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Generation of full-length cDNA BVDV genomic clone in pGEM4. The final step in the construction of pVVNADL consisted of cloning the ClaIDraIII DNA fragment from pBV18-F2 into ClaI-DraIII-digested pSD-MLU3.
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Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV c...
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... of the BVDV genome. pBV18-F2 consists of a T7 RNA polymerase promoter abutted to the viral 5 UTR and adjacent polyprotein open reading frame sequences encoding the viral structural proteins. The second plasmid (pSD-MLU3) encompasses the nonstructural protein region and the 3 UTR of the BVDV genome followed by a SacII restriction endonuclease site (Fig. 1). These plasmids were used to join the two halves of the viral genome to give rise to a genomic-length construct termed pVVNADL (Fig. ...
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... sequences encoding the viral structural proteins. The second plasmid (pSD-MLU3) encompasses the nonstructural protein region and the 3 UTR of the BVDV genome followed by a SacII restriction endonuclease site (Fig. 1). These plasmids were used to join the two halves of the viral genome to give rise to a genomic-length construct termed pVVNADL (Fig. ...
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... patterns of premature transcrip- tion termination, without improvement in full-length RNA transcription (data not shown). In contrast, pVVNADL DNA linearized at position 2828 (RsrII) or position 5621 (SalI) of the BVDV genome gave rise to high yields of discrete-sized RNA by runoff transcription with virtually no heterogeneous abor- tive products (Fig. 2, lanes 1 and 2). Although the T7 RNA polymerase is highly processive for elongation of shorter tran- scripts, certain secondary structures in nascent transcripts can act as termination signals (31). The probability of such fortu- itous events increases with transcript length. The presence of discrete subgenomic length RNA molecules was observed in ...
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... The first strategy in the development of candidate CSF DIVA vaccines is the development of a recombinant chimeric vaccine. Among the most promising candidates are chimeric pestiviruses based on the infectious cDNA clones of CSFV or BVDV [46][47][48][49]. ...
Classical swine fever (CSF), caused by CSF virus (CSFV), is one of the most devastating viral epizootic diseases of swine in many countries. To control the disease, highly efficacious and safe live attenuated vaccines have been used for decades. However, the main drawback of these conventional vaccines is the lack of differentiability of infected from vaccinated animals (DIVA concept). Advances in biotechnology and our detailed knowledge of multiple basic science disciplines have facilitated the development of effective and safer DIVA vaccines to control CSF. To date, two types of DIVA vaccines have been developed commercially, including the subunit vaccines based on CSFV envelope glycoprotein E2 and chimeric pestivirus vaccines based on infectious cDNA clones of CSFV or bovine viral diarrhea virus (BVDV). Although inoculation of these vaccines successfully induces solid immunity against CSFV, none of them could ideally meet all demands regarding to safety, efficacy, DIVA potential, and marketability. Due to the limitations of the available choices, researchers are still striving towards the development of more advanced DIVA vaccines against CSF. This review summarizes the present status of candidate CSFV vaccines that have been developed. The strategies and approaches revealed here may also be helpful for the development of new-generation vaccines against other diseases.
... Viruses 2020, 12 To study the special characteristics of BuPV in detail, a robust reverse genetics system (RGS) is essential. For other pestiviruses, such as CSFV or BVDV, many RGSs based on cDNA copies of the viral genome cloned into plasmid or bacterial artificial chromosome (BAC) vectors have already been reported [15][16][17][18][19][20]. Furthermore, recombinant pestiviruses were generated by full-length genome RT-PCR-based amplification and direct RNA generation from the amplicons without cloning steps [21]. ...
Reverse genetics systems are powerful tools for functional studies of viral genes or for vaccine development. Here, we established DNA-launched reverse genetics for the pestivirus Bungowannah virus (BuPV), where cDNA flanked by a hammerhead ribozyme sequence at the 5′ end and the hepatitis delta ribozyme at the 3′ end was placed under the control of the CMV RNA polymerase II promoter. Infectious recombinant BuPV could be rescued from pBuPV-DNA-transfected SK-6 cells and it had very similar growth characteristics to BuPV generated by conventional RNA-based reverse genetics and wild type BuPV. Subsequently, DNA-based ERNS deleted BuPV split genomes (pBuPV∆ERNS/ERNS)—co-expressing the ERNS protein from a separate synthetic CAG promoter—were constructed and characterized in vitro. Overall, DNA-launched BuPV genomes enable a rapid and cost-effective generation of recombinant BuPV and virus mutants, however, the protein expression efficiency of the DNA-launched systems after transfection is very low and needs further optimization in the future to allow the use e.g., as vaccine platform.
... Among the most promising marker vaccines candidates against CSFV are chimeric pestiviruses based on infectious cDNA clones of CSFV or BVDV (Meyers et al., 1996a(Meyers et al., , 1996bMoormann et al., 1996;Ruggli et al., 1996;Vassilev et al., 1997). ...
... Apart from this construct, several chimeric pestiviruses have been described so far, and some of them have been extensively studied in the target species (de Smit et al., 2001b;Rasmussen et al., 2007;Reimann et al., 2004;Vassilev et al., 1997). Examples are "flc11" and "flc9" that showed potential in the studies of different working groups de Smit et al., 2001b;Eble et al., 2012). ...
Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. These vaccines have usually outstanding efficacy and safety but lack differentiability of infected from vaccinated animals (DIVA or marker strategy). In contrast, the first generation of E2 subunit marker vaccines shows constraints in efficacy, application, and production. To overcome these limitations, new generations of marker vaccines are developed. A wide range of approaches have been tried including recombinant vaccines, recombinant inactivated vaccines or subunit vaccines, vector vaccines, and DNA/RNA vaccines. During the last years, especially attenuated deletion vaccines or chimeric constructs have shown potential. At present, especially two new constructs have been intensively tested, the adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine candidate “rAdV-SFV-E2” and the pestivirus chimera “CP7_E2alf”. The later was recently licensed by the European Medicines Agency. Under field conditions, all marker vaccines have to be accompanied by a potent test system. Particularly this point shows still weaknesses and it is important to embed vaccination in a well-established vaccination strategy and a suitable diagnostic workflow.
In summary, conventional vaccines are a standard in terms of efficacy. However, only vaccines with DIVA will allow improved eradication strategies e.g. also under emergency vaccination conditions in free regions. To answer this demand, new generations of marker vaccines have been developed and add now to the tool box of CSF control.
... In this study, the full-length RNA genome of a field isolate of CSFV was amplified by RT-PCR in six overlapping fragments and assembled into full-length CSFV cDNA using overlap extension PCR. This did not require sequential cloning and sub-cloning steps using restriction enzymes as reported earlier (Vassilev et al., 1997;Park et al., 2012) or modified RT-PCR as described for bovine viral diarrhea virus (Jones et al., 2006) and hepatitis C virus (Lu et al., 2005). Although, full-length amplification of different pestiviruses genome in a single step has been described (Rasmussen et al., 2008(Rasmussen et al., , 2010, however, not reproducible and reflected that this was developed with high concentrations of purified full-length viral RNA which is not always possible with field isolates where relatively lower virus titer is common (Puig et al., 2002;Shiffman et al., 2003). ...
To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.
... Studies on the BVDV RNA replication process were significantly facilitated by DNA copies (cDNAs) of the viral genome that enabled the production of infectious viral RNA by in vitro transcription (e.g., see Meyers et al., 1996;Vassilev et al., 1997;Mendez et al., 1998). Thus, reverse-genetics studies of the BVDV 39 NTR revealed a bipartite organization of this region of the viral genome. ...
Infections with bovine viral diarrhea virus (BVDV) have a huge economic impact on cattle production and reproduction worldwide. A key factor for BVDV surveillance and eventual eradication is to efficiently detect infections and to monitor herd immunity. In this study, we generated a stable, bi-cistronic BVDV that encodes the enhanced green fluorescent protein (EGFP) in addition to the viral proteins. Applying this recombinant virus, a new flow-cytometry based virus neutralization test was established that enables an accurate and reliable detection of field virus infected and vaccinated animals. The test, which is simple and fast, is expected to support novel, effective screening procedures in eradication and vaccination programs.
... To date, all infectious cDNA clones that are routinely used for molecular studies have been established from the moderately virulent Alfort/187 strain of CSFV [22]; the C-strain, which is the most well-known and widely used attenuated vaccine strain in the world [19]; the highly virulent Eystrup strain; and the avirulent Riems/IVI vaccine strain [16]. Most of the novel modified CSF vaccines, including deletion mutants, chimeric viruses, and replicons, have been developed by constructing cDNA clones of CSFV and BVDV [18, 19,22,30]. These have the potential for inducing immunity to an extent similar to that of conventional live attenuated vaccines, and present the possibility of discriminating vaccinated from infected animals. ...
Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.
... Cells were maintained at 37°C in a 5% CO 2 incubator. The cytopathic (cp) strain of bovine viral diarrhea virus (BVDV), NADL, was generated through the use of a cDNA clone, pNADLp15A [40], supplied graciously by Ruben Donis, Center for Disease Control (CDC, Atlanta, GA). ...
Very little is known about BVDV NS4B, a protein of approximately 38 kDa. However, a missense mutation in NS4B has been implicated in changing BVDV from a cytopathic to noncytopathic virus, suggesting that NS4B might play a role in BVDV pathogenesis. Though this is one possible function, it is also likely that NS4B plays a role in BVDV genome replication. For example, BVDV NS4B interacts with NS3 and NS5A, implying that NS4B is part of a complex, which contains BVDV replicase proteins. Other possible BVDV NS4B functions can be inferred by analogy to hepatitis C virus (HCV) NS4B protein. For instance, HCV NS4B remodels host membranes to form the so-called membranous web, the site for HCV genome replication. Finally, HCV NS4B is membrane-associated, implying that HCV NS4B may anchor the virus replication complex to the membranous web structure. Unlike its HCV counterpart, we know little about the subcellular distribution of BVDV NS4B protein. Further, it is not clear whether NS4B is localized to host membrane alterations associated with BVDV infection.
We show first that release of infectious BVDV correlates with the kinetics of BVDV genome replication in infected cells. Secondly, we found that NS4B subcellular distribution changes over the course of BVDV infection. Further, BVDV NS4B is an integral membrane protein, which colocalizes mainly with the Golgi compartment when expressed alone or in the context of BVDV infection. Additionally, BVDV induces host membrane rearrangement and these membranes contain BVDV NS4B protein. Finally, NS4B colocalizes with replicase proteins NS5A and NS5B proteins, raising the possibility that NS4B is a component of the BVDV replication complex. Interestingly, NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity.
These results show that BVDV NS4B is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDV genome replication. On the basis of NS4B Colocalization with NS5A and NS5B, we conclude that NS4B protein is an integral component of the BVDV replication complex.
... Durch unterschiedliche Antikörperantworten zwischen markervakzinierten und infizierten Tieren sind diese dann entweder nur im E2-ELISA oder im E2-und E RNS - ELISA positiv (de Smit, 2000; Floegel-Niesmann, 2003; Moormann, Bouma et al., 2000b; van Aarlen, 2003)Bognar, 1963; Lin, Shieh et al., 1974; Lin, 1981; Olah & Palatka, 1967; Zhou, 1980) Bouma, de Smit et al., 2000; de Smit, Bouma et al., 2000; Moormann, Bouma et al., 2000), Laevens et al., 2001; Dewulf, Laevens et al., 2004; Dewulf, Koenen et al., 2005; Ziegler & Kaden, 2002) Schweinepocken Virus (Hahn, Park et al., 2001; Hammond, McCoy et al., 2000; Hammond, Jansen et al., 2001a; Hammond, Jansen et al., 2001b; Hammond, Jansen et al., 2003; Hammond & Johnson, 2005; Hooft, I, de et al., 1996; Konig, Lengsfeld et al., 1995; Mulder, de Jong et al., 1995; Peeters, Bienkowska et al., 1997; van Zijl, Wensvoort et al., 1991; ). Die Anwendung E2- exprimierender viraler Vektoren führte in einigen Fällen bereits nach einmaliger Immunisierung von Schweinen zum vollständigen Schutz vor einer KSPV-Infektion (Hammond, McCoy et al., 2000; Hooft, I, de et al., 1996) Moormann, Van Gennip et al., 1996; Ruggli, Tratschin et al., 1996; Vassilev, Collett et al., 1997). Verschiedene chimäre Pestiviren aus BVDV und KSPV, bei denen entweder das E2-oder das E RNS -Protein ausgetauscht wurde, sind bereits beschrieben worden (de Smit, Bouma et al., 2001b; Kaden & Riebe, 2001; Kaden, Lange et al., 2004c)Epitop ist eines der zentralen KSPV-Epitope im E2, gegen welches wichtige neutralisierenden Antikörper gebildet werden (Lin, Lin et al., 2000; Qi, Liu et al., 2008; Qi, Zhang et al., 2009)ELISA beruht (Holinka, Fernandez-Sainz et al., 2009; Kortekaas, Vloet et al., 2009; Lin, Lin et al., 2000; Qi, Liu et al., 2008; Qi, Zhang et al., 2009) Within the European Union (EU), a stamping out strategy is implemented f or CSFV outbreak situations. ...
... However, the immune response after vaccination with the E2-subunit vaccine is delayed, and pigs have to be vaccinated twice for a sufficient protection (Depner et al., 2001;). Facilitated by the establishment of infectious cDNA clones of classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) ( Moorman et al., 1996; Rüggli et al., 1996; Vassilev et al., 1997) trans-complemented deletion mutants (Wjdjoatmodjo et al., 2000; Maurer et al., 2005;) and chimeric pestiviruses (van Gennip et al., 2000;, König et al.,2007a, Wehrle et al., 2007, Rasmussen et al., 2007) are the most promising candidates for the development of new, attenuated DIVA vaccines. Serological diagnostics of pestivirus infections are often based on the detection of antibodies directed against the non-structural protein NS3 or the envelope proteins E2 and E rns . ...
Classical swine fever (CSF) is one of the most important diseases of pigs. Although prophylactic vaccination is banned within the European Union, emergency vaccination, allowing differentiation of vaccinated from infected animals, is an option for disease control. Up to now, these strategies are based on antibody detection. In this context, conventional modified live vaccines are not suitable. A promising perspective could be genetic differentiation of vaccinated from infected animals where field virus strains are differentiated from vaccine viruses by sequence differences. This concept could also be used with marker vaccines. To this end, a set of real-time reverse transcription-polymerase chain reaction (RT-PCR) assays was developed and validated. Specific primers and probes were designed for detection of the C-strain "Riems" vaccine virus or the chimeric marker vaccine candidate CP7_E2alf. A heterologous internal positive control was also included. The assays were then multiplexed to detect simultaneously either CSF field virus, C-strain "Riems", and the internal control or CSF field virus, CP7_E2alf, and the internal control. To validate both systems, samples from vaccination/challenge trials were tested. Only samples from vaccinated animals were found to be positive, while all samples from wild type virus-infected animals and a broad test panel of different pestiviruses were negative. Field application of the "C-strain Riems" specific assay was proven with wild boar samples from surveillance programs in Germany and France. In conclusion, ready-to-use RT-PCR sets are presented as reliable tools for genetic differentiation of vaccinated from infected animals for CSFV eradication strategies.
... Translation of the single ORF is initiated capindependently at an internal ribosomal entry site (IRES) located in the 5 -NTR and the resulting viral polyprotein is processed into mature viral proteins including N pro , C, E rns , E1, E2, p7, NS2/3, NS4A, NS4B, NS5A and NS5B (Lindenbach and Rice, 2003). Over the past decade, several recombinant viruses have been successfully derived from the infectious full length cDNA clones of different BVDV strains such as NADL, CP7, Oregon and SD1 (Vassilev et al., 1997;Meyers et al., 1996a;Kümmerer et al., 1998;Fan and Bird, 2008a). Based on these systems, numerous mutant viruses have been created to investigate viral life cycle and pathogenesis, however if BVDV is suitable for stable expression of heterologous genes has not been investigated systematically. ...
... Reverse genetics of the positive, single-stranded RNA viruses provides a powerful tool for investigation of many aspects of the viral life cycle or pathogenesis. Although several infectious full length cDNA clones have been constructed (Mendez et al., 1998;Meyers et al., 1996a;Moormann et al., 1996;Vassilev et al., 1997;Kümmerer et al., 1998;Fan and Bird, 2008a) and numerous virus mutants have been created for investigation of viral replication and pathogenesis, the potential use of BVDV as a viral vector was not investigated. The first concern regarding BVDV as a viral vector is that BVDV genome, as a single-stranded RNA molecule, is not stable in host cells and the heterologous gene might not be maintained stably in viral genome. ...
Bovine viral diarrhea virus (BVDV) is a group of small enveloped viruses with a single-stranded, positive-oriented RNA genome of approximately 12.3 kb. BVDV genome directs the production of a viral polyprotein that is subsequently cleaved to release the mature viral proteins. To explore the potential of using BVDV as viral vector for stable expression of heterologous genes, eGFP2A was inserted in between N(pro) and C genes of a noncytopathic type-I BVDV strain SD1. eGFP2A was designed with eGFP protein in frame fused to the N terminus of the foot-and-mouth disease virus 2A protease. This strategy promised not only the correct processing of both viral N(pro) and C protein but also releasing of the chimeric protein from the nascent viral polyprotein. The recombinant reporter virus was successfully rescued in MDBK cells. In vitro study showed that eGFP2A protein, as expected, was expressed and processed properly from the nascent viral polyprotein. The reporter virus was similar to wt SD1 in viral RNA replication and protein expression and comparable to wt SD1 in growth kinetics except that this virus had a peak virus titer approximately 0.5 log(10) lower and a maximum yield about 4h later than wt SD1. In summary, these results indicated that BVDV is a suitable viral vector for stable expression of heterologous genes when inserted in between N(pro) and C genes.
... Construction of BVDV mutants and sequence analysis. BVDV mutants were generated using a reverse genetics system established previously (42,75). The introduction of mutations was confirmed by reverse transcription (RT)-PCR and sequence analysis. ...
The alpha/beta interferon (IFN-α/β) system is the first line of defense against viral infection and a critical link between
the innate and adaptive immune responses. IFN-α/β secretion is the hallmark of cellular responses to acute RNA virus infections.
As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-α/β response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under
others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces
IFN-α/β very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-α/β response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-α/β antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-α/β suppression. A catalytically active mutant with a change at a
conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-α/β production, confirming its involvement
in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-α/β induction, but also implicate the amino-terminal domain of the protein in this function.
