Differentiation of C-strain “Riems” or CP7_E2alf vaccinated animals from animals infected by classical swine fever virus field strains using real-time RT-PCR
“…In order to assess the specificity of each differential PCR design, a template mixture containing 10 4 copies of C-strain, CBR/93 and UK2000/7.1 RNA was analyzed using the differential genotype 1.1 real-time RT-PCR developed for the Riemser C-strain [17,18] together with quantification standards or differential real-time qRT-PCR assays developed specifically to detect the challenge strains. The specificity of the genotype 1.1 assay depends on a single nucleotide at the 3’end of each primer, and, as expected, only produced an amplicon from the C-strain template and not from the other viral strains (Figure 1A).…”
Section: Resultsmentioning
confidence: 99%
“…The sensitivity of the differential assays for the challenge strains were evaluated using a standard curve prepared from a serial 10-fold dilution of viral RNA from the corresponding viral strain that had been quantified using a pan-CSFV PCR [27]. Although having a less sensitive detection limit than the 10 copies/assay reported for the genotype 1.1 assay [17], the challenge strain differential assays allowed reliable quantification of template RNA down to 10 2 copies/assay (Table 1). Figure 1
Specificity of differential PCR assays for detection of C-strain (C-str), CBR/93 and UK2000/7.1 (UK) RNA templates.…”
Section: Resultsmentioning
confidence: 99%
“…Leifer and colleagues developed a real-time RT-PCR assay to specifically detect the C-strain for use as a genetic DIVA test in circumstances where this vaccine might be used [17]. This assay was subsequently modified to accommodate nucleotide variations found in different C-strain vaccine batches and now also detects all genotype 1.1 CSFV strains, including the C-strain [18].…”
BackgroundControl of classical swine fever (CSF) by vaccination ideally requires that field strain infection can be detected irrespective of the vaccination status of the herd. To inform on the usefulness of molecular tests compatible with genetic Differentiation of Infected from Vaccinated Animals (DIVA) principles when using live-attenuated vaccines, tonsil homogenates from a vaccination-challenge experiment were analyzed using a differential real-time qRT-PCR for the C-strain vaccine or real-time qRT-PCR assays developed to specifically detect the challenge strains used.ResultsIn animals with high or moderate levels of blood viraemia, which were not, or not fully, protected by vaccination, challenge virus RNA was readily detected in tonsil homogenates. In three out of the seven vaccinated animals that had high or moderate viraemia, the vaccine strain RNA also could be detected but at lower levels. Lower but varying levels of challenge and/or vaccine virus RNA were detected in tonsil homogenate samples from animals with no or low-level viraemia, and in groups solely consisting of such animals, no transmission of infection to naïve in-contact animals occurred. In one group of animals that were vaccinated 3 days prior to challenge, viraemia levels varied from high to absent and transmission of challenge virus to naïve in-contact animals occurred. The DIVA assay revealed challenge virus in all tonsil homogenates from this group, even in those animals that did not have viraemia and were protected from clinical disease by vaccination. Such animals, particularly in a low biosecurity/informal farm setting, could constitute a risk for disease control in the field.ConclusionsGenetic DIVA testing is useful for detecting the presence of field virus infection especially in non-viraemic animals without overt clinical signs but which are incompletely protected by vaccination. Such tests could particularly be useful to inform decisions prior to and during cessation of a control strategy that employs vaccination.
“…In order to assess the specificity of each differential PCR design, a template mixture containing 10 4 copies of C-strain, CBR/93 and UK2000/7.1 RNA was analyzed using the differential genotype 1.1 real-time RT-PCR developed for the Riemser C-strain [17,18] together with quantification standards or differential real-time qRT-PCR assays developed specifically to detect the challenge strains. The specificity of the genotype 1.1 assay depends on a single nucleotide at the 3’end of each primer, and, as expected, only produced an amplicon from the C-strain template and not from the other viral strains (Figure 1A).…”
Section: Resultsmentioning
confidence: 99%
“…The sensitivity of the differential assays for the challenge strains were evaluated using a standard curve prepared from a serial 10-fold dilution of viral RNA from the corresponding viral strain that had been quantified using a pan-CSFV PCR [27]. Although having a less sensitive detection limit than the 10 copies/assay reported for the genotype 1.1 assay [17], the challenge strain differential assays allowed reliable quantification of template RNA down to 10 2 copies/assay (Table 1). Figure 1
Specificity of differential PCR assays for detection of C-strain (C-str), CBR/93 and UK2000/7.1 (UK) RNA templates.…”
Section: Resultsmentioning
confidence: 99%
“…Leifer and colleagues developed a real-time RT-PCR assay to specifically detect the C-strain for use as a genetic DIVA test in circumstances where this vaccine might be used [17]. This assay was subsequently modified to accommodate nucleotide variations found in different C-strain vaccine batches and now also detects all genotype 1.1 CSFV strains, including the C-strain [18].…”
BackgroundControl of classical swine fever (CSF) by vaccination ideally requires that field strain infection can be detected irrespective of the vaccination status of the herd. To inform on the usefulness of molecular tests compatible with genetic Differentiation of Infected from Vaccinated Animals (DIVA) principles when using live-attenuated vaccines, tonsil homogenates from a vaccination-challenge experiment were analyzed using a differential real-time qRT-PCR for the C-strain vaccine or real-time qRT-PCR assays developed to specifically detect the challenge strains used.ResultsIn animals with high or moderate levels of blood viraemia, which were not, or not fully, protected by vaccination, challenge virus RNA was readily detected in tonsil homogenates. In three out of the seven vaccinated animals that had high or moderate viraemia, the vaccine strain RNA also could be detected but at lower levels. Lower but varying levels of challenge and/or vaccine virus RNA were detected in tonsil homogenate samples from animals with no or low-level viraemia, and in groups solely consisting of such animals, no transmission of infection to naïve in-contact animals occurred. In one group of animals that were vaccinated 3 days prior to challenge, viraemia levels varied from high to absent and transmission of challenge virus to naïve in-contact animals occurred. The DIVA assay revealed challenge virus in all tonsil homogenates from this group, even in those animals that did not have viraemia and were protected from clinical disease by vaccination. Such animals, particularly in a low biosecurity/informal farm setting, could constitute a risk for disease control in the field.ConclusionsGenetic DIVA testing is useful for detecting the presence of field virus infection especially in non-viraemic animals without overt clinical signs but which are incompletely protected by vaccination. Such tests could particularly be useful to inform decisions prior to and during cessation of a control strategy that employs vaccination.
“…It also gives an indication regarding genetic variation and quasispecies evolution (Li et al, 2006). Moreover, it can be useful for the differentiation of infected from vaccinated animals in scenarios involving (emergency) vaccination (Leifer et al, 2009 …”
Classical swine fever (CSF) has caused significant economic losses in industrialized pig production, and is still present in some European countries. Recent CSF outbreaks in Europe were mainly associated with strains of genogroup 2 (subgroup 2.3). Although there are extensive datasets regarding 2.3 strains, there is very little information available on longer fragments or whole classical swine fever virus (CSFV) genomes. Furthermore, there are no detailed analyses of the molecular epidemiology of CSFV wild boar isolates available. Nevertheless, complete genome sequences are supportive in phylogenetic analyses, especially in affected wild boar populations. Here, German CSFV strains of subgroup 2.3 were fully sequenced using two different approaches: (i) a universal panel of CSFV primers that were developed to amplify the complete genome in overlapping fragments for chain-terminator sequencing; and (ii) generation of a single full-length amplicon of the CSFV genome obtained by long-range RT-PCR for deep sequencing with next-generation sequencing technology. In total, five different strains of CSFV subgroup 2.3 were completely sequenced using these newly developed protocols. The approach was used to study virus spread and evolutionary history in German wild boar. For the first time, the results of our study clearly argue for the possibility of a long-term persistence of genotype 2.3 CSFV strains in affected regions at an almost undetectable level, even after long-term oral vaccination campaigns with intensive monitoring. Hence, regional persistence in wild boar populations has to be taken into account as an important factor in the continual outbreaks in affected areas.
“…Previously reported real-time RT-PCR assays for discriminating wildtype CSFV from the Riems vaccine-strain have been established in the EU [12,13] and an assay for differentiating between wild type and the K-LOM vaccine-strain CSFV in Korea [14]. In China, a two-step real time RT-PCR assay to distinguish wild-type CSFV from the HCLVstrain vaccine based on nucleotide differences at the probe binding site and a one-step real time RT-PCR assay (wt-rRTPCR) using a Minor Groove Binding (MGB) probe for detection of mutations in wild-types have also been described [15].…”
Monoclonal antibodies WH211 and WH303 are in routine laboratory use for detecting Classical Swine Fever Virus (CSFV) or its major envelope E2 protein. While E2 is recognized as the most immunogenic protein, it is also the most variable and often not recognized by specific monoclonal against this protein. The aim of this study was to compare the detection sensitivity of WH211 and WH303 antibodies for CSFV GPE -strain via two well-known assays, Surface Plasmon Resonance Biosensor and Western Blot Assays. Both WH211 and WH303 Abs which specifically known to detect E2 gene (gp55) of CSFV at different recognition sites (epitope) were used as ligands to detect the GPE -strain (analytes). GPE − strain showed interaction with monoclonal antibodies at highest dilution of 1:1000 (v/v) in SPR assay. At lowest dilution (1:10), the interaction of GPE − strain with immobilized monoclonal antibody WH211 showed more than two-fold increase (163.5 RU) than the interaction with monoclonal antibody WH303 (60.0 RU). This study documented profound preference for WH211 as the target for CSFV E2 gene by having higher sensitivity towards GPE − strain but with lesser affinity. E2 epitope of GPE − strain was found undetectable when blotted with WH303 at the investigated dilutions as compared to WH211. The findings indicated a 2500-fold higher SPR sensitivity in detection in comparison to Western Blot and the limit of detection of GPE − strain by Western Blot could not be achieved beyond 1:10 dilution of monoclonal antibodies.Therefore, SPR approach could overcome the risk of GPE − vaccine strain from being invisible for identification. Although, WH303 was unable to recognize this strain by Western Blot, both WH211 and WH303 were applicable as a sensitive detection ligand for GPE -strain of CSFV using SPR analysis.
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