RESUMO
In order to inhibit pathogenic complications and to enhance animal and poultry growth, antibiotics have been extensively used for many years. Antibiotics applications not only affect target pathogens but also intestinal beneficially microbes, inducing long-lasting changes in intestinal microbiota associated with diseases. The application of antibiotics also has many other side effects like, intestinal barrier dysfunction, antibiotics residues in foodstuffs, nephropathy, allergy, bone marrow toxicity, mutagenicity, reproductive disorders, hepatotoxicity carcinogenicity, and antibiotic-resistant bacteria, which greatly compromise the efficacy of antibiotics. Thus, the development of new antibiotics is necessary, while the search for antibiotic alternatives continues. Probiotics are considered the ideal antibiotic substitute; in recent years, probiotic research concerning their application during pathogenic infections in humans, aquaculture, poultry, and livestock industry, with emphasis on modulating the immune system of the host, has been attracting considerable interest. Hence, the adverse effects of antibiotics and remedial effects of probiotics during infectious diseases have become central points of focus among researchers. Probiotics are live microorganisms, and when given in adequate quantities, confer good health effects to the host through different mechanisms. Among them, the regulation of host immune response during pathogenic infections is one of the most important mechanisms. A number of studies have investigated different aspects of probiotics. In this review, we mainly summarize recent discoveries and discuss two important aspects: (1) the application of probiotics during pathogenic infections; and (2) their modulatory effects on the immune response of the host during infectious and non-infectious diseases.
Assuntos
Doenças Transmissíveis/etiologia , Suscetibilidade a Doenças , Imunomodulação , Probióticos/administração & dosagem , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biomarcadores , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/metabolismo , Doenças Transmissíveis/terapia , Gerenciamento Clínico , Suscetibilidade a Doenças/imunologia , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologiaRESUMO
BACKGROUND: Vesicular stomatitis (VS) is an acute, highly contagious and economically important zoonotic disease caused by the vesicular stomatitis virus (VSV). There is a need for effective and safe stable recombinant vaccine for the control of the disease. The human type 5 replication-defective adenovirus expression vector is a good way to construct recombinant vaccines. RESULTS: Three recombinant adenoviruses (rAd) were successfully constructed that expressed the VSV Indiana serotype glycoprotein (VSV-IN-G), VSV New Jersey serotype glycoprotein (VSV-NJ-G), and the G fusion protein (both serotypes of G [VSV-IN-G-NJ-G]) with potentiality to induce protective immunity. G proteins were successfully expressed with good immunogenicity. The rAds could induce the production of VSV antibodies in mice, and VSV neutralizing antibodies in goats, respectively. The neutralizing antibody titers could reach 1:32 in mice and 1:64 in goats. The rAds induced strong lymphocyte proliferation in mice and goats, which was significantly higher compared to the negative control groups. CONCLUSIONS: The three rAds constructed in the study expressed VSV-G proteins and induced both humoral and cellular immune responses in mice and goats. These results lay the foundation for further studies on the use of rAds in vaccines expressing VSV-G.
Assuntos
Glicoproteínas de Membrana/imunologia , Estomatite Vesicular/prevenção & controle , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular New Jersey/imunologia , Proteínas do Envelope Viral/imunologia , Adenoviridae , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Feminino , Doenças das Cabras/imunologia , Doenças das Cabras/prevenção & controle , Doenças das Cabras/virologia , Cabras , Imunidade Celular , Imunidade Humoral , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Vacinas Sintéticas/imunologia , Estomatite Vesicular/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/imunologiaRESUMO
Senecavirus A (SVA), which is associated with porcine vesicular disease and high mortality in neonatal piglets, is a small non-enveloped RNA virus and a member of Picornaviridae family. An emerging SVA strain, named SVA CH/FuJ/2017, was isolated from vesicular liquid and vesicular lesion tissue from piglets with vesicular disease in Fujian province, China. In our study, the complete genome sequence of SVA CH/FuJ/2017 strain has been determined. The viral genome was 7285â¯nt in length. The homology analysis indicated that the gene sequences of polyprotein and VP1 in SVA CH/FuJ/2017 shared highest nucleotide identities with American SVA isolates; and polyprotein showed the highest similarity with American SVA isolates. The phylogenetic analysis based on polyprotein and VP1 nucleotide sequences indicated that SVA CH/FuJ/2017 was closely related to American SVA isolates. The results revealed that the novel SVA strain was closely related to those SVA strains that were isolated in America. Hence, the retrospective study is important for tracing the probable origin of China SVA strains.
Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Infecções por Picornaviridae/veterinária , Picornaviridae/classificação , Picornaviridae/genética , Doença Vesicular Suína/epidemiologia , Doença Vesicular Suína/virologia , Animais , Linhagem Celular , China/epidemiologia , Doenças Transmissíveis Emergentes/história , Efeito Citopatogênico Viral , Genes Virais , Genoma Viral , História do Século XXI , Filogenia , Picornaviridae/isolamento & purificação , Picornaviridae/fisiologia , Suínos , Doença Vesicular Suína/históriaRESUMO
Foot-and-mouth disease virus (FMDV) is the causative agent of Foot-and-mouth disease (FMD), which is an acute and highly contagious disease affecting pigs, cattle and other cloven-hoofed animals. Several studies have shown that FMDV has evolved multiple strategies to evade the host innate immune response, but the underlying mechanisms for immune evasion are still not fully understood. In the current research, we have demonstrated that FMDV utilizes its non-structural protein 2B to sabotage the host immune response. Over-expression of the FMDV 2B inhibited Poly(I:C)-induced or SeV-triggered up-regulation of IFN-ß, IL-6 as well as ISG15. When HEK293T cells were transfected with FMDV 2B, the phosphorylation of TBK1 and IRF3 was inhibited. Co-immunoprecipitation and pull-down experiments indicated that FMDV 2B protein could interact with host RIG-I and MDA5. Moreover, FMDV 2B also inhibited the expression of the RIG-I and MDA5. Thus, FMDV 2B negatively regulates the RLR-mediated IFN-ß induction by targeting RIG-I and MDA5.
Assuntos
Vírus da Febre Aftosa/metabolismo , Interferon beta/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/metabolismo , Fosforilação , Receptores Imunológicos , Transdução de Sinais , Transfecção , Regulação para CimaRESUMO
TRAF family member-associated NF-κB activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)-(IκB) kinase ε (IKKε) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN production in HEK293T cells. Mechanistically, EMCV 3C protease (EMCV 3C) cleaved TANK and disrupted the TANK-TBK1-IKKε-IRF3 complex, which resulted in the reduction in IRF3 phosphorylation and type I IFN production. Taken together, our findings demonstrate that EMCV adopts a novel strategy to evade host innate immune responses through cleavage of TANK.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Cisteína Endopeptidases/metabolismo , Vírus da Encefalomiocardite/enzimologia , Quinase I-kappa B/antagonistas & inibidores , Fator Regulador 3 de Interferon/antagonistas & inibidores , Interferon Tipo I/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteases Virais 3C , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cães , Deleção de Genes , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/química , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Mesocricetus , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
To study the outcome of natural selection using phylogenetic trees, we analyzed full-length genome sequences of porcine teschovirus (PTV). PTV belongs to the family Picornaviridae and has a positive-stranded RNA genome, the replication of which is carried out by the error-prone viral RNA-dependent RNA polymerase. The viral RNA encodes a single polyprotein that is cleaved into structural (i.e., L, VP4, VP2, VP3 and VP1) and nonstructural proteins (i.e., 2A, 2B, 2C, 3A, 3B, and 3C). A high degree of genetic diversity was found based on the pairwise nucleotide distances and on the mean ratio of the number of nonsynonymous (dN) and synonymous (dS) substitutions (dN/dS) in the structural genes. Conversely, the diversity of the nonstructural genes was lower. The differences in genetic diversity between the structural and nonstructural genomic regions were likely due to strong purifying selection; consequently, the estimates of phylogenies were also discordant among these genes. In particular, maximum-likelihood and Bayesian methods generated short-branched trees when loci that are under strong purifying selection were used. These findings indicate that even in an RNA virus with an intrinsically high mutation rate, a strong purifying selection will curb genetic diversity and should be considered an important source of bias in future studies based on phylogenetic methods.
Assuntos
Genoma Viral/genética , Teschovirus/genética , Evolução Molecular , Genes Virais/genética , Variação Genética/genética , Filogenia , Recombinação Genética/genética , Alinhamento de Sequência , Teschovirus/isolamento & purificaçãoRESUMO
Astrocyte elevated gene-1 (AEG-1) has been reported to regulate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and is also regulated by it. This study investigated how AEG-1 participates in the survival pathway of motor neurons in amyotrophic lateral sclerosis (ALS). We found reduced levels of AEG-1 in ALS motor neurons, both in vivo and in vitro, compared to wild type controls. Moreover, AEG-1 silencing demonstrated inhibition of the PI3K/Akt pathway and increased cell apoptosis. Additionally, the PI3K/Akt pathway in mSOD1 cells was unresponsive under serum deprivation conditions compared to wtSOD1 cells. These results suggest that AEG-1 deficiency, together with the inhibited PI3K/Akt pathway was associated with decreased viability of ALS motor neurons. However, the mRNA levels of AEG-1 were still lower in mSOD1 cells compared to the control groups, though the signaling pathway was activated by application of a PI3-K activator. This suggests that in ALS motor neurons, some unknown interruption exists in the PI3K/Akt/CREB/AEG-1 feedback loop, thus attenuating the protection by this signaling pathway. Together, these findings support that AEG-1 is a critical factor for cell survival, and the disrupted PI3K/Akt/CREB/AEG-1cycle is involved in the death of injured motor neurons and pathogenesis of ALS.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Proteínas de Membrana/metabolismo , Neurônios Motores/patologia , Transdução de Sinais/fisiologia , Esclerose Lateral Amiotrófica/genética , Animais , Apoptose/genética , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião de Mamíferos , Feminino , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/patologia , Superóxido Dismutase/genéticaRESUMO
TRAF family member-associated NF-κB activator (TANK) is a negative regulator of canonical NF-κB signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-κB activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-κB signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-κB activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cisteína Endopeptidases/metabolismo , Vírus da Encefalomiocardite/enzimologia , NF-kappa B/metabolismo , Proteólise , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Sequência de Aminoácidos , Cisteína Endopeptidases/genética , Equartevirus/enzimologia , Vírus da Febre Aftosa/enzimologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Vírus da Síndrome Respiratória e Reprodutiva Suína/enzimologia , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Proteínas Virais/genéticaRESUMO
Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. A nanoPCR assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (PRV). Three pairs of primers for nanoPCR developed in this study were selected from conserved regions of PRV, producing specific amplicons of 431 bp (gB), 316 bp (gE), and 202 bp (gG). The sensitivity of this assay using purified plasmid constructs containing the specific gene fragments was 100-1000 fold higher than conventional PCR. The PRV nanoPCR assay did not amplify porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine teschovirus, or African swine fever virus but produced three bands of expected size with PRV and two bands of expected size with the gene-deleted PRV-Bartha-K61. Of 110 clinical samples collected from seven provinces in China, 53% and 48% were positive for wild-type PRV according to the nanoPCR assay and virus isolation, respectively.
Assuntos
Deleção de Genes , Herpesvirus Suídeo 1/classificação , Herpesvirus Suídeo 1/genética , Nanopartículas , Reação em Cadeia da Polimerase/métodos , Vacinas contra Pseudorraiva/genética , Virologia/métodos , Animais , China , Primers do DNA/genética , Herpesvirus Suídeo 1/isolamento & purificação , Sensibilidade e Especificidade , Suínos , Proteínas do Envelope Viral/genéticaRESUMO
A detailed analysis of the Ns1/Vp1Vp2 genome region of the porcine parvovirus (PPV) strains isolated from vaccinated animals was performed. We found many inconsistencies in the phylogenetic trees of these viral isolates, such as low statistical support and strains with long branches in the phylogenetic trees. Thus, we used distance-based and phylogenetic methods to distinguish de facto recombinants from spurious recombination signals. We found a mosaic virus in which the Ns1 gene was acquired from one PPV clade and the Vp1Vp2 gene was acquired from a distinct phylogenetic clade. We also described the interclade mosaic structure of the Vp1Vp2 gene of a reference strain. If recombination is an adaptive mechanism over the course of PPV evolution, we would likely observe increasing numbers of chimeric strains over time. However, when the PPV sequences isolated from 1964 to 2011 were analysed, only two chimeric strains were detected. Thus, PPV recombination is an independent event, resulting from close contact between animals housed in high-density conditions.
Assuntos
Parvovirus Suíno/genética , Animais , Evolução Molecular , Genoma Viral , Dados de Sequência Molecular , Parvovirus Suíno/classificação , Filogenia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Recombinação Genética , Sus scrofa , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Newcastle disease virus (NDV) Italien, a velogenic strain, is an oncolytic virus that is considered to be a potential agent for antitumor viral therapy. We constructed three helper plasmids expressing the NP, P and L genes of NDV Italien based on the eukaryotic expression plasmid pcDNA3.1(+). The minigenome consisting of the 3' leader and 5' trailer regions of NDV Italien flanking a reporter gene encoding firefly luciferase was constructed to examine the efficacy of the three helper plasmids in viral genome replication and transcription. After co-transfection of BSR-T7/5 cells with the three helper plasmids and the minigenome plasmid, replication of minigenome RNA was evaluated by determining luciferase activity. In the minigenome rescue system, expression of the reporter gene was detected. Our results indicate that the three proteins NP, P, and L are correctly expressed and can assemble into a functional ribonucleoprotein complex that effectively directs the transcription of minigenome RNA.
Assuntos
Genética Microbiana/métodos , Genoma Viral , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus da Doença de Newcastle/genética , Plasmídeos , Virologia/métodos , Animais , Linhagem Celular , Cricetinae , Vírus Oncolíticos/genética , Vírus Oncolíticos/crescimento & desenvolvimento , Transfecção , Replicação ViralRESUMO
Porcine parvovirus (PPV) is the major causative agent in a syndrome of reproductive failure in swine. Much has been learned about the structure and function of PPV in recent years, but nothing is known about the epitopes of the structural protein VP1, which is an important antigen of PPV. In this study, the monoclonal antibody C4 against VP1 of PPV was prepared and was used to biopan a 12-mer phage peptide library three times. The selected phage clones were identified by ELISA and then sequencing. The amino acid sequences detected by phage display were analyzed, and a mimic immuno-dominant epitope was identified. The epitope of VP1 is located in the N-terminal and contains the role amino acid sequence R-K-R. Immunization of mice indicated that the phage-displayed peptide induces antibodies against PPV. This study shows that peptide mimotopes have potential as alternatives to the complex antigens currently used for diagnosis of PPV infection or for development of vaccines.
Assuntos
Infecções por Parvoviridae/veterinária , Parvovirus Suíno/imunologia , Doenças dos Suínos/virologia , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Mapeamento de Epitopos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Parvovirus Suíno/química , Parvovirus Suíno/genética , Biblioteca de Peptídeos , Alinhamento de Sequência , Suínos , Doenças dos Suínos/imunologia , Proteínas Estruturais Virais/genéticaRESUMO
A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.
Assuntos
Vírus da Cinomose Canina/classificação , Vírus da Cinomose Canina/isolamento & purificação , Cinomose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Primers do DNA/genética , Vírus da Cinomose Canina/genética , Cães , Feminino , Masculino , Sensibilidade e EspecificidadeRESUMO
To establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of infectious bursal disease virus (IBDV), four primers specific to six regions of the VP3 gene were designed; the VP3 region was selected because it is a conserved part of the IBDV genome. After amplification in an isothermal water bath for 70 min, samples containing IBDV generated the expected ladder-like products while other viruses generated no product. The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation. The assay was significantly more sensitive than normal gel-based RT-PCR. Because it is specific and simple, the RT-LAMP assay can be widely applied in clinical laboratories for rapid detection of IBVD.
Assuntos
Vírus da Doença Infecciosa da Bursa , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologia , Embrião de Galinha , Galinhas/virologia , Primers do DNA , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologia , Sensibilidade e Especificidade , Análise de Sequência de DNA , Proteínas Estruturais Virais/genéticaRESUMO
A rapid immune colloidal gold test strip for detecting 1 lineage of the H5 subtype of highly pathogenic avian influenza was developed based on membrane chromatography with monoclonal antibodies (mAbs) against hemagglutinin (HA) of the H5 subtype of Avian influenza virus (AIV). Bagg albino C mice that were immunized with AIV A/Goose/Guangdong/3/96(H5N1) yielded 4 hybridomas secreting mAbs (1A6, 1B8, 3C4, and 8F6) specific for HA. Monoclonal antibody 3C4 was conjugated with colloidal gold as the detector antibody. On the test strip, 1A6, in combination with rabbit anti-H5N1 polyclonal antibody, was used as the capture complex at the "test line," and goat antimouse immunoglobulin G antibody was used as the capture antibody at the "control line." The strip test correctly detected the H5 subtype of AIV in known positive samples and did not react with the standard antigens of AIV H1-H4 or H6-H14, or the antigens of Newcastle disease virus, Infectious bursal disease virus, Infectious bronchitis virus, or Avian infectious laryngotracheitis virus. When 483 clinical specimens suspected of infection with H5N1 were tested, results from the strip test were nearly identical with those from the HA/hemagglutination inhibition test, reverse transcription polymerase chain reaction, and a commercial enzyme-linked immunosorbent assay kit. Because the newly developed strip was specific, rapid, and sensitive, it should be useful for diagnosing the H5 subtype of AIV and for investigating its epidemiology. The test strip is only reliable for the lineage of H5N1. As for the other strains of AIV, further experiments will be needed.
Assuntos
Aves/virologia , Virus da Influenza A Subtipo H5N1/classificação , Influenza Aviária/virologia , Animais , Antígenos Virais , Linhagem Celular Tumoral , Embrião de Galinha , Galinhas , Humanos , Virus da Influenza A Subtipo H5N1/genética , Mieloma Múltiplo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
Porcine reproductive and respiratory syndrome is rapidly gaining worldwide importance as one of the most economically significant diseases of swine. The antibody of Porcine reproductive and respiratory syndrome virus (PRRSV) is detected currently by the combined use of an enzyme-linked immunosorbent assay, serum neutralization test, immunoperoxidase monolayer assay, indirect immunofluorescent antibody test. These methods are time-consuming and require specialized equipment operated by trained technicians. The purpose of this study was to evaluate a simple strip assay (based on a chromatographic and immunogold system) for specific detection of PRRSV antibody in swine sera. This "immunochromatographic strip" test uses Escherichia coli-expressed viral recombinant membrane protein antigen in combination with recombinant nucleocapsid protein as capture protein for detecting antibodies against PRRSV. In this study, the performance of this assay was evaluated with sera from both clinical samples and experimentally infected piglets. Detection by immunochromatographic strip test was compared with detection by a standard, available commercially, indirect enzyme-linked immunosorbent assay and an immunoperoxidase monolayer assay. The immunochromatographic test strip detected antibodies in sera known to contain antibodies to PRRSV in 95.7% sensitivity of samples from pigs infected experimentally and 98.6% sensitivity of clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was 97.8% and 98.2% for clinical and experimental serum samples, respectively.