scholarly journals Characterization of a Cytopathogenic Reporter CSFV

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1209
Author(s):  
Carina Maria Reuscher ◽  
Lisa Schmidt ◽  
Anette Netsch ◽  
Benjamin Lamp
Keyword(s):  
Reverse Genetics ◽  
Classical Swine Fever ◽  
Genetically Engineered ◽  
Rna Recombination ◽  
Live Vaccines ◽  
Diarrhea Virus ◽  
Persistent Infections ◽  
Cost Efficient ◽  
Species Specific ◽  
Viral Diarrhea Virus

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

scholarly journals CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
O. Isken ◽  
A. Postel ◽  
B. Bruhn ◽  
E. Lattwein ◽  
P. Becher ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Temporal Control ◽  
Rna Recombination ◽  
Diarrhea Virus ◽  
Nonstructural Protein 2 ◽  
Persistent Infections ◽  
Cellular Cofactor ◽  
Viral Diarrhea Virus

ABSTRACTPestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCEOnly noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

scholarly journals Porcine complement regulatory protein CD46 is a major receptor for atypical porcine pestivirus but not for classical swine fever virus

2021 ◽  
Author(s):  
Gökce Nur Cagatay ◽  
Aleksandra Antos ◽  
Oliver Suckstorff ◽  
Olaf Isken ◽  
Norbert Tautz ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Regulatory Protein ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Bovine Viral Diarrhea ◽  
Complement Regulatory Protein ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus ◽  
Entry Mechanism

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig expressing and areas of non-expressing cells and one single cell line revealed not to express any CD46pig, respectively. The CD46pig deficient porcine lymphoma cells, known to facilitate CSFV replication, was the only porcine cell line non-permissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized. IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in absence of CD46pig. Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

scholarly journals Characterization of Helper Virus-Independent Cytopathogenic Classical Swine Fever Virus Generated by an In Vivo RNA Recombination System

2005 ◽  
Vol 79 (4) ◽  
pp. 2440-2448 ◽  
Author(s):  
Andreas Gallei ◽  
Till Rümenapf ◽  
Heinz-Jürgen Thiel ◽  
Paul Becher
Keyword(s):  
Classical Swine Fever Virus ◽  
Nonstructural Protein ◽  
Classical Swine Fever ◽  
Helper Virus ◽  
Fever Virus ◽  
Tissue Culture Cell ◽  
Rna Recombination ◽  
Diarrhea Virus ◽  
Recombination System

ABSTRACT Molecular analyses revealed that most cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In contrast to bovine viral diarrhea virus (BVDV), cp classical swine fever virus (CSFV) field isolates were rarely detected and always represented helper virus-dependent subgenomes. To investigate RNA recombination in more detail, we recently established an in vivo system allowing the efficient generation of recombinant cp BVDV strains in cell culture after transfecting a synthetic subgenomic and nonreplicatable transcript into cells being infected with noncp BVDV (A. Gallei, A. Pankraz, H.-J. Thiel, and P. Becher, J. Virol. 78:6271-6281, 2004). Using an analogous approach, the first helper virus-independent cp CSFV strain (CP G1) has now been generated by RNA recombination. Accordingly, this study demonstrates the applicability of RNA recombination for designing new viral RNA genomes. The genomic RNA of CP G1 has a calculated size of 18.139 kb, almost 6 kb larger than all previously described CSFV genomes. It contains cellular sequences encoding a polyubiquitin fragment directly upstream of the nonstructural protein NS3 coding gene together with a duplication of viral sequences. CP G1 induces a cytopathic effect on different tissue culture cell lines from pigs and cattle. Subsequent analyses addressed growth kinetics, expression of NS3, and genetic stability of CP G1.

scholarly journals Protection of Piglets with Maternally Derived Antibodies from Sows Inoculated with an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns)

Pathogens ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 608
Author(s):  
SeEun Choe ◽  
Jihye Shin ◽  
Ki-Sun Kim ◽  
Sok Song ◽  
Ra Mi Cha ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Classical Swine Fever Virus ◽  
Protective Efficacy ◽  
Classical Swine Fever ◽  
Antibody Detection ◽  
Passive Immunity ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

Here, we investigated the protective efficacy provided by passive immunity induced by a classical swine fever (Flc-LOM-BErns) vaccine with the newly developed DIVA (Differentiating Infected from Vaccinated Animals) function. Ten pigs (aged 40–60 days) with maternally derived antibodies (MDAs) obtained from sows inoculated with the Flc-LOM-BErns vaccine were challenged with virulent classical swine fever virus (CSFV). Pigs with an MDA titer of 6 log2 induced by the Flc-LOM-BErns vaccine were fully protected against virulent CSFV challenge but not the pigs with an MDA titer under 5 log2. In addition, Flc-LOM-BErns vaccine-derived MDAs successfully differentiated vaccinated pigs by bovine viral diarrhea virus (BVDV) Erns/CSFV Erns antibody detection, functioning as a DIVA.

scholarly journals Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates

2010 ◽  
Vol 30 (12) ◽  
pp. 1053-1057 ◽  
Author(s):  
Sandra Arenhart ◽  
Fernando V. Bauermann ◽  
Fernanda S.F. Vogel ◽  
Rudi Weiblen ◽  
Eduardo F. Flores
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Persistent Infections ◽  
Fetal Infection ◽  
Viral Diarrhea Virus ◽  
Antigenic Profile ◽  
Virus Isolates

Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV - may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.

scholarly journals Nonhomologous RNA Recombination in Bovine Viral Diarrhea Virus: Molecular Characterization of a Variety of Subgenomic RNAs Isolated during an Outbreak of Fatal Mucosal Disease

1999 ◽  
Vol 73 (7) ◽  
pp. 5646-5653 ◽  
Author(s):  
Paul Becher ◽  
Michaela Orlich ◽  
Matthias König ◽  
Heinz-Jürgen Thiel
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Sequence Similarity ◽  
Bovine Viral Diarrhea ◽  
Nucleotide Sequencing ◽  
Rna Recombination ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Mucosal Disease ◽  
Subgenomic Rnas ◽  
Viral Diarrhea Virus

ABSTRACT Four bovine viral diarrhea virus type 2 (BVDV-2) pairs consisting of cytopathogenic (cp) and noncp BVDV-2 were isolated during an outbreak of mucosal disease. Comparative sequence analysis showed that the four noncp BVDV-2 isolates were almost identical. For the cp BVDV-2 isolates, viral subgenomic RNAs were shown by Northern blot to have a length of about 8 kb, which is about 4.3 kb shorter than the genome of noncp BVDV. Cytopathogenicity and the expression of NS3 were both strictly correlated to the presence of viral subgenomic RNAs. By reverse transcription-PCR, Southern blot analysis, and nucleotide sequencing, a set of 11 unique subgenomes was identified with up to 5 different subgenomes isolated from one animal. To our knowledge, this is the first report on isolation of a set of pestiviral subgenomes from individual animals. Common features of the BVDV-2 subgenomic RNAs include (i) deletion of most of the genomic region encoding the structural proteins, as well as the nonstructural proteins p7 and NS2, and (ii) insertion of cellular (poly)ubiquitin coding sequences. Three subgenomes also comprised 15 to 75 nucleotides derived from the 5′ part of the NS2 gene. Comparisons of the obtained nucleotide sequences revealed that the different BVDV-2 subgenomes evolved from the respective noncp BVDV-2 by RNA recombination. The presence of short regions of sequence similarity at several crossing-over sites suggests that base pairing between the nascent RNA strand and the acceptor RNA template facilitates template switching of the BVDV RNA-dependent RNA polymerase.

scholarly journals Special Issue: Bovine Viral Diarrhea Virus and Related Pestiviruses

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1181
Author(s):  
Helle Bielefeldt-Ohmann
Keyword(s):  
Classical Swine Fever ◽  
Bovine Viral Diarrhoea Virus ◽  
Border Disease Virus ◽  
Diarrhea Virus ◽  
Border Disease ◽  
Diarrhoea Virus ◽  
Positive Strand Rna ◽  
Viral Diarrhea Virus

The genus Pestivirus, encompassing small positive-strand RNA viruses in the family Flaviviridae, comprises four viruses of very significant economic impact to the cattle, swine and sheep industries worldwide: bovine viral diarrhoea virus (BVDV) type 1 and type 2, classical swine fever virus (CSFV) and border disease virus (BDV) [...]

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