indicating a recent infection in at least 2 animals. CPXV-typical pock morphologic features on the CAM could be visualized after homogenized liver and spleen of the animal with the highest titer was passaged 3 times. Infected CAM and original organ tissues (liver and spleen) showed CPXV by PCR and subsequent sequencing. The corresponding HA ORF displayed perfect homology to the viruses isolated from the elephant and the animal keeper. We report CPXV infection in humans transmitted from an elephant, with rats as a probable source of the elephant’s infection (Figure, panel A). Although the animal keeper was infected by direct contact with the elephant, the exact transmission route from rat to elephant remains unclear. Nevertheless, rats have proven to be a natural reservoir for CPXV (4,7), and infections persisting for >3 weeks were shown for other rodents (8). No data about CPXV prevalence in rats are available, and no data for CPXV isolates from rats have been published in Germany. Therefore, further studies on rats as CPXV reservoir are needed to estimate the potential risk for infection among humans and exotic animals. Zoo and circus animals, especially elephants, seem to be highly susceptible to generalized CPXV infections. Although modified vaccinia virus Ankara was authorized in Germany to be used in vaccinating exotic animals (9), this case highlights the need for increased efforts toward general vaccination of potentially susceptible exotic animals in Europe. The sequence identity of the HA ORFs also demonstrates a low mutation rate of CPXV after it crosses species barriers. As the Figure, panel B, infers, there is a phylogenetic difference between CPCV GuWi and CPXV from a human patient living in the same geographic area (CPXV #2), which indicates the cocirculation of >1 CPXV variant (9,10). Considering the extremely high virus load in infected animals, the broad host range of CPXV, and the abandoned vaccination against smallpox, this case emphasizes the risk among humans of acquiring CPXV infection (6). It also highlights the need for increased awareness regarding clinical features of orthopoxvirus infections and the importance of developing new antiviral drugs against orthopoxviruses.

New Adenovirus in Bats, Germany

We tested 55 deceased vespertilionid bats of 12 species from southern Germany for virus infections. A new adenovirus was isolated from tissue samples of 2 Pipistrellus pipistrellus bats, which represents the only chiropteran virus isolate found in Europe besides lyssavirus (rabies virus). Evidence was found for adenovirus transmission between bats.

Management of pregnant women infected with Ebola virus in a treatment centre in Guinea, June 2014

We report two cases of confirmed Ebola virus disease in pregnant women, who presented at the Médecins Sans Frontières Ebola treatment centre in Guéckédou. Despite the very high risk of death, both pregnant women survived. In both cases the critical decision was made to induce vaginal delivery. We raise a number of considerations regarding the management of Ebola virus-infected pregnant women, including the place of amniocentesis and induced delivery, and whether certain invasive medical acts are justified.

Unique human immune signature of Ebola virus disease in Guinea

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4+ and CD8+ T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Novel Paramyxoviruses in Free-Ranging European Bats

The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats experimentally infected with Hendra virus. Our findings indicate the presence of bat-born paramyxoviruses in geographic areas free of fruit bat species and therefore emphasize a possible virus–host co-evolution in European bats. Since these novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk of the emergence of zoonotic paramyxoviruses in Europe needs to be considered.