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Thomas Bruun Rasmussen
Department of Virus and Microbiological Special Diagnostics, Statens Serum Institut, 2300 Copenhagen, Denmark

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Journal article
Published: 04 June 2021 in Viruses
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Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera—i.e., Pedacovirus, Nyctacovirus, and Myotacovirus—that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates—including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus—present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

ACS Style

Christina Lazov; Graham Belsham; Anette Bøtner; Thomas Rasmussen. Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark. Viruses 2021, 13, 1073 .

AMA Style

Christina Lazov, Graham Belsham, Anette Bøtner, Thomas Rasmussen. Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark. Viruses. 2021; 13 (6):1073.

Chicago/Turabian Style

Christina Lazov; Graham Belsham; Anette Bøtner; Thomas Rasmussen. 2021. "Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark." Viruses 13, no. 6: 1073.

Preprint content
Published: 07 May 2021
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Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene. The infected mink recovered and after free-testing of the mink, the animals remained seropositive. During follow-up studies, after a period of more than 2 months without virus detection, over 75% of tested animals scored positive again for SARS-CoV-2 RNA. Whole genome sequencing showed that the virus circulating during this re-infection was most closely related to the virus identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies after re-infection than at free-testing. Thus, following recovery from an initial infection, seropositive mink rapidly became susceptible to re-infection by SARS-CoV-2.

ACS Style

Thomas Bruun Rasmussen; Jannik Fonager; Charlotte Sværke Jørgensen; Ria Lassaunière; Anne Sofie Hammer; Michelle Lauge Quaade; Anette Boklund; Louise Lohse; Bertel Strandbygaard; Morten Rasmussen; Thomas Yssing Michaelsen; Sten Mortensen; Anders Fomsgaard; Graham J. Belsham; Anette Bøtner. Infection, recovery and re-infection of farmed mink with SARS-CoV-2. 2021, 1 .

AMA Style

Thomas Bruun Rasmussen, Jannik Fonager, Charlotte Sværke Jørgensen, Ria Lassaunière, Anne Sofie Hammer, Michelle Lauge Quaade, Anette Boklund, Louise Lohse, Bertel Strandbygaard, Morten Rasmussen, Thomas Yssing Michaelsen, Sten Mortensen, Anders Fomsgaard, Graham J. Belsham, Anette Bøtner. Infection, recovery and re-infection of farmed mink with SARS-CoV-2. . 2021; ():1.

Chicago/Turabian Style

Thomas Bruun Rasmussen; Jannik Fonager; Charlotte Sværke Jørgensen; Ria Lassaunière; Anne Sofie Hammer; Michelle Lauge Quaade; Anette Boklund; Louise Lohse; Bertel Strandbygaard; Morten Rasmussen; Thomas Yssing Michaelsen; Sten Mortensen; Anders Fomsgaard; Graham J. Belsham; Anette Bøtner. 2021. "Infection, recovery and re-infection of farmed mink with SARS-CoV-2." , no. : 1.

Journal article
Published: 21 April 2021 in Viruses
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Atypical porcine pestivirus (APPV) was first discovered in North America in 2015 and was later shown to be associated with congenital tremor (CT) in piglets. CT is an occasional challenge in some Danish sow herds. Therefore, we initiated an observational case control study to clarify a possible relationship between CT and APPV in Danish pig production. Blood samples were collected from piglets affected by CT (n = 55) in ten different sow herds and from healthy piglets in five sow herds without a history of CT piglets (n = 25), as well as one sow herd with a sporadic occurrence of CT (n = 5). APPV was detected by RT-qPCR in all samples from piglets affected by CT and in three out of five samples from piglets in the herd with a sporadic occurrence of CT. In the herds without a history of CT, only one out of 25 piglets were positive for APPV. In addition, farmers or veterinarians in CT-affected herds were asked about their experience of the issue. CT is most often seen in gilt litters, and a substantial increase in pre-weaning mortality is only observed in severe cases. According to our investigations, APPV is a common finding in piglets suffering from CT in Denmark.

ACS Style

Kasper Pedersen; Charlotte Kristensen; Bertel Strandbygaard; Anette Bøtner; Thomas Rasmussen. Detection of Atypical Porcine Pestivirus in Piglets from Danish Sow Herds. Viruses 2021, 13, 717 .

AMA Style

Kasper Pedersen, Charlotte Kristensen, Bertel Strandbygaard, Anette Bøtner, Thomas Rasmussen. Detection of Atypical Porcine Pestivirus in Piglets from Danish Sow Herds. Viruses. 2021; 13 (5):717.

Chicago/Turabian Style

Kasper Pedersen; Charlotte Kristensen; Bertel Strandbygaard; Anette Bøtner; Thomas Rasmussen. 2021. "Detection of Atypical Porcine Pestivirus in Piglets from Danish Sow Herds." Viruses 13, no. 5: 717.

Communication
Published: 07 April 2021 in Viruses
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European bat lyssavirus type 1 (EBLV-1) is the causative agent for almost all reported rabies cases found in European bats. In recent years, increasing numbers of available EBLV-1 full genomes and their phylogenetic analyses helped to further elucidate the distribution and genetic characteristics of EBLV-1 and its two subtypes, namely EBLV-1a and EBLV-1b. Nonetheless, the absence of full-genome sequences from regions with known detections of EBLV-1 still limit the understanding of the phylogeographic relations between viruses from different European regions. In this study, a set of 21 archived Danish EBLV-1 samples from the years 1985 to 2009 was processed for the acquisition of full-genome sequences using a high-throughput sequencing approach. Subsequent phylogenetic analysis encompassing all available EBLV-1 full genomes from databases revealed the Danish sequences belong to the EBLV-1a subtype and further highlighted the distinct, close phylogenetic relationship of Danish, Dutch and German isolates in this region. In addition, the formation of five putative groups nearly exclusively formed by Danish isolates and the overall increased resolution of the EBLV-1a branch indicate a higher genetic diversity and spatial segregation for this sublineage than was previously known. These results emphasize the importance of phylogenetic analyses of full-genome sequences of lyssaviruses for genetic geography.

ACS Style

Sten Calvelage; Conrad Freuling; Anthony Fooks; Dirk Höper; Denise Marston; Lorraine McElhinney; Thomas Rasmussen; Stefan Finke; Martin Beer; Thomas Müller. Full-Genome Sequences and Phylogenetic Analysis of Archived Danish European Bat Lyssavirus 1 (EBLV-1) Emphasize a Higher Genetic Resolution and Spatial Segregation for Sublineage 1a. Viruses 2021, 13, 634 .

AMA Style

Sten Calvelage, Conrad Freuling, Anthony Fooks, Dirk Höper, Denise Marston, Lorraine McElhinney, Thomas Rasmussen, Stefan Finke, Martin Beer, Thomas Müller. Full-Genome Sequences and Phylogenetic Analysis of Archived Danish European Bat Lyssavirus 1 (EBLV-1) Emphasize a Higher Genetic Resolution and Spatial Segregation for Sublineage 1a. Viruses. 2021; 13 (4):634.

Chicago/Turabian Style

Sten Calvelage; Conrad Freuling; Anthony Fooks; Dirk Höper; Denise Marston; Lorraine McElhinney; Thomas Rasmussen; Stefan Finke; Martin Beer; Thomas Müller. 2021. "Full-Genome Sequences and Phylogenetic Analysis of Archived Danish European Bat Lyssavirus 1 (EBLV-1) Emphasize a Higher Genetic Resolution and Spatial Segregation for Sublineage 1a." Viruses 13, no. 4: 634.

Short communication
Published: 16 March 2021 in Journal of Virological Methods
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Several reverse genetics systems for viral haemorrhagic septicaemia virus (VHSV) have been developed over the last decade. These systems have been based on genotype Ia, IVa and IVb isolates and have used the fish cell line EPC, which is less susceptible to some VHSV isolates belonging to genotype I and genotypes II and III. While developing a reverse genetics system in our laboratories for VHSV genotype Ib, we realized that the isolate in interest (SE SVA 1033 9C) did not grow in EPC cells and it was necessary to adapt the reverse genetics protocols to the BF-2 fish cell line. This cell line is very sensitive to high temperatures and is therefore not compatible with the original protocols based on the use of recombinant vaccinia virus (vTF7-3) as a provider of the T7 RNA polymerase (T7-RNAP) to the system, which includes incubation periods at 37 °C. Transfection efficiency was assessed in BF-2 cells using a reporter plasmid and it showed to be highest when using Lipofectamine™ 3000 compared to other transfection reagents. A luciferase assay was performed to determine the optimal activity of T7-RNAP in BF-2 cells with different amounts of vTF7-3. We successfully recovered recombinant VHSV (rVHSV) in BF-2 cells by reducing the incubation time at 37 °C after transfection to both 3 and 6 h. Another strategy we attempted successfully was to transfect mammalian BHK-21 cells, which are routinely used to propagate vTF7-3, and after the 37 °C incubation period, a BF-2 cell suspension was added hypothesizing that the virions formed in the transfected mammalian cells would infect the subsequently added fish cells at 15 °C incubation over the following days. We have successfully recovered rVHSV from both BHK-21 with a BF-2 cells suspension as well as a new protocol for VHSV reverse genetics in BF-2 cells has been established.

ACS Style

Anna Luiza Farias Alencar; Argelia Cuenca; Niels Jørgen Olesen; Thomas Bruun Rasmussen. Technical challenges in the development of reverse genetics for a viral haemorrhagic septicaemia virus (VHSV) genotype Ib isolate: Alternative cell lines and general troubleshooting. Journal of Virological Methods 2021, 292, 114132 .

AMA Style

Anna Luiza Farias Alencar, Argelia Cuenca, Niels Jørgen Olesen, Thomas Bruun Rasmussen. Technical challenges in the development of reverse genetics for a viral haemorrhagic septicaemia virus (VHSV) genotype Ib isolate: Alternative cell lines and general troubleshooting. Journal of Virological Methods. 2021; 292 ():114132.

Chicago/Turabian Style

Anna Luiza Farias Alencar; Argelia Cuenca; Niels Jørgen Olesen; Thomas Bruun Rasmussen. 2021. "Technical challenges in the development of reverse genetics for a viral haemorrhagic septicaemia virus (VHSV) genotype Ib isolate: Alternative cell lines and general troubleshooting." Journal of Virological Methods 292, no. : 114132.

Journal article
Published: 12 January 2021 in Animals
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SARS-CoV-2 infection is the cause of COVID-19 in humans. In April 2020, SARS-CoV-2 infection in farmed mink (Neovision vision) occurred in the Netherlands. The first outbreaks in Denmark were detected in June 2020 in three farms. A steep increase in the number of infected farms occurred from September and onwards. Here, we describe prevalence data collected from 215 infected mink farms to characterize spread and impact of disease in infected farms. In one third of the farms, no clinical signs were observed. In farms with clinical signs, decreased feed intake, increased mortality and respiratory symptoms were most frequently observed, during a limited time period (median of 11 days). In 65% and 69% of farms, virus and sero-conversion, respectively, were detected in 100% of sampled animals at the first sampling. SARS-CoV-2 was detected, at low levels, in air samples collected close to the mink, on mink fur, on flies, on the foot of a seagull, and in gutter water, but not in feed. Some dogs and cats from infected farms tested positive for the virus. Chickens, rabbits, and horses sampled on a few farms, and wildlife sampled in the vicinity of the infected farms did not test positive for SARS-CoV-2. Thus, mink are highly susceptible to infection by SARS-CoV-2, but routes of transmission between farms, other than by direct human contact, are unclear.

ACS Style

Anette Boklund; Anne Sofie Hammer; Michelle Lauge Quaade; Thomas Bruun Rasmussen; Louise Lohse; Bertel Strandbygaard; Charlotte Sværke Jørgensen; Ann Sofie Olesen; Freja Broe Hjerpe; Heidi Huus Petersen; Tim Kåre Jensen; Sten Mortensen; Francisco F. Calvo-Artavia; Stine Kjær Lefèvre; Søren Saxmose Nielsen; Tariq Halasa; Graham J. Belsham; Anette Bøtner. SARS-CoV-2 in Danish Mink Farms: Course of the Epidemic and a Descriptive Analysis of the Outbreaks in 2020. Animals 2021, 11, 164 .

AMA Style

Anette Boklund, Anne Sofie Hammer, Michelle Lauge Quaade, Thomas Bruun Rasmussen, Louise Lohse, Bertel Strandbygaard, Charlotte Sværke Jørgensen, Ann Sofie Olesen, Freja Broe Hjerpe, Heidi Huus Petersen, Tim Kåre Jensen, Sten Mortensen, Francisco F. Calvo-Artavia, Stine Kjær Lefèvre, Søren Saxmose Nielsen, Tariq Halasa, Graham J. Belsham, Anette Bøtner. SARS-CoV-2 in Danish Mink Farms: Course of the Epidemic and a Descriptive Analysis of the Outbreaks in 2020. Animals. 2021; 11 (1):164.

Chicago/Turabian Style

Anette Boklund; Anne Sofie Hammer; Michelle Lauge Quaade; Thomas Bruun Rasmussen; Louise Lohse; Bertel Strandbygaard; Charlotte Sværke Jørgensen; Ann Sofie Olesen; Freja Broe Hjerpe; Heidi Huus Petersen; Tim Kåre Jensen; Sten Mortensen; Francisco F. Calvo-Artavia; Stine Kjær Lefèvre; Søren Saxmose Nielsen; Tariq Halasa; Graham J. Belsham; Anette Bøtner. 2021. "SARS-CoV-2 in Danish Mink Farms: Course of the Epidemic and a Descriptive Analysis of the Outbreaks in 2020." Animals 11, no. 1: 164.

Journal article
Published: 01 November 2020 in Journal of General Virology
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Border disease virus (BDV) envelope glycoprotein E2 is required for entry into cells and is a determinant of host tropism for sheep and pig cells. Here, we describe adaptive changes in the BDV E2 protein that modify virus replication in pig cells. To achieve this, two BDV isolates, initially collected from a pig and a sheep on the same farm, were passaged in primary sheep and pig cells in parallel with a rescued variant of the pig virus derived from a cloned full-length BDV cDNA. The pig isolate and the rescued virus shared the same amino acid sequence, but the sheep isolate differed at ten residues, including two substitutions in E2 (K771E and Y925H). During serial passage in cells, the viruses displayed clear selectivity for growth in sheep cells; only the cDNA-derived virus adapted to grow in pig cells. Sequencing revealed an amino acid substitution (Q739R) in the E2 domain DA of this rescued virus. Adaptation at the same residue (Q739K/Q739R) was also observed after passaging of the pig isolate in sheep cells. Use of reverse genetics confirmed that changing residue Q739 to R or K (each positively charged) was sufficient to achieve adaptation to pig cells. Furthermore, this change in host tropism was suppressed if Q739R was combined with K771E. Another substitution (Q728R), conferring an additional positive charge, acquired during passaging, restored the growth of the Q739R/K771E variant. Overall, this study provided evidence that specific, positively charged, residues in the E2 domain DA are crucial for pig-cell tropism of BDV.

ACS Style

Ulrik Fahnøe; Yu Deng; Nana A. Davids; Louise Lohse; Jens Bukh; Graham J. Belsham; Thomas Bruun Rasmussen. Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells. Journal of General Virology 2020, 101, 1170 -1181.

AMA Style

Ulrik Fahnøe, Yu Deng, Nana A. Davids, Louise Lohse, Jens Bukh, Graham J. Belsham, Thomas Bruun Rasmussen. Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells. Journal of General Virology. 2020; 101 (11):1170-1181.

Chicago/Turabian Style

Ulrik Fahnøe; Yu Deng; Nana A. Davids; Louise Lohse; Jens Bukh; Graham J. Belsham; Thomas Bruun Rasmussen. 2020. "Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells." Journal of General Virology 101, no. 11: 1170-1181.

Journal article
Published: 10 October 2019 in Viruses
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Vaccines against classical swine fever have proven very effective in protecting pigs from this deadly disease. However, little is known about how vaccination impacts the selective pressures acting on the classical swine fever virus (CSFV). Here we use high-throughput sequencing of viral genomes to investigate evolutionary changes in virus populations following the challenge of naïve and vaccinated pigs with the highly virulent CSFV strain “Koslov”. The challenge inoculum contained an ensemble of closely related viral sequences, with three major haplotypes being present, termed A, B, and C. After the challenge, the viral haplotype A was preferentially located within the tonsils of naïve animals but was highly prevalent in the sera of all vaccinated animals. We find that the viral population structure in naïve pigs after infection is very similar to that in the original inoculum. In contrast, the viral population in vaccinated pigs, which only underwent transient low-level viremia, displayed several distinct changes including the emergence of 16 unique non-synonymous single nucleotide polymorphisms (SNPs) that were not detectable in the challenge inoculum. Further analysis showed a significant loss of heterogeneity and an increasing positive selection acting on the virus populations in the vaccinated pigs. We conclude that vaccination imposes a strong selective pressure on viruses that subsequently replicate within the vaccinated animal.

ACS Style

Ulrik Fahnøe; Anders Gorm Pedersen; Camille Melissa Johnston; Richard J. Orton; Dirk Höper; Martin Beer; Jens Bukh; Graham J. Belsham; Thomas Bruun Rasmussen. Virus Adaptation and Selection Following Challenge of Animals Vaccinated against Classical Swine Fever Virus. Viruses 2019, 11, 932 .

AMA Style

Ulrik Fahnøe, Anders Gorm Pedersen, Camille Melissa Johnston, Richard J. Orton, Dirk Höper, Martin Beer, Jens Bukh, Graham J. Belsham, Thomas Bruun Rasmussen. Virus Adaptation and Selection Following Challenge of Animals Vaccinated against Classical Swine Fever Virus. Viruses. 2019; 11 (10):932.

Chicago/Turabian Style

Ulrik Fahnøe; Anders Gorm Pedersen; Camille Melissa Johnston; Richard J. Orton; Dirk Höper; Martin Beer; Jens Bukh; Graham J. Belsham; Thomas Bruun Rasmussen. 2019. "Virus Adaptation and Selection Following Challenge of Animals Vaccinated against Classical Swine Fever Virus." Viruses 11, no. 10: 932.

Journal article
Published: 11 September 2019 in Viruses
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African swine fever (ASF) is a severe disease of suids caused by African swine fever virus (ASFV). Its dsDNA genome (170–194 kbp) is scattered with homopolymers and repeats as well as inverted-terminal-repeats (ITR), which hamper whole-genome sequencing. To date, only a few genome sequences have been published and only for some are data on sequence quality available enabling in-depth investigations. Especially in Europe and Asia, where ASFV has continuously spread since its introduction into Georgia in 2007, a very low genetic variability of the circulating ASFV-strains was reported. Therefore, only whole-genome sequences can serve as a basis for detailed virus comparisons. Here, we report an effective workflow, combining target enrichment, Illumina and Nanopore sequencing for ASFV whole-genome sequencing. Following this approach, we generated an improved high-quality ASFV Georgia 2007/1 whole-genome sequence leading to the correction of 71 sequencing errors and the addition of 956 and 231 bp at the respective ITRs. This genome, derived from the primary outbreak in 2007, can now serve as a reference for future whole-genome analyses of related ASFV strains and molecular approaches. Using both workflow and the reference genome, we generated the first ASFV-whole-genome sequence from Moldova, expanding the sequence knowledge from Eastern Europe.

ACS Style

Jan H. Forth; Leonie F. Forth; Jacqueline King; Oxana Groza; Alexandra Hübner; Ann Sofie Olesen; Dirk Höper; Linda K. Dixon; Christopher L. Netherton; Thomas Bruun Rasmussen; Sandra Blome; Anne Pohlmann; Martin Beer. A Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever Virus Whole-Genome Sequences. Viruses 2019, 11, 846 .

AMA Style

Jan H. Forth, Leonie F. Forth, Jacqueline King, Oxana Groza, Alexandra Hübner, Ann Sofie Olesen, Dirk Höper, Linda K. Dixon, Christopher L. Netherton, Thomas Bruun Rasmussen, Sandra Blome, Anne Pohlmann, Martin Beer. A Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever Virus Whole-Genome Sequences. Viruses. 2019; 11 (9):846.

Chicago/Turabian Style

Jan H. Forth; Leonie F. Forth; Jacqueline King; Oxana Groza; Alexandra Hübner; Ann Sofie Olesen; Dirk Höper; Linda K. Dixon; Christopher L. Netherton; Thomas Bruun Rasmussen; Sandra Blome; Anne Pohlmann; Martin Beer. 2019. "A Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever Virus Whole-Genome Sequences." Viruses 11, no. 9: 846.

Journal article
Published: 11 September 2018 in Viruses
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Bat populations harbour a multitude of viruses; some of these are pathogenic or potentially pathogenic in other animals or humans. Therefore, it is important to monitor the populations and characterize these viruses. In this study, the presence of coronaviruses (CoVs) in different species of Danish bats was investigated using active surveillance at different geographical locations in Denmark. Faecal samples were screened for the presence of CoVs using pan-CoV real-time RT-PCR assays. The amplicons, obtained from five different species of bats, were sequenced. Phylogenetic analysis revealed a species-specific clustering with the samples from Myotis daubentonii, showing a close resemblance to coronavirus sequences obtained from the same species of bat in Germany and the United Kingdom. Our results show, for the first time, that multiple, distinct alphacoronaviruses are present in the Danish bat populations.

ACS Style

Christina M. Lazov; Mariann Chriél; Hans J. Baagøe; Esben Fjederholt; Yu Deng; Engbert A. Kooi; Graham J. Belsham; Anette Bøtner; Thomas Bruun Rasmussen. Detection and Characterization of Distinct Alphacoronaviruses in Five Different Bat Species in Denmark. Viruses 2018, 10, 486 .

AMA Style

Christina M. Lazov, Mariann Chriél, Hans J. Baagøe, Esben Fjederholt, Yu Deng, Engbert A. Kooi, Graham J. Belsham, Anette Bøtner, Thomas Bruun Rasmussen. Detection and Characterization of Distinct Alphacoronaviruses in Five Different Bat Species in Denmark. Viruses. 2018; 10 (9):486.

Chicago/Turabian Style

Christina M. Lazov; Mariann Chriél; Hans J. Baagøe; Esben Fjederholt; Yu Deng; Engbert A. Kooi; Graham J. Belsham; Anette Bøtner; Thomas Bruun Rasmussen. 2018. "Detection and Characterization of Distinct Alphacoronaviruses in Five Different Bat Species in Denmark." Viruses 10, no. 9: 486.

Methodology article
Published: 09 August 2018 in BMC Genomics
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Direct molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection. In this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order. This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.

ACS Style

Camille Melissa Johnston; Ulrik Fahnøe; Graham J. Belsham; Thomas Bruun Rasmussen. Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping. BMC Genomics 2018, 19, 1 -9.

AMA Style

Camille Melissa Johnston, Ulrik Fahnøe, Graham J. Belsham, Thomas Bruun Rasmussen. Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping. BMC Genomics. 2018; 19 (1):1-9.

Chicago/Turabian Style

Camille Melissa Johnston; Ulrik Fahnøe; Graham J. Belsham; Thomas Bruun Rasmussen. 2018. "Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping." BMC Genomics 19, no. 1: 1-9.

Journal article
Published: 01 August 2018 in Veterinary Microbiology
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Since 2014, African swine fever virus (ASFV) has been spreading within Eastern Europe. Within affected regions, the virus has infected some farms with high biosecurity and a marked seasonality of outbreaks in domestic pigs has been observed. ASFV transmission from stable flies, Stomoxys calcitrans, has previously been shown both mechanically and via ingestion of whole flies. Hence, blood-feeding flies may offer one explanation for the introductions into high biosecurity farms and for the observed seasonality. The aim of this study was to further elucidate the potential role of stable flies in ASFV transmission. Different parts of flies were analyzed for the presence of viral DNA and infectious virus at different time points following in vitro feeding of the flies on blood from an ASFV-infected pig. Using qPCR, ASFV DNA was detectable in mouth parts of flies for at least 12 h and remained in head and body samples from the flies for up to three days following feeding. Infectious virus was detected in fly body samples prepared at 3 h and 12 h after feeding. The presence of infectious ASFV in stable flies following feeding on viremic blood means that such flies are capable of transporting infectious virus. The detection of ASFV DNA in the flies for up to three days following feeding suggests that qPCR analysis of blood-feeding flies during ASFV outbreaks could be a useful method to elucidate the role of these flies in ASFV transmission under field conditions.

ACS Style

Ann Sofie Olesen; Mette Frimodt Hansen; Thomas Bruun Rasmussen; Graham J. Belsham; Rene Bødker; Anette Bøtner. Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder. Veterinary Microbiology 2018, 222, 25 -29.

AMA Style

Ann Sofie Olesen, Mette Frimodt Hansen, Thomas Bruun Rasmussen, Graham J. Belsham, Rene Bødker, Anette Bøtner. Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder. Veterinary Microbiology. 2018; 222 ():25-29.

Chicago/Turabian Style

Ann Sofie Olesen; Mette Frimodt Hansen; Thomas Bruun Rasmussen; Graham J. Belsham; Rene Bødker; Anette Bøtner. 2018. "Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder." Veterinary Microbiology 222, no. : 25-29.

Short communication
Published: 29 July 2018 in Journal of Virological Methods
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African swine fever (ASF) is an important disease of domestic pigs and wild boar. The disease is caused by African swine fever virus (ASFV). In 2014, ASFV was introduced into Eastern Europe, and it has since then continued to spread within various Eastern European countries. Investigating differences in sequences between ASFV isolates may be a valuable tool to understand differences in virulence among them, however currently, no complete genome sequences of the viruses responsible for the Eastern European outbreaks have been reported. In this study, the complete genome sequence of a highly virulent ASFV was determined directly from erythrocyte-associated nucleic acids obtained from a pig experimentally infected with an isolate from Poland (ASFV POL/2015/Podlaskie). The sequence (ca. 189 kb) of this recent European ASFV showed 95 nt differences (99.95 % identity) from the ASFV Georgia 2007/1 genome. The complete sequence of ASFV/Pol/2015/Podlaskie should assist further studies on the genetic diversity and evolution of the European ASFVs.

ACS Style

Ann Sofie Olesen; Louise Lohse; Marlene Danner Dalgaard; Grzegorz Woźniakowski; Graham J. Belsham; Anette Bøtner; Thomas Bruun Rasmussen. Complete genome sequence of an African swine fever virus (ASFV POL/2015/Podlaskie) determined directly from pig erythrocyte-associated nucleic acid. Journal of Virological Methods 2018, 261, 14 -16.

AMA Style

Ann Sofie Olesen, Louise Lohse, Marlene Danner Dalgaard, Grzegorz Woźniakowski, Graham J. Belsham, Anette Bøtner, Thomas Bruun Rasmussen. Complete genome sequence of an African swine fever virus (ASFV POL/2015/Podlaskie) determined directly from pig erythrocyte-associated nucleic acid. Journal of Virological Methods. 2018; 261 ():14-16.

Chicago/Turabian Style

Ann Sofie Olesen; Louise Lohse; Marlene Danner Dalgaard; Grzegorz Woźniakowski; Graham J. Belsham; Anette Bøtner; Thomas Bruun Rasmussen. 2018. "Complete genome sequence of an African swine fever virus (ASFV POL/2015/Podlaskie) determined directly from pig erythrocyte-associated nucleic acid." Journal of Virological Methods 261, no. : 14-16.

Rapid communication
Published: 07 June 2018 in Transboundary and Emerging Diseases
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Within Eastern Europe, African swine fever virus (ASFV) has unexpectedly spread to farms with high biosecurity. In an attempt to explain this process, pigs were allowed to ingest flies that had fed on ASFV‐spiked blood, which had a realistic titre for an infected pig. Some of the pigs became infected with the virus. Thus, ingestion of blood‐sucking flies, having fed on ASFV‐infected wild boar before entering stables, represents a potential route for disease transmission.

ACS Style

Ann Sofie Olesen; Louise Lohse; Mette Frimodt Hansen; Anette Boklund; Tariq Halasa; Graham J. Belsham; Thomas Bruun Rasmussen; Anette Bøtner; Rene Bødker. Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans ). Transboundary and Emerging Diseases 2018, 65, 1152 -1157.

AMA Style

Ann Sofie Olesen, Louise Lohse, Mette Frimodt Hansen, Anette Boklund, Tariq Halasa, Graham J. Belsham, Thomas Bruun Rasmussen, Anette Bøtner, Rene Bødker. Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans ). Transboundary and Emerging Diseases. 2018; 65 (5):1152-1157.

Chicago/Turabian Style

Ann Sofie Olesen; Louise Lohse; Mette Frimodt Hansen; Anette Boklund; Tariq Halasa; Graham J. Belsham; Thomas Bruun Rasmussen; Anette Bøtner; Rene Bødker. 2018. "Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans )." Transboundary and Emerging Diseases 65, no. 5: 1152-1157.

Journal article
Published: 01 November 2017 in Veterinary Microbiology
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In 2014, African swine fever virus (ASFV) was introduced into the Baltic states and Poland. Since then, the disease has continued to spread within these regions, and recently, cases were reported in the Czech Republic and Romania. Currently, there is an increasing risk of ASFV introduction into Western Europe. Hence, there is an urgent need to assess current contingency plans. For this purpose, knowledge of modes-of-transmission and clinical outcome in pigs infected with new European ASFV strains is needed. In the present study, two experiments were conducted in pigs using an isolate of ASFV from Poland (designated here POL/2015/Podlaskie/Lindholm). In both studies, pigs were inoculated intranasally with the virus and contact pigs were exposed to the experimentally infected pigs, either directly (contact within and between pens) or by air. Pigs exposed to the virus by intranasal inoculation, by direct contact to infected animals and by aerosol developed acute disease characterized by viremia, fever and depression. Infectious virus was first detected in blood obtained from the inoculated pigs and then sequentially among the within-pen, between-pen and air-contact pigs. ASFV DNA and occasionally infectious virus was found in nasal-, oral-, and rectal swabs obtained from the pigs, and ASFV DNA was detected in air samples. No anti-ASFV antibodies were detected in sera. In conclusion, the study shows that the currently circulating strain of ASFV can be efficiently transmitted via direct contact and by aerosols. Also, the results provide quantitative transmission parameters and knowledge of infection stages in pigs infected with this ASFV.

ACS Style

Ann Sofie Olesen; Louise Lohse; Anette Boklund; Tariq Halasa; Carmina Gallardo; Zygmunt Pejsak; Graham Belsham; Thomas Bruun Rasmussen; Anette Bøtner. Transmission of African swine fever virus from infected pigs by direct contact and aerosol routes. Veterinary Microbiology 2017, 211, 92 -102.

AMA Style

Ann Sofie Olesen, Louise Lohse, Anette Boklund, Tariq Halasa, Carmina Gallardo, Zygmunt Pejsak, Graham Belsham, Thomas Bruun Rasmussen, Anette Bøtner. Transmission of African swine fever virus from infected pigs by direct contact and aerosol routes. Veterinary Microbiology. 2017; 211 ():92-102.

Chicago/Turabian Style

Ann Sofie Olesen; Louise Lohse; Anette Boklund; Tariq Halasa; Carmina Gallardo; Zygmunt Pejsak; Graham Belsham; Thomas Bruun Rasmussen; Anette Bøtner. 2017. "Transmission of African swine fever virus from infected pigs by direct contact and aerosol routes." Veterinary Microbiology 211, no. : 92-102.

Short report
Published: 25 November 2014 in Virology Journal
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In the frame of active lyssavirus surveillance in bats, oropharyngeal swabs from German (N = 2297) and Danish (N = 134) insectivorous bats were investigated using a newly developed generic pan-lyssavirus real-time reverse transcriptase PCR (RT-qPCR). In total, 15 RT-qPCR positive swabs were detected. Remarkably, sequencing of positive samples did not confirm the presence of bat associated lyssaviruses but revealed nine distinct novel rhabdovirus-related sequences. Several novel rhabdovirus-related sequences were detected both in German and Danish insectivorous bats. The results also prove that the novel generic pan-lyssavirus RT-qPCR offers a very broad detection range that allows the collection of further valuable data concerning the broad and complex diversity within the family Rhabdoviridae.

ACS Style

Melina Fischer; Conrad M Freuling; Thomas Müller; Juliane Schatz; Thomas Bruun Rasmussen; Mariann Chriél; Anne Balkema-Buschmann; Martin Beer; Bernd Hoffmann; Melina Fischer. Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats. Virology Journal 2014, 11, 196 .

AMA Style

Melina Fischer, Conrad M Freuling, Thomas Müller, Juliane Schatz, Thomas Bruun Rasmussen, Mariann Chriél, Anne Balkema-Buschmann, Martin Beer, Bernd Hoffmann, Melina Fischer. Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats. Virology Journal. 2014; 11 (1):196.

Chicago/Turabian Style

Melina Fischer; Conrad M Freuling; Thomas Müller; Juliane Schatz; Thomas Bruun Rasmussen; Mariann Chriél; Anne Balkema-Buschmann; Martin Beer; Bernd Hoffmann; Melina Fischer. 2014. "Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats." Virology Journal 11, no. 1: 196.

Journal article
Published: 19 September 2014 in Virology
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Classical swine fever virus (CSFV) strain “Koslov” is highly virulent with a mortality rate of up to 100% in pigs. In this study, we modified non-functional cDNAs generated from the blood of Koslov virus infected pigs by site-directed mutagenesis, removing non-synonymous mutations step-by-step, thereby producing genomes encoding the consensus amino acid sequence. Viruses rescued from the construct corresponding to the inferred parental form were highly virulent, when tested in pigs, with infected animals displaying pronounced clinical symptoms leading to high mortality. The reconstruction therefore gave rise to a functional cDNA corresponding to the highly virulent Koslov strain of CSFV. It could be demonstrated that two single amino acid changes (S763L and P968H) in the surface structural protein E2 resulted in attenuation in the porcine infection system while another single amino acid change within the nonstructural protein NS3 (D2183G) reduced virus growth within cells in vitro.

ACS Style

Ulrik Fahnøe; Anders Gorm Pedersen; Peter Christian Risager; Jens Nielsen; Graham Belsham; Dirk Höper; Martin Beer; Thomas Bruun Rasmussen. Rescue of the highly virulent classical swine fever virus strain “Koslov” from cloned cDNA and first insights into genome variations relevant for virulence. Virology 2014, 468-470, 379 -387.

AMA Style

Ulrik Fahnøe, Anders Gorm Pedersen, Peter Christian Risager, Jens Nielsen, Graham Belsham, Dirk Höper, Martin Beer, Thomas Bruun Rasmussen. Rescue of the highly virulent classical swine fever virus strain “Koslov” from cloned cDNA and first insights into genome variations relevant for virulence. Virology. 2014; 468-470 ():379-387.

Chicago/Turabian Style

Ulrik Fahnøe; Anders Gorm Pedersen; Peter Christian Risager; Jens Nielsen; Graham Belsham; Dirk Höper; Martin Beer; Thomas Bruun Rasmussen. 2014. "Rescue of the highly virulent classical swine fever virus strain “Koslov” from cloned cDNA and first insights into genome variations relevant for virulence." Virology 468-470, no. : 379-387.

Journal article
Published: 26 June 2014 in Genome Announcements
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The complete genome sequence of the genotype 2.2 classical swine fever virus strain Bergen has been determined; this strain was originally isolated from persistently infected domestic pigs in the Netherlands and is characterized to be of low virulence.

ACS Style

Ulrik Fahnøe; Louise Lohse; Paul Becher; Thomas Bruun Rasmussen. Complete Genome Sequence of Classical Swine Fever Virus Genotype 2.2 Strain Bergen. Genome Announcements 2014, 2, e00483-14 .

AMA Style

Ulrik Fahnøe, Louise Lohse, Paul Becher, Thomas Bruun Rasmussen. Complete Genome Sequence of Classical Swine Fever Virus Genotype 2.2 Strain Bergen. Genome Announcements. 2014; 2 (3):e00483-14.

Chicago/Turabian Style

Ulrik Fahnøe; Louise Lohse; Paul Becher; Thomas Bruun Rasmussen. 2014. "Complete Genome Sequence of Classical Swine Fever Virus Genotype 2.2 Strain Bergen." Genome Announcements 2, no. 3: e00483-14.

Journal article
Published: 17 February 2014 in Veterinary Microbiology
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Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does not include any CSFV components. In the present study, the DIVA vaccine properties of CP7_E2gif were evaluated in comparison to the conventional live attenuated Riemser C-strain vaccine. Sera and tonsil samples obtained from pigs immunised with these two vaccines were analysed. No viral RNA was found in serum after vaccination with CP7_E2gif, whereas some serum samples from C-strain vaccinated animals were positive. In both vaccinated groups, individual viral RNA-positive tonsil samples were detected in animals euthanised between 7 and 21 days post vaccination. Furthermore, serum samples from these animals, together with archival samples from pigs vaccinated with CP7_E2gif and subsequently CSFV challenged, were analysed for specific antibodies using ELISAs and for homologous neutralising antibodies. In animals vaccinated with CP7_E2gif, neutralising antibodies were detected from day 10. However, the sera remained negative for anti-CSFV E2-specific antibodies whereas pigs vaccinated with C-strain seroconverted against CSFV by 14 days after vaccination, as determined by a CSFV-E2 specific blocking ELISA. One week after subsequent CSFV challenge, a strong anti-CSFV E2 reaction was detected in CP7_E2gif vaccinated pigs and anti-Erns antibodies were detected from 10 days after infection. In conclusion, CP7_E2gif has the potential to be used as a DIVA vaccine in combination with detection of anti-CSFV E2-specific antibodies.

ACS Style

Tanya von Rosen; Desislava Rangelova; Jens Nielsen; Thomas Bruun Rasmussen; Åse Uttenthal. DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif. Veterinary Microbiology 2014, 170, 224 -231.

AMA Style

Tanya von Rosen, Desislava Rangelova, Jens Nielsen, Thomas Bruun Rasmussen, Åse Uttenthal. DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif. Veterinary Microbiology. 2014; 170 (3-4):224-231.

Chicago/Turabian Style

Tanya von Rosen; Desislava Rangelova; Jens Nielsen; Thomas Bruun Rasmussen; Åse Uttenthal. 2014. "DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif." Veterinary Microbiology 170, no. 3-4: 224-231.

Journal article
Published: 01 January 2013 in BMC Genomics
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Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.

ACS Style

Thomas Bruun Rasmussen; Peter Christian Risager; Ulrik Fahnøe; Martin Barfred Friis; Graham J Belsham; Dirk Höper; Ilona Reimann; Martin Beer. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA. BMC Genomics 2013, 14, 819 -819.

AMA Style

Thomas Bruun Rasmussen, Peter Christian Risager, Ulrik Fahnøe, Martin Barfred Friis, Graham J Belsham, Dirk Höper, Ilona Reimann, Martin Beer. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA. BMC Genomics. 2013; 14 (1):819-819.

Chicago/Turabian Style

Thomas Bruun Rasmussen; Peter Christian Risager; Ulrik Fahnøe; Martin Barfred Friis; Graham J Belsham; Dirk Höper; Ilona Reimann; Martin Beer. 2013. "Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA." BMC Genomics 14, no. 1: 819-819.