Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge
The Bovine respiratory syncytial virus (BRSV) causes bronchiolitis and interstitial pneumonia, predominantly in calves, and is a major cause of bovine respiratory disease worldwide. In humans, BRSV is paralleled by the closely related Human respiratory syncytial virus (HRSV), an important cause of r...
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| Formato: | Second cycle, A1N, A1F or AXX |
| Lenguaje: | Inglés Inglés |
| Publicado: |
2011
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| Acceso en línea: | https://stud.epsilon.slu.se/2213/ |
| _version_ | 1855570403524608000 |
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| author | Blodörn, Krister |
| author_browse | Blodörn, Krister |
| author_facet | Blodörn, Krister |
| author_sort | Blodörn, Krister |
| collection | Epsilon Archive for Student Projects |
| description | The Bovine respiratory syncytial virus (BRSV) causes bronchiolitis and interstitial pneumonia, predominantly in calves, and is a major cause of bovine respiratory disease worldwide. In humans, BRSV is paralleled by the closely related Human respiratory syncytial virus (HRSV), an important cause of respiratory disease, most severe in infants.
The clinical signs and pathology during RSV infection is caused, not only by the direct effects of viral replication, but also by the response of the host immune system. The immunopathology of RSV has long obfuscated our understanding of the disease, and development of effective treatment and vaccines will be very difficult until greater knowledge is gained.
One of the components of the immune system that has come into focus in RSV research the last few years, is the Toll-like receptor 4 (TLR4). The TLR4 receptor is well known as the receptor that binds lipopolysaccaride (LPS), and initiates the host response to bacterial infection. Recently, it has been shown that the fusion protein of RSV also interacts with, and up-regulates the expression of, the TLR4 receptor. Whether this has a predominantly protective effect, as would be expected from an immune response, or if it is mainly detrimental to the host, remains to be determined.
The objective of this work was to develop an assay for quantification of TLR4 mRNA in clinical samples and to determine if TLR4 mRNA in bronchoalveolar lavage (BAL) cell samples could be used as a marker of protection during experimental BRSV infection.
Two one-step quantitative real-time PCR systems were developed and optimized in this work. One for detection of TLR4 mRNA, and the other for detection of the housekeeping gene product 28S rRNA. The assays showed good efficiency as well as intra- and inter-assay reproducibility. Furthermore, BAL cell samples collected during an experimental vaccinal challenge were used to evaluate the level of TLR4 mRNA expression in relation to detected BRSV RNA. When the results were analyzed, it appeared that TLR4 mRNA quantification can not be used as a marker of protection against BRSV infection after previous vaccination.
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| format | Second cycle, A1N, A1F or AXX |
| id | RepoSLU2213 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés Inglés |
| publishDate | 2011 |
| publishDateSort | 2011 |
| record_format | eprints |
| spelling | RepoSLU22132012-04-20T14:17:33Z https://stud.epsilon.slu.se/2213/ Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge Blodörn, Krister Animal physiology and biochemistry Veterinary science and hygiene - General aspects Animal diseases The Bovine respiratory syncytial virus (BRSV) causes bronchiolitis and interstitial pneumonia, predominantly in calves, and is a major cause of bovine respiratory disease worldwide. In humans, BRSV is paralleled by the closely related Human respiratory syncytial virus (HRSV), an important cause of respiratory disease, most severe in infants. The clinical signs and pathology during RSV infection is caused, not only by the direct effects of viral replication, but also by the response of the host immune system. The immunopathology of RSV has long obfuscated our understanding of the disease, and development of effective treatment and vaccines will be very difficult until greater knowledge is gained. One of the components of the immune system that has come into focus in RSV research the last few years, is the Toll-like receptor 4 (TLR4). The TLR4 receptor is well known as the receptor that binds lipopolysaccaride (LPS), and initiates the host response to bacterial infection. Recently, it has been shown that the fusion protein of RSV also interacts with, and up-regulates the expression of, the TLR4 receptor. Whether this has a predominantly protective effect, as would be expected from an immune response, or if it is mainly detrimental to the host, remains to be determined. The objective of this work was to develop an assay for quantification of TLR4 mRNA in clinical samples and to determine if TLR4 mRNA in bronchoalveolar lavage (BAL) cell samples could be used as a marker of protection during experimental BRSV infection. Two one-step quantitative real-time PCR systems were developed and optimized in this work. One for detection of TLR4 mRNA, and the other for detection of the housekeeping gene product 28S rRNA. The assays showed good efficiency as well as intra- and inter-assay reproducibility. Furthermore, BAL cell samples collected during an experimental vaccinal challenge were used to evaluate the level of TLR4 mRNA expression in relation to detected BRSV RNA. When the results were analyzed, it appeared that TLR4 mRNA quantification can not be used as a marker of protection against BRSV infection after previous vaccination. Bovint respiratoriskt syncytialt virus (BRSV) orsakar bronkiolit och interstitiell pneumoni, framförallt hos kalvar, och är en betydande orsak till respiratoriskt lidande hos nötkreatur över hela världen. Lika betydelsefullt på humansidan är det nära besläktade Humant respiratoriskt syncytialt virus (HRSV), som orsakar sjukdom liknande BRSV, framförallt hos spädbarn. Kliniska symtom och patologi vid RSV-infektion är inte bara en direkt följd av virusets replikation, utan även en effekt av värdens immunsvar. Denna immunopatologi har länge höljt patogenesen för RSV i ett dunkel, och utvecklandet av effektiva behandlingar och vacciner kommer att bli svårt tills en djupare förståelse erhållits. En av de komponenter i immunförsvaret som kommit i blickfånget de senaste åren inom RSV-forskning, är Toll-like receptor 4 (TLR4). TLR4 är välkänd för att binda lipopolysackarid (LPS) från Gram-negativa bakterier, och därmed initiera immunförsvaret vid bakteriella infektioner. Nya studier har visat att även RS-virusets fusionsprotein interagerar med TLR4, och uppreglerar uttrycket av denna receptor på infekterade cellers yta. Hurvida detta har övervägande skyddande effekt, som man skulle förvänta sig av ett immunsvar, eller om denna uppreglering har huvudsakligen negativa effekter för värden, är inte klarlagt. Syftet med denna studie var att utveckla en analysmetod för att kvantifiera uttrycket av TLR4 i kliniska prover, samt att avgöra om nivån av TLR4 mRNA i cellprover från bronchoalveolar lavage (BAL) kunde användas som en markör för hur väl skyddat djuret är mot experimentell BRSV infektion. Två kvantitativa realtids PCR-metoder (qPCR) utvecklades och optimerades i detta projekt. En av dessa qPCR-metoder kvantifierar uttrycket av TLR4 mRNA, den andra 28S rRNA. De uppmätta värdena av 28S rRNA användes för att standardisera resultaten för TLR4 mRNA och BRSV-titer. Båda dessa qPCR-metoder uppvisade god effektivitet och reproducerbarhet. Dessutom analyserades BAL-prover, som samlats in från kalvar som deltog i ett vaccinförsök, för att utvärdera uttrycket av TLR4 mRNA i förhållande till BRSV-titer. När dessa resultat analyserades verkade det som om nivån TLR4 mRNA inte utgör någon bra markör för graden av skydd mot BRSV-infektion efter tidigare vaccination. 2011-01-26 Second cycle, A1N, A1F or AXX NonPeerReviewed application/pdf eng https://stud.epsilon.slu.se/2213/1/blodorn_k_110128.pdf Blodörn, Krister, 2011. Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge. Second cycle, A1N, A1F or AXX ( AXX). Uppsala: (VH) > Dept. of Biomedical Sciences and Veterinary Public Health (until 231231) <https://stud.epsilon.slu.se/view/divisions/OID-713.html> urn:nbn:se:slu:epsilon-6-414 eng |
| spellingShingle | Animal physiology and biochemistry Veterinary science and hygiene - General aspects Animal diseases Blodörn, Krister Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge |
| title | Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge |
| title_full | Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge |
| title_fullStr | Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge |
| title_full_unstemmed | Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge |
| title_short | Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge |
| title_sort | development of a real-time rt-pcr for quantification of bovine tlr4 mrna and evaluation of its use during a brsv vaccine challenge |
| topic | Animal physiology and biochemistry Veterinary science and hygiene - General aspects Animal diseases |
| url | https://stud.epsilon.slu.se/2213/ https://stud.epsilon.slu.se/2213/ |