Browsing by Author "Lemon, Ken"

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    Assessment of Rapid MinION Nanopore DNA Virus Meta-Genomics Using Calves Experimentally Infected with Bovine Herpes Virus-1
    (MDPI, 2022-08-24) Esnault, Gaelle; Earley, Bernadette; Cormican, Paul; Waters, Sinead M.; Lemon, Ken; Cosby, S. Louise; Lagan, Paula; Barry, Thomas; Reddington, Kate; McCabe, Matthew S.
    Bovine respiratory disease (BRD), which is the leading cause of morbidity and mortality in cattle, is caused by numerous known and unknown viruses and is responsible for the widespread use of broad-spectrum antibiotics despite the use of polymicrobial BRD vaccines. Viral metagenomics sequencing on the portable, inexpensive Oxford Nanopore Technologies MinION sequencer and sequence analysis with its associated user-friendly point-and-click Epi2ME cloud-based pathogen identification software has the potential for point-of-care/same-day/sample-to-result metagenomic sequence diagnostics of known and unknown BRD pathogens to inform a rapid response and vaccine design. We assessed this potential using in vitro viral cell cultures and nasal swabs taken from calves that were experimentally challenged with a single known BRD-associated DNA virus, namely, bovine herpes virus 1. Extensive optimisation of the standard Oxford Nanopore library preparation protocols, particularly a reduction in the PCR bias of library amplification, was required before BoHV-1 could be identified as the main virus in the in vitro cell cultures and nasal swab samples within approximately 7 h from sample to result. In addition, we observed incorrect assignment of the bovine sequence to bacterial and viral taxa due to the presence of poor-quality bacterial and viral genome assemblies in the RefSeq database used by the EpiME Fastq WIMP pathogen identification software.
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    ATAC-Seq identifies regions of open chromatin in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus
    (BMC (Springer nature), 2021-01-06) Johnston, Dayle; JaeWoo, Kim; Taylor, Jeremy F.; Earley, Bernadette; McCabe, Matthew S.; Lemon, Ken; Duffy, Catherine; McMenamy, Michael; Cosby, S. Louise; Waters, Sinéad M.
    Background: Bovine Respiratory Syncytial Virus (BRSV) is a cause of Bovine Respiratory Disease (BRD). DNA-based biomarkers contributing to BRD resistance are potentially present in non-protein-coding regulatory regions of the genome, which can be determined using ATAC-Seq. The objectives of this study were to: (i) identify regions of open chromatin in DNA extracted from bronchial lymph nodes (BLN) of healthy dairy calves experimentally challenged with BRSV and compare them with those from non-challenged healthy control calves, (ii) elucidate the chromatin regions that were differentially or uniquely open in the BRSV challenged relative to control calves, and (iii) compare the genes found in regions proximal to the differentially open regions to the genes previously found to be differentially expressed in the BLN in response to BRSV and to previously identified BRD susceptibility loci. This was achieved by challenging clinically healthy Holstein-Friesian calves (mean age 143 ± 14 days) with either BRSV inoculum (n = 12) or with sterile phosphate buffered saline (PBS) (n = 6) and preparing and sequencing ATACSeq libraries from fresh BLN tissues. Results: Using Diffbind, 9,144 and 5,096 differentially accessible regions (P < 0.05, FDR < 0.05) were identified between BRSV challenged and control calves employing DeSeq2 and EdgeR, respectively. Additionally, 8,791 chromatin regions were found to be uniquely open in BRSV challenged calves. Seventy-six and 150 of the genes that were previously found to be differentially expressed using RNA-Seq, were located within 2 kb downstream of the differentially accessible regions, and of the regions uniquely open in BRSV challenged calves, respectively. Pathway analyses within ClusterProfiler indicated that these genes were involved in immune responses to infection and participated in the Th1 and Th2 pathways, pathogen recognition and the anti-viral response. There were 237 differentially accessible regions positioned within 40 previously identified BRD susceptibility loci. Conclusions: The identified open chromatin regions are likely to be involved in the regulatory response of gene transcription induced by infection with BRSV. Consequently, they may contain variants which impact resistance to BRD that could be used in breeding programmes to select healthier, more robust cattle.
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    Co-infection status of novel parvovirus’s (PPV2 to 4) with porcine circovirus 2 in porcine respiratory disease complex and porcine circovirus-associated disease from 1997 to 2012
    (Wiley, 2020-10-18) Lagan Tregaskis, Paula; Staines, Anthony; Gordon, Alan; Sheridan, Pauline; McMenamy, Michael; Duffy, Catherine; Collins, P. J.; Mooney, Mark H.; Lemon, Ken
    As global pig health diseases, porcine respiratory disease complex (PRDC) and porcine circovirus-associated disease (PCVAD) generate substantial economic losses despite pigs been vaccinated against the primary causative virus, highlighting the importance of understanding virome interactions and specifically co-factor infections. Established primary endemic pathogens for PRDC include porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSv) and swine influenza virus (SIV), and PCV2 aetiology in interaction with other co-infecting viruses can result in PCVAD. Porcine parvovirus (PPV) 1 is a well-characterized virus with an available vaccine preventing reproductive failure in sows. However, whilst novel PPV 2 to 7 viruses have been identified since 2001, their viral pathogenic potential in clinical and subclinical disease remains to be determined. Therefore, this study has sought to develop a better understanding of their potential role as associated co-infections in PRDC and PCVAD by examining archival samples for the presence of PCV2 and the novel parvoviruses PPV2-4 from clinically diseased pigs across production age stages. Epidemiologically, the novel PPV2 was found to be the most prevalent within the fattener age group with PPV2-4 statistically associated with pig respiratory disease and enteric ulcers. Additionally, statistical modelling by latent class analysis (LCA) on veterinary pathology scored pigs found a clustering co-factor association between PPV2 and PCV2, suggesting the novel PPV may be involved in PRDC and PCVAD. Phylogenetic analysis of novel PPVs revealed the PPV2 capsid evolution to be diverged from the original strains with a low nucleotide homology of 88%–96% between two distinct clades. These findings determine that novel PPV 2–4 viruses are statistically associated as co-infectors in a diseased pig population, and significantly detected PPV2 clustering co-infection frequency with PCV2 in PRDC and PCVAD diseased pigs through LCA analysis.
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    Elucidation of the Host Bronchial Lymph Node miRNA Transcriptome Response to Bovine Respiratory Syncytial Virus
    (Frontiers Media, 2021-04-22) Johnston, Dayle; Earley, Bernadette; McCabe, Matthew S.; Kim, Jaewoo; Taylor, Jeremy F.; Lemon, Ken; McMenamy, Michael; Duffy, Catherine; Cosby, S. Louise; Waters, Sinéad M.
    Bovine respiratory disease (BRD) causes substantial morbidity and mortality, affecting cattle of all ages. One of the main causes of BRD is an initial inflammatory response to bovine respiratory syncytial virus (BRSV). MicroRNAs are novel and emerging noncoding small RNAs that regulate many biological processes and are implicated in various inflammatory diseases. The objective of the present study was to elucidate the changes in the bovine bronchial lymph node miRNA transcriptome in response to BRSV following an experimental viral challenge. Holstein-Friesian calves were either administered a challenge dose of BRSV (103:5 TCID50/ml 15 ml) (n = 12) or were mock inoculated with sterile phosphate buffered saline (n = 6). Daily scoring of clinical signs was performed and calves were euthanized at day 7 post-challenge. Bronchial lymph nodes were collected for subsequent RNA extraction and sequencing (75 bp). Read counts for known miRNAs were generated using the miRDeep2 package using the UMD3.1 reference genome and the bovine mature miRNA sequences from the miRBase database (release 22). EdgeR was used for differential expression analysis and Targetscan was used to identify target genes for the differentially expressed (DE) miRNAs. Target genes were examined for enriched pathways and gene ontologies using Ingenuity Pathway Analysis (Qiagen). Multi-dimensional scaling (MDS) based on miRNA gene expression changes, revealed a clearly defined separation between the BRSV challenged and control calves, although the clinical manifestation of disease was only mild. One hundred and nineteen DE miRNAs (P < 0.05, FDR < 0.1, fold change > 1.5) were detected between the BRSV challenged and control calves. The DE miRNAs were predicted to target 465 genes which were previously found to be DE in bronchial lymph node tissue, between these BRSV challenged and control calves. Of the DE predicted target genes, 455 had fold changes that were inverse to the corresponding DE miRNAs. There were eight enriched pathways among the DE predicted target genes with inverse fold changes to their corresponding DE miRNA including: granulocyte and agranulocyte adhesion and diapedesis, interferon signalling and role of pathogen recognition receptors in recognition of bacteria and viruses. Functions predicted to be increased included: T cell response, apoptosis of leukocytes, immune response of cells and stimulation of cells. Pathogen recognition and proliferation of cytotoxic T cells are vital for the recognition of the virus and its subsequent elimination.
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    Genome analyses of species A rotavirus isolated from various mammalian hosts in Northern Ireland during 2013−2016
    (Oxford Univerity Press, 2023-07-04) Lagan, Paula; Mooney, Mark H.; Lemon, Ken
    Rotavirus group A (RVA) is the most important cause of acute diarrhoea and severe dehydration in young mammals. Infection in livestock is associated with significant mortality and economic losses and, together with wildlife reservoirs, acts as a potential source of zoonotic transmission. Therefore, molecular surveillance of circulating RVA strains in animal species is necessary to assess the risks posed to humans and their livestock. An RVA molecular epidemiological surveillance study on clinically diseased livestock species revealed high prevalence in cattle and pigs (31 per cent and 18 per cent, respectively) with significant phylogenetic diversity including a novel and divergent ovine artiodactyl DS-1-like constellation G10-P[15]-I2-R2-C2-M2-A11-N2-T6-E2-H3. An RVA gene reassortment occurred in an RVA asymptomatic pig and identified as a G5-P[13] strain, and a non-structural protein (NSP)2 gene had intergenomically reassorted with a human RVA strain (reverse zoonosis) and possessed a novel NSP4 enterotoxin E9 which may relate to the asymptomatic RVA infection. Analysis of a novel sheep G10-P[15] strain viral protein 4 gene imparts a putative homologous intergenic and interspecies recombination event, subsequently creating the new P[15] divergent lineage. While surveillance across a wider range of wildlife and exotic species identified generally negative or low prevalence, a novel RVA interspecies transmission in a non-indigenous pudu deer (zoo origin) with the constellation of G6-P[11]12-R2-C2-M2-A3-N2-T6-E2-H3 was detected at a viral load of 11.1 log10 copies/gram. The detection of novel emerging strains, interspecies reassortment, interspecies infection, and recombination of RVA circulating in animal livestock and wildlife reservoirs is of paramount importance to the RVA epidemiology and evolution for the One Health approach and post-human vaccine introduction era where highly virulent animal RVA genotypes have the potential to be zoonotically transmitted.
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    Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020–2022
    (American Society for Microbiology, 2023-06-26) Byrne, Alexander M. P.; James, Joe; Mollett, Benjamin C.; Meyer, Stephanie M.; Lewis, Thomas; Czepiel, Magdalena; Seekings, Amanda H.; Mahmood, Sahar; Thomas, Saumya S.; Ross, Craig S.; Byrne, Dominic J. F.; McMenamy, Michael; Bailie, Valerie; Lemon, Ken; Hansen, Rowena D. E.; Falchieri, Marco; Lewis, Nicola S.; Reid, Scott M.; Brown, Ian H.; Banyard, Ashley C.
    Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020–2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes. Following a small number of detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021–2022 saw another European H5 HPAIV epizootic that dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk, and whether incidents of lateral spread can be defined over independent incursions of infections from wild birds. This provides key supporting data for mitigation activities.
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    Needle-free delivery of measles virus vaccine to the lower respiratory tract of non-human primates elicits optimal immunity and protection
    (Nature Research, 2017-08-01) de Swart, Rik L.; de Vries, Rory D.; Rennick, Linda J.; van Amerongen, Geert; McQuaid, Stephen; Verburgh, R. Joyce; Yuksel, Selma; de Jong, Alwin; Lemon, Ken; Nguyen, D. Tien; Ludlow, Martin; Osterhaus, Albert D.M.E.; Duprex, W. Paul
    Needle-free measles virus vaccination by aerosol inhalation has many potential benefits. The current standard route of vaccination is subcutaneous injection, whereas measles virus is an airborne pathogen. However, the target cells that support replication of liveattenuated measles virus vaccines in the respiratory tract are largely unknown. The aims of this study were to assess the in vivo tropism of live-attenuated measles virus and determine whether respiratory measles virus vaccination should target the upper or lower respiratory tract. Four groups of twelve cynomolgus macaques were immunized with 104 TCID50 of recombinant measles virus vaccine strain Edmonston-Zagreb expressing enhanced green fluorescent protein. The vaccine virus was grown in MRC-5 cells and formulated with identical stabilizers and excipients as used in the commercial MVEZ vaccine produced by the Serum Institute of India. Animals were immunized by hypodermic injection, intra-tracheal inoculation, intra-nasal instillation, or aerosol inhalation. In each group six animals were euthanized at early time points post-vaccination, whereas the other six were followed for 14 months to assess immunogenicity and protection from challenge infection with wild-type measles virus. At early time-points, enhanced green fluorescent protein-positive measles virus-infected cells were detected locally in the muscle, nasal tissues, lungs, and draining lymph nodes. Systemic vaccine virus replication and viremia were virtually absent. Infected macrophages, dendritic cells and tissueresident lymphocytes predominated. Exclusive delivery of vaccine virus to the lower respiratory tract resulted in highest immunogenicity and protection. This study sheds light on the tropism of a live-attenuated measles virus vaccine and identifies the alveolar spaces as the optimal site for respiratory delivery of measles virus vaccine.