BackgroundDetection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD.ResultsA mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT).ConclusionsThe mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.
Ovine herpesvirus 2 (OvHV-2) is a lymphotropic gammaherpesvirus that asymptomatically infects most sheep, but causes malignant catarrhal fever in cattle, bison, pigs and deer. There is no permissive cell culture system but OvHV-2-infected T lymphocytes can be cultured from diseased animals. We showed that the OvHV-2 genome was in a circular conformation in sheep peripheral blood mononuclear cells and that the latency-associated ORF73 was transcribed, while expression of the productive cycle genes ORF9 (DNA polymerase) and ORF50 (R-transactivator) was barely detectable, suggestive of latency. Doxorubicin treatment of these cells induced the appearance of linear viral DNA and transcription of productive cycle genes along with several viral unique genes. In contrast, cultured T cells from diseased cattle and rabbits contained a mixture of circular and linear genome configurations indicative of a mixture of latently- and productively-infected cells. Most of the OvHV-2 unique genes were transcribed in these cells but ORF50 expression was only seen after doxorubicin treatment indicating a 'leaky' latent pattern of gene expression. 5-azacytidine treatment increased the proportion of circular DNA and inhibited the expression of most of the OvHV-2 unique genes except Ov2.5 (vIL-10) and Ov4.5 (Bcl-2 homologue) in the cattle cell line. These studies provide key insights into the differences in OvHV-2 gene expression in cells from reservoir and susceptible species and, for the first time, an in vitro system for studying the latent and productive phases of the OvHV-2 virus life cycle.
An innovative approach was tested to treat cat allergy in humans by vaccinating cats with Fel-CuMV (HypoCatTM), a vaccine against the major cat allergen Fel d 1 based on virus-like particles derived from cucumber mosaic virus (CuMV-VLPs). Upon vaccination, cats develop neutralizing antibodies against the allergen Fel d 1, which reduces the level of reactive allergen, thus lowering the symptoms or even preventing allergic reactions in humans. The combined methodological field study included ten cat-allergic participants who lived together with their cats (n = 13), that were immunized with Fel-CuMV. The aim was to determine methods for measuring a change in allergic symptoms. A home-based provocation test (petting time and organ specific symptom score (OSSS)) and a general weekly (or monthly) symptom score (G(W)SS) were used to assess changes in allergic symptoms. The petting time until a pre-defined level of allergic symptoms was reached increased already early after vaccination of the cats and was apparent over the course of the study. In addition, the OSSS after provocation and G(W)SS recorded a persistent reduction in symptoms over the study period and could serve for long-term assessment. Hence, the immunization of cats with HypoCatTM (Fel-CuMV) may have a positive impact on the cat allergy of the owner, and changes could be assessed by the provocation test as well as G(W)SS.
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