Biology subjects

Larsen, L. E.

Publications and source records attributed to Larsen, L. E..

13 recordsLinked to original sources

bioRxiv2026

Genetic diversity and phylogeography of swine influenza A virus in 2013-2022 in Europe using a harmonized genotyping nomenclature

Swine Influenza A virus (swIAV) poses constant health threat for both humans and animals. This study comprehensively analysed the genetic diversity and evolution of European swIAVs from 2013 to 2022. To address existing classification gaps, an easily accessible full-genome genotyping tool based on Nextclade was developed alongside a global nomenclature system. Application of this tool to European sequence data revealed unprecedented genetic diversity, successfully identifying 150 swIAV genotypes mixing segments of both swine and human origin. Bayesian phylogeographic models, combined with economic data, identified that the trade of live weaner pigs for fattening drove the transboundary spread of swIAVs across Europe. The study detected repeated human-to-swine reverse-zoonotic events across multiple European countries. These findings clearly highlight the role of commercial pig movements in viral dissemination and emphasize the critical importance of harmonized genomic surveillance to monitor swIAV viral evolution to inform global pre-pandemic preparedness strategies.

microbiology↗

bioRxiv2026

Two epidemics, one genotype, different outcomes: evolutionary changes of Avian Influenza H5N1, genotype EA-2024-DI

Since 2020, high pathogenicity avian influenza H5Nx viruses of clade 2.3.4.4b have become enzootic in Europe, causing recurrent epidemic waves characterized by extensive reassortment events. Here, we describe the emergence of a single high-fitness genotype (EA-2024-DI) that has driven two consecutive waves, evolving into distinct sub-lineages. While its circulation is ongoing, during the 2025-2026 wave it caused an unprecedented number of cases in wild birds. Using phylodynamic analyses of a large dataset of genomic sequences, we compared the spatial diffusion and host transmission pattern of the EA-2024-DI sub-lineages across the three most recent epidemic waves (2023-2024, 2024-2025 and 2025-2026). We show that the genotype has persisted over time and has spread primarily through wild Anseriformes, but with a marked change in the transmission patterns between the different waves and a shift in the epicenter from Eastern to Central Europe, the latter having emerged as an important hub for virus diffusion throughout Europe. Our results reveal a recent increase in the frequency of viruses from wild and domestic mammals carrying mutations enhancing virus replication in mammalian hosts, highlighting the importance of proactive monitoring of this group of hosts to better understand its role in the virus ecology and evolution.

evolutionary biology↗

bioRxiv2026

Mapping Tissue-Specific Protease Dynamics in the Pig Respiratory Tract During Influenza A Virus Infection

Influenza A virus (IAV) is a zoonotic pathogen capable of infecting diverse avian and mammalian hosts, causing seasonal epidemics and occasional global pandemics in humans. Viral entry requires proteolytic activation of hemagglutinin (HA). While serine proteases such as TMPRSS2, and HAT are known HA activators, the respiratory tract harbours additional proteases whose contributions to infection remain unclear. Dysregulation of these proteases can enhance viral replication, tissue damage, and inflammation, highlighting the need for a systems-level view of the proteolytic landscape. Here, we use high-throughput, proteome-wide proteomics and N-terminomics to identify 112 host proteases across the nasal mucosa, trachea, and lung. We monitor and validate 28 proteases with targeted proteomics and microfluidic qPCR, representing a comprehensive degradome analysis in the respiratory tract of the highly translational pig model of influenza infection. We show that protease abundance and activity were highly tissue-specific: while the nasal mucosa showed selective activation of broad- and narrow-specificity proteases alongside robust antiviral responses, the trachea exhibited modest modulation with subtle shifts in protease-inhibitor balance, and the lung maintained predominantly active proteases despite lower viral loads but severe tissue damage, indicative of immune-mediated pathology. Sequence motif analysis revealed distinct cleavage preferences across tissues, indicating differential protease processing across the studied respiratory tissues in antiviral pathways, antigen processing, and tissue remodelling. Several identified proteases, including ST14, KLKB1, PRSS8, and LGMN, were increased and functionally active upon infection, suggesting roles in viral processing and host immune regulation. Collectively, our results define a spatially organised proteolytic network that shapes tissue-specific antiviral host responses and contributes to H1N1 influenza pathogenesis.

immunology↗

bioRxiv2025

Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract

Several pathogens are known to affect the respiratory tract of pigs resulting in decreased health and welfare. Virological investigations and the use of metagenomic sequencing of samples allowed the identification of new viruses in pigs, such as porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). The pathogenicity of PRV1 has been investigated experimentally by one research group, whereas SOV is yet to be studied. In this experimental trial, groups of weaners were inoculated with a European isolate of PRV1 (n = 8), a pool of SOV RNA-positive clinical nasal swab material (n = 8) or culture medium serving as controls (n = 4). Four days post inoculation (DPI), two controls and four pigs from each of the PRV1 and SOV groups were euthanized and necropsied. All SOV inoculated pigs tested negative for SOV at DPI 4 and therefore four of these pigs were transferred to the stable with the PRV1- infected pigs to assess direct-contact transmission. Nasal swab samples were collected at regular intervals after challenge and blood samples were collected at DPI 0, 4, and 14. At 14 DPI, the remaining pigs (n = 10) were euthanized and necropsied. Nasal swab samples and tissues from the respiratory tract were examined for PRV1 RNA by reverse transcriptase real- time PCR and blood samples were investigated for PRV1 antibodies by ELISA. Respiratory tissues were also evaluated macro- and microscopically and selected tissues were investigated for in situ detection of PRV1 mRNA by RNAscope. No clinical signs, except for nasal discharge, were observed in any of the pigs. PRV1-shedding was observed from DPI 2 to 11 with peaks between DPI 4 and 7, and PRV1 was transmitted horizontally to all direct-contact pigs. The highest viral load was detected in the upper respiratory tract, i.e. nose, upper and lower trachea compared to the lower respiratory tract, i.e., bronchioles, and alveoli. Generally, a chronic tracheitis at 4 DPI, developing into chronic, erosive tracheitis at 14 DPI was observed in the PRV1 groups and was supported by in situ detection of PRV1 by RNAscope. Three pigs also developed mild, bronchointerstitial pneumonia at 14 DPI. In conclusion, these results showed that PRV1 is a primary porcine respiratory pathogen. Author summaryRespiratory diseases in pigs impair porcine health and welfare and are often caused by a complex interaction between pathogens. We investigated the pathogenesis of two recently discovered viruses: porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). An experimental model in weaner pigs showed that PRV1 is capable of efficient replication, horizontal transmission and caused pathological manifestations mainly in the upper respiratory tract. The lesions consisted of chronic, erosive tracheitis in all PRV1 infected pigs, resulting in a breakage of the tracheal epithelial barrier, and a few pigs developed bronchointerstitial pneumonia. These findings confirm that PRV1 should be considered a primary porcine respiratory pathogen, but in contrast, SOV did not result in a successful infection, and the reasons why remain to be elucidated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/666573v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d93a11org.highwire.dtl.DTLVardef@17341e9org.highwire.dtl.DTLVardef@1ec4ca4org.highwire.dtl.DTLVardef@9bd42f_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract created with Biorender.com

microbiology↗

bioRxiv2024

Rapid Surge of Reassortant A(H1N1) Influenza Viruses in Danish Swine and their Zoonotic Potential

In 2018, a single detection of a novel reassortant swine influenza A virus (swIAV) was made in Denmark. The hemagglutinin (HA) of the virus was from the H1N1 pandemic 2009 (H1N1pdm09) lineage and the neuraminidase (NA) from the H1N1 Eurasian avian-like swine lineage (H1N1av). By 2022, the novel reassortant virus (H1pdm09N1av) constituted 27 % of swIAVs identified through the Danish passive swIAV surveillance program. Sequencing detected two H1pdm09N1av genotypes; Genotype 1 contained an internal gene cassette of H1N1pdm09 origin, Genotype 2 differed by carrying an NS gene segment of H1N1av origin. The internal gene cassette of Genotype 2 became increasingly dominant, not only in the H1pdm09N1av population, but also in other Danish enzootic swIAV subtypes. Phylogenetic analysis of the HA genes from H1pdm09N1av viruses revealed a monophyletic source, a higher substitution rate compared to other H1N1pdm09 viruses and genetic differences with human seasonal and other swine adapted H1N1pdm09 viruses. Correspondingly, H1pdm09N1av viruses were antigenically distinct from human H1N1pdm09 vaccine viruses. Both H1pdm09N1av genotypes transmitted between ferrets by direct contact, but only Genotype 1 was capable of efficient aerosol transmission. The rapid spread of H1pdm09N1av viruses in Danish swine herds is concerning for swine and human health. Their zoonotic threat is highlighted by the limited pre-existing immunity observed in the human population, aerosol transmission in ferrets and the finding that the internal gene cassette of Genotype 2 was present in the first two zoonotic infections ever detected in Denmark.

genomics↗

bioRxiv2024

tDCS Cranial Nerve Co-Stimulation: Unveiling Brainstem Pathways Involved in Trigeminal Nerve Direct Current Stimulation in Rats

The effects of transcranial direct current stimulation (tDCS) are typically attributed to the polarization of cortical neurons by the weak electric fields it generates in the cortex. However, emerging evidence indicates that certain tDCS effects may be mediated through the co-stimulation of peripheral or cranial nerves, particularly the trigeminal nerve (TN), which projects to critical brainstem nuclei that regulate the release of various neurotransmitters throughout the central nervous system. Despite this, the specific pathways involved remain inadequately characterized. In this study, we examined the effects of acute transcutaneous TN direct current stimulation (TN-DCS) on tonic (i.e. mean spike rate and spike rate over time) and phasic (number of bursts, spike rate per burst, burst duration, and inter-burst interval) activities while simultaneously recording single-neuron activity across three brainstem nuclei in rats: the locus coeruleus (LC), dorsal raphe nucleus (DRN), and median raphe nucleus (MnRN). We found that TN-DCS significantly modulated tonic activity in the LC, with notable interactions between stimulation amplitude, polarity, and time epoch affecting mean spike rates. Similar effects were observed in the DRN regarding tonic activity. Further, phasic activity in the LC was influenced by TN-DCS, with changes in burst number, duration, and inter-burst intervals linked to stimulation parameters. Conversely, MnRN tonic activity following TN-DCS remained unchanged. Importantly, xylocaine administration to block TN abolished the effects on tonic activities in both the LC and DRN. These results suggest that tDCS effects may partially arise from indirect modulation of the TN, leading to altered neuronal activity in DRN and LC. Besides, the differential changes in tonic and phasic LC activities underscore their complementary roles in mediating TN-DCS effects on higher cortical regions. This research bears significant translational implications, providing mechanistic insights that could enhance the efficacy of tDCS applications and deepen our understanding of its neurophysiological effects.

neuroscience↗

bioRxiv2024

Photopharmacological activation of adenosine A1receptor signaling suppresses seizures in a mousemodel for temporal lobe epilepsy

Up to 30% of epilepsy patients suffer from drug-resistant epilepsy (DRE). The search for innovative therapies is therefore important to close the existing treatment gap in these patients. The adenosinergic system possesses potent anticonvulsive effects, mainly through the adenosine A1 receptor (A1R). However, clinical application of A1R agonists is hindered by severe systemic side effects. To achieve local modulation of A1Rs, we employed a photopharmacological approach using a caged version of the A1R agonist N6-cyclopentyladenosine, termed cCPA. We performed the first in vivo study with intracerebroventricularly (ICV) administered cCPA to investigate the potential to uncage sufficient amounts of cCPA in the hippocampus by local illumination in order to suppress hippocampal excitability and seizures in mice. Using hippocampal evoked potential recordings, we showed a reduction in hippocampal neurotransmission after photo-uncaging of cCPA, similar to that obtained with ICV injection of CPA. Furthermore, in the intrahippocampal kainic acid mouse model for DRE, photo-uncaging of CPA in the epileptic hippocampus resulted in a strong suppression of seizures. Finally, we demonstrated that intrahippocampal photo-uncaging of CPA resulted in less impairment of motor performance in the rotarod test compared to ICV administration of CPA. These results provide a proof of concept for photopharmacological A1R modulation as an effective precision treatment for DRE.

neuroscience↗

bioRxiv2024

Feedback Control of Neuronal Excitability and Epileptiform Bursting using a Photocaged Adenosine A1 Agonist

Adenosine is a potent regulator of neurotransmission and neuronal excitability through activation of Gi protein-coupled adenosine A1 receptors (A1Rs). It has gained interest as a potential anticonvulsant due to its endogenous involvement in ending ongoing seizure activity. A recently developed coumarin-caged derivative of the A1R agonist N6-cyclopentyl-adenosine (CPA), cCPA, was used for photo-uncaging of CPA with millisecond flashes of 405 nm light. At population level, CPA reduces Schaffer Collateral stimulated extracellular dendritic field potentials (FPs) in the CA1 region of the hippocampus with an ED50 of 44.1{+/-}2.8 nM and a Hill coefficient of 3. Response onset is CPA dependent and takes less than seconds, while recovery is CPA independent with a time constant of around 20 minutes. A closed-loop feedback system used the amplitude of evoked dendritic FPs to photorelease CPA and was able to control FP amplitude to user defined levels between 10% and 90% of baseline level. In the acute elevated potassium model of epilepsy raising extracellular K+ to 8.5 mM enhances neuronal excitability and induces regularly occurring epileptiform bursts, but FPs evoked with low intensity could still continuously monitor excitability without interfering with bursting. In this model the closed-loop system that controlled CPA release, was able to suppress epileptiform bursting, while maintaining an acceptable level of functional neurotransmission. Including in the control algorithm a second parameter that combined population spike amplitude and number of population spikes, enabled the system to automatically find a level of functional neurotransmission that was just below the threshold for multiple spiking and epileptiform bursting. The combination of photopharmacological adenosinergic modulation with real-time FP monitoring provides a first step towards closed-loop precision treatment for diseases related to neuronal hyperexcitability such as epilepsy.

neuroscience↗

bioRxiv2024

The avian and human influenza A virus receptors sialic acid (SA)-α2,3 and SA-α2,6 are widely expressed in the bovine mammary gland

An outbreak of H5N1 highly pathogenic influenza A virus (HPIAV) has been detected in dairy cows in the United States. Influenza A virus (IAV) is a negative-sense, single-stranded, RNA virus that has not previously been associated with widespread infection in cattle. As such, cattle are an extremely under-studied domestic IAV host species. IAV receptors on host cells are sialic acids (SAs) that are bound to galactose in either an 2,3 or 2,6 linkage. Human IAVs preferentially bind SA-2,6 (human receptor), whereas avian IAVs have a preference for 2,3 (avian receptor). The avian receptor can further be divided into two receptors: IAVs isolated from chickens generally bind more tightly to SA-2,3-Gal-{beta}1,4 (chicken receptor), whereas IAVs isolated from duck to SA-2,3-Gal-{beta}1,3 (duck receptor). We found all receptors were expressed, to a different degree, in the mammary gland, respiratory tract, and cerebrum of beef and/or dairy cattle. The duck and human IAV receptors were widely expressed in the bovine mammary gland, whereas the chicken receptor dominated the respiratory tract. In general, only a low expression of IAV receptors was observed in the neurons of the cerebrum. These results provide a mechanistic rationale for the high levels of H5N1 virus reported in infected bovine milk and show cattle have the potential to act as a mixing vessel for novel IAV generation. Graphical abstractCreated with Biorender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/592326v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d6493org.highwire.dtl.DTLVardef@1b60585org.highwire.dtl.DTLVardef@1247e69org.highwire.dtl.DTLVardef@e75da1_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Diverse fox circovirus (Circovirus canine) variants circulate at high prevalence in grey wolves (Canis lupus)

Canine circoviruses (CanineCV) have a worldwide distribution in dogs and are occasionally detected in wild carnivorans, indicating their ability for cross-species transmission. However, fox circovirus, a lineage of CanineCV, has been identified exclusively in wild canids. We analyzed spleen samples from 159 grey wolves from the Northwest Territories, Canada, to investigate the molecular epidemiology of CanineCV and formulate hypotheses about virus ecology and evolution. Overall, 72 out of 159 (45.3%) animals tested positive. Virus prevalence was similar between males and females, adults and juveniles, and across the investigated years and locations. CanineCV infection was not associated with a poor body condition. While the percentage of co-infections with canine parvoviruses, investigated in a previous study, was high (63/72, 87.5%), the rate of parvovirus infection in CanineCV-negative animals was significantly lower (42/87, 48.3%, {chi}2 = 27.03, p < 0.001), and CanineCV infection was associated with a 7.5- and 2.4-fold increase in the risk of acquiring canine parvovirus 2 or canine bufavirus infections, respectively (odds ratios: 3.5-16.9 and 1.3-5.8). Although common risk factors cannot be ruled out, this suggests that CanineCV may facilitate parvoviral super-infections. Sequencing revealed high CanineCV genetic diversity, further exacerbated by recombination. Of the 69 sequenced strains, 87.5% were fox circoviruses, five were related to a fox circovirus-like recombinant strain, and one belonged to a distant lineage. In the phylogenetic analysis, the virus sequences were distributed according to sampling locations, with some viruses being geographically restricted. Different clades of viruses were identified in the same areas and over multiple years (2007-2019), indicating the co-existence of multiple endemic lineages in the investigated area. Phylogenetic analysis of all available complete fox circovirus genomes (32 from foxes and 15 from wolves from North America and Europe) demonstrated four lineages, each including sequences from this study. Within each lineage, strains segregated geographically and not by host. This implies that, although multiple lineages co-exist, viruses do not frequently move between locations. Finally, viruses from Europe and North America were mixed, indicating that the origin of the four lineages might predate the segregation of European and American wolf and fox populations. Given the high prevalence and diversity of fox circoviruses in wolves, these animals should be considered reservoir hosts for these viruses. Although we cannot exclude a lower susceptibility of dogs, the lack of fox circovirus in dogs could be due to environmental circumstances that prevented its spread to dogs. Given the high diversity and wild host specificity, we presume a long-lasting association between fox circovirus and canine hosts and hypothesize a higher likelihood of transmission from dogs to wild animals than vice versa. Further studies should investigate other sympatric wild species and additional locations to explore the possible existence of additional maintenance hosts and the reasons behind the marked difference in cross-species transmission dynamics among CanineCV lineages.

microbiology↗

bioRxiv2023

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

microbiology↗

bioRxiv2023

Modulation of locus coeruleus neurons and strong release of noradrenaline during acute hippocampal seizures in rats

The locus coeruleus (LC), a brainstem nucleus, is the sole source of noradrenaline in the neocortex, hippocampus and cerebellum. Noradrenaline is a powerful neuromodulator involved in the regulation of excitability and plasticity of large-scale brain networks. In this study, we assessed the activity of locus coeruleus neurons and changes in noradrenergic transmission during acute hippocampal seizures evoked with perforant path stimulation. LC neurons were recorded in anesthetized rats using a multichannel electrophysiology probe and were identified based on electrophysiological characteristics or optogenetic tagging. The majority of LC neurons (55%) were inhibited during seizures, while only a subset of LC neurons (28%) was excited during seizures. Topographic analysis of multi-unit activity showed anatomical separation of neurons that were excited and inhibited during seizures. Changes in hippocampal noradrenaline transmission during seizures were assessed using a fluorescent biosensor for noradrenaline, GRABNE2m, in combination with fiber photometry in both anesthetized and awake rats. Our results indicate that acute electrically evoked hippocampal seizures are associated with strong changes in LC unit activity and strong and consistent time-locked release of noradrenaline. Understanding the role of mass release of noradrenaline during hippocampal seizures is likely to be important to understand seizure pathophysiology.

neuroscience↗

bioRxiv2020

Co-circulation of multiple influenza A variants in swine harboring genes from seasonal human and swine influenza viruses

Since the influenza pandemic in 2009, there has been an increased focus on swine influenza A virus (swIAV) surveillance. This paper describes the results of the surveillance of swIAV in Danish swine from 2011 to 2018. In total, 3800 submissions were received with a steady increase in swIAV positive submissions, reaching 56% in 2018. Ten different swIAV subtypes were detected. Full genome sequences were obtained from 129 swIAV positive samples. Altogether, 17 different circulating genotypes were identified including novel reassortants and subtypes harboring human seasonal IAV gene segments. The phylogenetic analysis revealed substantial genetic drift and also evidence of positive selection occurring mainly in antigenic sites of the hemagglutinin protein and confirmed the presence of a swine divergent cluster among the H1pdm09Nx viruses. The results provide essential data for the control of swIAV in pigs and for early detection of novel swIAV strains with zoonotic potential.

microbiology↗