Entwicklung und Evaluation einer diagnostischen Methode zum Nachweis von BoHV-1 während der Akutphase.

• Forschungsstelle: BLV
• Projektnummer: 1.18.03
• Projekttitel Englisch: Development and evaluation of a diagnostic tool for the detection of BoHV-1 during the acute phase.

Schlüsselwörter (Deutsch)

Bovines Herpesvirus Typ 1 (BoHV-1), Infektiöse Bovine Rhinotracheitis (IBR), Quantitative PCR (qPCR), Loop-mediated isothermal AMPlification (LAMP), Recombinase Polymerase Amplification (RPA).

Schlüsselwörter (Englisch)

Bovine Herpesvirus type 1 (BoHV-1), Infectious Bovine Rhinotracheitis (IBR), Quantitative PCR (qPCR), Loop-mediated isothermal AMPlification (LAMP), Recombinase Polymerase Amplification (RPA).

Kurzbeschreibung (Deutsch)

Das Bovine Herpesvirus Typ 1 (BoHV-1) ist ein Alphaherpes-Virus, das die Infektiöse Bovine Rhinotracheitis (IBR) und die Infektiöse pustulöse Vulvovaginitis/Balanoposthitis (IPV/IBP) verursacht. Das Virus hat die Eigenschaft nach einer kurzen akuten Infektionsphase im Wirt eine latente Infektion zu etablieren. Serologisch kann das Virus erst nach der Reaktion des Immunsystems indirekt nachgewiesen werden jedoch nicht in der Akutphase. Für den Nachweis von BoHV-1 DNA bei akuten Infektionen soll ein auf isothermaler Amplifikation basierter Test etabliert werden. Der einfach konzipierte Test sollte von technischem Personal in standardmässig ausgerüsteten Diagnostiklabors durchgeführt werden können. Der Verzicht auf technisch anspruchsvolle Geräte würde eine markante Steigerung des Probendurchsatzes erlauben. Im Falle eines Ausbruchs könnte so schnell und mit der nötigen Kapazität diagnostisch reagiert werden.

Kurzbeschreibung (Englisch)

Bovine Herpesvirus typ 1 (BoHV-1) belongs to the subfamily of the Alphaherpesvirinae and is the causative agent of infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanopostthitis (IPV/IBP). The virus is able to establish a latent infection in the host upon a short acute phase. Serological tests for the detection of BoHV-1 are only useful after the reaction of the immune system but not during the acute infection. Therefore, a diagnostic tool for BoHV-1 during the acute phase based on isothermal amplification of viral DNA will be developed and evaluated. Various methods for the read out will be evaluated. Of note, the test procedure might be performed by technical staff in diagnostics laboratories with standard equipment. As no specialized equipment is necessary, the throughput of samples would be very high, which is an important factor in the event of an outbreak.

Projektziele (Deutsch)

During the first year, quantitative PCR for detection of BoHV-1 DNA will be established and several sets of primers for LAMP and RPA will be designed. The sensitivity and specificity of the reactions will be evaluated with DNA extracted from viral cell culture supernatant. During the second year, the LAMP based assay will be compared to the qPCR based assay using clinical samples as well as with cell cultured virus. Furthermore, simple methods for DNA extraction from nasal swabs and blood samples will be evaluated. During the second year, parallel to the comparative application of LAMP and qPCR based assays on clinical samples, we plan and organize a field study of the assay to test the reality situation. This schedule would allow to carefully evaluate potential collaboration partners and to apply for a follow-up project covering the field study. In the case the development of the assay would not be advanced enough, the planning could be postponed for a maximum of half a year. The choice of the collaboration partners will be based on our network connections and on the current epidemic situation of BoHV-1 at the time point of planning. The field study is meant to be executed as a parallel assay to the conventional method. As such, the results of both methods could be compared and the performance of the LAMP based method could be evaluated.  

Projektziele (Englisch)

The current tools to diagnose infections of BoHV-1 include the detection of virus specific antibodies by ELISA and the detection of viral genome by PCR. While the detection of antibodies indicates a former infection, the detection of viral DNA allows conclusions concerning the presence of virus or even the presence of replicating virus in the sampled animal. In contrast to the detection of virus specific antibodies, which can be detected only five to ten days post infection, viral DNA can be amplified already at day 1 and up to approximately day 14 post infection. Therefore, the detection of BoHV-1 in acute phase at early time points of infection is most easily accomplished by nucleic acid amplification.

Amplification of genomes by PCR for direct detection of DNA viruses is a valuable and frequently applied method. BoHV-1 specific PCR assays are described by mean of conventional as well as quantitative methods (Fuchs 1999, Claus 2005, Wernike 2011). However isothermal amplifications (Loop-mediated isothermal AMPlification, LAMP; Rolling Circle Amplification, RCA; Recombinase Polymerase Amplification, RPA) are described as alternative methods for the specific amplification and detection of DNA (Kim 2011). These methods might be equally sensitive as PCR assays. Indeed, a careful evaluation of the primers for a LAMP reaction should lead to an assay of high sensitivity and specificity. Several diagnostic tests for DNA viruses engaging LAMP (Luo 2011, Livingstone 2016) and RNA viruses engaging RT-LAMP (Shirato 2014, Kurosaki 2016) are published. Isothermal amplification methods are also described for detection of herpesvirus DNA. A LAMP-based diagnostic assay was developed for Gallid herpesvirus 1 (Ou 2012) and a RPA-based assay for Cyprinid herpesvirus 3 (Presscott 2016). A LAMP-based assay specifically for the detection of Indian isolates of BoHV-1 in semen was recently published (Pawar 2015).

Abstract (Deutsch)

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Abstract (Englisch)

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