RESUMEN

In this study, a specific and sensitive method for simultaneous detection of human astrovirus, human rotavirus, norovirus, sapovirus and enteric adenovirus associated with acute enteritis was developed, based on the specific dual priming oligonucleotide (DPO) system and the sensitive high-performance liquid chromatography (HPLC) analysis. The DPO system-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) combined with HPLC assay was more sensitive than agarose gel electrophoresis analysis and real-time SYBR Green PCR assay, and showed a specificity of 100% and sensitivity of 96%-100%. The high sensitivity and specificity of the assay indicates its great potential to be a useful tool for the accurate diagnosis of enteric virus infections.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Enteritis/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virosis/diagnóstico , Humanos , Sensibilidad y Especificidad

RESUMEN

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine parvovirus (BPV) DNA. Four primers were designed to recognize six distinct regions on the target DNA based on a highly conserved sequence in the VP2 region of the BPV genome. The optimized LAMP reaction conditions were 8 mM Mg²âº, 1.2 mM betaine, and an incubation at 63°C for 45 min. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The detection limit of the LAMP assay was 9 copies of BPV-DNA and was 100 times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the BPV LAMP assay was highly specific without any cross-reactivity with other related viruses. The LAMP assay was evaluated further using 59 field samples and the results were comparable to conventional PCR. The LAMP assay is a simple, rapid and economic detection method; it can provide a useful technique suitable for detection of BPV infection in both field conditions and laboratory settings.

Asunto(s)

Bocavirus/aislamiento & purificación , Enfermedades de los Bovinos/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones por Parvoviridae/veterinaria , Medicina Veterinaria/métodos , Animales , Bocavirus/genética , Bovinos , Enfermedades de los Bovinos/virología , Cartilla de ADN/genética , ADN Viral/genética , Infecciones por Parvoviridae/virología , Sensibilidad y Especificidad

RESUMEN

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

Asunto(s)

Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/prevención & control , Lacticaseibacillus casei/inmunología , Oncorhynchus mykiss/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Administración Oral , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones por Birnaviridae/prevención & control , Enfermedades de los Peces/virología , Inmunidad Mucosa , Inmunoglobulina M/sangre , Virus de la Necrosis Pancreática Infecciosa/inmunología , Pruebas de Neutralización , Oncorhynchus mykiss/virología , Proteínas Recombinantes de Fusión/inmunología , Carga Viral , Vacunas Virales/administración & dosificación

RESUMEN

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.

Asunto(s)

Infecciones por Coronavirus/veterinaria , Lactobacillus/genética , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Enfermedades de los Porcinos/inmunología , Proteínas del Envoltorio Viral/inmunología , Administración Oral , Animales , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus , Femenino , Expresión Génica , Inmunidad Mucosa , Lactobacillus/metabolismo , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/administración & dosificación , Proteínas de la Nucleocápside/genética , Virus de la Diarrea Epidémica Porcina/genética , Glicoproteína de la Espiga del Coronavirus , Porcinos , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunología

RESUMEN

The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

Asunto(s)

Formación de Anticuerpos/inmunología , Proteínas de la Cápside/inmunología , Lactococcus lactis/metabolismo , Vacunas contra Rotavirus/inmunología , Sus scrofa/virología , Vacunación , Administración Oral , Animales , Western Blotting , Línea Celular , Proliferación Celular , Electroforesis en Gel de Poliacrilamida , Femenino , Inmunoensayo , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/prevención & control

RESUMEN

Porcine epidemic diarrhea virus (PEDV) LJB/03 strain was isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Heilongjiang Province, and was identified by immunofluorescence test, immunelectronmicroscopy, RT-PCR and indirect ELISA assay. Characteristics of the virus culture and the methods of improvement of virus titer were explored. The results showed that the virus had the typical appearance of the coronavirus. Analysis of the nucleotide sequences of RT-PCR products revealed 98% homology with the reference strains. Indirect immunofluorescence assay showed a significant presence of green fluorescence, and an average P/N ratio of 7.6 by indirect ELISA assay. Taken together, these tests showed positive isolation of PEDV. Using the virus plaque purification cloning methods established in the test, the purified PEDV large plaque and small plaque were obtained, and the large plaque and small plaque titers were measured with significant difference. These results provide potential for the application of PEDV on the basis of the biological features of isolated virus.

Asunto(s)

Infecciones por Coronavirus/veterinaria , Virus de la Diarrea Epidémica Porcina/crecimiento & desarrollo , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Enfermedades de los Porcinos/virología , Animales , Técnicas de Cultivo de Célula , China/epidemiología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Epidemias , Heces/virología , Virus de la Diarrea Epidémica Porcina/genética , Porcinos , Enfermedades de los Porcinos/epidemiología , Cultivo de Virus

RESUMEN

Lactobacillus casei 393 was selected as a bacterial carrier for the expression of Porcine Parvovirus (PPV) protective antigen VP2 protein. The gene encoding PPV VP2 protein was cloned into the Lactobacillus casei surface expression vector pPG, and then the constructed recombinant vector pPG-VP2 was electrotransformed into Lactobacillus casei 393 generating the recombinant system pPG-VP2/L. casei393 expressing PPV VP2 protein. The recombinant strain was induced by 2% Lactose in MRS and about 74kD protein was detected with SDS-PAGE. The result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescent test showed that the expressed protein was secreted on the cell surface Lactobacillus casei.

Asunto(s)

Antígenos Virales/metabolismo , Proteínas de la Cápside/metabolismo , Lacticaseibacillus casei/metabolismo , Proteínas Virales/metabolismo , Animales , Antígenos Virales/genética , Western Blotting , Proteínas de la Cápside/genética , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Lacticaseibacillus casei/genética , Parvovirus Porcino/genética , Parvovirus Porcino/metabolismo , Plásmidos/genética , Proteínas Recombinantes/metabolismo , Porcinos/virología , Transformación Genética , Proteínas Virales/genética