Establishment of a multiplex real-time PCR assay for the detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of bovine herpesvirus type 1

Bovine herpesvirus type 1 (BoHV-1), an alphaherpesvirus, is a major
pathogen of cattle causing different syndromes such as infectious
bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis
(IPV) and infectious balanoposthitis (IBP). BoHV-1 control programs
have been initiated in several European countries including
Germany. One of the major components is the vaccination with
inactivated or attenuated glycoprotein E (gE)-deleted live marker
vaccines. The aim of this study was the development of a triplex
real-time polymerase chain reaction (qPCR) assay for the sensitive,
specific and reliable BoHV-1 detection. A BoHV-1-specific
glycoprotein D (gD) assay was combined with a gE-specific qPCR
system for differentiation between wild-type strains and
gE-negative vaccine virus strains. Finally, an internal control
based on amplification of the bovine beta-actin gene was introduced
to verify efficient DNA extraction and PCR amplification. The
analytical sensitivity of the triplex BoHV-1 qPCR enables the
detection of 10 genome copies per reaction. Furthermore, the
sensitivity of the newly developed qPCR assay was compared to an
OIE-validated qPCR and the “gold standard” method of virus
isolation in cell culture using 10-fold dilution series of BoHV-1
in extended semen as well as in cell culture medium. For all
preparations, the tested qPCR assays showed comparable results and
the sensitivity of the triplex qPCR was equal or even greater than
that of virus isolation. A broad spectrum of reference strains and
field isolates was detected reliably. The specificity of the test
was confirmed using nasal swabs, semen and different organ
materials of BoHV-1-negative cattle. Bovine herpesviruses type 2, 4
and 5 and further ruminant herpesviruses, namely bubaline
herpesvirus (BuHV-1), caprine herpesvirus type 1 and cervine
herpesvirus type 1 and 2 (CvHV-1, -2) were tested as well. The
close genetic and serological relationship between these viruses
combined with their ability to infect bovines may interfere with
diagnostics resulting in false-positive results. All non-BoHV-1
herpesviruses were negative in the gD-specific assay, while BuHV-1,
CvHV-1 and -2 were tested positive by the gE-specific qPCR.
Consequently, the triplex qPCR offers for the first time the
possibility to detect some related herpesviruses and distinguish
them from BoHV-1 in addition to the simultaneous differentiation of
BoHV-1 wild-type and gE-deleted vaccine strains.