scholarly journals Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation

2019 ◽  
Vol 93 (18) ◽  
Author(s):  
D. Dubrau ◽  
S. Schwindt ◽  
O. Klemens ◽  
H. Bischoff ◽  
N. Tautz
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Rna Replication ◽  
Fever Virus ◽  
Genome Packaging ◽  
Gain Of Function ◽  
Diarrhea Virus ◽  
Virion Production ◽  
Virion Morphogenesis ◽  
Virion Formation

ABSTRACTFor members of theFlaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCEForFlaviviridaemembers, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

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 Analysis of hepatitis C virus/classical swine fever virus chimeric 5′NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication

2003 ◽  
Vol 84 (7) ◽  
pp. 1761-1769 ◽  
Author(s):  
Chantal B. E. M. Reusken ◽  
Tim J. Dalebout ◽  
Peter Eerligh ◽  
Peter J. Bredenbeek ◽  
Willy J. M. Spaan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Rna Replication ◽  
Fever Virus ◽  
Viral Rna

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 Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

2009 ◽  
Vol 90 (12) ◽  
pp. 2923-2928 ◽  
Author(s):  
Ming Xiao ◽  
Yujing Wang ◽  
Zailing Zhu ◽  
Jialin Yu ◽  
Lingzhu Wan ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Border Disease Virus ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Diarrhea Virus ◽  
Border Disease

An internal ribosome entry site (IRES) present in the 5′ untranslated region (UTR) promotes translation of classical swine fever virus (CSFV) genomes. Using an in vitro system with monocistronic reporter RNA containing the CSFV 5′UTR, this study found that CSFV NS5A decreased CSFV IRES-mediated translation in a dose-dependent manner. Deletion analysis showed that the region responsible for repressing CSFV IRES activity might cover aa  390–414, located in the C-terminal half of CSFV NS5A. Triple and single alanine-scanning mutagenesis revealed that the inhibitory effect on CSFV IRES-directed translation mapped to the K399, T401, E406 and L413 residues of NS5A. These important amino acids were also found to be present in the NS5A proteins of bovine viral diarrhea virus (BVDV)-1, BVDV-2, border disease virus and hepatitis C virus, indicating that NS5A may play an important role in the switch from translation to replication in these viruses.

Molecular chaperone Jiv promotes the RNA replication of classical swine fever virus

Virus Genes ◽  
2017 ◽  
Vol 53 (3) ◽  
pp. 426-433 ◽  
Author(s):  
Kangkang Guo ◽  
Haimin Li ◽  
Xuechao Tan ◽  
Mengmeng Wu ◽  
Qizhuang Lv ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Rna Replication ◽  
Fever Virus

scholarly journals Serum neutralization as a differential serological test for classical swine fever virus and other pestivirus infections

1999 ◽  
Vol 51 (5) ◽  
pp. 403-408 ◽  
Author(s):  
J.C.M. Paredes ◽  
E.A.S. Oliveira ◽  
L.G. Oliveira ◽  
J.C.A. Rosa ◽  
P.M. Roehe
Keyword(s):  
Classical Swine Fever Virus ◽  
Serological Test ◽  
Sampling Strategy ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Border Disease Virus ◽  
Serum Samples ◽  
Diarrhea Virus ◽  
Serum Neutralization ◽  
Border Disease

Serum neutralization tests (SN) were performed against classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV) and border disease virus (BDV) on samples of swine serum collected for screening of antibodies to CSFV, in order to determine the SN value as a differential serological test. Ninety-nine sera out of a sample of 16,664 were positive for antibodies to pestiviruses in an ELISA test which did not distinguish antibodies to different pestiviruses. When submitted to SN, 81 sera were positive for CSFV antibodies only. In 17 sera, crossreactive antibodies to either CSFV, BVDV or BDV were detected. In most of these sera (13 out of 17) the differences between SN titres against the three viruses were not sufficient to estimate which was the most likely antibody-inducing virus. It was concluded that, for the SN to be useful in such differentiation, it is essential to examine a sample which must include a representative number of sera from the same farm where suspect animals were detected. When isolated serum samples are examined, such as those obtained with the sampling strategy adopted here, the SN may give rise to inconclusive results.

scholarly journals Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1655
Author(s):  
Yu-Liang Huang ◽  
Denise Meyer ◽  
Alexander Postel ◽  
Kuo-Jung Tsai ◽  
Hsin-Meng Liu ◽  
...  
Keyword(s):  
Disease Virus ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Conformational Epitope ◽  
Cross Reactivity ◽  
Fever Virus ◽  
Site Directed Mutagenesis ◽  
Border Disease Virus ◽  
Diarrhea Virus ◽  
Border Disease

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

scholarly journals Cytopathogenic and Noncytopathogenic RNA Replicons of Classical Swine Fever Virus

1999 ◽  
Vol 73 (9) ◽  
pp. 7787-7794 ◽  
Author(s):  
Christian Moser ◽  
Peter Stettler ◽  
Jon-Duri Tratschin ◽  
Martin A. Hofmann
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Rna Replication ◽  
Virus Genome ◽  
Helper Virus ◽  
Fever Virus ◽  
Regulatory Function ◽  
Transfected Cells ◽  
Cell Extracts

ABSTRACT To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding Npro, C, Erns, E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.

scholarly journals Cytopathogenicity of Classical Swine Fever Virus Correlates with Attenuation in the Natural Host

2008 ◽  
Vol 82 (19) ◽  
pp. 9717-9729 ◽  
Author(s):  
Andreas Gallei ◽  
Sandra Blome ◽  
Stefanie Gilgenbach ◽  
Norbert Tautz ◽  
Volker Moennig ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Neutralizing Antibodies ◽  
Defense Mechanisms ◽  
Animal Experiments ◽  
Classical Swine Fever ◽  
Helper Virus ◽  
Natural Host ◽  
Fever Virus ◽  
Diarrhea Virus ◽  
Culture Cells

ABSTRACT For the important livestock pathogens classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), cytopathogenic (cp) and non-cp viruses are distinguished according to the induction of apoptosis in infected tissue culture cells. However, it is currently unknown whether cp CSFV differs from non-cp CSFV with regard to virulence in the acutely infected host. In this study, we generated helper virus-independent CSFV Alfort-Jiv, which encompasses sequences encoding domain Jiv-90 of cellular J-domain protein interacting with viral protein (Jiv). Expanding the knowledge of BVDV, our results suggest that Jiv acts as a regulating cofactor for the nonstructural (NS) protein NS2 autoprotease of CSFV and initiates NS2-3 cleavage in trans. For Alfort-Jiv, the resulting expression of large amounts of NS3 correlated with increased viral RNA synthesis and viral cytopathogenicity. Moreover, both cp Alfort-Jiv and the parental non-cp CSFV strain Alfort-p447 efficiently replicate in cell culture. Animal experiments demonstrated that in contrast to parental non-cp Alfort-p447, infection with cp Alfort-Jiv did not cause disease in pigs but induced high levels of neutralizing antibodies, thus elucidating that cp CSFV is highly attenuated in its natural host. In contrast to virulent Alfort-p447, the attenuated CSFV strain Alfort-Jiv induces the expression of cellular Mx protein in porcine PK-15 cells. Accordingly, the remarkable difference between cp and non-cp CSFV with regard to the ability to cause classical swine fever in pigs correlates with different effects of cp and non-cp CSFV on cellular antiviral defense mechanisms.

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