ABSTRACT The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species. IMPORTANCE We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent.
During the compulsory vaccination programme against bluetongue virus serotype 1 (BTV-1) in Corsica (France) in 2014, a BTV strain belonging to a previously uncharacterized serotype (BTV-27) was isolated from asymptomatic goats. The present study describes the detection and molecular characterization of two additional distinct BTV-27 variants found in goats in Corsica in 2014 and 2015. The full coding genome of these two novel BTV-27 variants show high homology (90-93 % nucleotide/93-95 % amino acid) with the originally described BTV-27 isolate from Corsican goats in 2014. These three variants constitute the novel serotype BTV-27 ('BTV-27/FRA2014/v01 to v03'). Phylogenetic analyses with the 26 other established BTV serotypes revealed the closest relationship to BTV-25 (SWI2008/01) (80 % nucleotide/86 % amino acid) and to BTV-26 (KUW2010/02) (73-74 % nucleotide/80-81 % amino acid). However, highest sequence homologies between individual segments of BTV-27/FRA2014/v01-v03 with BTV-25 and BTV-26 vary. All three variants share the same segment 2 nucleotype with BTV-25. Neutralization assays of anti-BTV27/FRA2014/v01-v03 sera with a reassortant virus containing the outer capsid proteins of BTV-25 (BTV1VP2/VP5 BTV25) further confirmed that BTV-27 represents a distinct BTV serotype. Relationships between the variants and with BTV-25 and BTV-26, hypotheses about their origin, reassortment events and evolution are discussed.
Five avian paramyxoviruses of serotype 8 (APMV-8) were isolated during a study monitoring wild birds in Kazakhstan in 2013 and each was further characterized. The viruses were isolated from three White-fronted geese (Anser albifrons), one Whooper swan (Cygnus cygnus), and one Little stint (Calidris minuta). Before our study, only two complete APMV-8 sequences had been reported worldwide since their discovery in the USA and Japan in the 1970s. We report the complete genome sequences of the newly detected viruses and analyze the genetic evolution of the APMV-8 viruses over four decades.
Die Next Generation Sequencing (NGS)-Technologie hat sich in den vergangenen Jahren rasant entwickelt und findet in der Gegenwart breite Anwendung in der Grundlagenforschung und anwendungsorientierten Forschung. Dazu zahlen unter anderem Transkriptom- und Translatom-Analysen, die Bestimmung von regulatorischen Elementen und die Analyse von RNA-Sekundarstrukturen. Ziel dieser Arbeit war es, das Potential der NGS-Technologie (i) fur die Identifizierung von Erregern bei Haufungen von Krankheitsfallen oder wahrend Krankheitsausbruchen bei Versagen der klassischen Diagnostikverfahren und (ii) fur die Identifikation moglicher Virulenz-relevanter Sequenzpolymorphismen bei Komplettgenomvergleich zu erproben. Dazu wurde bereits im Vorfeld ein valider und sensitiver Workflow fur die Sequenzierung und Datenanalyse entwickelt und bei der Metagenomanalyse eines neu entdeckten Virus aus der Familie Bornaviridae gepruft. Mittels der im Anschluss generierten Volllangengenomsequenzen dieses Virus konnte der molekular-epidemiologische Zusammenhang von Infektionen beim Bunthornchen (Sciurus variegatoides) und drei Fallen von todlich verlaufener Enzephalitis bei Hornchenzuchtern bewiesen werden. Die Bestimmung der Komplettgenome des in Deutschland nachgewiesenen Porcine Epidemic Diarrhea Virus (PEDV) mittels NGS konnte deutliche Sequenzunterschiede zu den in den USA zirkulierenden hochvirulenten Stammen dieses Virus aufzeigen. Somit lieferte der Sequenzvergleich der generierten Volllangengenomsequenzen Hinweise auf potentielle Virulenzmarker der unterschiedlichen Virusstamme in den USA und Deutschland. Weitere Untersuchungen zu Zusammenhangen von Sequenzvarianten und Virulenz wurden fur das das Bovine Virusdiarrhoe-Virus Genotyp 2c (BVDV-2c), das Kuhpockenvirus (CPXV) und das Virus der klassischen Schweinepest (CSFV) durchgefuhrt. Dabei konnte fur einen hochvirulenten, in Deutschland zirkulierenden BVDV-2c Stamm eine Mischung von verschiedenen Genomstrukturvarianten detektiert werden. Die Mehrheit dieser Varianten trug eine Duplikation im p7/NS2 kodierenden Bereich, die vermutlich im Zusammenhang mit der beobachteten erhohten Virulenz steht. Diese Untersuchungen zeigten daneben auch, dass die Wahl des Datenanalyse-Algorithmus das Ergebnis beeinflusst. So wurden per de novo Assembly die entsprechenden Strukturvarianten detektieren, wahrend diese mittels Referenzmapping unentdeckt blieben. Fur CPXV wurden Isolate unterschiedlicher Wirtsherkunft, das heist aus Feldmaus Microtus arvalis und Wanderratte Rattus norvegicus, untersucht und die Genomunterschiede mit dem in Zellkultur beobachtetem A-type inclusion body-Phanotyp in Zusammenhang gebracht. Des Weiteren wurden mittels Sequenzvergleich Deletions/-Insertionsereignisse in Virulenzfaktor-Genen detektiert die vermutlich im Zusammenhang mit der Virulenz dieser Isolate im naturlichen und zufalligen Wirt stehen. Abschliesend wurden Viruspopulationen von verschiedenen CSFV-Stammen in Schweinen charakterisiert. Die Zusammensetzung der Viruspopulationen im Inokulum, sowie akut-letal und chronisch infizierten Schweinen lieferte keine Hinweise auf bestimmte Genomregionen, die diese unterschiedlichen Krankheitsverlaufe hervorrufen konnten. Zusammenfassend zeigten die Ergebnisse der vorliegenden Arbeit das grose Potential der NGS-Technologie fur eine detaillierte Viruscharakterisierung, die die Basis fur die Identifikation moglicher Virulenzmarker darstellt, die anschliesend mittels reverser Genetik-Verfahren gepruft werden mussen, und fur die Entwicklung neuer diagnostischer Nachweisverfahren sowie die Prufung molekular-epidemiologischer Zusammenhange zoonotischer Virusubertragungen. Dieses Potential der NGS-Plattform wird zukunftig Eingang in die Routinediagnostik finden und damit zu einer Beschleunigung und Verfeinerung der molekularen Virus- und anderen Erregerdiagnostik fuhren.
In February 2013, very severe acute clinical symptoms were observed in calves, heifers, and dairy cattle in several farms in North Rhine Westphalia and Lower Saxony, Germany. Deep sequencing revealed the coexistence of three distinct genome variants within recent highly virulent bovine viral diarrhea virus type 2 (BVDV-2) isolates. While the major portion (ca. 95%) of the population harbored a duplication of a 222-nucleotide (nt) segment within the p7-NS2-encoding region, the minority reflected the standard structure of a BVDV-2 genome. Additionally, unusual mutations were found in both variants, within the highly conserved p7 protein and close to the p7-NS2 cleavage site. Using a reverse genetic system with a BVDV-2a strain harboring a similar duplication, it could be demonstrated that during replication, genomes without duplication are generated de novo from genomes with duplication. The major variant with duplication is compulsorily escorted by the minor variant without duplication. RNA secondary structure prediction allowed the analysis of the unique but stable mixture of three BVDV variants and also provided the explanation for their generation. Finally, our results suggest that the variant with duplication plays the major role in the highly virulent phenotype.This study emphasizes the importance of full-genome deep sequencing in combination with manual in-depth data analysis for the investigation of viruses in basic research and diagnostics. Here we investigated recent highly virulent bovine viral diarrhea virus isolates from a 2013 series of outbreaks. We discovered a unique special feature of the viral genome, an unstable duplication of 222 nucleotides which is eventually deleted by viral polymerase activity, leading to an unexpectedly mixed population of viral genomes for all investigated isolates. Our study is of high importance to the field because we demonstrate that these insertion/deletion events allow another level of genome plasticity of plus-strand RNA viruses, in addition to the well-known polymerase-induced single nucleotide variations which are generally considered the main basis for viral adaptation and evolution.
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