Development of a TaqMan assay for sensitive detection of all pestiviruses infecting cattle, including the emerging HoBi-like strains

Abstract

A real-time RT-PCR assay based on the TaqMan technology was developed for rapid and sensitive detection of pestiviruses infecting cattle, i.e., bovine viral diarrhea virus (BVDV) 1, BVDV-2, and the emerging HoBi-like pestiviruses. The assay was linear and reproducible, being able to detect as few as 10 copies of viral RNA. By real-time RT-PCR analysis of 986 biological samples collected from cattle herd with clinical signs suggestive of pestivirus infection and from animals recruited in a pestivirus surveillance programme, 165 pestivirus positive samples were detected, including 6 specimens, 2 nasal swabs, and 4 EDTA-blood samples, that tested negative by a gel-based RT-PCR assay targeting the 5'UTR. The developed TaqMan assay represents a new reliable and effective tool for rapid and sensitive diagnosis of infections caused by all pestiviruses circulating in cattle, thus being useful for extensive surveillance programs in geographic areas where HoBi-like pestiviruses are co-circulating with BVDV-1 and BVDV-2.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus

“…Several PCR-based methods have been developed for sensitive and rapid detection of BVDV in clinical samples (Bhudevi and Weinstock, 2003;Young et al, 2006;La Rocca and Sandvik, 2009;Yan et al, 2011;Fan et al, 2012;Zhang et al, 2014;Losurdo et al, 2015), but only few methods are currently available for unambiguous discrimination between BVDV-1 and BVDV-2 (Sullivan and Akkina, 1995;Letellier and Kerkhofs, 2003;Baxi et al, 2006;LeBlanc et al, 2010). The emergence of HoBi-like pestivirus that causes clinical pictures overlapping those induced by BVDV-1 and BVDV-2 (Bauermann et al, 2012(Bauermann et al, , 2013(Bauermann et al, , 2014, posed several concerns as for the ability of existing diagnostic methods to efficiently detect this emerging group of pestiviruses (Schirrmeier et al, 2004).…”

“…The panpestivirus RT-PCR developed by Vilcek et al (1994), which is commonly used for BVDV molecular screening, fails to detect or detects with low efficiency HoBi-like strains due to the presence of a mismatch at the 3 end of primer 324 that prevents the correct primer annealing. Other conventional and real-time RT-PCR protocols are able to detect the novel pestivirus but do not provide any virus characterisation, which is helpful to assess virus epidemiology (Elvander et al, 1998;Letellier et al, 1999;Gaede et al, 2005;Hoffmann et al, 2006;Losurdo et al, 2015). A Taq-Man assay that was claimed to be specific for HoBi-like pestivirus was recently developed (Liu et al, 2008), but this assay could not discriminate simultaneously BVDV-1 and BVDV-2 and showed partial cross-reaction with high-titre BVDV-2 samples (Decaro et al, 2012b(Decaro et al, , 2013b.…”

“…On the other hand, the nested PCR approach established by Sullivan and Akkina (1995) misclassifies HoBi-like viruses as BVDV-2 (Decaro et al, 2012b). In order to detect all pestiviruses infecting cattle using a single tool, a novel real-time RT-PCR assay has been recently developed (Losurdo et al, 2015). In addition, a nested PCR (nPCR) protocol has been set up for pestivirus typing (Decaro et al, 2012b).…”

HoBi-Like Pestivirus and Its Impact on Cattle Productivity

“…The farmer was convinced to carry out an eradication programme aimed to detect and slaughter PI animals. For this purpose, all the 752 animals of the herd were bled by the farm personnel and EDTA blood samples were submitted to nested‐PCR (Decaro et al., ) and real‐time RT‐PCR assays (Losurdo et al., ; Mari et al., ) for detection and characterization of ruminant pestiviruses. A total of 9 PI animals were detected that were immediately slaughtered.…”

“…Virological investigations were carried out on a total of 780 animals that were sampled twice at 30 days apart by collecting EDTA blood samples. All blood samples were tested for HoBi‐like pestivirus by nested‐PCR (Decaro et al., ) and real‐time RT‐PCR (Losurdo et al., ; Mari et al., ) assays. Animals were considered PI when they were found to be viremic at both sampling times; in contrast, acutely infected (AI) animals were viremic only at the first sampling, while after 30 days, they were expected to become virus negative.…”