High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

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.

Highly pathogenic avian influenza A(H5N1) virus infection in foxes with PB2-M535I identified as a novel mammalian adaptation, Northern Ireland, July 2023.

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.

Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020-2022.

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.

Whole blood transcriptome analysis in dairy calves experimentally challenged with bovine herpesvirus 1 (BoHV-1) and comparison to a bovine respiratory syncytial virus (BRSV) challenge.

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.

A Review of UK-Registered and Candidate Vaccines for Bovine Respiratory Disease.

Vaccination is widely regarded as a cornerstone in animal or herd health and infectious disease management. Nineteen vaccines against the major pathogens implicated in bovine respiratory disease are registered for use in the UK by the Veterinary Medicines Directorate (VMD). However, despite annual prophylactic vaccination, bovine respiratory disease is still conservatively estimated to cost the UK economy approximately £80 million per annum. This review examines the vaccine types available, discusses the surrounding literature and scientific rationale of the limitations and assesses the potential of novel vaccine technologies.

Elucidation of the Host Bronchial Lymph Node miRNA Transcriptome Response to Bovine Respiratory Syncytial Virus.

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.

Messenger RNA biomarkers of Bovine Respiratory Syncytial Virus infection in the whole blood of dairy calves.

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.

ATAC-Seq identifies regions of open chromatin in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus.

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.

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.

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.

Experimental challenge with bovine respiratory syncytial virus in dairy calves: bronchial lymph node transcriptome response.

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.

Detection of influenza D virus in bovine respiratory disease samples, UK.

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.

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV.

Molecular detection of kobuviruses in livestock in Northern Ireland and the Republic of Ireland.

Kobuviruses have been detected in a wide range of mammals including cats, dogs, pigs, cattle, goats, sheep and bats. Kobuviruses have been detected in symptomatic and asymptomatic animals; however, the clinical significance of infection in animals is still unclear. To date, there is no information regarding kobuvirus prevalence in livestock in Ireland. This study reports the first detection of kobuviruses in pigs, bovines and ovines using quantitative PCR. In this study, mesenteric lymph node was collected from cattle (n = 57), pigs (n = 53) and sheep (n = 50) from farms in Northern Ireland and the Republic of Ireland, from animals which had been submitted by private veterinary practitioners from 2009 to 2011 for routine post mortem and clinico-pathological examination. Kobuviruses were detected in 14 cows (24.5%), 5 pigs (9.4%) and 1 sheep (2%). Phylogenetic analysis of Irish kobuviruses from cattle and pigs revealed that the isolates clustered according to their host species. Interestingly, the sheep kobuvirus clustered with bovine kobuviruses detected in this study and other published kobuvirus strains. The data presented in this study contributes to the understanding of the epidemiology of these viruses in animals and to the genetic diversity that these viruses possess.

The development of an accelerated reverse-transcription loop mediated isothermal amplification for the serotype specific detection of bluetongue virus 8 in clinical samples.

In 2006 bluetongue virus serotype 8 (BTV 8) was identified for the first time in the Netherlands causing a major epidemic in sheep and cattle that quickly spread to neighbouring Belgium, Germany and beyond to France and the UK. This resulted in severe animal health and welfare problems as well as substantial economic losses to the agrifood industries of these countries. Given that the early diagnosis of BTV infection 'in-the-field' is extremely useful to its subsequent management and control, this study was established to design a novel, sensitive and rapid nucleic acid diagnostic test for the serotype-specific detection of BTV 8, which could be used without the use of advanced laboratory support and equipment. Primers for the detection of BTV 8 were based on genome segment 2 of the virus, the VP2 gene. The assay was assessed using a full panel of BTV reference strains and clinical samples. Positive amplification was observed using a fluorescent detection reagent. The sensitivity of the RT-LAMP assay was 102 copies of RNA. The assay did not amplify the closely related orbivirus EHDV. This novel RT-LAMP offers a sensitive, specific and rapid method of detecting BTV 8. The approach is inexpensive and easy to use and could potentially be used in a 'pen-side' setting 'in the field' or by smaller less well-equipped laboratories in developing countries.

Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and Torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain.

In this study we detail the detection and genetic analysis of a novel porcine boca-like virus (PBo-likeV) in archival sera and tissue samples from pigs from farms in Great Britain. We also investigate the distribution of porcine circovirus type 2 (PCV2) genotypes and Torque teno sus virus (TTSuV) genogroups 1 and 2 in combination with this novel PBo-likeV. PBo-likeV was detected in over 70% of all tissues investigated. Over 24% of all tissues recovered from PMWS-affected animals had all viruses present and 25% of tissues recovered from non-PMWS-affected pigs were positive for all 4 viruses.

Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus.

A novel real-time PCR strategy was applied to simultaneously detect and to discriminate low-pathogenic lentogenic and virulent meso/velogenic Newcastle disease virus (NDV). The pathotyping is achieved by a three-step semi-nested PCR. A pre-amplification of the cleavage site (CS) region of the F gene is followed by a two-level duplex real-time PCR directly targeting the CS, combining detection and pathotyping in a single tube. A wide range of NDV isolates spanning all genotypes were successfully detected and pathotyped. Clinical samples from outbreaks in Sweden in 2010 that were positive by the novel PCR method were also successfully pathotyped. The method is time-saving, reduces labour and costs and provides opportunities for rapid diagnosis at remote locations and in the field. Since the same strategy was also recently applied to avian influenza virus pathotyping, it shows promise of finding broad utility in diagnostics of infectious diseases caused by different RNA viruses in various hosts.

Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland.

We report the isolation in cell cultures of two novel bocavirus species in pigs from farms in Northern Ireland with clinical postweaning multisystemic wasting syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3) and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4 have been sequenced, respectively. PBoV3 and PBoV4 show nucleotide homology to other known bocaviruses in swine and other organisms. Open reading frame (ORF) analysis has shown that these viruses have a third small ORF, equivalent to the NP1 ORF that distinguishes the bocaviruses from other parvoviruses. A panel of porcine field sera was screened by indirect immunofluorescence against both viruses. Of the 369 samples analysed, 32 (8.7%) and 35 (9.5%) sera were seropositive for PBoV3 and PBoV4 respectively, thus providing serological evidence of the exposure of swine in the field to bocavirus-like viruses. To date, the clinico-pathological significance of these novel swine bocaviruses, as primary pathogens or as immunosuppresive triggers for other infectious agents, is undetermined.

Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay.

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 × 10¹ to 2 × 108 copies and amplification time was approximately 2 h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5' untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications.

Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe.

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2x10(1) to 2x10(10). The assay is rapid with an amplification time just over 2h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications.

Phylogenetic analysis of porcine circovirus type 2 (PCV2) pre- and post-epizootic postweaning multisystemic wasting syndrome (PMWS).

The porcine circovirus type 2 (PCV2) genome encodes three major open reading frames (ORFs) encoding the replicase proteins (ORF1), the viral capsid protein (ORF2), and a protein with suggested apoptotic activity (ORF3). Previous phylogenetic analyses of complete genome sequences of PCV2 from GenBank have demonstrated 95-100% intra-group nucleotide sequence identity. However, although these isolates were readily grouped into clusters and clades, there was no correlation between the occurrence of specific PCV2 genotypes and the geographic origin or health status of the pig. In the present study, a unique dataset from a field study spanning the years pre and post the recognition of postweaning multisystemic wasting syndrome (PMWS) in Sweden was utilized. Using this dataset it was possible to discriminate three Swedish genogroups (SG1-3) of PCV2, of which SG1 was recovered from a pig on a healthy farm ten years before the first diagnosis of PMWS in Sweden. The SG1 PCV2/ORF2 gene sequence has been demonstrated to exhibit a high genetic stability over time and has subsequently only been demonstrated in samples from pigs on nondiseased farms. In contrast, SG2 was almost exclusively found on farms that had only recently broken down with PMWS whereas the SG3 genogroup predominated in pigs from PMWS-affected farms. These results further support the results obtained from earlier in vitro and in vivo experimental models and suggest the association of specific PCV2 genogroups with diseased and nondiseased pigs in the field.