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In January 2020, increased mortality was reported in a small broiler breeder flock in County Fermanagh, Northern Ireland. Gross pathological findings included coelomitis, oophoritis, salpingitis, visceral gout, splenomegaly, and renomegaly. Clinical presentation included inappetence, pronounced diarrhoea, and increased egg deformation. These signs, in combination with increased mortality, triggered a notifiable avian disease investigation. High pathogenicity avian influenza virus (HPAIV) was not suspected, as mortality levels and clinical signs were not consistent with HPAIV. Laboratory investigation demonstrated the causative agent to be a low-pathogenicity avian influenza virus (LPAIV), subtype H6N1, resulting in an outbreak that affected 15 premises in Northern Ireland. The H6N1 virus was also associated with infection on 13 premises in the Republic of Ireland and six in Great Britain. The close genetic relationship between the viruses in Ireland and Northern Ireland suggested a direct causal link whereas those in Great Britain were associated with exposure to a common ancestral virus. Overall, this rapidly spreading outbreak required the culling of over 2 million birds across the United Kingdom and the Republic of Ireland to stamp out the incursion. This report demonstrates the importance of investigating LPAIV outbreaks promptly, given their substantial economic impacts.
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Galinhas , Surtos de Doenças , Fazendas , Vírus da Influenza A , Influenza Aviária , Doenças das Aves Domésticas , Aves Domésticas , Animais , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Surtos de Doenças/veterinária , Reino Unido/epidemiologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Irlanda/epidemiologia , Galinhas/virologia , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/genética , Vírus da Influenza A/classificação , Aves Domésticas/virologia , FilogeniaRESUMO
Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.
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Swine influenza A virus (swIAV) is one of the main viral pathogens responsible for respiratory disease in farmed pigs. While outbreaks are often epidemic in nature, increasing reports suggest that continuous, endemic infection of herds is now common. The move towards larger herd sizes and increased intensification in the commercial pig industry may promote endemic infection; however, the impact that intensification has on swIAV infection dynamics and evolution is unclear. We carried out a longitudinal surveillance study for over 18 months on two enzootically infected, intensive, indoor, and multi-site pig production flows. Frequent sampling of all production stages using individual and group sampling methods was performed, followed by virological and immunological testing and whole-genome sequencing. We identified weaned pigs between 4 and 12-weeks old as the main reservoir of swIAV in the production flows, with continuous, year-round infection. Despite the continuous nature of viral circulation, infection levels were not uniform, with increasing exposure at the herd level associated with reduced viral prevalence followed by subsequent rebound infection. A single virus subtype was maintained on each farm for the entire duration of the study. Viral evolution was characterised by long periods of stasis punctuated by periods of rapid change coinciding with increasing exposure within the herd. An accumulation of mutations in the surface glycoproteins consistent with antigenic drift was observed, in addition to amino acid substitutions in the internal gene products as well as reassortment exchange of internal gene segments from newly introduced strains. These data demonstrate that long-term, continuous infection of herds with a single subtype is possible and document the evolutionary mechanisms utilised to achieve this.
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We report cases of mammalian infection with highly pathogenic avian influenza (HPAI) virus A(H5N1) clade 2.3.4.4b in Northern Ireland. Two common gulls (Larus canus) and two red fox kits (Vulpes vulpes), were found dead in close vicinity. Comparison of viral whole genome sequences obtained from the animals identified a novel mammalian adaptation, PB2-M535I. Analysis of genetic sequences from other recent mammalian infections shows that this mutation has arisen on at least five occasions in three European countries since April 2023.
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Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Aviária , Animais , Raposas , Virus da Influenza A Subtipo H5N1/genética , Irlanda do Norte/epidemiologia , Vírus da Influenza A/genética , FilogeniaRESUMO
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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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. IMPORTANCE High-pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors, having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the United Kingdom has experienced two successive outbreaks of H5 HPAIV. While H5N8 HPAIV was predominant during the 2020-2021 outbreak, other H5 subtypes were also detected. The following year, there was a shift in the subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through the thorough utilization of whole-genome sequencing, it was possible to track and characterize the genetic evolution of these H5 HPAIVs in United Kingdom poultry and wild birds. This enabled us to assess the risk posed by these viruses at the poultry-wild bird and the avian-human interfaces and to investigate the potential lateral spread between infected premises, a key factor in understanding the threat to the commercial sector.
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Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Aviária , Animais , Humanos , Influenza Aviária/epidemiologia , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A/genética , Animais Selvagens , Aves , Reino Unido/epidemiologia , Aves Domésticas , Variação Genética , FilogeniaRESUMO
Bovine herpesvirus 1 (BoHV-1), is associated with several clinical syndromes in cattle, among which bovine respiratory disease (BRD) is of particular significance. Despite the importance of the disease, there is a lack of information on the molecular response to infection via experimental challenge with BoHV-1. The objective of this study was to investigate the whole-blood transcriptome of dairy calves experimentally challenged with BoHV-1. A secondary objective was to compare the gene expression results between two separate BRD pathogens using data from a similar challenge study with BRSV. Holstein-Friesian calves (mean age (SD) = 149.2 (23.8) days; mean weight (SD) = 174.6 (21.3) kg) were either administered BoHV-1 inoculate (1 × 107/mL × 8.5 mL) (n = 12) or were mock challenged with sterile phosphate buffered saline (n = 6). Clinical signs were recorded daily from day (d) -1 to d 6 (post-challenge), and whole blood was collected in Tempus RNA tubes on d six post-challenge for RNA-sequencing. There were 488 differentially expressed (DE) genes (p < 0.05, False Discovery rate (FDR) < 0.10, fold change ≥2) between the two treatments. Enriched KEGG pathways (p < 0.05, FDR <0.05); included Influenza A, Cytokine-cytokine receptor interaction and NOD-like receptor signalling. Significant gene ontology terms (p < 0.05, FDR <0.05) included defence response to virus and inflammatory response. Genes that are highly DE in key pathways are potential therapeutic targets for the treatment of BoHV-1 infection. A comparison to data from a similar study with BRSV identified both similarities and differences in the immune response to differing BRD pathogens.
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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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Doenças dos Bovinos , Herpesvirus Bovino 1 , Nanoporos , Vírus , Animais , Antibacterianos , Bovinos , Genômica , Herpesvirus Bovino 1/genética , Metagenômica/métodos , Vírus/genéticaRESUMO
White chick hatchery disease is an emerging disease of broiler chicks with which the virus, chicken astrovirus, has been associated. Adult birds typically show no obvious clinical signs of infection, although some broiler breeder flocks have experienced slight egg drops. Substantial decreases in hatching are experienced over a two-week period, with an increase in mid-to-late embryo deaths, chicks too weak to hatch and pale, runted chicks with high mortality. Chicken astrovirus is an enteric virus, and strains are typically transmitted horizontally within flocks via the faecal-oral route; however, dead-in-shell embryos and weak, pale hatchlings indicate vertical transmission of the strains associated with white chick hatchery disease. Hatch levels are typically restored after two weeks when seroconversion of the hens to chicken astrovirus has occurred. Currently, there are no commercial vaccines available for the virus; therefore, the only means of protection is by good levels of biosecurity. This review aims to outline the current understanding regarding white chick hatchery disease in broiler chick flocks suffering from severe early mortality and increased embryo death in countries worldwide.
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Infecções por Astroviridae/veterinária , Avastrovirus , Galinhas , Doenças Transmissíveis Emergentes/veterinária , Doenças das Aves Domésticas , Criação de Animais Domésticos , Animais , Infecções por Astroviridae/fisiopatologia , Infecções por Astroviridae/prevenção & controle , Infecções por Astroviridae/virologia , Avastrovirus/isolamento & purificação , Doenças Transmissíveis Emergentes/fisiopatologia , Doenças Transmissíveis Emergentes/prevenção & controle , Doenças Transmissíveis Emergentes/virologia , Progressão da Doença , Doenças das Aves Domésticas/fisiopatologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologiaRESUMO
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 non-coding 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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Bovine Respiratory Syncytial Virus (BRSV) is a primary viral cause of Bovine Respiratory Disease (BRD) in young calves, which is responsible for substantial morbidity and mortality. Infection with BRSV induces global gene expression changes in respiratory tissues. If these changes are observed in tissues which are more accessible in live animals, such as whole blood, they may be used as biomarkers for diagnosis of the disease. Therefore, the objective of the current study was to elucidate the whole blood transcriptomic response of dairy calves to an experimental challenge with BRSV. Calves (Holstein-Friesian) were either administered BRSV inoculate (103.5 TCID50/ml × 15 ml) (n = 12) or sterile phosphate buffered saline (n = 6). Clinical signs were scored daily and whole blood was collected in Tempus RNA tubes immediately prior to euthanasia, at day 7 post-challenge. RNA was extracted from blood and sequenced (150 bp paired-end). The sequence reads were aligned to the bovine reference genome (UMD3.1) and EdgeR was subsequently employed for differential gene expression analysis. Multidimensional scaling showed that samples from BRSV challenged and control calves segregated based on whole blood gene expression changes, despite the BRSV challenged calves only displaying mild clinical symptoms of the disease. There were 281 differentially expressed (DE) genes (p < 0.05, FDR < 0.1, fold change > 2) between the BRSV challenged and control calves. The top enriched KEGG pathways and gene ontology terms were associated with viral infection and included "Influenza A", "defense response to virus", "regulation of viral life cycle" and "innate immune response". Highly DE genes involved in these pathways may be beneficial for the diagnosis of subclinical BRD from blood samples.
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Biomarcadores/sangue , Doenças dos Bovinos/diagnóstico , Regulação da Expressão Gênica , RNA Mensageiro/genética , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino/genética , Animais , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/genética , Doenças dos Bovinos/virologia , RNA Mensageiro/sangue , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , TranscriptomaRESUMO
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 ATAC-Seq 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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Doenças dos Bovinos , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Bovino , Animais , Bovinos , Doenças dos Bovinos/genética , Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Linfonodos , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino/genéticaRESUMO
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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Infecções por Circoviridae , Coinfecção , Síndrome Respiratória e Reprodutiva Suína , Doenças dos Suínos , Animais , Infecções por Circoviridae/complicações , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/veterinária , Circovirus , Coinfecção/veterinária , Feminino , Parvovirus Suíno/genética , Filogenia , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.
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Coinfecção/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Sistema Respiratório/virologia , Infecções Respiratórias/virologia , Animais , Linhagem Celular , Feminino , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Pulmão/virologia , Mucosa Respiratória/virologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Genética Reversa , Sigmodontinae , Proteína Vermelha FluorescenteRESUMO
Bovine Respiratory Disease (BRD) is the leading cause of mortality in calves. The objective of this study was to examine the response of the host's bronchial lymph node transcriptome to Bovine Respiratory Syncytial Virus (BRSV) in a controlled viral challenge. Holstein-Friesian calves were either inoculated with virus (103.5 TCID50/ml × 15 ml) (n = 12) or mock challenged with phosphate buffered saline (n = 6). Clinical signs were scored daily and blood was collected for haematology counts, until euthanasia at day 7 post-challenge. RNA was extracted and sequenced (75 bp paired-end) from bronchial lymph nodes. Sequence reads were aligned to the UMD3.1 bovine reference genome and differential gene expression analysis was performed using EdgeR. There was a clear separation between BRSV challenged and control calves based on gene expression changes, despite an observed mild clinical manifestation of the disease. Therefore, measuring host gene expression levels may be beneficial for the diagnosis of subclinical BRD. There were 934 differentially expressed genes (DEG) (p < 0.05, FDR <0.1, fold change >2) between the BRSV challenged and control calves. Over-represented gene ontology terms, pathways and molecular functions, among the DEG, were associated with immune responses. The top enriched pathways included interferon signaling, granzyme B signaling and pathogen pattern recognition receptors, which are responsible for the cytotoxic responses necessary to eliminate the virus.
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Doenças dos Bovinos/genética , Bovinos/virologia , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino/fisiologia , Transcriptoma , Animais , Brônquios/metabolismo , Brônquios/virologia , Bovinos/genética , Doenças dos Bovinos/virologia , Interações Hospedeiro-Patógeno , Linfonodos/metabolismo , Linfonodos/virologia , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologiaRESUMO
Influenza D is a newly described virus of cattle, pigs and small ruminants first detected in North America during 2011. Cattle have been shown to be the main viral reservoir and mounting evidence indicates that infection with influenza D may contribute to the development of bovine respiratory disease. The virus has been detected across the United States, Europe and Asia. To date, influenza D has not been reported in the UK. During the winter and spring of 2017/2018, we performed molecular testing of cattle submitted for post-mortem examination where respiratory disease signs were present. We detected influenza D virus in 8.7% of cases, often as the sole viral agent and always in conjunction with bacterial co-infection with one or more agents. Viral RNA was present in both the upper and lower respiratory tract and pathological changes in lung tissues were observed alongside signs of concurrent bacterial infections. Sequencing of one UK isolate revealed that it is similar to viruses from the Republic of Ireland and Italy.
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Doenças dos Bovinos/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Infecções Respiratórias/veterinária , Thogotovirus/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/virologia , Feminino , Masculino , Irlanda do Norte/epidemiologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Prevalência , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologiaRESUMO
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 live-attenuated 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 tissue-resident 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.
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UNLABELLED: Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies. IMPORTANCE: Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.
Assuntos
Proteínas de Fluorescência Verde/análise , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Genótipo , Proteínas de Fluorescência Verde/genética , Humanos , Lactente , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Sistema Respiratório/virologia , Sigmodontinae , Coloração e Rotulagem , VirulênciaRESUMO
UNLABELLED: Although live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MV(EZ). Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMV(EZ)EGFP(1), rMV(EZ)EGFP(3), and rMV(EZ)EGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMV(EZ)EGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMV(EZ)EGFP(1) and rMV(EZ)EGFP(6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMV(EZ)EGFP(3) was functionally equivalent to the commercial MV(EZ)-containing vaccine. Intramuscular vaccination of macaques with rMV(EZ)EGFP(3) resulted in the identification of EGFP(+) cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV. IMPORTANCE: Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicates in vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Macrófagos/imunologia , Macrófagos/virologia , Vacina contra Sarampo/imunologia , Vírus do Sarampo/imunologia , Músculos/imunologia , Animais , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Macaca fascicularis , Masculino , Vacina contra Sarampo/administração & dosagem , Vacina contra Sarampo/genética , Coloração e Rotulagem , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
Paramyxoviruses, including measles virus (MV), human metapneumovirus (HMPV), human respiratory syncytial virus (HRSV) and canine distemper virus (CDV), are transmitted via the respiratory route. Despite their close phylogenetic relationship, the pathogenesis of these viruses is very different. To study viral tropism and replication ex vivo, a protocol for the inflation of lungs with low-melting-point agarose mixed with culture medium was established. Lung slices were prepared and remained viable in culture at 37°C for at least 7 days. Lung slices obtained from different animal species were infected with recombinant paramyxoviruses expressing enhanced green fluorescent protein (EGFP). Progression of infection was monitored in real time by detection of EGFP fluorescence. Flow cytometric analysis of cells emigrating into the culture medium proved to be a valuable tool for qualitative and quantitative assessment of infection over time. MV replicated optimally in lung slices of macaques, HMPV and HRSV in lung slices of cotton rats, and CDV in lung slices obtained from dogs and ferrets. In conclusion, these ex vivo lung slice cultures are discriminatory models for the study of respiratory virus infections and can be used to inform the design of future in vivo experiments, thereby reducing numbers of animals required.