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In the fall of 2019, a fatal encephalitis outbreak led to the deaths of >200 European hedgehogs (Erinaceus europaeus) in England. We used next-generation sequencing to identify a novel arterivirus with a genome coding sequence of only 43% similarity to existing GenBank arterivirus sequences.
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Arterivirus , Encefalitis , Animales , Brotes de Enfermedades , Inglaterra/epidemiología , ErizosRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious threat to the pig industry worldwide. Increasing evidence suggests that microevolution within a quasispecies population can give rise to high sequence heterogeneity in PRRSV; potentially impacting the pathogenicity of the virus. Here, we report on micro-evolutionary events taking place within the viral quasispecies population in lung and lymph node 3 days post infection (dpi) following experimental in vivo infection with the prototypical Lelystad PRRSV (LV). Sequence analysis revealed 16 high frequency single nucleotide variants (SNV) or differences from the reference LV genome which are assumed to be representative of the consensus inoculum genome. Additionally, 49 other low frequency SNVs were also found in the inoculum population. At 3 dpi, a total of 9 and 10 SNVs of varying frequencies could already be detected in the LV population infecting the lung and lymph nodes, respectively. Interestingly, of these, three and four novel SNVs emerged independently in the two respective tissues when compared to the inoculum. The remaining variants, though already present at lower frequencies in the inoculum, were positively selected and their frequency increased within the quasispecies population. Hence, we were able to determine directly from tissues infected with PRRSV the repertoire of genetic variants within the viral quasispecies population. Our data also suggest that microevolution of these variants is rapid and some may be tissue-specific.
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Evolución Molecular , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos/virología , Animales , Variación Genética , Genotipo , Pulmón/virología , Ganglios Linfáticos/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificaciónRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215-06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-α and IFN-α in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-γ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.
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Citocinas/genética , Ganglios Linfáticos/virología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Transcriptoma , Animales , Citocinas/metabolismo , Mediastino/virología , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/virología , ARN Viral/genética , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Porcinos , VirulenciaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS), an important viral disease of swine caused by PRRS virus (PRRSV) was first confirmed in Nepal in 2013. Since then, the virus has spread throughout the country and has now become endemic affecting the pig production nationally. However, molecular characterization of circulating strains has not been done in Nepal yet. In the present study, serum samples were collected from outbreak areas of different districts of Nepal and samples positive for PRRSV by ELISA were sent to Animal and Plant Health Agency (APHA), United Kingdom for sequence analysis. Out of 35 samples that were sent to APHA, only one sample was found positive by PCR and subjected to sequence analysis based on ORF5, ORF7 and Nsp2. The results from the phylogenetic analysis demonstrated that the PRRSV strain belongs to PRRSV-2 and lineage 8 strain. The sequences from the Nepalese PRRSV strain revealed a high degree of similarity with the strains isolated from India, China and Vietnam, with the closest genetic relatedness to the Indian isolates from 2020 and 2018. This is the first study on molecular characterization of PRRS virus circulating in Nepal. Further studies on strains circulating in Nepal are very essential to understand the virus diversity, its spread and evolution.
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BACKGROUND: Porcine reproductive and respiratory syndrome is a highly infectious disease of swine caused by PRRS virus (PRRSV). OBJECTIVES: To evaluate the prevalence of PRRSV antibodies in the four districts of hilly and terai regions of Nepal. Toassess the farm characteristics through a questionnaire interview of farmersregarding management practices and PRRS. METHODS: A cross-sectional study was conducted from July 2020 to June 2021 to determine the sero-prevalence of PRRSV in pigs. A total of 180 porcine serum samples were collected from 23 pig farms and tested for PRRSV antibodies by ELISA. Alongside, farm characteristics were also assessed through questionnaire to determine the level of biosecurity measures in the farm, knowledge of the disease and possible control mechanisms. RESULTS: Out of 180 samples, 37 were tested positive resulting the overall sero-prevalence of 20.5%. There was significant association between different districts (p < 0.05) and PRRS prevalence. Prevalence of PRRSV antibody was found higher in Kaski district (10.5%) followed by Sunsari (8.8%) district. Based on age groups, highest prevalence was found in age groups of above 18 months (9.4%), followed by 13-18 months age groups (7.7%). Regarding the knowledge level of the disease, 43% of the farmers responded that they have heard about the disease. Biosecurity practices in the farm was found very poor where only 40% of the farms had disinfectant at the entrance of the farm and 25% pig farmers were found using separate boots while dealing with pigs. CONCLUSIONS: The findings of this study reveal the presence of PRRSV antibodies in pigs of Nepal. In addition poor biosecurity measures, management practices and poor knowledge level about the disease among farmers highly affect in the control and prevention of disease thereby affecting the pig production and productivity. Therefore, government should develop and implement effective control measures and biosecurity programs.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Porcinos , Animales , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , Granjas , Prevalencia , Estudios Transversales , Nepal/epidemiología , Anticuerpos Antivirales , Enfermedades de los Porcinos/epidemiologíaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response.
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Vacunas contra Herpesvirus , Inmunogenicidad Vacunal , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Proteínas de la Matriz Viral , Proteínas no Estructurales Virales , Vacunas contra Herpesvirus/inmunología , Vacunas Atenuadas/inmunología , Linfocitos T/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vectores Genéticos , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Animales , Porcinos , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Little is known how the virus subverts the innate immune response to initiate its replication in alveolar macrophages. Large-scale transcriptional responses of macrophages with different levels of susceptibility to PRRSV infection were compared over 30 h of infection. This study demonstrates a rapid and intense host transcriptional remodelling during the early phase of the replication of the virus which correlates with transient repression of type-I interferon transcript as early as 8 h post-infection. These results support the suggestion from previous studies that host innate immune response inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-I interferon transcriptional response.
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Interacciones Huésped-Patógeno/genética , Interferón Tipo I/genética , Macrófagos Alveolares/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Transcripción Genética , Replicación Viral , Animales , Regulación de la Expresión Génica , Inmunidad Innata/genética , Macrófagos Alveolares/virología , Síndrome Respiratorio y de la Reproducción Porcina/genética , PorcinosRESUMEN
The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Línea Celular , Epítopos/genética , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Pruebas de Neutralización , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/terapia , Proteínas Proto-Oncogénicas c-bcl-6/genética , Porcinos , Proteína bcl-X/genéticaAsunto(s)
Calostro/virología , Infecciones por Deltaretrovirus/diagnóstico , Virus de la Leucemia Bovina , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Bovinos , Infecciones por Deltaretrovirus/inmunología , Infecciones por Deltaretrovirus/virología , Ensayo de Inmunoadsorción Enzimática , Femenino , Virus de la Leucemia Bovina/genética , Virus de la Leucemia Bovina/inmunología , Embarazo , Reino UnidoRESUMEN
Monocytes are considered refractory to porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) infection. However, monocytes are only short-lived in blood, being able to differentiate into macrophages and dendritic cells (DC). It was therefore merited to revisit PRRSV-1 interaction with monocytes, particularly those treated with cytokines influencing monocyte biology. Thus, several factors were screened, particularly those modulating monocyte differentiation and expression of putative PRRSV-1 receptors (CD169 and CD163). M-CSF, known to stimulate macrophage differentiation, did not increase their susceptibility to PRRSV-1. Nor did GM-CSF or IL-4, known drivers for monocyte-derived DC (MoDC) differentiation. In contrast, monocyte treatment with IL-10 or the corticosteroid, dexamethasone, known to be potent suppressors of monocyte differentiation, was correlated with increased susceptibility to PRRSV-1 infection. While this effect was strongly correlated to CD163 and CD169 expression, our data suggest that receptor expression is not the only factor driving successful infection of PPRSV-1 in monocytes.
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Dexametasona/farmacología , Interleucina-10/farmacología , Monocitos/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Células Cultivadas , Medios de Cultivo , Monocitos/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/clasificación , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Porcinos , Cultivo de Virus , Replicación ViralRESUMEN
Porcine epidemic diarrhoea virus, strain CV777, was initially characterized in 1978 as the causative agent of a disease first identified in the UK in 1971. This coronavirus has been widely distributed among laboratories and has been passaged both within pigs and in cell culture. To determine the variability between different stocks of the PEDV strain CV777, sequencing of the full-length genome (ca. 28kb) has been performed in 6 different laboratories, using different protocols. Not surprisingly, each of the different full genome sequences were distinct from each other and from the reference sequence (Accession number AF353511) but they are >99% identical. Unique and shared differences between sequences were identified. The coding region for the surface-exposed spike protein showed the highest proportion of variability including both point mutations and small deletions. The predicted expression of the ORF3 gene product was more dramatically affected in three different variants of this virus through either loss of the initiation codon or gain of a premature termination codon. The genome of one isolate had a substantially rearranged 5´-terminal sequence. This rearrangement was validated through the analysis of sub-genomic mRNAs from infected cells. It is clearly important to know the features of the specific sample of CV777 being used for experimental studies.
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Infecciones por Coronavirus/virología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virus de la Diarrea Epidémica Porcina/genética , Análisis de Secuencia de ARN/métodos , Enfermedades de los Porcinos/virología , Animales , Secuencia de Bases , Evolución Molecular , Genoma Viral , Sistemas de Lectura Abierta , Filogenia , Mutación Puntual , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , ARN Viral/química , ARN Viral/genética , Eliminación de Secuencia , PorcinosRESUMEN
Hepatitis E virus (HEV) and porcine reproductive and respiratory syndrome virus (PRRSV) and are both globally prevalent in the pig population. While HEV does not cause clinical disease in pigs, its zoonotic potential has raised concerns in the food safety sector. PRRS has become endemic in the United Kingdom (UK) since its introduction in 1991, and continues to cause considerable economic losses to the swine industry. A better understanding of the current prevalence and diversity of PRRSV and HEV in the UK, and their potential association, is needed to assess risks and target control measures appropriately. This study used plasma, tonsil, and cecal content samples previously collected from pigs in 14 abattoirs in England and Northern Ireland to study the prevalence of several pathogens including PRRSV and HEV. The diversity of PRRSV strains detected in these samples was analyzed by sequencing open reading frame 5 (ORF5), revealing no substantial difference in PRRSV strains from these clinically unaffected pigs relative to those from clinical cases of disease in the UK. Despite the potential immuno-modulatory effect of PRRSV infection, previously demonstrated to affect Salmonella and HEV shedding profiles, no significant association was found between positive PRRSV status and positive HEV status.
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Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/epidemiología , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificación , Sus scrofa/virología , Enfermedades de los Porcinos/epidemiología , Animales , Coinfección/epidemiología , Coinfección/veterinaria , Coinfección/virología , Virus de la Hepatitis E/genética , Sistemas de Lectura Abierta , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Prevalencia , Análisis de Secuencia de ADN , Porcinos , Enfermedades de los Porcinos/virología , Reino Unido/epidemiología , Esparcimiento de VirusRESUMEN
Porcine reproductive and respiratory syndrome viruses (PRRSV) present a wide phenotypic and genetic diversity. Experimental infections have demonstrated viral replication, including highly pathogenic strains (HP-PRRSV), in primary lymphoid organs such as the thymus. However, studies of the bone marrow are scarce but necessary to help elucidate the immunobiology of PRRSV strains of differing virulence. In this study, whereas viral RNA was detected within the bone marrow of animals experimentally infected with both low virulent Lelystad (LV) and 215-06 PRRSV-1 strains and with the highly virulent SU1-bel strain, PRRSV positive cells were only occasionally detected in one SU1-bel infected animal. PRRSV RNA levels were associated to circulating virus with the highest levels detected in LV-infected pigs. At 3 dpi, a decrease in the proportion of haematopoietic tissue and number of erythroid cells in all infected groups was associated with an increase in TUNEL or cleaved caspase 3 labelling and higher counts of myeloid cells compared to control. The expression of IL-1α and IL-6 was elevated at the beginning of the infection in all infected animals. The expression of TNF-α was increased at the end of the study in all infected groups with respect to control. Different PRRSV-1 strains induced, presummably by indirect mechanisms and independently of viral load and strain virulence, moderate and sustained hypoplasia of erythroid cells and myeloid cell hyperplasia at early stages of infection. These changes were paralleled by a peak in the local expression of IL-1α, IL-6 and TNF-α in all infected groups.
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Síndrome Respiratorio y de la Reproducción Porcina/patología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Animales , Médula Ósea/patología , Médula Ósea/virología , Citocinas/inmunología , Células Eritroides/patología , Células Eritroides/virología , Hiperplasia/patología , Hiperplasia/veterinaria , Hiperplasia/virología , Masculino , Células Mieloides/patología , Células Mieloides/virología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Porcinos , Carga Viral , Virulencia , Replicación ViralRESUMEN
PRRS control is hampered by the inadequacies of existing vaccines to combat the extreme diversity of circulating viruses. Since immune clearance of PRRSV infection may not be dependent on the development of neutralising antibodies and the identification of broadly-neutralising antibody epitopes have proven elusive, we hypothesised that conserved T cell antigens represent potential candidates for development of a novel PRRS vaccine. Previously we had identified the M and NSP5 proteins as well-conserved targets of polyfunctional CD8 and CD4 T cells. To assess their vaccine potential, peptides representing M and NSP5 were encapsulated in hydrophobically-modified chitosan particles adjuvanted by incorporation of a synthetic multi-TLR2/TLR7 agonist and coated with a model B cell PRRSV antigen. For comparison, empty particles and adjuvanted particles encapsulating inactivated PRRSV-1 were prepared. Vaccination with the particulate formulations induced antigen-specific antibody responses, which were most pronounced following booster immunisation. M and NSP5-specific CD4, but not CD8, T cell IFN-γ reactivity was measurable following the booster immunisation in a proportion of animals vaccinated with peptide-loaded particles. Upon challenge, CD4 and CD8 T cell reactivity was detected in all groups, with the greatest responses being detected in the peptide vaccinated group but with limited evidence of an enhanced control of viraemia. Analysis of the lungs during the resolution of infection showed significant M/NSP5 specific IFN-γ responses from CD8 rather than CD4 T cells. Vaccine primed CD8 T cell responses may therefore be required for protection and future work should focus on enhancing the cross-presentation of M/NSP5 to CD8 T cells.
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Antígenos Virales/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Linfocitos T/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos/inmunología , Quitosano/química , Péptidos/administración & dosificación , Péptidos/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos , Vacunas Virales/química , Vacunas Virales/normasRESUMEN
Porcine Epidemic Diarrhea Virus (PEDV) is a member of the genus Alphacoronavirus, in the family Coronaviridae, of the Nidovirales order and outbreaks of porcine epidemic diarrhoea (PED) were first recorded in England in the 1970s. Intriguingly the virus has since successfully made its way around the globe, while seemingly becoming extinct in parts of Europe before its recent return from Northern America. In this review we are re-evaluating the spread of PEDV, its biology and are looking at lessons learnt from both failure and success. While a new analysis of PEDV genomes demonstrates a wider heterogeneity of PEDV than previously anticipated with at least five rather than two genotypes, biological features of the virus and its replication also point towards credible control strategies to limit the impact of this re-emerging virus.
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Infecciones por Coronavirus/veterinaria , Virus de la Diarrea Epidémica Porcina/genética , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/virología , Animales , Enfermedades Transmisibles Emergentes/veterinaria , Brotes de Enfermedades , Europa (Continente)/epidemiología , Variación Genética , Genotipo , Salud Global , Filogenia , Virus de la Diarrea Epidémica Porcina/clasificación , Porcinos , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/transmisiónRESUMEN
Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection.
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Outbreaks of porcine epidemic diarrhea (PED) were first recorded in England in the 1970s and continued to be confirmed until 2002. Retrospective analysis of archived material from one of the last confirmed cases in England in the year 2000 demonstrates the previous existence of a very diverse PED virus strain. Following the outbreaks of PED in North America in 2013, there has been renewed interest in phylogenetic analysis of sequences from PEDV strains worldwide. There is a gap in the available sequence data between the mid 1980s and the mid 2000s. This work is an example of how this gap can be at least partially filled by the examination of archived material.
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Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two distinct viruses, type 1 (PRRSV-1) and type 2 (PRRSV-2). Atrophy of the thymus in PRRSV-2 infected piglets has been associated with a loss of thymocytes. The present study aimed to evaluate the impact of PRRSV-1 strains of differing virulence on the thymus of infected piglets by analysing the histomorphometry, the presence of apoptotic cells and cells producing cytokines. Thymic samples were taken from animals experimentally infected (with LV, SU1-bel, and 215-06 strains) or mock inoculated animals at 3, 7 and 35days post-infection (dpi) and processed for histopathological and immunohistochemical analyses. PRRSV antigen was detected in the thymus from 3dpi until the end of the study in all virus-infected animals with the highest numbers of infected cells detected in SU1-bel group. The histomorphometry analysis and counts of CD3(+) thymocytes in the thymic cortex displayed significant differences between strains at different time-points (p≤0.011), with SU1-bel group showing the most severe changes at 7dpi. Cell death displayed statistically significant increase in the cortex of all infected animals, with SU1-bel group showing the highest rate at 3 and 7dpi. The number of cells immunostained against IL-1α, TNF-α and IL-10 were predominantly detected in the medulla (p≤0.01). An increase in the number of TNF-α and IL-10 positive cells was observed in LV and SU-1bel groups. Our results demonstrate that different PRRSV-1 strains induced depletion of the thymic cortex due to apoptosis of thymocytes and that the most severe depletion was associated with the highly virulent SU1-bel strain.
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Atrofia/virología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Timocitos/virología , Timo/virología , Animales , Antígenos Virales/metabolismo , Atrofia/fisiopatología , Muerte Celular , Citocinas/metabolismo , Linfocitos/citología , Linfocitos/virología , Síndrome Respiratorio y de la Reproducción Porcina/fisiopatología , Especificidad de la Especie , Porcinos , Timocitos/citología , Timo/patologíaRESUMEN
The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-α and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development.
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We report here the complete genome of the pathogenic eastern European subtype 3 porcine reproductive and respiratory syndrome virus (PRRSV) strain SU1-Bel, sequenced directly from a pig lymph node. While sharing substantial sequence similarity with other subtype 3 strains, SU1-Bel is found to harbor unique indels and contain putative novel subgenomic RNAs.