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1.  The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro 
PLoS Pathogens  2012;8(3):e1002598.
Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.
Author Summary
Virus particles of members of the Flaviviridae consist of an inner complex of viral RNA genome and core protein that together form the nucleocapsid, and an outer lipid layer containing the viral glycoproteins. Functional analyses of core protein of the classical swine fever virus (CSFV), a pestivirus related to hepatitis C virus (HCV), led to the observation that crippling mutations or even complete deletion of the core gene were compensated by single amino acid substitutions in the helicase domain of non-structural protein 3 (NS3). NS3 is well conserved among the Flaviviridae and acts as protease and helicase. In addition to its essential role in RNA replication, NS3 apparently organizes the incorporation of RNA into budding virus particles. Characterization of core deficient CSFV particles (Vp447Δc) revealed that the lack of core had no effect with regard to thermostability, size, density, and morphology. Vp447Δc was fully attenuated in the natural host. Our results provide evidence that core protein is not essential for virus assembly. Hence, Vp447Δc might help to explain the enigmatic existence of GB viruses -A and -C, close relatives of HCV that do not encode an apparent core protein.
PMCID: PMC3310793  PMID: 22457622
2.  Cytopathogenicity of Classical Swine Fever Virus Correlates with Attenuation in the Natural Host▿  
Journal of Virology  2008;82(19):9717-9729.
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.
PMCID: PMC2546992  PMID: 18653456
3.  CD46 Is a Cellular Receptor for Bovine Viral Diarrhea Virus 
Journal of Virology  2004;78(4):1792-1799.
Various monoclonal antibodies (MAbs) that recognize cell surface proteins on bovine cells were previously shown to efficiently block infection with bovine viral diarrhea virus (BVDV) (C. Schelp, I. Greiser-Wilke, G. Wolf, M. Beer, V. Moennig, and B. Liess, Arch. Virol. 140:1997-2009, 1995). With one of these MAbs, a 50- to 58-kDa protein was purified from calf thymus by immunoaffinity chromatography. Microchemical analysis of two internal peptides revealed significant sequence homology to porcine and human CD46. The cDNA of bovine CD46 (CD46bov) was cloned and further characterized. Heterologously expressed CD46bov was detected by the MAb used for purification. A putative function of CD46bov as a BVDV receptor was studied with respect to virus binding and susceptibility of nonpermissive cells. While the expression of CD46bov correlated well with the binding of [3H]uridine-labeled BVDV, the susceptibility of cells nonpermissive for BVDV was not observed. However, the expression of CD46bov resulted in a significant increase in the susceptibility of porcine cells to BVDV. These results provide strong evidence that CD46bov serves as a cellular receptor for BVDV.
PMCID: PMC369467  PMID: 14747544
4.  Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay 
Journal of Clinical Microbiology  1999;37(4):1049-1056.
Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the IgG class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA. By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections.
PMCID: PMC88648  PMID: 10074525

Results 1-4 (4)