Viperin inhibits classical swine fever virus replication by interacting with viral nonstructural 5A protein

Classical swine fever virus (CSFV) is a single-stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon-inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK-15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co-immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N-terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV-induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.     

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

The nonstructural protein 2 (NS2) of classical swine fever virus (CSFV) is a self-splicing ribozyme wherein the precursor protein NS2-3 is cleaved, and the cleavage efficiency of NS2-3 is crucial to the replication of viral RNA. However, the proteolytic activity of NS2 autoprotease may be achieved through a cellular chaperone called J-domain protein interacting with viral protein (Jiv) or its fragment Jiv90, as evidence suggests that Jiv is required for the proper functioning of the NS2 protein of bovine viral diarrhea virus. Hence, the expression of Jiv may be correlated with the replication efficiency of CSFV RNA. We investigated the expression levels of Jiv and viral RNA in CSFV-infected cells and tissues using Real-time RT-PCR or Western blot analysis. The obtained results show that Jiv90 possibly plays an important role in the lifecycle of CSFV because the distribution of Jiv90 protein shows a positive correlation with the viral load of CSFV. Furthermore, the overexpression or knockdown of Jiv90 in swine cells can also significantly promote or decrease the viral load, respectively. The detection of Flow cytometry shows that the overexpression of Jiv90 prolongs the G1 phase of cell cycles but has no effect on apoptosis. These findings are likely to be of benefit in clarifying the pathogenesis of the CSFV.     

Identification of a linear epitope on the capsid protein of classical swine fever virus

The capsid (C) protein of Classical swine fever virus (CSFV) is proposed to play an essential role in the replication and translation of the viral RNA. In this study, a monoclonal antibody (mAb) directed against the C protein was generated with the recombinant C protein expressed in Escherichia coli as immunogen. IFA and IPMA analysis showed that the native C protein of CSFV virions was reactive to the mAb. By truncating the C protein, we identified a linear epitope recognized by the mAb, corresponding to amino acids (61)TQDGLYHNKN(70) of the CSFV C protein, which is well conserved among pestiviruses. Laser confocal analysis showed that the C protein mainly locates in the cellular nucleoplasm and nucleolus of PK-15 cells. The results have implications for further study of CSFV replication.     

African swine fever virus replication in porcine lymphocytes

Purified preparation of porcine lymphocytes were infected with three isolates of virulent African swine fever virus (ASFV). Electron microscopy showed the presence of small numbers of mature virus particles in degenerating cells. The titres of infective virus released were low and reached a maximum by 24 h after infection.     

Phylogenetic analysis of classical swine fever virus in Taiwan

Summary. Two envelope glycoprotein (E^rns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the E^rns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (E^rns–E2) displayed better resolution than that based on the complete open reading frame.     

Evidence of natural recombination in classical swine fever virus

Classical swine fever (CSF) virus, one member of the family Flaviviridae is the pathogen of CSF, an economically important and highly contagious disease of pigs. Although homologous recombination has been demonstrated in many other members of the family, it is unknown whether there is recombination in natural populations of CSFV. To detect possible recombination events, we performed a phylogenetic analysis of 25 full-length CSFV strains isolated all over the world. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning. A mosaic virus, CSFV 39 (AF407339) isolated in China was found. And its two putative parental-like strains CSFV Shimen (AF333000) and GXWZ02 (AY367767) were identified. Our work revealed that homologous recombination occurred in natural CSFV populations, generating genetic diversity. This would provide some insights for the role homologous recombinant plays in CSFV evolution.     

Identification of an NTPase motif in classical swine fever virus NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.     

Interaction between Core protein of classical swine fever virus with cellular IQGAP1 protein appears essential for virulence in swine

Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155]. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues ²⁰⁷ATI²⁰⁹ (I), ²¹⁰VVE²¹² (II), ²¹³GVK²¹⁵ (III), or ²³²GLYHN²³⁶ (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle.     

Use of recombinant protein E2 of the classical swine fever virus for immunization of animals

Recombinant major surface glycoprotein E2 from virulent Shimen strain of classical swine fever virus (CSFV) has been tested for immunogenicity in animal immunization experiments. Immunization of 3-month-old piglets with 200 micrograms of recombinant protein protected the animals from lethal challenge with virulent CSFV strain. CSFV-specific antibody detection test based on competitive ELISA has been developed using the recombinant E2 protein. The test can evaluate specific antibody levels after subunit vaccination with recombinant E2 after immunization with live vaccine based on attenuated CSFV strain.     

Phylogenetic comparison of classical swine fever virus in China.

An N-terminal fragment of the E2 gene of classical swine fever (CSF) virus encoding major immunogenic sites was amplified by RT-PCR directly from 110 clinical specimens representing 109 epizootic sites during the last decade in China. Phylogenetic relationships between these viruses as well as 20 reference strains were determined by comparison of their nucleotide sequences. A phylogenetic tree showed that 103 of the 110 field viruses (93.6%) were clustered within group 2 and subdivided into three subgroups, while the remaining seven viruses (6.4%), along with two Chinese reference strains, Shimen and HCLV (attenuated vaccine strain), were clustered into subgroup 1.1 within group 1. However, none of the Chinese CSF viruses were members of subgroup 1.2 (represented by reference strain Brescia). This is the first report on the distribution of CSF virus genotypes in China. Results indicated that CSF viruses predominating in recent epizootics within China are genetically divergent from the reference strain Shimen and the vaccine strain HCLV.     

Analysis of synonymous codon usage in classical swine fever virus

Using the complete genome sequences of 35 classical swine fever viruses (CSFV) representing all three genotypes and all three kinds of virulence, we analyzed synonymous codon usage and the relative dinucleotide abundance in CSFV. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in CSFV. Furthermore, we observed that the relative abundance of dinucleotides in CSFV is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as the subgenotypes and aromaticity, also influence the codon usage variation among the genomes of CSFV. This study represents the most comprehensive analysis to date of CSFV codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Construction of recombinant swinepox viruses and expression of the classical swine fever virus E2 protein

To explore the swinepox virus (SPV) as a potential live vector for immunization, a vector was developed for the construction of a recombinant SPV carrying foreign genes. In this system, a foreign gene placed under the strong vaccinia virus promoter P(11) can be inserted into the viral thymidine kinase (TK) gene, and the recombinant virus can be isolated in a non-selective medium by the co-expression of E. coli lacZ gene. Compared with the wild type virus, the TK(-)recombinant SPV showed a modest level of attenuation in porcine cells while more attenuation was observed in monkey or human cells. Using this system, a recombinant virus expressing the E2 glycoprotein of classical swine fever virus (CSFV) was produced. Engineered with the gX signal sequence of the pseudorabies virus, and transmembrane domain of E2, the E2 protein was expressed as a dimeric form in the cytoplasm of the infected cells.     

Effects of the interactions of classical swine fever virus Core protein with proteins of the SUMOylation pathway on virulence in swine

Here we have identified host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme that interact with classical swine fever virus (CSFV) Core protein. Five highly conserved lysine residues (K179, K180, K220, K221, and K246) within the CSFV Core were identified as putative SUMOylation sites. Analysis of these interactions showed that K179A, K180A, and K221A substitutions disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9 binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A, K221A) harboring those substitutions were attenuated in swine. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo.     

Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice

Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections.     

Propagation of classical swine fever virus in vitro circumventing heparan sulfate-adaptation

Amplification of natural virus isolates in permanent cell lines can result in adaptation, in particular enhanced binding to heparan sulfate (HS)-containing glycosaminoglycans present on most vertebrate cells. This has been reported for several viruses, including the pestivirus classical swine fever virus (CSFV), the causative agent of a highly contagious hemorrhagic disease in pigs. Propagation of CSFV in cell culture is essential in virus diagnostics and research. Adaptation of CSFV to HS-binding has been related to amino acid changes in the viral E(rns) glycoprotein, resulting in viruses with altered replication characteristics in vitro and in vivo. Consequently, a compound blocking the HS-containing structures on cell surfaces was employed to monitor conversion from HS-independency to HS-dependency. It was shown that the porcine PEDSV.15 cell line permitted propagation of CSFV within a limited number of passages without adaptation to HS-binding. The selection of HS-dependent CSFV mutants was also prevented by propagation of the virus in the presence of DSTP 27. The importance of these findings can be seen from the altered ratio of cell-associated to secreted virus upon acquisition of enhanced HS-binding affinity, a phenotype proposed previously to be related to virulence in the natural host.     

The selection pressure analysis of classical swine fever virus envelope protein genes Erns and E2

Classical swine fever virus, one member of the family Flaviviridae, is the pathogen of CSF, an economically important and highly contagious disease of pigs. Knowledge of virus genes under positive selection pressure can help identify molecular determinants of virulence or pathogenesis without prior knowledge of the mechanisms governing virulence and pathogenesis and clarify the driving force of classical swine fever virus evolution. The positive selection pressure acting on envelope protein genes E(rns), E1 and E2 of classical swine fever virus were assessed and a site-by-site analysis of the d(N)/d(S) ratio was performed, to identify specific codons undergoing diversifying positive selection. Whilst no significant evidence for positive selection was observed in E1, four positively selected sites (208 in E(rns) and 72, 75, and 200 in E2) were identified. The positively selected site (208) of E(rns) corresponds to one of the amino acid substitutions (Ser to Arg) found in an HS-binding CSFV variant. The mutant at the positively selected site (75) is located within an O-glycosylation motif and altered the predicted glycosylation pattern. In addition, Thr at the positively selective site 200 are directly involved with mAb WH308 with which CS vaccine strain does not react, unlike most of the virulent CSFV strains.     

Detection of classical swine fever virus in the ovaries of experimentally infected sows

Six sows were infected intranasally with a Korean isolate of classical swine fever virus (CSFV). The distribution of virus in ovarian tissues was then assessed for 21 days by in-situ hybridization and immunohistochemistry. CSFV was detected in the ovaries between 7 and 21 days post-inoculation (dpi) by both methods, but the labelling was particularly intense and widespread at 7 dpi. CSFV nucleic acid and antigen were located almost exclusively within the cytoplasm of cells shown by haematoxylin and eosin staining to be macrophages, which were numerous in atretic follicles. Small numbers of CSFV nucleic acid-positive cells with distinctly round morphology and oval nuclei, resembling monocytes, were also observed in the blood vessels of sows at 7 and 14 dpi. CSFV nucleic acid and antigen were not observed in primordial, primary or secondary follicles from infected sows at 7, 14 or 21 dpi. The results suggest that CSFV replicates in circulating peripheral monocytes and gains access to ovarian tissues from the bloodstream, and that this contributes to the distribution of CSFV in macrophages throughout the atretic follicles.     

Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine

Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics in primary porcine macrophage cell cultures, the major target cell of CSFV in vivo, it was markedly attenuated in swine. All RB-C22v-infected pigs survived infection remaining clinically normal in contrast to the 100% mortality observed for BICv-infected animals. Comparative pathogenesis studies demonstrated a delay in RB-C22v spread to, and decreased replication in the tonsils, a 10(2) to 10(7) log10 reduction in virus titers in lymphoid tissues and blood, and an overall delay in generalization of infection relative to BICv. Notably, RB-C22v-infected animals were protected from clinical disease when challenged with pathogenic BICv at 3, 5, 7, and 21 days post-RB-C22v inoculation. Viremia, viral replication in tissues, and oronasal shedding were reduced in animals challenged at 7 and 21 DPI. Notably BICv-specific RNA was not detected in tonsils of challenged animals. These results indicate that a carboxyl-terminal domain of E1 glycoprotein affects virulence of CSFV in swine, and they demonstrate that mutation of this domain provides the basis for a rationally designed and efficacious live-attenuated CSF vaccine.