Removal of a N-linked glycosylation site of classical swine fever virus strain Brescia Erns glycoprotein affects virulence in swine

E(rns) glycoprotein, along with E(1) and E(2), is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E(rns) is a heavily glycosylated protein involved in several functions, including virus attachment and entry to target cells, production of neutralizing antibodies, and virulence. The role of added glycans to CSFV strain Brescia E(rns) on virus virulence was assessed in swine. A panel of virus mutants was constructed and used to investigate whether the removal of each of seven putative glycosylation sites in the E(rns) glycoprotein would affect viral virulence in swine. Only N269A/Q substitution rendered attenuated viruses (N1v/N1Qv) that, unlike BICv and other mutants, produced a transient infection in swine characterized by mild symptoms and decreased virus shedding. Notably, N1v efficiently protected swine from challenge with virulent BICv at 3 and 21 days post-infection suggesting that glycosylation of E(rns) could be modified for development of CSF live-attenuated vaccines.     

The classic swine fever virus (CSFV) core protein can enhance de novo-initiated RNA synthesis by the CSFV polymerase NS5B

Study of the classical swine fever virus (CSFV) replication is challenging because it is a BSL-3-Ag pathogen that requires specialized facilities. We developed a cell-based assay in human embryonic kidney 293T cells that can quantify the activities of NS5B, the CSFV RNA-dependent RNA polymerase. The 5BR assay uses transiently-expressed CSFV NS5B to produce RNAs that activate the RIG-I-mediated signaling pathway to result in reporter protein production. Upon co-expression of the CSFV core protein, we observed enhancement of the CSFV RdRp activity. The CSFV core and NS5B proteins could co-immunoprecipitate with each other and co-localize in cells, when visualized by confocal microscopy. Analyses of combinations of RdRps and capsid proteins from different viruses demonstrated that the CSFV core could enhance the CSFV NS5B activity in a virus species-specific manner. Studies of truncated versions of CSFV core demonstrated that the first 30 residues of core protein are dispensable for interaction with the CSFV NS5B. Purified core protein could enhance RNA synthesis by the purified NS5B in vitro, with the increase being in the synthesis of the de novo-initiated RNA. These results demonstrate that the CSFV core protein can regulate the mechanism of RNA synthesis by the CSFV RdRp.     

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.     

Alteration of the N-linked glycosylation condition in E1 glycoprotein of Classical Swine Fever Virus strain Brescia alters virulence in swine

E1, along with E^rns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previously we showed that glycosylation status of virulent CSFV strain Brescia E2 or E^rns affects virus virulence. Here, the three putative glycosylation sites of E1 were serially removed by means of site directed mutagenesis of a CSFV Brescia infectious clone (BICv) and their effect on virulence assessed in swine. Removal of all three putative glycosylation sites in E1, at CSFV positions N500, N513 and N594, yielded nonviable progeny, while single or dual site mutants excluding N594 were viable. Individual N594A (E1.N3 virus) or combined N500A/N513A (E1.N1N2 virus) substitutions resulted in BICv attenuation. Furthermore infection with E1.N3 or E1.N1N2 viruses efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. As previously observed with E^rns and E2 and here with E1 data suggest that modification of glycosylation patterns could be used for developing CSFV live-attenuated vaccines.     

Classical swine fever virus NS5B-GFP fusion protein possesses an RNA-dependent RNA polymerase activity

Summary.  RNA-dependent RNA polymerase (RdRp) is the replicase of positive-strand RNA viruses. Expression and characterization of the replicase are the first steps in the elucidation of the virus replication mechanism. We expressed nonstructural protein 5B (NS5B) of classical swine fever virus (CSFV) as a fusion protein with green fluorescent protein (GFP) in porcine kidney cells (PK-15 cells), natural host cells of CSFV. The expressed CSFV NS5B-GFP fusion protein possessed RdRp activity. By fluorescence microscope it was observed that the density of the fusion protein near cytoplasmic membranes was higher than that in other parts of cells. This was in contrast to the distribution of the GFP alone which was uniformly distributed throughout the cytoplasm. The GFP is a signal for the location of NS5B in a host cell that allows in vitro and in vivo investigation of the distribution of plus-strand RNA virus RdRp. Received September 24, 2001; accepted March 23, 2002 Published online July 19, 2002     

Mutational analysis of the GDD sequence motif of classical swine fever virus RNA-dependent RNA polymerases

To define the function of the GDD motif of the RNA-dependent RNA polymerase (RdRp) of classical swine fever virus (CSFV), single amino acid substitutions were introduced into the CSFV NS5B. All substitutions within the GDD motif were detrimental to the polymerase activity, the binding activity and the terminal nucleotidyl transferase activity of the NS5B protein. It was also found that the wild-type NS5B had higher RdRp activity with Mg(+2) than with Mn(+2) whereas some mutants worked better with Mn(+2) than with Mg(+2), suggesting that substitutions within the GDD motif modified the enzyme cation preferences and the GDD sequence of CSFV NS5B might be involved in polymerase-metal interaction. Therefore, the GDD amino acid sequence is important for the function of CSFV RdRp.     

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.     

Mutations in the carboxyl terminal region of E2 glycoprotein of classical swine fever virus are responsible for viral attenuation in swine

We have previously reported [Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L. 2005a. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J. Virol. 79, 3787-3796] that chimeric virus 319.1v containing the E2 glycoprotein gene from Classical Swine Fever Virus (CSFV) vaccine strain CS with the genetic background of highly virulent CSFV strain Brescia (BICv) was markedly attenuated in pigs. To identify the amino acids mediating 319.1v attenuation a series of chimeric viruses containing CS E2 residues in the context of the Brescia strain were constructed. Chimera 357v, containing CS E2 residues 691 to 881 of CSFV polyprotein was virulent, while chimera 358v, containing CS E2 residues 882 to 1064, differing in thirteen amino acids from BICv, was attenuated in swine. Single or double substitutions of those amino acids in BICv E2 to CS E2 residues did not affect virulence. Groups of amino acids were then substituted in BICv E2 to CS E2 residues. Mutant 32v, with six substitutions between residues 975 and 1059, and mutant 33v, with six substitutions between 955 and 994, induced disease indistinguishable from BICv. Mutant 31v, with seven substitutions between residues 882 and 958, induced a delayed onset of lethal disease. Amino acids abrogating BICv virulence were then determined by progressively introducing six CS residues into 31v. Mutant 39v, containing nine residue substitutions, was virulent. Mutant 40v, containing ten residue substitutions, induced mild disease. Mutant 42v, containing twelve substitutions, and mutant 43v, with an amino acid composition identical to 358v, were attenuated in swine indicating that all substitutions were necessary for attenuation of the highly virulent strain Brescia. Importantly, 358v protected swine from challenge with virulent BICv at 3 and 28 days post-infection.     

Phylogenetic analysis of recent isolates of classical swine fever virus from Colombia

The ability to discriminate between different classical Swine fever virus (CSFV) isolates is a prerequisite for identifying the possible origin of an outbreak. To determine the relatedness between Colombian isolates from different geographical regions, genetic sequences of the glycoprotein E2 and the 5'UTR of CSFV were amplified by PCR, sequenced and compared with reference strains of different genetic grouping. The viruses originated from classical swine fever (CSF) outbreaks in Colombia during 1998-2002. All viruses characterized belonged to genogroup 1 and were members of the subgroup 1.1. The results indicate that the outbreaks from the year 2002 are caused by a strain related to the virus CSF/Santander, isolated in 1980, suggesting that the current CSF outbreaks are the consequence of a single strain that continues to circulate in the field. For the first time, an association between isolates from outbreaks in Colombia in the 1990s was established with a virus isolate from Brazil, indicating a possible origin of the virus causing the outbreak.     

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.     

Origin and evolution of viruses causing classical swine fever in Cuba

We have analyzed the origin and evolution of viruses from the classical swine fever (CSF) epidemic that affects Cuba since 2001 by nucleotide sequencing of regions within the E2 glycoprotein and the NS5B (polymerase) genes. The sequence of 190 nucleotides from E2 gene was determined for 10 CSF viruses isolated at different locations of the island, and used for phylogenetic analyses, including sequences from viruses of the 1993--1997 epizootic, previously determined, as well as those from representatives of the different CSFV genotypes. The phylogenetic tree obtained indicates that viruses circulating at present belong to the subgroup 1.2 and are closely related to those isolated during the 1993--1997 epizootic, including the strain Margarita used for vaccine potency tests in Cuba. However, the pattern of evolution revealed by these analyses was different than that observed previously, in which western isolates were almost identical to Margarita strain, while eastern isolates showed a higher level of genetic diversification. In this case, all the viruses analyzed grouped in an independent, define cluster that is closely related, albeit distinguishable, from that of Margarita-related viruses that previously circulated in the western part of Cuba. In addition, the 2001--2003 viruses showed a branched pattern with a level of sequence diversification similar to that observed in the eastern 1993--1997 viruses. Interestingly, a significant fraction (about 54%) of the mutations found in the E2 sequence led to amino acid replacements. This high rate of non-synonymous mutations was not found in the previous Cuban epizootic and has not been reported for other CSF outbreaks. In spite of these amino acid replacements, no antigenic changes were observed in the reactivity of different isolates with CSFV-specific MAbs and polyclonal sera. The phylogenetic tree derived from 409 nucleotides of NS5B gene of seven isolates and Margarita strain, was consistent with that obtained from E2 sequences. In this region, encoding a non-structural protein, a low level of fixation of non-synonymous mutations was observed. The results obtained suggests that epidemiological factors affecting CSFV spread during the current epizootic in Cuba can favour the fixation of non-synonymous mutation in the E2 gene, which could be associated with a lower severity in the clinical signs developed by most of the affected animals.     

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.     

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.     

Development of a live attenuated antigenic marker classical swine fever vaccine

Until recently strategies for controlling Classical Swine Fever Virus (CSFV) involve either prophylactic vaccination or non-vaccination with elimination of infected herds depending on the epidemiological situation of the affected geographical area. Marker vaccines allowing distinction between naturally infected from vaccinated swine could complement “stamping out” measures. Here we developed a double antigenic marker live attenuated CSFV strain FlagT4v obtained by combining two genetic determinants of attenuation. FlagT4v harbors a positive antigenic marker, synthetic Flag® epitope, introduced via a 19mer insertion in E1 glycoprotein; and a negative marker resulting from mutations of the binding site of monoclonal antibody WH303 (mAbWH303) epitope in E2 glycoprotein. Intranasal or intramuscular administration of FlagT4v protected swine against virulent CSFV Brescia strain at early (2 or 3 days), and late (28 days) time post-inoculation. FlagT4v induced antibody response in pigs reacted strongly against the Flag® epitope but failed to inhibit binding of mAbWH303 to a synthetic peptide representing the WH303 epitope. These results constitute a proof-of-concept for rationally designing a CSFV antigenically marked live attenuated virus.     

Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303 epitope in CSFV virulent strain Brescia (BICv) to obtain the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). In vitro growth of mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) was similar to parental BICv, while mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and reduced plaque size. In vivo, T1v, T2v or T3v induced lethal disease, T4v induced mild and transient disease and T5v induced mild clinical signs. Protection against BICv challenge was observed at 3 and 21 days post-T5v infection. These results indicate that E2 residues TAVSPTTLR play a significant role in CSFV virulence.     

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.     

Phylogenetic Comparison and Molecular Epidemiology of Classical Swine Fever Virus

The genetic diversity of classical swine fever virus (CSFV) was studied by RT-PCR amplification and sequencing of a 409 bp fragment of the NS5B polymerase region. A total of 106 viruses isolated from 20 countries over a period of 52 years (1945–1997) were included in the phylogenetic study. The results showed that the viruses could be divided into two main groups. Group 1 consisted of Asian and South American isolates from the 1980s, as well as of old European and American isolates. Group 2 consisted mostly of recent European viruses from the 1980s and 1990s, and was further divided into three subgroups largely according to geographic origin and/or year of isolation. Five 1997 CSFV isolates from Germany, Netherlands and Italy clustered together indicating a common origin for these outbreaks, but two other 1997 isolations in different regions of Germany are likely due to different epidemiological events. The results show that the NS5B region of the genome gives a good resolution for phylogenetic studies of CSFV. Molecular epidemiology based on nucleotide sequence diversity is a useful tool for tracing virus spread and for developing disease control strategies.