Agent Of Classical Swine Fever Research Articles

Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a disease notifiable to the Office International des Epizooties (OIE). A live marker vaccine would be the ultimate choice for controlling CSF, which enables serological and genetic differentiation of vaccine from wild type CSFV. Recently, a marker vaccine CP7_E2alf has been reported [Koenig, P., Lange, E., Reimann, I., Beer, M., 2007. CP7_E2alf: a safe and efficient marker vaccine strain for oral immunisation of wild boar against classical swine fever virus (CSFV). Vaccine 25, 3391–3399]. A vaccine-specific TaqMan real-time RT-PCR assay was developed and evaluated, and a second, wild type-specific assay was modified from an established one in such a way that both can be performed in two wells side-by-side in a microplate in a single run. The detection limit is 50 viral RNA copies per reaction for the vaccine-specific assay, and 20 copies per reaction for the wild type assay. The two assays have been shown to be highly specific and reproducible, with potential application for genetic differentiation of wild type CSFV from the marker vaccine CP7_E2alf in wild boar vaccination programs.

The Npro product of classical swine fever virus interacts with IκBα, the NF-κB inhibitor

Classical swine fever virus (CSFV) belongs to the genus Pestivirus and is the causative agent of classical swine fever, a haemorrhagic disease of pigs. The virus replicates in host cells without activating interferon (IFN) production and has been reported to be an antagonist of double-stranded RNA-induced apoptosis. The N-terminal protease (N(pro)) of CSFV is responsible for this evasion of the host innate immune response. In order to identify cellular proteins that interact with the N(pro) product of CSFV, a yeast two-hybrid screen of a human library was carried out, which identified IkappaBalpha, the inhibitor of NF-kappaB, a transcription factor involved in the control of apoptosis, the immune response and IFN production. The N(pro)-IkappaBalpha interaction was confirmed using yeast two-hybrid analysis and additional co-precipitation assays. It was also shown that N(pro) localizes to both the cytoplasmic and nuclear compartments in stably transfected cells and in CSFV-infected cells. Following stimulation by tumour necrosis factor alpha, PK-15 cell lines expressing N(pro) exhibited transient nuclear accumulation of pIkappaBalpha, but no effect of CSFV infection on IkappaBalpha localization or NF-kappaB p65 activation was observed.

Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5′-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5 copies/μL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China.

Characterization of classical swine fever virus entry by using pseudotyped viruses: E1 and E2 are sufficient to mediate viral entry

Classical swine fever virus (CSFV) is the causative agent of classical swine fever. Its envelope comprises glycoproteins E rns, E1, and E2. In this study, we showed that the unmodified CSFV glycoproteins could incorporate into the HIV core to generate an infectious CSFV pseudotyped virus. The infection was specific to several porcine cell lines, and could be neutralized by anti-E2 monoclonal antibodies (mAbs) completely and by anti-E rns mAbs partially, indicating that this pseudotyped virus can mimic the early infection steps of parental CSFV. To investigate the specific role of each envelope protein involved in viral entry, a series of pseudotyped viruses were generated bearing CSFV glycoproteins in various combinations. It was found that specific infectivity was also achieved with non-E rns pseudotyped virus carrying E1 and E2 glycoproteins. This indicated that E1 and E2 are sufficient to mediate CSFV entry, and E rns is not indispensable in this process.