Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus

The neutralization immunofluorescence test (NIFT), currently used for detecting neutralizing antibodies (NAbs) against classical swine fever virus (CSFV), is time-consuming. Here, a simplified neutralization test based on enhanced green fluorescent protein (EGFP)-tagged CSFV (EGFP-NT) was developed for direct detection of anti-CSFV NAbs without immunostaining. The relative sensitivity and specificity between EGFP-NT and NIFT or blocking enzyme-linked immunosorbent assay (ELISA) were both 100%. The NAb titers by EGFP-NT and the blocking rates by blocking ELISA showed a good correlation. Classical swine fever (CSF) is an economically important disease of pigs worldwide caused by classical swine fever virus (CSFV), generally a noncytopathogenic virus (1, 2). A neutralization test (NT), either neutralization immunofluorescence test (NIFT) (3) or neutralization peroxidase-linked assay (NPLA) (4), is the gold standard for detection of neutralizing antibodies (NAbs) against CSFV. NPLA can be used for serological discrimination of CSFV from other pestiviruses (5-7). However, these tests are labor-intensive and time-consuming due to the necessary incubation and staining procedures. It would be convenient to use CSFV tagged with a fluorescent molecule to detect NAbs directly in unfixed cells. Therefore, the present study aimed to develop a simplified NT for rapid detection of anti-CSFV NAbs in sera.First, the enhanced green fluorescent protein (EGFP)-tagged CSFV, EGFP-CSFV, was generated from the pEGFP-CSFV, in which the EGFP gene was inserted between amino acids 13 and 14 of the N pro protein of CSFV in pBRCISM, a full-length infectious cDNA clone of the highly virulent CSFV strain Shimen (CSFV-Shimen) (8). The cDNA-derived virus was rescued and identified essentially as described previously (8). The observation of fluorescent foci and detection by the CSFV Antigen Test kit (Idexx, Switzerland), reverse transcription-PCR (RT-PCR), and indirect immunofluorescence assay (IFA) showed that the marker virus EGFP-CSFV was successfully rescued and the EGFP insertion in the recombinant virus was stable (data not shown). Importantly, the replication kinetics of EGFP-CSFV was similar to that of CSFV-Shimen (Fig. 1), indicating that insertion of the EGFP gene did not affect virus replication.The NT based on EGFP-CSFV (EGFP-NT) was performed in 96-well flat-bottom nonpyrogenic polystyrene culture plates (Corning, NY, USA) as described previously (9). Unlike the NIFT based on CSFV-Shimen, EGFP-NT entailed direct examination under a fluorescence microscope (Nikon TE200; Nikon, Japan) and CSFV-specific NAb titers were determined rapidly. The CSFV-specific NAb titers were expressed as the reciprocal of the highest dilution that inhibited the infection of PK-15 cells in 50% of the culture wells. The NT results were validated using positive and negative reference sera. A back titration was mounted each time when performing NT.A number of reference swine sera were used to evaluate the specificity and sensitivity of EGFP-NT. Experimental s...

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“…2). The peak antibody titers were Ͼ384, which corresponded to a blocking rate of 70 to 80%, consistent with the data reported by Sun et al (11).…”

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confidence: 91%

Simplified Serum Neutralization Test Based on Enhanced Green Fluorescent Protein-Tagged Classical Swine Fever Virus

Shen

Sun

et al. 2013

J Clin Microbiol

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“…The serum samples were also tested by SNT for the PRV-and CSFVspecific serum neutralizing antibodies (NAbs) as described previously (17,18). The titers of PRV-and CSFV-specific serum NAbs were determined and expressed as the reciprocal of the highest dilution at which infection of the PK-15 cells was inhibited in 50% of the culture wells.…”

Section: Figmentioning

confidence: 99%

“…It has been shown that E2 is highly immunogenic and can provide protection for pigs against CSF (18,24). To develop a virus vector-based bivalent vaccine, it is important that the recombinant virus still retains the growth ability and immunogenicity of the vector virus.…”

Section: Figmentioning

confidence: 99%

“…Several CSF marker vaccines, such as pSFV1CS-E2 (25), pcdSW (26), or rAdV-E2 (18), have been shown to provide incomplete protection from lethal CSFV challenge. In contrast, rPRVTJ-delgE/gI-E2 showed a much greater efficacy, since it was able to provide complete protection even when administered as a single dose, which was comparable to that of rAdV-SFV-E2 (24) or CP7_E2alf (27).…”

Section: Figmentioning

confidence: 99%

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Generation and Efficacy Evaluation of a Recombinant Pseudorabies Virus Variant Expressing the E2 Protein of Classical Swine Fever Virus in Pigs

Wang

Yuan

et al. 2015

Clin Vaccine Immunol

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Classical swine fever (CSF) is an economically important infectious disease of pigs caused by classical swine fever virus (CSFV). Pseudorabies (PR), which is caused by pseudorabies virus (PRV), is another important infectious disease of pigs and other animals. Coinfections of pigs with PRV and CSFV occur occasionally in the field. The modified live vaccine Bartha-K61 strain has played an important role in the control of PR in many countries, including China. Since late 2011, however, increasing PR outbreaks caused by an emerging PRV variant have been reported in Bartha-K61-vaccinated swine populations on many farms in China. Previously, we generated a gE/gI-deleted PRV (rPRVTJ-delgE) based on this PRV variant, which was shown to be safe and can provide rapid and complete protection against lethal challenge with the PRV variant in pigs. Here, we generated a new recombinant PRV variant expressing the E2 gene of CSFV (rPRVTJ-delgE/gI-E2) and evaluated its immunogenicity and efficacy in pigs. The results showed that rPRVTJ-delgE/gI-E2 was safe for pigs, induced detectable anti-PRV and anti-CSFV neutralizing antibodies, and provided complete protection against the lethal challenge with either the PRV TJ strain or the CSFV Shimen strain. The data indicate that rPRVTJ-delgE/gI-E2 is a promising candidate bivalent vaccine against PRV and CSFV coinfections.

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“…The differential detection of CSFV vaccine and wild-type viruses is of great significance for clinical detection of CSFV, eradication of CSF in breeding swine farms and purity test of CSFV vaccines (Sun et al, 2011a;Sun et al, 2011b;Sun et al, 2010a;Sun et al, 2010b;Tu et al, 2001;Zhu et al, 2009). Taking into account the limited sensitivity of conventional RT-PCR and the restriction endonuclease cleavage of PCR amplicons after the RT-PCR reaction is laborious, time consuming and easy to get contaminated, therefore, it is essential to establish a differential diagnostic technique with high sensitivity (Dewulf et al, 2004;Haegeman et al, 2006;Hoffmann et al, 2009;Vilcek & Belak, 1998).…”

Section: Introductionmentioning

confidence: 99%

Development and Application of a RT-NestPCR Assay for Differential Detection of C-Strain and Wild-Type Viruses of Classical Swine Fever Virus

Wang¹,

Yi-bao²,

Cong³

et al. 2013

SAR

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Due to the urgent need of differentiation of infected from vaccinated animals in control and eradication of classical swine fever (CSF) and the shortcomings of current differential diagnostic tools, this study is aiming to establish a RT-nestPCR assay for differential detection of wild-type viruses and lapinized Chinese vaccine strain (C-strain) of classical swine fever virus (CSFV) of high sensitivity. Two pairs of CSFV-specific primers were designed in the conservative regions of NS5B (a non-structural protein encoded by the CSFV genome, which performs the RNA dependent RNA polymerase activity) and 3′ un-translated regions (3′-UTR) to encompass the T-rich insertion uniquely existing in the 3′-UTR of C-strain genome. Thus the amplification fragment of C-strain is longer than that of the wild-type viruses for it contains the T-rich insertion region. Two pairs of primers were used in combination and the wild-type viruses and C-strain of CSFV could be detected and accurately distinguished with a high sensitivity through super fine resolution (SFR) argarose gel electrophoresis that displays the different lengths of the amplicons. The detection limit of the C-strain and Shimen strain were respectively 4.5×10 -2 pg and 3.2×10 -2 pg of viral RNA. The results of the specificity test showed that this method can detect different strains of CSFV without amplifying other non-CSFV pathogens. The results of the detection of 400 clinical samples indicated that 16 samples were CSFV positive in total; in which 4 samples were C-Strain positive and 12 were wild-type CSFV positive. The total CSFV positive rate was 4%. The detection results of the 14 batches of C-Strain vaccines showed that all samples displayed bands of C-Strain amplicons in the SFR argarose gel electrophoresis and all vaccines were free of wild-type virus contamination. In conclusion, the RT-nestPCR assay established in the present study could supply a sensitive and specific test method for distinguishing wild-type CSFV infected animals from those vaccinated with C-strain vaccines in the field.

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Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus

Cited by 37 publications

References 21 publications

Simplified Serum Neutralization Test Based on Enhanced Green Fluorescent Protein-Tagged Classical Swine Fever Virus

Simplified Serum Neutralization Test Based on Enhanced Green Fluorescent Protein-Tagged Classical Swine Fever Virus

Generation and Efficacy Evaluation of a Recombinant Pseudorabies Virus Variant Expressing the E2 Protein of Classical Swine Fever Virus in Pigs

Development and Application of a RT-NestPCR Assay for Differential Detection of C-Strain and Wild-Type Viruses of Classical Swine Fever Virus

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