Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protects Pigs against PCV2b Challenge and Induces Serum Neutralizing Antibody Response against CSFV.

Porcine circovirus type 2 (PCV2) is endemic worldwide. PCV2 causes immunosuppressive infection. Co-infection of pigs with other swine viruses, such as pseudorabies virus (PRV) and classical swine fever virus (CSFV), have fatal outcomes, causing the swine industry significant economic losses in many if not all pig-producing countries. Currently available inactivated/modified-live/vectored vaccines against PCV2/CSFV/PRV have safety and efficacy limitations. To address these shortcomings, we have constructed a triple gene (thymidine kinase, glycoprotein E [gE], and gG)-deleted (PRVtmv) vaccine vector expressing chimeric PCV2b-capsid, CSFV-E2, and chimeric Erns-fused with bovine granulocytic monocyte-colony stimulating factor (Erns-GM-CSF), designated as PRVtmv+, a trivalent vaccine. Here we compared this vaccine's immunogenicity and protective efficacy in pigs against wild-type PCV2b challenge with that of the inactivated Zoetis Fostera Gold PCV commercial vaccine. The live PRVtmv+ prototype trivalent subunit vaccine is safe and highly attenuated in pigs. Based on PCV2b-specific neutralizing antibody titers, viremia, viral load in lymphoid tissues, fecal-virus shedding, and leukocyte/lymphocyte count, the PRVtmv+ yielded better protection for vaccinated pigs than the commercial vaccine after the PCV2b challenge. Additionally, the PRVtmv+ vaccinated pigs generated low to moderate levels of CSFV-specific neutralizing antibodies.

Base-catalysed 18F-labelling of trifluoromethyl ketones. Application to the synthesis of 18F-labelled neutrophil elastase inhibitors.

A new method for the fluorine-18 labelling of trifluoromethyl ketones has been developed. This method is based on the conversion of a-COCF3 functional group to a difluoro enol silyl ether followed by halogenation and fluorine-18 labelling. The utility of this new method was demonstrated by the synthesis of fluorine-18 labelled neutrophil elastase inhibitors, which are potentially useful for detection of inflammatory disorders.

Comparative Analysis of Tunisian Sheep-like Virus, Bungowannah Virus and Border Disease Virus Infection in the Porcine Host.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

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

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.

Trifluoromethylthiolation, Trifluoromethylation, and Arylation Reactions of Difluoro Enol Silyl Ethers.

This study reports a new application area of difluoro enol silyl ethers, which can be easily obtained from trifluoromethyl ketones. The main focus has been directed to the electrophilic fluoroalkylation and arylation methods. The trifluoromethylthiolation of difluoro enol silyl ethers can be used for the construction of a novel trifluoromethylthio-α,α-difluoroketone (-COCF2SCF3) functionality. The -CF2SCF3 moiety has interesting properties due to the electron-withdrawing, albeit lipophilic, character of the SCF3 group, which can be combined with the high electrophilicity of the difluoroketone motif. The methodology could also be extended to difluoro homologation of the trifluoromethyl ketones using the Togni reagent. In addition, we presented a method for transition-metal-free arylation of difluoro enol silyl ethers based on hypervalent iodines.

Inactivation of Classical Swine Fever Virus in Porcine Serum Samples Intended for Antibody Detection.

Shipping of serum samples that were taken from pigs infected with classical swine fever (CSF) virus is frequently requested with the objective of serological analyses, not only for diagnostic purposes but also for exchange of reference materials that are used as control material of diagnostic assays. On the basis of the fact that an outbreak with CSF is associated with enormous economic losses, biological safety during the exchange of reference material is of great importance. The present study aimed to establish a pragmatic approach for reliable CSF virus (CSFV) inactivation in serum without impairing antibody detection. Considering the fact that complement inactivation through heating is routinely applied, the basic idea was to combine heat treatment with the dilution of serum in a detergent containing buffer in order to facilitate the inactivation process. The results show that treatment of serum samples with phosphate buffered saline-Tween20 (final concentration = 0.15%) along with incubation at 56 °C for 30 min inactivated CSFV and such treatment with ≤ 0.25% PBS-Tween20 does not impair subsequent antibody detection by ELISA or virus neutralization test. This minimizes the risk of virus contamination and represents a valuable contribution to a safer CSF diagnosis on a national and international level.

Characterization of the Humoral Immune Response Induced after Infection with Atypical Porcine Pestivirus (APPV).

Atypical porcine pestivirus (APPV) is a widely distributed pathogen causing congenital tremor (CT) in piglets. So far, no data are available regarding the humoral immune response against APPV. In this study, piglets and their sows from an affected herd were tested longitudinally for viral genome and antibodies. APPV genome was detected in the majority of the piglets (14/15) from CT affected litters. Transient infection of gilts was observed. Kinetics of Erns- and E2-specific antibodies and their neutralizing capacity were determined by recently (Erns) and newly (E2) developed antibody ELISAs and virus neutralization assays. Putative maternally derived antibodies (MDA) were detected in most piglets, but displayed only low to moderate neutralizing capacity (ND50 ≤ 112). Horizontal APPV transmission occurred when uninfected and infected piglets were mingled on the flat deck. Horizontally infected piglets were clinically inapparent and showed only transient viremia with subsequently consistently high E2 antibody levels. For piglets from CT affected litters, significantly lower neutralizing antibody titers were observed. Results indicate that E2 represents the main target of neutralizing antibodies. Characterization of the humoral immune response against APPV will help to provide valuable serological diagnosis, to understand the epidemiology of this novel pathogen, and to implement tailored prevention strategies.

Reemergence of Classical Swine Fever, Japan, 2018.

In September 2018, classical swine fever reemerged in Japan after 26 years, affecting domestic pigs and wild boars. The causative virus belongs to the 2.1 subgenotype, which caused repeated outbreaks in eastern and Southeast Asia. Intensive surveillance of swine and vaccination of wild boars will help control and eradicate this disease in Japan.

Further characterization of bovine hepacivirus: Antibody response, course of infection, and host tropism.

Bovine hepacivirus (BovHepV) is a recently added member to the growing genus Hepacivirus within the family Flaviviridae. Animal hepaciviruses are rarely characterized so far. Apart from norway rat hepacivirus which represents a promising HCV surrogate model, only equine hepaciviruses have been studied to some extent. BovHepV has been initially identified in bovine samples and was shown to establish persistent infections in cattle. However, consequences of those chronic infections, humoral immune response and the possibility of an extended host spectrum have not been explored so far. Therefore, we here investigated (a) the presence of anti-NS3-antibodies and viral RNA in cattle herds in Germany, (b) the course of infection in cattle, and (c) the host tropism including zoonotic potential of bovine hepaciviruses. Our results show that 19.9% of investigated bovine serum samples had antibodies against BovHepV. In 8.2% of investigated samples, viral RNA was detected. Subsequent genetic analysis revealed a novel genetic cluster of BovHepV variants. For 25 selected cattle in a BovHepV positive herd the presence of viral genomic RNA was monitored over one year in two to three months intervals by RT-PCR in order to discriminate acute versus persistent infection. In persistently infected animals, no serum antibodies were detected. Biochemical analyses could not establish a link between BovHepV infection and liver injury. Apart from a single sample of a pig providing a positive reaction in the antibody test, neither BovHepV-specific antibodies nor viral RNA were detected in porcine, equine or human samples implying a strict host specificity of BovHepV.