To identify genetic determinants of classicalswinefevervirus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2glycoprotein in swinevirulence.
E2 is one of the three envelope glycoproteins of classicalswinefevervirus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classicalswinefever live attenuated vaccines.
In fact, for protein production understanding cellular metabolism is essential (Morokuma et al., 2017) and for large scale protein production, baculovirus system could be an alternative source. Also, for large scale foreign protein production, the baculovirus expression system is a cheap, quick and easy method (Chico E., 2000). For expression of protein in eukaryotic system, it is one of the most efficient and popular tools (Miao et al., 2006). Therefore, here, we demonstrated ways to create an effective method for producing vaccines against CSFV in pig industry by cloning the E2 gene. The successful delivery of E2 baculovirus in BmN cells and silkworm larvae, confirmed by SDS-PAGE and western blot open up ways for large scale vaccine production.
Subunit recombinant vaccines against classicalswinefevervirus (CSFV) are a promising alternative to overcome practical and biosafety issues with inactivated vaccines. One of the strategies in evaluation under field conditions is the use of a new marker E2-based vaccine produced in the milk of adenovirally transduced goats. Previously we had demonstrated the efficacy of this antigen, which conferred early protection and long-lasting immunity in swine against CSFV infection. Here, we have used a sim- pler downstream process to obtain and formulate the recombinant E2glycoprotein expressed in the mammary gland. The ex- pression levels reached approximately 1.7 mg/ml, and instead of chromatographic separation of the antigen, we utilized a clarifi- cation process that eliminates the fat content, retains a minor amount of caseins, and includes an adenoviral inactivation step that improves the biosafety of the final formulation. In a vaccination and challenge experiment in swine, different doses of the E2 antigen contained within the clarified whey generated an effective immune response of neutralizing antibodies that protected all of the animals against a lethal challenge with CSFV. During the immunization and after challenge, the swine were monitored for adverse reactions related to the vaccine or symptoms of CSF, respectively. No adverse reactions or clinical signs of disease were observed in vaccinated animals, in which no replication of CSFV could be detected after challenge. Overall, we consider that the simplicity of the procedures proposed here is a further step toward the introduction and implementation of a commercial sub- unit vaccine against CSF.
Classicalswinefever is a highly contagious disease of swine caused by classicalswinefevervirus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classicalswinefevervirus (CSFV). Two B-cell linear epitopes rE2-ba from the E2glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865) and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716), were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified- pathogen-free (SPF) piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope- vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10 6 TCID 50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without
Generation of VVR. The regions of the CSFV polyprotein expressed by the different VVR are schematically shown in Fig. 1. For the expression of E0, the putative internal signal se- quence localized immediately upstream of the E0 N terminus was included. While the exact start of this signal peptide is not known, sequence comparison with other signal sequences (33) suggested the use of a construct starting with aa 250 (alanine) of the CSFV polyprotein. VVR encoding E1 or E2 contained the PRV-SP upstream of the respective CSFV sequences to assure translocation of recombinant CSFV proteins into the lumen of the endoplasmic reticulum. When PRV-SP was used, effective translocation of CSFV glycoproteinE2 occurred in cells infected with PRV-CSFV recombinants (18). In order to obtain heterodimerization, an additional VVR encoding E1 together with E2 was generated. Translational stop codons at the 3 9 ends of inserted CSFV nucleotide sequences were in- troduced to prevent the synthesis of fusion proteins.
cells (20, 41, 56). Unfortunately, there is only limited information available on the receptors and pathways involved in CSFV binding and entry. Further investigations are necessary to clarify the im- pact of the alanine at position 830 of E2 on virus infection of swine cells. E2 is exposed on the outer surface of the virus and is the most immunodominant protein inducing the major neutralizing anti- bodies in infected pigs (58). The amino acid at position 830 is located in the N-terminal region of E2, and this region is the min- imal domain required for binding to pig antibodies generated during CSFV infection (29, 61). Recent studies support the im- portance of this domain for the pathogenicity of CSFV. In the Brescia strain, amino acid substitutions in this domain, including the amino acid at position 830, attenuated virulence in pigs (37). In NS4B, the amino acid substitutions at positions 2475 and 2563 have not yet been reported to be involved in virulence. On the basis of the predicted topology of CSFV NS4B, the residues at positions 2475 and 2563 are located in the N-terminal and C-ter- minal transmembrane domains, respectively. A recent study on the biosynthesis of the CSFV nonstructural proteins showed that NS4B is mostly found attached to NS5A (27). The role of trans- membrane domains of CSFV NS4B in the virus life cycle is not well understood, while these domains of hepatitis C virus (HCV) are reported to play a role in viral replication (15). Importantly, a study identified a putative Toll/interleukin-1 receptor-like do- main in the C-terminal region of CSFV NS4B. Mutations in this domain of NS4B in the highly virulent CSFV Brescia backbone, immediately downstream of residue 2563 identified here, resulted in an attenuated phenotype along with enhanced activation of Toll-like receptor 7 (TLR-7)-induced genes (7). NS4B also har- bors a nucleoside triphosphatase (NTPase) motif. The NTPase activity is an absolute requirement for CSFV replication, but there is no evidence yet that modulating the NTPase activity may affect virulence (10). The 2 amino acids of NS4B identified in the present study are not located within the NTPase motif but clearly deter- mine the replication efficiency of the virus in vivo and in vitro. These substitutions may enhance the efficiency of viral RNA rep- lication by influencing the stability of the replicase complex and/or the interaction of the replicase complex with host factors in swine. However, the function of NS4B in the replication complex
Pseudorabies (PR) or Aujeszky’s disease (AD), caused by pseu- dorabies virus (PRV), also known as suid herpesvirus 1 (SHV-1), is another economically important viral disease of pigs and other animals in many regions, especially in many developing countries (5, 6). The disease is characterized by high mortality in newborn pigs, respiratory illness in growing pigs, and abortions and still- births in sows (5). PRV belongs to the Alphaherpesvirinae subfam- ily of the Herpesviridae family and has a number of features that make it an attractive candidate for a viral vector (7). The PRV genome is approximately 145 kb and composed of a unique long (UL) region, a unique short (US) region, large inverted repeat sequences, internal repeats (IRs), and terminal repeats (TRs). There exist many nonessential regions, such as genes coding for thymidine kinase (TK), gE, gG, gC, protein kinase (PK), ribonu- cleotide reductase (RR), and dUTPase. This means that these genes can be deleted or replaced by heterogeneous genes without affecting the in vitro and/or in vivo replication in most cases, in- stead resulting in reduced virulence in animals. Thus, PRV can be used to develop economical and promising vectored vaccines. A number of PRV recombinants vectored by several gene-deleted vaccines were generated to express foreign genes (7–12).
Classicalswinefever, also called hog cholera or European swinefever, is a viral disease leading to severe economic losses worldwide (34). The causative agent is classicalswinefevervirus (CSFV) (17, 18, 28, 30), a member of the Pestivirus genus, family Flaviviridae (4, 7, 29). Other members of this genus are bovine viral diarrhea virus and border disease virus of sheep (4). The positive-stranded RNA genome comprises a single long open reading frame (15, 19). Viral gene expression occurs by translation of this open reading frame into a large polypro- tein which is processed co- and posttranslationally by virus- as well as host cell-encoded proteases (23, 27, 35). The 5 9 -termi- nal part of the pestiviral RNA encodes the structural proteins, namely, the capsid protein C and three envelope glycoproteins (E0, E1, and E2) (30). E0 and E2 induce virus-neutralizing antibodies (32, 33); both glycoproteins have been shown to induce protective immunity in the natural host (9, 12, 22, 31). The E0 glycoprotein forms a disulfide-bonded homodimer with an apparent molecular mass of about 97 kDa. The corre- sponding monomers have slightly differing molecular masses (44 and 48 kDa, respectively), probably due to variation in glycosylation. Each monomer consists of 227 amino acids and corresponds to residues 268 to 494 of the CSFV polyprotein (15, 23). This indicates that about 50% of the molecular mass of the mature E0 glycoprotein is made up of carbohydrates. E0 is apparently not attached to the membrane by a transmem- brane helix, and a considerable portion of the protein is actu- ally secreted from infected cells (23). The mechanism by which E0 is bound to the virion surface is yet to be elucidated (32). Only recently, an additional function of E0 was discovered (8, 24). Comparative amino acid sequence analysis with sensitive search algorithms revealed the existence of sequence features in the deduced primary structure of E0 characteristic of a family of fungal and plant RNases (24). Subsequent biochem- ical analysis of E0 glycoprotein purified from cells infected with CSFV demonstrated that E0 actually possesses RNase activity (24).
Classicalswinefevervirus (CSFV), bovine viral diarrhea virus (BVDV), and border disease virus belong to the genus Pestivirus within the family Flaviviridae (10). The viruses are structurally, antigenically, and genetically closely related. BVDV and border disease virus can infect ruminants and pigs. CSFV infections are restricted to pigs (6). Pestiviruses are small, enveloped, positive-stranded RNA viruses (23). The ge- nome of pestiviruses varies in length from 12.5 to 16.5 kb (1, 2, 7, 17, 19, 25, 26, 28, 32) and contains a single large open reading frame (ORF) (1, 7, 8, 17, 26). The ORF is translated into a polyprotein which is processed into mature proteins by viral and host cell proteases (30). The envelope of the pestivi- rus virion contains three glycoproteins, E rns , E1, and E2 (35).
The E2 protein of classicalswinefevervirus (CSFV) is an envelope glycoprotein that is involved in virus attachment and entry. To date, the E2-interacting cellular proteins and their involvement in viral replication have been poorly documented. In this study, thioredoxin 2 (Trx2) was identified to be a novel E2-interacting partner using yeast two-hybrid screening from a porcine macrophage cDNA library. Trx2 is a mitochondrion-associated protein that participates in diverse cellular events. The Trx2-E2 interaction was further confirmed by glutathione S-transferase (GST) pulldown, in situ proximity ligation, and laser confocal assays. The thioredoxin domain of Trx2 and the asparagine at position 37 (N37) in the E2 protein were shown to be critical for the interaction. Silencing of the Trx2 expression in PK-15 cells by small interfering RNAs significantly promotes CSFV replica- tion, and conversely, overexpression of Trx2 markedly inhibits viral replication of the wild-type (wt) CSFV and to a greater ex- tent that of the CSFV N37D mutant, which is defective in binding Trx2. The wt CSFV but not the CSFV N37D mutant was shown to reduce the Trx2 protein expression in PK-15 cells. Furthermore, we demonstrated that Trx2 increases nuclear factor kappa B (NF- B) promoter activity by promoting the nuclear translocation of the p65 subunit of NF- B. Notably, activation of the NF-B signaling pathway induced by tumor necrosis factor alpha (TNF-␣) significantly inhibits CSFV replication in PK-15 cells, whereas blocking the NF-B activation in Trx2-overexpressing cells no longer suppresses CSFV replication. Taken together, our findings reveal that Trx2 inhibits CSFV replication via the NF- B signaling pathway.
Pestiviruses are the etiologic agents of economically impor- tant diseases of animals in many countries worldwide. Accord- ing to the host animals from which the viruses originate, pres- ently known pestivirus isolates have been grouped into three different species which together form one genus within the family Flaviviridae. A new taxonomy with four virus species has been proposed that is based on the results of sequence com- parison studies and takes into account that pestiviruses infect different host species. Pestiviruses predominantly found in ru- minants are two types of bovine viral diarrhea virus and border disease virus. These viruses are responsible for a variety of syndromes characterized by different clinical symptoms (1, 18, 32). Classicalswinefevervirus (CSFV), formerly named hog cholera virus, represents the fourth species and is causative for classicalswinefever (CSF), a severe disease that results in high morbidity and mortality of infected swine (18, 32). Acute CSF is characterized by pyrexia and leukopenia. Diseased animals show anorexia and diarrhea and in late stage central nervous disorders, hemorrhages in the skin, mucosa, and inner organs. Like all pestiviruses, CSFV is immunosuppressive during acute infection. A characteristic feature detected early after infection of swine is a dramatic decrease of peripheral B cells (30). Infection with CSFV variants of high virulence mostly leads to death of the affected animal. However, recent CSFV outbreaks in Europe resulted predominantly from viruses apparently in- ducing milder and chronic forms of the disease (18, 32).
washed twice with EMEM without FBS and antibiotics (EMEM). The cells were preincubated at 37°C for 30 min with 100 l of EMEM plus different concen- trations of heparin. A 100-l volume of a dilution of a virus stock in EMEM was added to the wells, mixed, and incubated as described above. When the virus solution was added, the concentration of heparin in the wells was diluted twofold. The concentration used in the text and figures hereafter corresponds to this diluted concentration (the concentration at which inhibition actually is mea- sured). After 30 min, the virus was removed and the wells were washed twice with 0.5 ml of EMEM and supplied with overlay medium (see above). Cells were grown for 18 h at 37°C, and infectious centers (hereafter referred to as plaques) were detected by immune staining with E2-specific MAb3. Positive plaques in a well were counted with a microscope. When more than about 250 plaques per well were present, a minimum of 100 plaques in a fixed area (at a magnification of ⫻40) were counted to calculate the total number of plaques in these wells. The percent inhibition was calculated using the formula 100 ⫻ [1 ⫺ (e/c)], where c is the average number of plaques in duplicate or triplicate wells to which no heparin was added (control well) and e is the average number of plaques in duplicate or triplicate wells to which heparin was added. Percent infection com- pared to control wells was calculated using the formula 100 ⫻ (e/c). For all virus samples, the percent inhibition was determined at two different multiplicities of infection. For all samples, no significantly different percentages were measured when different amounts of viruses were tested for inhibition by heparin. There- fore, percentages measured at the highest multiplicity of infection are presented. Relative plaque sizes of viruses were scored in wells to which no heparin was added after 48 h of growth under methylcellulose.
To assess the presence of VGIv and BICv, and to study the expression of IL-6, sections (thickness, 4 m) were obtained from each of the triplicate cryopre- served tissue samples and were fixed with acetone for 10 min at ⫺ 20°C. After fixation, tissue sections were incubated at room temperature (RT) for 90 min in blocking buffer containing 2% (wt/vol) bovine serum albumin (Sigma, St. Louis, MO) and 20% (vol/vol) normal bovine serum (Gibco-Invitrogen, Carlsbad, CA) in phosphate-buffered saline (PBS). Either primary MAb WH303 against CSFV E2 (6) or an anti-swine IL-6 MAb (R&D Systems, Minneapolis, MN) was diluted in blocking buffer and incubated with tissue sections overnight at 4°C in a humid chamber. After five washes with PBS at RT, tissue sections were incubated for 90 min at 37°C with the appropriate secondary antibodies, goat anti-mouse isotype- specific IgG labeled with either Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes-Invitrogen, Carlsbad, CA), diluted in blocking buffer. Following this incubation, tissue sections were washed five times with PBS at RT, counter- stained with TOPRO-iodide 642/661 (Molecular Probes) for 5 min at RT, washed as before, mounted, and examined in a Leica scanning confocal micro- scope (TCS2; Leica Microsystems, Bannockburn, IL). Data were collected uti- lizing an appropriate control lacking incubation with primary antibodies in order to determine channel crossover settings and negative background levels. The captured images were adjusted for contrast and brightness using Adobe Photo- shop software (Adobe, San Jose ´, CA).
Substitution of each cysteine residue at positions 5, 20, 24, 94, 123, and 171 to serine in BICv E1 yielded viable mutant viruses. Introduced mutations did not affect formation of E1-E2 heterodimers in infected cells or virus infectivity either in vitro or in vivo, suggesting that more than one disulfide link may contribute to the formation of E1 and E2 heterodimers and to the functions of these proteins. A similar observation has been made with other viruses; a single substitution of conserved Cys171 to Ser (Fig. 1), located in the putative trans- membrane domain of E1 protein, in the context of a BVDV E1- and E2-pseudotyped vesicular stomatitis virus, did not preclude formation of heterodimers or virus infectivity (13). It is possible that in these single-site mutants other interactions may contribute to formation of heterodimers and overall effi- ciency of the viral infection. Ronecker et al. (13) observed that substitutions of conserved Lys174Ala and Arg177Ala in BVDV E1 (Fig. 1) significantly reduced the formation of het- erodimers in cells infected with pseudotyped viruses, indicating that these charged amino acids in the transmembrane domains of BVDV E1 also contribute to the interaction with E2.
In comparison with most class I FPs, class II IFPs are shorter, less hydrophobic sequences (usually lacking aliphatic residues) and are comparatively enriched in aromatics (25). Besides these features, class II IFPs follow common patterns of interaction with membranes. These interactions are defined by the stabilization of the short loop conformation upon membrane association, shal- low insertion, and a high dose requirement for generating lipid bilayer perturbations (25). In this regard, the conformations ad- opted by representative synthetic peptides (compatible with ␤ -type structures), their capacities for inserting into lipid mono- layers and for partitioning from water solution into lipid bilayers, their membrane penetration depth, and their scored membrane- perturbing activity levels (Fig. 5 and 6) all support an IFP role for the CSFV E2 FPII sequence. The effects on these processes of the sequence alterations that interfere with virus production and in- fectivity add biological relevance to that notion.
Construction of CSF p7 mutant viruses. To assess the impor- tance of p7 for virus production, a series of recombinant CSFVs containing mutations in p7 were designed using the cDNA infec- tious clone of the Brescia strain (BICv) as a template. A total of 14 cDNA constructs containing sequential areas of three to six amino acid residues in the native p7 amino acid sequence substituted by alanine residues were constructed (Fig. 1). Infectious RNA was in vitro transcribed from each mutated full-length cDNA and used to transfect SK6 cells. Infectious virus was rescued from transfected cells by day 4 posttransfection using constructs p7.3, p7.4, p7.6, p7.7, p7.8, p7.10, p7.13, and p7.14 (depicted as white blocks in Fig. 1). In contrast, after three independent transfection proce- dures, p7.2, p7.5, p7.9, p7.11, and p7.12 constructs did not pro- duce infectious viruses (depicted as violet blocks in Fig. 1). Real- time RT-PCR analysis of total RNA extracted from these cells and cells transfected with p7⌬10-32, p7⌬15-51, and p7⌬33-51 constructs revealed genomic RNA replication, except for the p7 ⌬ 15-51 and p7 ⌬ 33-51 constructs (Table 1). In addition, immu- nohistochemistry and Western blot analysis (Fig. 2A) of trans- fected cell extracts showed comparable results showing a differen- tial expression of structural glycoproteinE2. As expected, p7 ⌬ 15-51 and p7 ⌬ 33-51 failed to produce any detectable levels of E2. p7.2-transfected cells present a very low level of expression of E2 only detectable by immunocytochemistry, whereas the levels of E2 in p7.5- and p7.12-transfected cells are intermediate relative to the levels observed in p7.9, p7.11, p7⌬10-32, and BICv cell extracts. Partial nucleotide sequencing of the rescued p7 mutated viruses was performed to ensure the presence of the predicted mutations.
corresponding sequence in pA187-1 to give pA187-CAT. By DNA sequencing, the CAT gene was confirmed to have been inserted in frame at nt 386 of the viral cDNA, 9 nt downstream of translation initiation codon ATG (Fig. 1) and to be flanked by the sequence AAT. This triplet corresponds to the fourth triplet of the viral ORF and was duplicated by treatment of the TfiI-cleaved plasmid DNA with Klenow enzyme (Fig. 1). In- fectious RNA was obtained by runoff transcription from SrfI- linearized pA187-CAT and was transfected into SK-6 cells as described before (22). After 30 h the cell culture supernatant was collected and passaged twice on SK-6 cells to obtain a virus stock (P2 stock) which had a titer of 10 7.7 50% tissue culture
When RNA from cells infected with cp CSFV was trans- fected into cells harboring a noncp helper virus, high amounts of genomic DI RNA could be detected on the Northern blot (Fig. 3C). The transfection experiments demonstrate that for all cp CSFV isolates, development of CPE requires a helper virus and correlates with the presence of the respective DI genomes. Therefore, CSFV Alfort/M, ATCC, and Steiermark each represent mixtures of a noncp helper virus and a cp DI. Genome structure of CSFV DI particles. The cp BVDV DI9 has a genome of the internal deletion type. The complete structural protein-coding region and the 5 9 part of the NS2-3 (p125) gene have been removed with respect to a full-length BVDV genome (33). Northern blot analysis had shown that the CSFV DI genomes are smaller than the BVDV CP9 DI genome (data not shown). For further analysis, cDNA cloning and sequencing were performed. RNA with a high content of DI genomes served as template for first-strand cDNA synthe- sis. Northern hybridization experiments had suggested that the deletions of the cp CSFV DI particles were located in the 59 half of the genome (data not shown). Therefore, specific oli- gonucleotides complementary to the pestivirus genome around positions 6000, 7000, 8000, and 9000 were used as cDNA prim- ers. Clones with CSFV-specific inserts were further analyzed by sequencing of the 5 9 and 3 9 ends. From comparison of the terminal sequences with the sequence of a full-length CSFV genome, putative DI particle-specific cDNA clones were iden- tified. DI particle-derived clones should meet the criterion that
The incorporation of the YFP-tagged structural Core pro- tein into particles could allow the application of such con- structs in live-virus imaging to monitor entry and egress kinet- ics. The YFP signal was easily detectable in transfected and infected cells, but we were unable to observe fluorescently labeled virions in the extracellular space (data not shown). Unfortunately, repeated propagation of CY X C viruses to ob- tain high virus titers failed because of genetic instability. As early as the third passage, revertants arose that showed wild- type genome organization. Intragenic homologous recombina- tion and selective pressure are a straightforward explanation for the rapid elimination of surplus YFP and Core genes. Attempts to stabilize a Core-YFP-Core-encoding virus by us- ing heterologous Core sequences from a BVDV NCP-7 (p447 BVDVcYc ) to avoid homologous recombination failed. Within three passages, a Core protein appeared that was slightly smaller than wild-type-sized Core. Inspection of the nucleotide sequence of the revertant revealed that the recom-