RESUMEN
Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.
Asunto(s)
Anticuerpos Neutralizantes/fisiología , Virus de la Parainfluenza 2 Humana/genética , Infecciones por Rubulavirus/virología , Adolescente , Distribución por Edad , Animales , Anticuerpos Antivirales/fisiología , Niño , Preescolar , Chlorocebus aethiops , Croacia/epidemiología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Femenino , Genotipo , Cobayas , Proteína HN/inmunología , Humanos , Lactante , Funciones de Verosimilitud , Persona de Mediana Edad , Virus de la Parainfluenza 2 Humana/clasificación , Virus de la Parainfluenza 2 Humana/inmunología , Filogenia , ARN Viral/química , ARN Viral/genética , Recurrencia , Infecciones por Rubulavirus/epidemiología , Infecciones por Rubulavirus/inmunología , Estaciones del Año , Alineación de Secuencia , Células VeroRESUMEN
The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.
Asunto(s)
Anticuerpos Antivirales/sangre , Inmunoglobulina G/sangre , Proteínas de la Nucleocápside/inmunología , Virus de la Parainfluenza 4 Humana/inmunología , Infecciones del Sistema Respiratorio/inmunología , Infecciones por Rubulavirus/inmunología , Saccharomyces cerevisiae/genética , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Niño , Preescolar , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Nucleocápside , Proteínas de la Nucleocápside/sangre , Proteínas de la Nucleocápside/genética , Virus de la Parainfluenza 2 Humana/química , Virus de la Parainfluenza 2 Humana/genética , Virus de la Parainfluenza 2 Humana/inmunología , Virus de la Parainfluenza 4 Humana/química , Virus de la Parainfluenza 4 Humana/genética , Estudios Prospectivos , Proteínas Recombinantes/inmunología , Infecciones del Sistema Respiratorio/virología , Saccharomyces cerevisiae/metabolismo , Estudios Seroepidemiológicos , Adulto JovenRESUMEN
Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.
Asunto(s)
Virus Defectuosos/genética , Genoma Viral , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Rubulavirus/genética , Animales , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interferones/genética , Interferones/inmunología , Infecciones por Rubulavirus/genética , Proteínas Virales/genéticaRESUMEN
Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones Virales del Ojo/diagnóstico , Pruebas de Inhibición de Hemaglutinación/veterinaria , Técnicas para Inmunoenzimas/veterinaria , Infecciones por Rubulavirus/diagnóstico , Rubulavirus/inmunología , Pruebas Serológicas/veterinaria , Enfermedades de los Porcinos/diagnóstico , Animales , Biomarcadores/sangre , Infecciones Virales del Ojo/sangre , Infecciones Virales del Ojo/inmunología , Infecciones Virales del Ojo/virología , Pruebas de Inhibición de Hemaglutinación/normas , Técnicas para Inmunoenzimas/normas , México , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Infecciones por Rubulavirus/sangre , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Pruebas Serológicas/normas , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virologíaRESUMEN
The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization.
Asunto(s)
Complemento C3b/inmunología , Proteína Cofactora de Membrana/inmunología , Virus de la Parotiditis/inmunología , Paperas/inmunología , Virus de la Parainfluenza 5/inmunología , Infecciones por Rubulavirus/inmunología , Animales , Células CHO , Línea Celular , Activación de Complemento , Complemento C4b/inmunología , Cricetinae , Cricetulus , Humanos , Paperas/virología , Pruebas de Neutralización , Infecciones por Rubulavirus/virologíaRESUMEN
Selected lymphocyte subpopulations were studied and the distribution of viral mRNA were investigated during acute and persistent porcine rubulavirus (PoRV-LPMV) infection in Vietnamese pot-bellied pigs. Six pigs infected with PoRV-LPMV at 17 days of age exhibited clinical signs 7-10 days post-inoculation (pi). One infected piglet died 11 days pi while the other five recovered around day 13 pi and survived until euthanasia on day 277 pi. Increased numbers of CD8+, CD4+ and CD2+ T cells were detected during the acute phase of infection while CD8+ cells were elevated throughout the infection, including during the persistent stage. Specific antibodies against the haemagglutinin-neuraminidase protein of PoRV-LPMV were detected during persistent infection. Although infectious virus could not be recovered from tissues from any of the infected pigs at necropsy 277 days pi, PoRV-LPMV mRNA was detected in lymph nodes, pancreas and central nervous system using a nested polymerase chain reaction technique. Continued lymphocyte interaction with viral RNA may be an important factor in promoting cellular and humoral responses during persistent PoRV-LPMV infection.
Asunto(s)
Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Infecciones por Rubulavirus/veterinaria , Rubulavirus/genética , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Rubulavirus/aislamiento & purificación , Rubulavirus/patogenicidad , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Porcinos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Parainfluenza viruses (PIV) have been generally disregarded as pathogens in spite of their importance in pediatric lower respiratory illness. Because PIVs account for 17% of hospitalized illness associated virus isolation, the development of PIV vaccine would be a major advance in preventing lower respiratory tract infection in infants and young children. We will review in detail several PIV vaccine candidates and recent newer approaches to PIV vaccine development. Intranasally administered bovine PIV3 (bPIV3) vaccine and cold-adapted PIV3 vaccine have been evaluated throughout the pediatric age spectrum. BPIV3 does not give a robust response to the heterotypic human strain although seroconversion rate to bPIV3 is 57-65%. However, bPIV3 vaccine is being used as an attenuated backbone for insertion of human PIV3 hemagglutinin-neuraminidase and fusion (F) proteins and a surface protein, F, of respiratory syncytial virus. The effectiveness of this vaccine against both PIV3 and RSV challenge has been demonstrated in African green monkeys. The cold-adapted PIV3 vaccine has been extensively evaluated and is safe and immunogenic in seronegative children with a seroconversion rate of 79%. These promising candidates deserve to enter into efficacy trials both for their ability to prevent PIV3 disease and as a model of protection against respiratory illness by mucosal vaccination.
Asunto(s)
Vacunas contra la Parainfluenza , Virus de la Parainfluenza 3 Humana/inmunología , Infecciones por Respirovirus/prevención & control , Adulto , Niño , Frío , Crup/prevención & control , Humanos , Lactante , Mutación , Vacunas contra la Parainfluenza/genética , Vacunas contra la Parainfluenza/inmunología , Virus de la Parainfluenza 1 Humana/inmunología , Virus de la Parainfluenza 2 Humana/inmunología , Virus de la Parainfluenza 3 Bovina/genética , Virus de la Parainfluenza 3 Bovina/inmunología , Virus de la Parainfluenza 3 Humana/genética , Virus de la Parainfluenza 4 Humana/inmunología , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/virología , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/prevención & control , Infecciones por Rubulavirus/virologíaRESUMEN
Hemagglutinin-neuraminidase (HN) from porcine rubulavirus La Piedad Michoacan (RvpLPM) is one of the most antigenic proteins known, and is responsible for virus-host cell interaction. We analyzed the amino acid sequence of HN, using computer-assisted techniques to identify B cell epitopes. From a pool of 18 possible antigenic peptides, we evaluated the antigenicity of the 2 peptides with the highest scores and the 1 with lowest score. Antibodies from RvpLPM-infected pigs recognized the synthesized HN-A, HN-B, and HN-R peptides (optical density [OD]: 0.33 +/- 0.02 for HN-A, 0.20 +/- 0.02 for HN-B, and 0.07 +/- 0.01 for HN-R); bovine serum albumin-coupled HN-A and HN-B induced rabbit anti-RvpLPM antibodies (OD: 0.39 +/- 0.01 for HN-A and 0.35 +/- 0.02 for HN-B). Loop 5 from the outer membrane protein, OmpC, from Salmonella typhi was replaced with HN-B; this protein was then expressed in Escherichia coli UH302. BALB/c mice were challenged intraperitoneally or orogastrically with the fusion protein expressed in E. coli and murine antibodies obtained from both types of administration inhibited virus-hemagglutinating activity, as did the antibodies from RvpLPM-infected swine. These results suggest that HN-A and HN-B are peptides involved in RvpLPM cell carbohydrate recognition, and could therefore be considered potential targets for vaccine and diagnostic procedures development.
Asunto(s)
Epítopos de Linfocito B/inmunología , Proteína HN/inmunología , Péptidos/inmunología , Infecciones por Rubulavirus/inmunología , Rubulavirus/inmunología , Algoritmos , Animales , Mapeo Epitopo , Proteína HN/química , Pruebas de Inhibición de Hemaglutinación , Hemaglutinación por Virus , Ratones , Ratones Endogámicos BALB C , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Infecciones por Rubulavirus/virología , Programas Informáticos , PorcinosRESUMEN
Professional antigen-presenting cells are critical components of both the innate and adaptive immune responses. Although dendritic cells (DCs) are generally thought to be the primary activators of naive T cells, macrophages have also been shown to fulfill this role. As with DCs, the capacity to induce optimal activation of T cells requires that macrophages undergo a process that results in the increased expression of costimulatory molecules, such as CD40, CD80, and CD86, and the production of cytokines. In this study we analyzed the effect of infection of macrophages generated from BALB/c mice with the paramyxovirus simian virus 5 (SV5). Here we have shown that bone marrow-derived macrophages (BMMs) are not productively infected at any multiplicity of infection tested. Analysis of activation markers revealed that SV5-infected BMMs robustly upregulated CD40 and modestly upregulated CD86, but did not upregulate the expression of CD80. Further, SV5-infected BMMs secreted low levels of interferon-beta and interleukin (IL)-12p40, but high levels of tumor necrosis factor-alpha and IL-6. Intriguingly, upregulation of these molecules on BMMs, unlike our previous results using bone marrow-derived dendritic cells, was not dependent on live virus. These findings provide evidence that different professional antigen-presenting cells can detect and respond to virus via distinct mechanisms.
Asunto(s)
Células Dendríticas/fisiología , Macrófagos/fisiología , Virus de la Parainfluenza 5 , Infecciones por Rubulavirus/inmunología , Transducción de Señal/fisiología , Animales , Antígenos CD40/biosíntesis , Línea Celular , Citocinas/biosíntesis , Activación de Macrófagos , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 9/fisiología , Proteínas Virales/análisisRESUMEN
Many enveloped RNA viruses recruit host cell proteins during assembly as a mechanism to limit antiviral effects of complement. Using viruses which incorporated CD46 alone, CD55 alone or both CD46 and CD55, we addressed the role of these two host cell regulators in limiting complement-mediated neutralization of Parainfluenza virus 5 (PIV5). PIV5 incorporated functional forms of both CD55 and CD46 into virions. PIV5 containing CD55 was highly resistant to complement-mediated neutralization, whereas CD46-containing PIV5 was as sensitive to neutralization as virus lacking both regulators. PIV5 infected cells had increased levels of cell surface CD55, which was further upregulated by exogenous treatment with tumor necrosis factor alpha. PIV5 derived from cells with higher CD55 levels was more resistant to complement-mediated neutralization in vitro than virus from control cells. We propose a role for virus induction of host cell complement inhibitors in defining virus growth and tissue tropism.
Asunto(s)
Antígenos CD55/genética , Proteínas del Sistema Complemento/inmunología , Regulación de la Expresión Génica , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/genética , Infecciones por Rubulavirus/virología , Replicación Viral , Animales , Antígenos CD55/metabolismo , Células CHO , Línea Celular , Activación de Complemento/inmunología , Cricetinae , Cricetulus , Humanos , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Pruebas de Neutralización , Infecciones por Rubulavirus/inmunologíaRESUMEN
The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.
Asunto(s)
Infecciones por Rubulavirus/veterinaria , Rubulavirus/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Especificidad de Anticuerpos , Antígenos Virales/inmunología , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Infecciones por Rubulavirus/inmunología , Porcinos/inmunologíaRESUMEN
The porcine immune system is unique in the expression of CD4+CD8+ (double-positive, DP) lymphocytes. These cells have been associated with immunological memory due to their gradual increase with age, the expression of memory phenotype and their ability to respond to recall viral antigen. This work analyzes the biological function of CD4+CD8- and CD4+CD8+ lymphocytes in the immune response to porcine rubulavirus (PRv). CD4+CD8- cells isolated from pigs 3 weeks after infection with porcine rubulavirus proliferated in response to homologous virus and generated lymphoblasts which were predominantly of the CD4+CD8+ phenotype, whereas stimulation with mitogen induced proliferation but did not switch the phenotype. CD4+CD8- lymphocytes isolated after 10 weeks of infection proliferated in response to phytohemagglutinin (PHA) but did not proliferate in response to homologous virus and did not change their phenotype, whereas CD4+CD8+ lymphocytes proliferated in response to PHA and to viral antigen. The cytokine profile of both lymphocyte populations showed the presence of IL-2 and IL-10 transcripts, quantitation demonstrated that CD4+CD8+ cells expressed mainly IL-10, whereas CD4+CD8- lymphocytes expressed primarily IL-2. Our results show that CD4+CD8- lymphocytes in the early phase of porcine rubulavirus infection can be converted to double-positive cells expressing IL-10 in an antigen-dependent manner, and that CD4+CD8- T-cells late in infection do not acquire CD8.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Rubulavirus/veterinaria , Rubulavirus/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Anticuerpos Monoclonales , Relación CD4-CD8 , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , ADN Viral/química , Densitometría/veterinaria , Epítopos de Linfocito T/inmunología , Citometría de Flujo/veterinaria , Interleucina-10/análisis , Interleucina-10/biosíntesis , Interleucina-2/análisis , Interleucina-2/biosíntesis , Activación de Linfocitos/inmunología , ARN Viral/química , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to porcine rubulavirus (La Piedad Michoacan Virus [LPMV]) in serum samples from pigs. The test, based on a monoclonal antibody against the LPMV hemagglutinin-neuraminidase glycoprotein, had a sensitivity of 99% and a specificity of 97%. The results of this test were in agreement with those obtained by an indirect ELISA and hemagglutination inhibition, indirect immunofluorescence, and virus neutralization tests. The blocking ELISA is considered the most suitable test for routine screening for antibodies against LPMV.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Rubulavirus/veterinaria , Rubulavirus/inmunología , Enfermedades de los Porcinos/diagnóstico , Animales , Anticuerpos Monoclonales , Línea Celular , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente Indirecta , Proteína HN/inmunología , Riñón , Ratones , Ratones Endogámicos BALB C , Infecciones por Rubulavirus/diagnóstico , Infecciones por Rubulavirus/inmunología , Sensibilidad y Especificidad , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/inmunologíaRESUMEN
"Blue eye" disease of pigs in Mexico is caused by porcine rubulavirus and characterized by infertility in sows and boars, nervous signs in young pigs, and corneal opacity in pigs of all ages. The pathogenesis of reproductive tract lesions in rubulavirus-infected boars has not previously been investigated. In a first experiment, four 9-month-old boars were inoculated with porcine rubulavirus and killed 5, 15, 30 or 45 days post-inoculation (pi). In a second experiment, four similar boars were inoculated with the same virus and two animals were killed on each of days 70 and 80 pi. Swelling of the head of the epididymis developed in all inoculated boars at approximately day 15 pi. Reduced spermatozoan motility and concentration were detected in semen samples collected from one boar from day 21 pi. At post-mortem examination, nodules were seen in the head of the epididymis of the boars killed 15, 30 or 45 days pi and the right testis of the pig killed 30 days pi was atrophic. Corresponding histopathological epididymal alterations included formation of spermatic granulomas and vacuolar degeneration of ductular epithelium. These lesions were associated with mononuclear cell infiltration and interstitial fibroplasia. Degeneration of seminiferous tubules and interstitial mononuclear cell infiltration were seen in the atrophic testis of the pig killed 30 days pi. There was fibrosis of the head of the epididymis in all boars killed 70 or 80 days pi and one of these animals also had right testicular atrophy associated with degeneration of seminiferous tubules, lymphocytic infiltration and giant cell formation. Porcine rubulavirus antigen was detected by immunofluorescence labelling in the head of the epididymis of the pigs killed 15, 30 or 45 days pi and in one animal killed on day 70 pi. These results indicate that porcine rubulavirus can cause severe epididymo-orchitis and reduced semen quality in sexually mature boars.
Asunto(s)
Infecciones por Rubulavirus/veterinaria , Rubulavirus , Enfermedades Testiculares/veterinaria , Animales , Antígenos Virales/análisis , Epidídimo/inmunología , Epidídimo/patología , Epidídimo/virología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Rubulavirus/inmunología , Rubulavirus/aislamiento & purificación , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/patología , Porcinos , Enfermedades Testiculares/inmunología , Enfermedades Testiculares/patología , Enfermedades Testiculares/virologíaRESUMEN
Numerous studies have demonstrated a critical role for T cell avidity in predicting in vivo efficacy. Even though the measurement of avidity is now a routine assessment for the analysis of effector and memory T cell populations, our understanding of how this property is controlled in vivo at both the population and individual cell levels is limited. Our previous studies have identified high avidity as a property of the initial effector population generated in mice following respiratory virus infection. As the response progresses, lower avidity cells appear in the effector pool. The studies described here investigate the mechanistic basis of this in vivo regulation of avidity. We present data supporting in vivo avidity modulation within the early high avidity responders that results in a population of lower avidity effector cells. Changes in avidity were correlated with decreased lck expression and increased sensitivity to lck inhibitors in effector cells present at late versus early times postinfection. The possibility of tuning within select individual effectors is a previously unappreciated mechanism for the control of avidity in vivo.
Asunto(s)
Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Virus de la Parainfluenza 5/inmunología , Infecciones por Rubulavirus/inmunología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Femenino , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Linfocitos T Citotóxicos/metabolismoRESUMEN
We sampled sera from 1013 non-vaccinated swine from four states in Mexico, Guanajuato, Jalisco, Michoacán and the Estado de Mexico, to analyse anti-porcine rubulavirus antibody titres against three different porcine rubulavirus isolates (PAC-4/1993, PAC-6/2001, and PAC-9/2003) using a hemagglutination inhibition assay. The results revealed that there were antigenic differences among the isolates assessed. In particular, the estimated correlation between the PAC-4/1993 and PAC-6/2001 (0.50) isolates and between the PAC-4/1993 and PAC-9/2003 isolates (0.56) displayed a moderate positive correlation. In contrast, there was a strong positive correlation between the PAC-6/2001 and PAC-9/2003 isolates (0.73). We also found that in the state of Guanajuato, PAC-4/1993 was the isolate that was most frequently identified; in Jalisco, the isolate was PAC-6/2001; and in Michoacán, the isolate was PAC-9/2003. By contrast, in the Estado de Mexico, all three isolates appeared to circulate with a low seroprevalence. In general, the analysed sera from the four states displayed a porcine rubulavirus serological prevalence ranging from 9% to 23.7%. These data indicate that there is not complete antibody cross-antigenicity among the three isolates, and the antigenic variations in the antibody response found in this study implies that the use of a monovalent vaccine would not generate complete protection against the different antigenic subtypes.
Asunto(s)
Antígenos Virales/genética , Infecciones por Rubulavirus/veterinaria , Rubulavirus/genética , Enfermedades de los Porcinos/virología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , Variación Genética , México/epidemiología , Rubulavirus/inmunología , Infecciones por Rubulavirus/epidemiología , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Estudios Seroepidemiológicos , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/inmunologíaRESUMEN
Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.
Asunto(s)
Anticuerpos Antivirales/sangre , Enfermedades de los Perros/prevención & control , Inmunización Secundaria , Virus de la Parainfluenza 5/inmunología , Infecciones por Rubulavirus/prevención & control , Infecciones por Rubulavirus/veterinaria , Vacunación , Animales , Anticuerpos Neutralizantes , Línea Celular , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/virología , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Virus de la Influenza A/química , Virus de la Parainfluenza 5/efectos de los fármacos , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
It has become clear that T cells with the potential to negatively regulate the immune response are normal constituents of the immune system. These cells often mediate their effects through the production of immunosuppressive factors. At present our understanding of how these cells are generated is limited. Here we report the presence of a population of IL-10-producing, virus-specific CD8+ T cells in the lungs of mice following acute respiratory infection. These cells were only found at minimal levels in the spleen and draining lymph node; instead they were restricted primarily to the infected lung tissue. A major finding from this study is demonstration that the ability to produce IL-10 can be acquired by IFNgamma-producing effector cells following entry into the infected lung. These studies suggest IL-10 production is the result of further differentiation of an antigen-specific CD8+ T cell that is governed by signals present in infected lung tissue.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Pulmón/citología , Virus de la Parainfluenza 5 , Infecciones por Rubulavirus/inmunología , Traslado Adoptivo , Animales , Antígenos Virales , Regulación de la Expresión Génica , Pulmón/inmunología , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos BALB C , Bazo/citologíaAsunto(s)
Avulavirus/inmunología , Pollos/virología , Psittaciformes/virología , Infecciones por Rubulavirus/veterinaria , Animales , Anticuerpos Antivirales/análisis , Pruebas de Hemaglutinación/veterinaria , Hemaglutinación por Virus , Israel , Infecciones por Rubulavirus/inmunología , Pruebas SerológicasRESUMEN
Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24-48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by 'cold-shocking' live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.