Publications by authors named "Graham J Belsham"

116 Publications

Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs.

Viruses 2021 11 22;13(11). Epub 2021 Nov 22.

Section of Veterinary Clinical Microbiology, Department of Veterinary and Animal Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.
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http://dx.doi.org/10.3390/v13112333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8623482PMC
November 2021

Infection, recovery and re-infection of farmed mink with SARS-CoV-2.

PLoS Pathog 2021 11 15;17(11):e1010068. Epub 2021 Nov 15.

Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark.

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 as well as the A22920T mutation, resulting in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those 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 in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2.
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http://dx.doi.org/10.1371/journal.ppat.1010068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8629378PMC
November 2021

Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms.

Front Microbiol 2021 25;12:698944. Epub 2021 Jun 25.

Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark.

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.
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http://dx.doi.org/10.3389/fmicb.2021.698944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8267889PMC
June 2021

Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark.

Viruses 2021 06 4;13(6). Epub 2021 Jun 4.

Department of Virus and Microbiological Special Diagnostics, Statens Serum Institut, 2300 Copenhagen, Denmark.

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., , , and -that the astroviruses may be new species in the genus , and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates-including two (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.
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http://dx.doi.org/10.3390/v13061073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229204PMC
June 2021

Foot-and-mouth disease viruses of the O/ME-SA/Ind-2001e sublineage in Pakistan.

Transbound Emerg Dis 2021 Nov 22;68(6):3126-3135. Epub 2021 May 22.

Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark.

The presence of foot-and-mouth disease virus (FMDV) of the O/ME-SA/Ind-2001e sublineage within Pakistan was initially detected in two samples collected during 2019. Analysis of further serotype O FMDVs responsible for disease outbreaks in 2019-2020 in the country has now identified the spread of this sublineage to 10 districts within two separate provinces in North-Eastern and North-Western Pakistan. Phylogenetic analysis indicates that these viruses are closely related to those circulating in Bhutan, Nepal and India. The VP1 coding sequences of these viruses from Pakistan belong to three distinct clusters, which may indicate multiple introductions of this virus sublineage, although the routes of introduction are unknown. Vaccine matching studies against O1 Manisa, O 3039 and O TUR/5/2009 support the suitability of existing vaccine strains to control current field outbreaks, but further studies are warranted to monitor the spread and evolution of the O/ME-SA/Ind-2001e sublineage in the region. (145 words).
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http://dx.doi.org/10.1111/tbed.14134DOI Listing
November 2021

Picornaviruses: A View from 3A.

Viruses 2021 03 11;13(3). Epub 2021 Mar 11.

Department of Veterinary and Animal Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark.

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3D (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.
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http://dx.doi.org/10.3390/v13030456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999760PMC
March 2021

Preliminary report of an outbreak of SARS-CoV-2 in mink and mink farmers associated with community spread, Denmark, June to November 2020.

Euro Surveill 2021 02;26(5)

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

In June-November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV-2-PCR-positive and approximately 27% (95% confidence interval (CI): 25-30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.
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http://dx.doi.org/10.2807/1560-7917.ES.2021.26.5.210009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863232PMC
February 2021

SARS-CoV-2 in Danish Mink Farms: Course of the Epidemic and a Descriptive Analysis of the Outbreaks in 2020.

Animals (Basel) 2021 Jan 12;11(1). Epub 2021 Jan 12.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark.

SARS-CoV-2 infection is the cause of COVID-19 in humans. In April 2020, SARS-CoV-2 infection in farmed mink () 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.
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http://dx.doi.org/10.3390/ani11010164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828158PMC
January 2021

SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.

Emerg Infect Dis 2021 Feb 18;27(2):547-551. Epub 2020 Nov 18.

Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink (Neovison vison) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.
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http://dx.doi.org/10.3201/eid2702.203794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853580PMC
February 2021

Animal Models for COVID-19: More to the Picture Than ACE2, Rodents, Ferrets, and Non-human Primates. A Case for Porcine Respiratory Coronavirus and the Obese Ossabaw Pig.

Front Microbiol 2020 25;11:573756. Epub 2020 Sep 25.

Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark.

The ongoing COVID-19 pandemic caused by infection with SARS-CoV-2 has created an urgent need for animal models to enable study of basic infection and disease mechanisms and for development of vaccines, therapeutics, and diagnostics. Most research on animal models for COVID-19 has been directed toward rodents, transgenic rodents, and non-human primates. The primary focus has been on the angiotensin-converting enzyme 2 (ACE2), which is a host cell receptor for SARS-CoV-2. Among investigated species, irrespective of ACE2 spike protein binding, only mild (or no) disease has occurred following infection with SARS-CoV-2, suggesting that ACE2 may be necessary for infection but is not sufficient to determine the outcome of infection. The common trait of all species investigated as COVID models is their healthy status prior to virus challenge. In contrast, the vast majority of severe COVID-19 cases occur in people with chronic comorbidities such as diabetes, obesity, and/or cardiovascular disease. Healthy pigs express ACE2 protein that binds the viral spike protein but they are not susceptible to infection with SARS-CoV-2. However, certain pig breeds, such as the Ossabaw pig, can reproducibly be made obese and show most aspects of the metabolic syndrome, thus resembling the more than 80% of the critically ill COVID-19 patients admitted to hospitals. We urge considering infection with porcine respiratory coronavirus of metabolic syndrome pigs, such as the obese Ossabaw pig, as a highly relevant animal model of severe COVID-19.
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http://dx.doi.org/10.3389/fmicb.2020.573756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545904PMC
September 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.

J Gen Virol 2020 11;101(11):1170-1181

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.

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.
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http://dx.doi.org/10.1099/jgv.0.001483DOI Listing
November 2020

Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants.

J Virol 2020 09 15;94(19). Epub 2020 Sep 15.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark

Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity. The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.
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http://dx.doi.org/10.1128/JVI.01119-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495388PMC
September 2020

Towards improvements in foot-and-mouth disease vaccine performance.

Authors:
Graham J Belsham

Acta Vet Scand 2020 May 20;62(1):20. Epub 2020 May 20.

Department of Veterinary and Animal Sciences, University of Copenhagen, 1870, Frederiksberg C, Denmark.

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals. Six (out of 7 that have been identified) different serotypes of the FMD virus continue to circulate in different parts of the world. Within each serotype there is also extensive diversity as the virus constantly changes. Vaccines need to be "matched" to the outbreak strain, not just to the serotype, to confer protection. Vaccination has been used successfully to assist in the eradication of the disease from Europe but is no longer employed there unless outbreaks occur. Thus the animal population in Europe, as in North America, is fully susceptible to the virus if it is accidentally (or deliberately) introduced. Almost 3 billion doses of the vaccine are made each year to control the disease elsewhere. Current vaccines are produced from chemically inactivated virus that has to be grown, on a large scale, under high containment conditions. The vaccine efficiently prevents disease but the duration of immunity is rather limited (about 6 months) and vaccination does not provide sterile immunity or block the development of carriers. Furthermore, the vaccine is quite unstable and a cold chain needs to be maintained to preserve the efficacy of the vaccine. This can be a challenge in the parts of the world where the disease is endemic. There is a significant interest in developing improved vaccines and significant progress in this direction has been made using a variety of approaches. However, no alternative vaccines are yet available commercially. Improved disease control globally is clearly beneficial to all countries as it reduces the risk of virus incursions into disease free areas.
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http://dx.doi.org/10.1186/s13028-020-00519-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240906PMC
May 2020

Potential routes for indirect transmission of African swine fever virus into domestic pig herds.

Transbound Emerg Dis 2020 Jul 23;67(4):1472-1484. Epub 2020 Mar 23.

Section of Veterinary Clinical Microbiology, Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Following its introduction into Georgia in 2007, African swine fever virus (ASFV) has become widespread on the European continent and in Asia. In many cases, the exact route of introduction into domestic pig herds cannot be determined, but most introductions are attributed to indirect virus transmission. In this review, we describe knowledge gained about different matrices that may allow introduction of the virus into pig herds. These matrices include uncooked pig meat, processed pig-derived products, feed, matrices contaminated with the virus and blood-feeding invertebrates. Knowledge gaps still exist, and both field studies and laboratory research are needed to enhance understanding of the risks for ASFV introductions, especially via virus-contaminated materials, including bedding and feed, and via blood-feeding, flying insects. Knowledge obtained from such studies can be applied to epidemiological risk assessments for the different transmission routes. Such assessments can be utilized to help predict the most effective biosecurity and control strategies.
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http://dx.doi.org/10.1111/tbed.13538DOI Listing
July 2020

Foot-and-mouth disease virus: Prospects for using knowledge of virus biology to improve control of this continuing global threat.

Virus Res 2020 05 29;281:197909. Epub 2020 Feb 29.

The Pirbright Institute, Pirbright, Woking, Surrey, GU24 0NF. UK.

Understanding of the biology of foot-and-mouth disease virus (FMDV) has grown considerably since the nucleotide sequence of the viral RNA was determined. The ability to manipulate the intact genome and also to express specific parts of the genome individually has enabled detailed analyses of viral components, both RNA and protein. Such studies have identified the requirements for specific functional elements for virus replication and pathogenicity. Furthermore, information about the functions of individual virus proteins has enabled the rational design of cDNA cassettes to express non-infectious empty capsid particles that can induce protective immunity in the natural host animals and thus represent new vaccine candidates. Similarly, attempts to block specific virus activities using antiviral agents have also been performed. However, currently, only the well-established, chemically inactivated FMDV vaccines are commercially available and suitable for use to combat this important disease of livestock animals. These vaccines, despite certain shortcomings, have been used very successfully (e.g. in Europe) to control the disease but it still remains endemic in much of Africa, southern Asia and the Middle East. Hence there remains a significant risk of reintroduction of the disease into highly susceptible animal populations with enormous economic consequences.
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http://dx.doi.org/10.1016/j.virusres.2020.197909DOI Listing
May 2020

Evidence for multiple recombination events within foot-and-mouth disease viruses circulating in West Eurasia.

Transbound Emerg Dis 2020 Mar 23;67(2):979-993. Epub 2019 Dec 23.

National Veterinary Institute, Technical University of Denmark, Lindholm, Denmark.

Phylogenetic studies on foot-and-mouth disease viruses (FMDVs) circulating in the West Eurasian region have largely focused on the genomic sequences encoding the structural proteins that determine the serotype. The present study has compared near-complete genome sequences of FMDVs representative of the viruses that circulate in this region. The near-complete genome sequences (ca. 7,600 nt) were generated from multiple overlapping RT-PCR products. These amplicons were from FMDVs belonging to serotypes O, A and Asia-1, including members of the O-PanAsia-II and the A-Iran05 lineages, and of Group-II and Group-VII (Sindh-08) within serotype Asia-1, which are currently predominant and widespread in West Eurasia. These new sequences were analysed together with other sequences obtained from GenBank. Comparison of different regions of the FMDVs genomes revealed evidence for multiple, inter-serotypic, recombination events between FMDVs belonging to the serotypes O, A and Asia-1. It is concluded from the present study that dramatic changes in virus sequences can occur in the field through recombination between different FMDV genomes. These analyses provide information about the ancestry of the serotype O, A and Asia-1 FMDVs that are currently circulating within the West Eurasian region.
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http://dx.doi.org/10.1111/tbed.13433DOI Listing
March 2020

Virus Adaptation and Selection Following Challenge of Animals Vaccinated against Classical Swine Fever Virus.

Viruses 2019 10 10;11(10). Epub 2019 Oct 10.

National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.

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.
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http://dx.doi.org/10.3390/v11100932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833067PMC
October 2019

Identification of plasticity and interactions of a highly conserved motif within a picornavirus capsid precursor required for virus infectivity.

Sci Rep 2019 08 13;9(1):11747. Epub 2019 Aug 13.

DTU National Veterinary Institute, Lindholm, Kalvehave, 4771, Denmark.

The picornavirus family includes poliovirus (PV) (genus: enterovirus), human rhinoviruses (enterovirus) and foot-and-mouth disease virus (FMDV) (aphthovirus). These are responsible for important human and animal health concerns worldwide including poliomyelitis, the common cold and foot-and-mouth disease (FMD) respectively. In picornavirus particles, the positive-sense RNA genome (ca. 7-9 kb) is packaged within a protein shell (capsid) usually consisting of three surface exposed proteins, VP1, VP2 and VP3 plus the internal VP4, which are generated following cleavage of the capsid precursor by a virus-encoded protease. We have previously identified a motif near the C-terminus of FMDV VP1 that is required for capsid precursor processing. This motif is highly conserved among other picornaviruses, and is also likely to be important for their capsid precursor processing. We have now determined the plasticity of residues within this motif for virus infectivity and found an important interaction between FMDV residue VP1 R188 within this conserved motif and residue W129 in VP2 that is adjacent in the virus capsid. The FMDV (VP1 R188A) mutant virus has only been rescued with the secondary substitution VP2 W129R. This additional change compensates for the defect resulting from the VP1 R188A substitution and restored both capsid precursor processing and virus viability.
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http://dx.doi.org/10.1038/s41598-019-48170-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692319PMC
August 2019

Diagnostic comparison of serum and EDTA-stabilized blood samples for the detection of foot-and-mouth disease virus RNA by RT-qPCR.

J Virol Methods 2019 08 13;270:120-125. Epub 2019 May 13.

National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark. Electronic address:

Foot-and-mouth disease (FMD) remains a globally important disease but there have only been occasional recent outbreaks in Europe, e.g. in the U.K. in 2001, U.K. 2007 and Bulgaria 2010/2011. However, this infection still poses a threat to Europe as the disease occurs close to its borders and incursions can occur through importation of contaminated animal products and through the air. To deal with a suspected outbreak, fast sampling, transportation and accurate laboratory diagnosis are critical; testing for FMDV is normally performed on epithelium samples or serum. Assessment of the use of stabilized blood in assays for FMDV RNA is useful as this sample material can be prepared on site for safe transportation and rapid analysis at the laboratory. Such samples are also collected for diagnosis of other diseases giving similar clinical signs. Testing serum and EDTA-stabilized blood samples from FMDV-infected cattle and pigs, using real time quantitative RT-PCR assays, yielded similar results. However, detection of FMDV RNA was less sensitive (about 10-fold) when using EDTA-stabilized blood compared to serum. Thus, diagnosis of FMD can be achieved using EDTA-stabilized blood samples in an outbreak situation on a herd basis, but serum is preferred at the single animal level for optimal sensitivity.
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http://dx.doi.org/10.1016/j.jviromet.2019.05.003DOI Listing
August 2019

Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

J Virol 2019 05 1;93(10). Epub 2019 May 1.

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain

Infection by viruses depends on a balance between capsid stability and dynamics. This study investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability and between thermostability and infectivity. In the FMDV capsid, a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essential role for virus infection of most of the 8 tested charged groups is not related to substantial changes in capsid protein expression or processing or in capsid assembly or stability against a thermally induced dissociation into pentamers. However, the positively charged side chains of R2018 and H3141, located at the interpentamer interfaces close to the capsid 2-fold symmetry axes, were found to be critical both for virus infectivity and for keeping the capsid in a state of weak thermostability. A charge-restoring substitution (N2019H) that was repeatedly fixed during amplification of viral genomes carrying deleterious mutations reverted both the lethal and capsid-stabilizing effects of the substitution H3141A, leading to a double mutant virus with close to normal infectivity and thermolability. H3141A and other thermostabilizing substitutions had no detectable effect on capsid resistance to acid-induced dissociation into pentamers. The results suggest that FMDV infectivity requires limited local stability around the 2-fold axes at the interpentamer interfaces of the capsid. The implications for the mechanism of genome uncoating in FMDV and the development of thermostabilized vaccines against foot-and-mouth disease are discussed. This study provides novel insights into the little-known structural determinants of the balance between thermal stability and instability in the capsid of foot-and-mouth disease virus and into the relationship between capsid stability and virus infectivity. The results provide new guidelines for the development of thermostabilized empty capsid-based recombinant vaccines against foot-and-mouth disease, one of the economically most important animal diseases worldwide.
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http://dx.doi.org/10.1128/JVI.02293-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498042PMC
May 2019

Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites.

PLoS Pathog 2019 01 18;15(1):e1007509. Epub 2019 Jan 18.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark.

Many picornaviruses cause important diseases in humans and other animals including poliovirus, rhinoviruses (causing the common cold) and foot-and-mouth disease virus (FMDV). These small, non-enveloped viruses comprise a positive-stranded RNA genome (ca. 7-9 kb) enclosed within a protein shell composed of 60 copies of three or four different capsid proteins. For the aphthoviruses (e.g. FMDV) and cardioviruses, the capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to generate VP0, VP3 and VP1 plus 2A. For enteroviruses, e.g. poliovirus, the capsid precursor is P1 alone, which is cleaved by the 3CD protease to generate just VP0, VP3 and VP1. The sequences required for correct processing of the FMDV capsid protein precursor in mammalian cells were analyzed. Truncation of the P1-2A precursor from its C-terminus showed that loss of the 2A peptide (18 residues long) and 27 residues from the C-terminus of VP1 (211 residues long) resulted in a precursor that cannot be processed by 3Cpro although it still contained two unmodified internal cleavage sites (VP0/VP3 and VP3/VP1 junctions). Furthermore, introduction of small deletions within P1-2A identified residues 185-190 within VP1 as being required for 3Cpro-mediated processing and for optimal accumulation of the precursor. Within this C-terminal region of VP1, five of these residues (YCPRP), are very highly conserved in all FMDVs and are also conserved amongst other picornaviruses. Mutant FMDV P1-2A precursors with single amino acid substitutions within this motif were highly resistant to cleavage at internal junctions. Such substitutions also abrogated virus infectivity. These results can explain earlier observations that loss of the C-terminus (including the conserved motif) from the poliovirus capsid precursor conferred resistance to processing. Thus, this motif seems essential for maintaining the correct structure of picornavirus capsid precursors prior to processing and subsequent capsid assembly; it may represent a site that interacts with cellular chaperones.
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http://dx.doi.org/10.1371/journal.ppat.1007509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338358PMC
January 2019

Transmission of Foot-and-Mouth Disease from Persistently Infected Carrier Cattle to Naive Cattle via Transfer of Oropharyngeal Fluid.

mSphere 2018 09 12;3(5). Epub 2018 Sep 12.

Foreign Animal Disease Research Unit, Plum Island Animal Disease Center, USDA-ARS, Greenport, New York, USA

Control and eradication of foot-and-mouth disease (FMD) are impeded by the existence of a persistent, subclinical phase of infection in ruminants; animals with this status are referred to as carriers. However, the epidemiological significance of these FMD virus (FMDV) carriers is uncertain. In the current investigation, the contagion associated with FMDV carrier cattle was investigated by exposure of susceptible cattle and pigs to oropharyngeal fluid (OPF) samples or tissues harvested from persistently infected cattle. Naive cattle were inoculated through intranasopharyngeal deposition of unprocessed OPF samples that had been collected from FMDV carriers at 30 days postinfection. These inoculated cattle developed clinical FMD, and the severity of disease they developed was similar to that of animals that had been infected with a high-titer inoculum. In contrast, pigs exposed via intraoropharyngeal inoculation of the same OPF samples or via ingestion of nasopharyngeal tissues harvested from the same cohort of persistently infected cattle did not develop FMD. These findings indicate that there is demonstrable contagion associated with FMDV carrier cattle despite the lack of evidence for transmission by direct contact. The findings presented herein provide novel information that should be considered for FMD risk mitigation strategies. Foot-and-mouth disease (FMD) is a viral disease of livestock with substantial impact on agricultural production and subsistence farming on a global scale. Control of FMD is impeded by the existence of a prolonged asymptomatic carrier phase during which infected cattle shed low quantities of infectious virus in oropharyngeal fluid (OPF) for months to years after infection. The epidemiological significance of FMD virus (FMDV) carriers is unresolved. However, the existence of the FMDV carrier state has substantial impact on international trade in animal products. The current investigation demonstrated that transfer of OPF from persistently infected FMDV carrier cattle to naive cattle led to fulminant clinical FMD. It was thus demonstrated that, although the risk for disease transmission under natural conditions is considered to be low, there is detectable contagion associated with FMDV carrier cattle. This finding is important for optimization of FMD risk mitigation strategies.
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http://dx.doi.org/10.1128/mSphere.00365-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135961PMC
September 2018

Detection and Characterization of Distinct Alphacoronaviruses in Five Different Bat Species in Denmark.

Viruses 2018 09 11;10(9). Epub 2018 Sep 11.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.

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 , 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.
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http://dx.doi.org/10.3390/v10090486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163574PMC
September 2018

A reply to "A comment on "Inter-laboratory study to characterize the detection of serum antibodies against porcine epidemic diarrhoea virus"".

Vet Microbiol 2018 10 13;224:118. Epub 2018 Aug 13.

National Veterinary Institute, Lindholm, Technical University of Denmark, Denmark. Electronic address:

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http://dx.doi.org/10.1016/j.vetmic.2018.08.015DOI Listing
October 2018

Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping.

BMC Genomics 2018 Aug 9;19(1):600. Epub 2018 Aug 9.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771, Kalvehave, Denmark.

Background: 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.

Results: 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.

Conclusions: This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.
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http://dx.doi.org/10.1186/s12864-018-4971-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085635PMC
August 2018

Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder.

Vet Microbiol 2018 Aug 19;222:25-29. Epub 2018 Jun 19.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vetmic.2018.06.010DOI Listing
August 2018

Complete genome sequence of an African swine fever virus (ASFV POL/2015/Podlaskie) determined directly from pig erythrocyte-associated nucleic acid.

J Virol Methods 2018 11 29;261:14-16. Epub 2018 Jul 29.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jviromet.2018.07.015DOI Listing
November 2018

Cleavages at the three junctions within the foot-and-mouth disease virus capsid precursor (P1-2A) by the 3C protease are mutually independent.

Virology 2018 09 26;522:260-270. Epub 2018 Jul 26.

National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave 4771, Denmark. Electronic address:

The foot-and-mouth disease virus capsid precursor, P1-2A, is cleaved by the 3C protease (3C) to VP0, VP3, VP1 and 2A. The P1-2A precursor (wt or mutant) was expressed alone or with 3C and processing of P1-2A was determined. The VP2 K217R and VP3 I2P substitutions (near the VP0/VP3 junction) strongly reduced the processing at this junction by 3C while the substitution VP2 K217E blocked cleavage. At the VP3/VP1 junction, the substitutions VP3 Q2221P and VP1 T1P each severely inhibited processing at this site. Blocking cleavage at either junction did not prevent processing elsewhere in P1-2A. These modifications were also introduced into full-length FMDV RNA; only wt and the VP2 K217R mutant were viable. Uncleaved VP0-VP3 and the processed products were observed within cells infected with the mutant virus. The VP0-VP3 was not incorporated into empty capsids or virus particles. The three junctions within P1-2A are processed by 3C independently.
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http://dx.doi.org/10.1016/j.virol.2018.07.010DOI Listing
September 2018

Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans).

Transbound Emerg Dis 2018 Oct 7;65(5):1152-1157. Epub 2018 Jun 7.

DTU National Veterinary Institute, Technical University of Denmark, Kgs. Lyngby, Denmark.

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.
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http://dx.doi.org/10.1111/tbed.12918DOI Listing
October 2018

Full-length genome sequences of porcine epidemic diarrhoea virus strain CV777; Use of NGS to analyse genomic and sub-genomic RNAs.

PLoS One 2018 1;13(3):e0193682. Epub 2018 Mar 1.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark.

Porcine epidemic diarrhoea virus, strain CV777, was initially characterized in 1978 as the causative agent of a disease first identified in the UK in 1971. This coronavirus has been widely distributed among laboratories and has been passaged both within pigs and in cell culture. To determine the variability between different stocks of the PEDV strain CV777, sequencing of the full-length genome (ca. 28kb) has been performed in 6 different laboratories, using different protocols. Not surprisingly, each of the different full genome sequences were distinct from each other and from the reference sequence (Accession number AF353511) but they are >99% identical. Unique and shared differences between sequences were identified. The coding region for the surface-exposed spike protein showed the highest proportion of variability including both point mutations and small deletions. The predicted expression of the ORF3 gene product was more dramatically affected in three different variants of this virus through either loss of the initiation codon or gain of a premature termination codon. The genome of one isolate had a substantially rearranged 5´-terminal sequence. This rearrangement was validated through the analysis of sub-genomic mRNAs from infected cells. It is clearly important to know the features of the specific sample of CV777 being used for experimental studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832266PMC
June 2018
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