A phylogenomic analysis of Marek's disease virus reveals independent paths to virulence in Eurasia and North America

Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10−5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV

Comparison of Porcine Epidemic Diarrhea Viruses from Germany and the United States, 2014

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they ‘chaperone’ p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes

Variegated squirrel bornavirus 1 in squirrels, Germany and the Netherlands

We screened squirrels in Germany and the Netherlands for the novel zoonotic variegated squirrel bornavirus 1 (VSBV-1). The detection of VSBV-1 in 11 squirrels indicates a considerable risk for transmission to humans handling those animals. Therefore, squirrels in contact with humans should routinely be tested for VSBV-1

Next-generation sequencing for molecular epidemiology and comparative virulence analyses of epizootic and zoonotic pathogens

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 zählen unter anderem Transkriptom- und Translatom-Analysen, die Bestimmung von regulatorischen Elementen und die Analyse von RNA-Sekundärstrukturen. Ziel dieser Arbeit war es, das Potential der NGS-Technologie (i) für die Identifizierung von Erregern bei Häufungen von Krankheitsfällen oder während Krankheitsausbrüchen bei Versagen der klassischen Diagnostikverfahren und (ii) für die Identifikation möglicher Virulenz-relevanter Sequenzpolymorphismen bei Komplettgenomvergleich zu erproben. Dazu wurde bereits im Vorfeld ein valider und sensitiver Workflow für die Sequenzierung und Datenanalyse entwickelt und bei der Metagenomanalyse eines neu entdeckten Virus aus der Familie Bornaviridae geprüft. Mittels der im Anschluss generierten Volllängengenomsequenzen dieses Virus konnte der molekular-epidemiologische Zusammenhang von Infektionen beim Bunthörnchen (Sciurus variegatoides) und drei Fällen von tödlich verlaufener Enzephalitis bei Hörnchenzüchtern 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 Stämmen dieses Virus aufzeigen. Somit lieferte der Sequenzvergleich der generierten Volllängengenomsequenzen Hinweise auf potentielle Virulenzmarker der unterschiedlichen Virusstämme in den USA und Deutschland. Weitere Untersuchungen zu Zusammenhängen von Sequenzvarianten und Virulenz wurden für das das Bovine Virusdiarrhoe-Virus Genotyp 2c (BVDV-2c), das Kuhpockenvirus (CPXV) und das Virus der klassischen Schweinepest (CSFV) durchgeführt. Dabei konnte für 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 erhöhten 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, während diese mittels Referenzmapping unentdeckt blieben. Für CPXV wurden Isolate unterschiedlicher Wirtsherkunft, das heißt aus Feldmaus Microtus arvalis und Wanderratte Rattus norvegicus, untersucht und die Genomunterschiede mit dem in Zellkultur beobachtetem A-type inclusion body-Phänotyp in Zusammenhang gebracht. Des Weiteren wurden mittels Sequenzvergleich Deletions/-Insertionsereignisse in Virulenzfaktor-Genen detektiert die vermutlich im Zusammenhang mit der Virulenz dieser Isolate im natürlichen und zufälligen Wirt stehen. Abschließend wurden Viruspopulationen von verschiedenen CSFV-Stämmen 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 Krankheitsverläufe hervorrufen könnten. Zusammenfassend zeigten die Ergebnisse der vorliegenden Arbeit das große Potential der NGS-Technologie für eine detaillierte Viruscharakterisierung, die die Basis für die Identifikation möglicher Virulenzmarker darstellt, die anschließend mittels reverser Genetik-Verfahren geprüft werden müssen, und für die Entwicklung neuer diagnostischer Nachweisverfahren sowie die Prüfung molekular-epidemiologischer Zusammenhänge zoonotischer Virusübertragungen. Dieses Potential der NGS-Plattform wird zukünftig Eingang in die Routinediagnostik finden und damit zu einer Beschleunigung und Verfeinerung der molekularen Virus- und anderen Erregerdiagnostik führen.Next Generation Sequencing (NGS)-technologies developed very fast in recent years and is used widely in current research areas. The aim of this study was to use NGS (i) for the identification of pathogens in outbreaks and (ii) for the identification of virulence-relevant sequencepolymorphisms when comparing whole genome sequences. Therefore, a previous developed workflow was used to identify a new virus of the family Bornaviridae. The generation of whole genome sequences elucidated the molecular epidemiological connection of infection of variegated squirrels (Sciurus variegatoides) and three human cases of fatal encephalitis. By generating the whole genome sequence of a Porcine Epidemic Diarrhea Virus (PEDV) in Germany it was possible to find difference compared to circulating high virulent strains in the USA. This led to potential virulence marker to distinguish strain in the USA and Germany. Connections between sequence variation and virulence were further investigated for the bovine viral diarrhea virus 2c (BVDV-2c), cowpox viruses (CPXV) and classical swine fever virus (CSFV). Here, for a highly virulent BVDV-2c strain a mixture of different genome structure variants could be found. The majority of these genomes harbors a duplication within the p7/NS2 coding region and might cause a high virulence. For CPXV virus isolated of different hosts were analyzed and a correlation between genome sequence and the A-type inclusion body phenotype could be found. Furthermore, several deletion/insertion events were detected which might influence the virulence of these strains. Finally, the virus population of CSFV strains in pigs was characterized. However, the population of the inoculum as well as of acute-lethal and chronically infected animals gave no indication that the virus itself causes the different types of disease outcome. In conclusion, this thesis shows the great potential of NGS for virus identification and characterization. Furthermore, it makes the identification of potential virulence marker possible which subsequently can be analyzed by reverse genetics

Investigations into the presence of nidoviruses in pythons

BACKGROUND: Pneumonia and stomatitis represent severe and often fatal diseases in different captive snakes. Apart from bacterial infections, paramyxo-, adeno-, reo- and arenaviruses cause these diseases. In 2014, new viruses emerged as the cause of pneumonia in pythons. In a few publications, nidoviruses have been reported in association with pneumonia in ball pythons and a tiger python. The viruses were found using new sequencing methods from the organ tissue of dead animals. METHODS: Severe pneumonia and stomatitis resulted in a high mortality rate in a captive breeding collection of green tree pythons. Unbiased deep sequencing lead to the detection of nidoviral sequences. A developed RT-qPCR was used to confirm the metagenome results and to determine the importance of this virus. A total of 1554 different boid snakes, including animals suffering from respiratory diseases as well as healthy controls, were screened for nidoviruses. Furthermore, in addition to two full-length sequences, partial sequences were generated from different snake species. RESULTS: The assembled full-length snake nidovirus genomes share only an overall genome sequence identity of less than 66.9% to other published snake nidoviruses and new partial sequences vary between 99.89 and 79.4%. Highest viral loads were detected in lung samples. The snake nidovirus was not only present in diseased animals, but also in snakes showing no typical clinical signs. CONCLUSIONS: Our findings further highlight the possible importance of snake nidoviruses in respiratory diseases and proof multiple circulating strains with varying disease potential. Nidovirus detection in clinical healthy individuals might represent testing during the incubation period or reconvalescence. Our investigations show new aspects of nidovirus infections in pythons. Nidoviruses should be included in routine diagnostic workup of diseased reptiles

Frequent intergenotypic recombination between the non-structural and structural genes is a major driver of epidemiological fitness in caliciviruses

The diversity of lagoviruses (Caliciviridae) in Australia has increased considerably in recent years. By the end of 2017, five variants from three viral genotypes were present in populations of Australian rabbits, while prior to 2014 only two variants were known. To understand the evolutionary interactions among these lagovirus variants, we monitored their geographical distribution and relative incidence over time in a continental-scale competition study. Within 3 years of the incursion of rabbit haemorrhagic disease virus 2 (RHDV2, denoted genotype GI.1bP-GI.2 [polymerase genotype]P-[capsid genotype]) into Australia, two novel recombinant lagovirus variants emerged: RHDV2-4e (genotype GI.4eP-GI.2) in New South Wales and RHDV2-4c (genotype GI.4cP-GI.2) in Victoria. Although both novel recombinants contain non-structural genes related to those from benign, rabbit-specific, enterotropic viruses, these variants were recovered from the livers of both rabbits and hares that had died acutely. This suggests that the determinants of host and tissue tropism for lagoviruses are associated with the structural genes, and that tropism is intricately connected with pathogenicity. Phylogenetic analyses demonstrated that the RHDV2-4c recombinant emerged independently on multiple occasions, with five distinct lineages observed. Both the new RHDV2-4e and -4c recombinant variants replaced the previous dominant parental RHDV2 (genotype GI.1bP-GI.2) in their respective geographical areas, despite sharing an identical or near-identical (i.e. single amino acid change) VP60 major capsid protein with the parental virus. This suggests that the observed replacement by these recombinants was not driven by antigenic variation in VP60, implicating the non-structural genes as key drivers of epidemiological fitness. Molecular clock estimates place the RHDV2-4e recombination event in early to mid-2015, while the five RHDV2-4c recombination events occurred from late 2015 through to early 2017. The emergence of at least six viable recombinant variants within a 2-year period highlights the high frequency of these events, detectable only through intensive surveillance, and demonstrates the importance of recombination in lagovirus evolution

Distribution and Genetic Diversity of Hepatitis E Virus in Wild and Domestic Rabbits in Australia

In 2020, Hepatitis E virus (HEV) was detected for the first time in Australian rabbits. To improve our understanding of the genetic diversity and distribution of the virus, 1635 rabbit liver samples from locations across Australia were screened via RT-qPCR for HEV. HEV genomes were amplified and sequenced from 48 positive samples. Furthermore, we tested 380 serum samples from 11 locations across Australia for antibodies against HEV. HEV was detected in rabbits from all states and territories, except the Northern Territory. Seroprevalence varied between locations (from 0% to 22%), demonstrating that HEV is widely distributed in rabbit populations across Australia. Phylogenetic analyses showed that Australian HEV sequences are genetically diverse and that HEV was likely introduced into Australia independently on several occasions. In summary, this study broadens our understanding of the genetic diversity of rabbit HEV globally and shows that the virus is endemic in both domestic and wild rabbit populations in Australia