A host's immune system can be invaded by mycotoxin deoxynivalenol (DON) poisoning and porcine circovirus type 2 (PCV2) infections, which affect the host's natural immune function. Pro-inflammatory cytokines, IL-1β and IL-6, are important regulators in the process of natural immune response, which participate in inflammatory response and enhance immune-mediated tissue damage. Preliminary studies have shown that DON promotes PCV2 infection by activating the MAPK signaling pathway. Here, we explored whether the mRNA expression of IL-1β and IL-6, induced by the combination of DON and PCV2, would depend on the MAPK signaling pathway. Specific pharmacological antagonists U0126, SP600125 and SB203580, were used to inhibit the activities of ERK, JNK and p38 in the MAPK signaling pathway, respectively. Then, the mRNA expression of IL-1β and IL-6 in PK-15 cells was detected to explore the effect of the MAPK signaling pathway on IL-1β and IL-6 mRNA induced by DON and PCV2. The results showed that PK-15 cells treated with DON or PCV2 induced the mRNA expression of IL-1β and IL-6 in a time- and dose-dependent manner. The combination of DON and PCV2 has an additive effect on inducing the mRNA expression of IL-1β and IL-6. Additionally, both DON and PCV2 could induce the mRNA expression of IL-1β and IL-6 via the ERK and the p38 MAPK signal pathways, while PCV2 could induce it via the JNK signal pathway. Taken together, our results suggest that MAPKs play a contributory role in IL-1β and IL-6 mRNA expression when induced by both DON and PCV2.
- Klíčová slova
- IL-1β, IL-6, MAPK, PCV2, deoxynivalenol,
- MeSH
- buněčné linie MeSH
- Circovirus * MeSH
- infekce viry čeledi Circoviridae genetika metabolismus MeSH
- interleukin-1beta genetika MeSH
- interleukin-6 genetika MeSH
- MAP kinasový signální systém účinky léků MeSH
- messenger RNA MeSH
- prasata MeSH
- trichotheceny toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- deoxynivalenol MeSH Prohlížeč
- interleukin-1beta MeSH
- interleukin-6 MeSH
- messenger RNA MeSH
- trichotheceny MeSH
Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 101 virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.
- Klíčová slova
- Beak and feather disease virus (BFDV), Real-time PCR,
- MeSH
- Circovirus genetika izolace a purifikace MeSH
- diagnostické techniky molekulární metody MeSH
- DNA primery genetika MeSH
- infekce viry čeledi Circoviridae diagnóza veterinární virologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nemoci ptáků diagnóza virologie MeSH
- oligonukleotidové sondy genetika MeSH
- ptáci MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- veterinární lékařství metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Názvy látek
- DNA primery MeSH
- oligonukleotidové sondy MeSH
A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.
- MeSH
- Circovirus klasifikace genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- infekce viry čeledi Circoviridae prevence a kontrola veterinární virologie MeSH
- nemoci prasat prevence a kontrola virologie MeSH
- prasata MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- virové vakcíny genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Názvy látek
- DNA primery MeSH
- virové vakcíny MeSH
A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5' end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10 mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. These results pave the way for a straightforward large-scale production of the recombinant PCV 2 capsid protein and its use as a diagnostic antigen or a PCV 2 subunit vaccine.
- MeSH
- Circovirus genetika imunologie metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- imunizace MeSH
- molekulární sekvence - údaje MeSH
- nemoci prasat diagnóza prevence a kontrola virologie MeSH
- prasata virologie MeSH
- protilátky virové krev MeSH
- rekombinantní proteiny genetika imunologie metabolismus MeSH
- sekvence aminokyselin MeSH
- syndrom multisystémového chřadnutí selat po odstavení diagnóza prevence a kontrola virologie MeSH
- virion metabolismus MeSH
- virové plášťové proteiny genetika imunologie metabolismus MeSH
- virové vakcíny aplikace a dávkování imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- protilátky virové MeSH
- rekombinantní proteiny MeSH
- virové plášťové proteiny MeSH
- virové vakcíny MeSH
Direct comparisons are important when assessing the value of DNA extraction methods for diagnostic virology as the inhibitors present and the efficiency of extraction vary with the target infectious agent as well as the species and the site from which the clinical sample was obtained. Three DNA extraction methods were compared for routine polymerase chain reaction (PCR) detection of beak and feather disease virus (BFDV) in whole blood and feather samples and of avian polyomavirus (APV) in feather samples. Boiling in Chelex 100 Resin was found to be the most sensitive method for detection of BFDV or APV DNA in both feather and blood samples. In combination with nested PCR it enabled detection of BFDV DNA in 13/13 positive whole blood samples and in 22/23 positive feather samples. It also enabled detection of APV in 31/31 samples detected as positive in this study. NucleoSpin kits enabled detection of BFDV DNA in only 9/13 blood samples and in 18/23 feather samples. The lower rate of BFDV DNA detection when using NucleoSpin kits was not a result of inhibition of PCR in most cases. The NucleoSpin Tissue Kit enabled detection of APV DNA in 29/31 feather samples. Inhibition of DNA amplification was observed when using the DNAzol Direct kit. Therefore, of the methods evaluated here, Chelex 100 Resin treatment of samples was the best option for routine testing for BFDV and APV DNA in clinical samples.
- MeSH
- Circovirus izolace a purifikace MeSH
- DNA virů izolace a purifikace MeSH
- infekce onkogenními viry veterinární virologie MeSH
- infekce viry čeledi Circoviridae virologie MeSH
- nemoci ptáků MeSH
- polymerázová řetězová reakce veterinární MeSH
- polyomavirové infekce veterinární virologie MeSH
- Polyomavirus izolace a purifikace MeSH
- Psittaciformes virologie MeSH
- senzitivita a specificita MeSH
- techniky amplifikace nukleových kyselin veterinární MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA virů MeSH
Eight captive-bred horned parakeets (Eunymphicus cornutus) and four captive-bred Major Mitchell cockatoos (Cacatua leadbeateri) from the same aviary tested positive for psittacine circovirus (PsCV) DNA in whole blood by nested-polymerase chain reaction (PCR). The chronic form of disease with feather fragility and loss was observed in three horned parakeets. Infection in other individuals was subclinical. Immunosuppression, either hematologically or as susceptibility to secondary infections, was not observed. Treatment consisted of the administration of beta-(1,3/1,6)-D-glucan from oyster mushroom (Pleurotus ostreatus). Excluding two accidentally dead parakeets, four out of the original six horned parakeets, and all Major Mitchell cockatoos were negative for PsCV DNA in whole blood in 7-9 mo after the treatment was started. Even though the absence of PsCV DNA in blood does not signify elimination of the virus from the whole organism, these preliminary results indicate a possible effect of beta-glucan in the treatment of PsCV infection. To the author's knowledge, this is the first report of PsCV in horned parakeets.
- MeSH
- antivirové látky terapeutické užití MeSH
- Circovirus klasifikace účinky léků fyziologie MeSH
- glukany terapeutické užití MeSH
- infekce viry čeledi Circoviridae farmakoterapie veterinární virologie MeSH
- kakaduové virologie MeSH
- nemoci ptáků farmakoterapie virologie MeSH
- papoušci s dlouhými ocasními pery virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antivirové látky MeSH
- epiglucan MeSH Prohlížeč
- glukany MeSH
Porcine circovirus 2 quantification by real-time PCR and determination of virus specific antibodies using a peptide based ELISA test was performed on a total of 400 serum samples. Samples for antibody measurement and virus quantification were obtained from a conventional pig farm with a clinical form of post-weaning multisystemic wasting syndrome. Samples were taken during the post-weaning period. Seven litters of weaned piglets (6-25 weeks) were systematically sampled at 2-3 week intervals. Porcine circovirus 2 specific antibodies were detected using the peptide based ELISA test. IgM antibodies were first detected at week 8 and reached their highest levels at week 12; IgG antibody appeared at week 10 and peaked at week 16 with an average titer at 1:3500. Although, the viral load peaked at week 10 (7x10(7) genomes copy/ml of sera), viral infection persisted through to adult age (10(5) genomes copy/ml of sera).
- MeSH
- Circovirus imunologie MeSH
- infekce viry čeledi Circoviridae krev imunologie veterinární MeSH
- nemoci prasat krev imunologie virologie MeSH
- prasata MeSH
- protilátky virové krev MeSH
- viremie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- protilátky virové MeSH
Three oligonucleotide primers for semi-nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV-1) and 2 (PCV-2) isolates. These primers were used to detect PCV-2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total. 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty-seven of them were positive for PCV-2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92-97% homology with other known PCV-2 strains and only 80-83% homology with PCV-1 strains.
- MeSH
- Circovirus klasifikace genetika izolace a purifikace MeSH
- DNA primery MeSH
- DNA virů genetika MeSH
- fylogeneze MeSH
- infekce viry čeledi Circoviridae epidemiologie veterinární MeSH
- lymfatické uzliny virologie MeSH
- nemoci prasat epidemiologie virologie MeSH
- nos virologie MeSH
- polymerázová řetězová reakce normy veterinární MeSH
- prasata virologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Názvy látek
- DNA primery MeSH
- DNA virů MeSH
