Genotypes Of Classical Swine Fever Virus Research Articles

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Molecular and Pathological Characterization of Classical Swine Fever Virus Genotype 2 Strains Responsible for the 2013-2018 Outbreak in Colombia.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

In vivo characterization of the superior fitness of classical swine fever virus genotype 2.1 to genotype 3.4

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease in pigs. In Taiwan, the emerging genotype 2.1 (G2.1) CSFV caused sporadic outbreaks in 1994 and replaced the previous G3.4 CSFV in the field. The shift of CSFV genotypes to G2 CSFV was also observed in several CSFV-affected countries. The present study aimed to explore the mechanism of the genotype shift of CSFV. Two groups of specific pathogen-free (SPF) pigs were first inoculated with either G2.1 or G3.4 CSFV (single-inoculated group) and housed together with naïve SPF pigs (cohabitating group). The results showed that peak viremia, viral loads in blood and tissues, and viral shedding of G2.1 were consistently higher than those of G3.4 CSFV in single-inoculated and cohabitating pigs. The phenomenon of superinfection exclusion (SIE), characterized by the prevention of secondary infection by a primary infection, was readily observed in CSFV single-inoculated pigs. Interestingly, coinfection of both genotypes of CSFV was observed in 3 out of 4 cohabitating pigs, while only one pig was infected with G2.1 CSFV alone. These findings suggest that the genetic shift in CSFV in the field may be in part the consequence of SIE.

The new emerging ovine pestivirus can infect pigs and confers strong protection against classical swine fever virus.

Several emerging pestiviruses have been reported lately, some of which have proved to cause disease. Recently, a new ovine pestivirus (OVPV), isolated from aborted lambs, with high genetic identity to classical swine fever virus (CSFV), has proved to induce reproductive disorders in pregnant ewes. OVPV also generated strong serological and molecular cross-reaction with CSFV. To assess the capacity of OVPV to infect swine, twelve piglets were infected either by intranasal or intramuscular route. Daily clinical evaluation and weekly samplings were performed to determine pathogenicity, viral replication and excretion and induction of immune response. Five weeks later, two pigs from each group were euthanized and tissue samples were collected to study viral replication and distribution. OVPV generated only mild clinical signs in the piglets, including wasting and polyarthritis. The virus was able to replicate, as shown by the RNA levels found in sera and swabs and persisted in tonsil for at least 5weeks. Viral replication activated the innate and adaptive immunity, evidenced by the induction of interferon-alpha levels early after infection and cross-neutralizing antibodies against CSFV, including humoural response against CSFV E2 and Erns glycoproteins. Close antigenic relation between OVPV and CSFV genotype 2.3 was detected. To determine the OVPV protection against CSFV, the OVPV-infected pigs were challenged with a highly virulent strain. Strong clinical, virological and immunological protection was generated in the OVPV-infected pigs, in direct contrast with the infection control group. Our findings show, for the first time, the OVPV capacity to infect swine, activate immunity, and the robust protection conferred against CSFV. In addition, their genetic and antigenic similarities, the close relationship between both viruses, suggest their possible coevolution as two branches stemming from a shared origin at the same time in two different hosts.

Investigation of congenital tremor associated with Classical swine fever virus genotype 2.2 in an organized pig farm in north-eastern India.

The present study describes an outbreak of Classical swine fever (CSF) in an organized pig farm followed by an episode of CSF virus (CSFV) associated congenital tremors in piglets. The outbreak was recorded in a newly procured herd of Hampshire pigs housed adjacent to the existing pigs of the farm. The recorded CSF outbreak caused a mortality of 100% in the newly procured and 54.28% in the existing herd. As the disease subsides, the clinically recovered boars were served naturally with Tamworth gilts. Though, the sows farrowed on usual gestation period, litters born to each sow showed congenital tremors and eventually died within 24 h of birth. Necropsy analysis of affected piglets was indicative of CSFV infection and was further confirmed using RT-PCR signifying a transplacental infection. The CSFV strains from the initial outbreak and post outbreak episode of congenital tremors were successfully isolated in PK-15 cells and detected in indirect FAT and RT-PCR. Phylogenetic analysis based on E2 gene and 5′NTR of CSFV grouped the isolates within the genotype 2.2 and revealed close resemblance with previously reported Indian isolates of CSFV genotype 2.2 origin. To the best of our knowledge, this is the first report of CSFV induced congenital form reported from India under natural conditions.

Development of a fluorescent probe-based real-time reverse transcription recombinase-aided amplification assay for the rapid detection of classical swine fever virus.

Classical swine fever (CSF), which is caused by the CSF virus (CSFV), remains one of the most economically important diseases of the global swine industry. Rapid and reliable detection of CSFV is critical for controlling CSF. In this study, a novel fluorescent probe-based real-time reverse transcription recombinase-aided amplification (rRT-RAA) assay, targeting a highly conserved position within the 5' non-translated region (5'NTR) among all CSFV genotypes, was developed for the detection of CSFV. The assay is highly specific to CSFV and does not cross react with other important viruses. Sensitivity analysis revealed that the assay could detect two 50% tissue culture infectious dose (TCID50 ) of CSFV RNA per reaction at 95% probability, which is comparable to that of a documentary reverse transcription quantitative PCR (RT-qPCR) assay for CSFV. The rRT-RAA assay exhibited good reproducibility, with intra- and inter-assay coefficient of variation values of<8.0%. Of the 135 samples (including 102 clinical tissue samples and 33 different cell culture isolates of CSFV), 50 and 52 samples were tested positive for CSFV by rRT-RAA and RT-qPCR, respectively. The coincidence rate between the two assays was 98.5% (133/135). Further linear regression analysis showed a significant correlation between the rRT-RAA and RT-qPCR assays with an R2 value of 0.8682. Interestingly, the amplification products of the rRT-RAA assay could be directly observed with naked eyes under a portable blue light imager, making it possible for an on-site testing. Our results indicate that the rRT-RAA assay is a robust diagnostic tool for the rapid detection of CSFV.

Genomic characteristics of classical swine fever virus strains of bovine origin according to primary and secondary sequence-structure analysis.

Background:Classical swine fever virus (CSFV), species member of the family Flaviviridae, is generally considered restricted to domestic and wild suids. The circulation of CFSV has been detected in cattle herds in China and India. Natural infection appeared associated with clinical signs in some cases.Aim:The secondary structures of the internal ribosome entry site in the 5′ untranslated region (UTR) were used for the genomic characterization of bovine strains.Methods:Sequences have been compared to the representative CSFV strains isolated from pigs, vaccines, and contaminants from porcine cell lines and an ovine strain isolated in Spain.Results:The observed sequences from cattle showed a genetic relatedness with live-attenuated vaccine strains used in pigs. Sequence characteristics of the Chinese strain S171 are genetically distant from the previously reported CSFV genotypes, suggesting a new outgroup in the species, described for the first time, and named CSFV-d. Other Chinese strains were genetically closely related to CSFV genotype a2 (Alfort type) pig strains. Indian strains, reported from the states of Tamil Nadu and Meghalaya, were genetically closely related to CSFV genotype a1 (Brescia type) and a5 pig strains, respectively.Conclusion:These preliminary observations are new and relevant in countries, where CSFV control and eradication strategies are applied.

The potential efficacy of the E2-subunit vaccine to protect pigs against different genotypes of classical swine fever virus circulating in Vietnam

PurposeTo date, many kinds of classical swine fever (CSF) vaccines have been developed to protect against this disease. However, the efficacy of these vaccines to protect the pig against field CSF strains needs to be considered, based on circulating strains of classical swine fever virus (CSFV).Materials and MethodsRecombinant E2-CSFV protein produced by baculovirus/insect cell system was analyzed by western blots and immunoperoxidase monolayer assay. The effect of CSFV-E2 subunit vaccines was evaluated in experimental pigs with three genotypes of CSFV challenge. Anti-E2 specific and neutralizing antibodies in experimental pigs were analyzed by blocking enzyme-linked immunosorbent assay and neutralization peroxidize-linked assay.ResultsThe data showed that CSFV VN91-E2 subunit vaccine provided clinical protection in pigs against three different genotypes of CSFV without noticeable clinical signs, symptoms, and mortality. In addition, no CSFV was isolated from the spleen of the vaccinated pigs. However, the unvaccinated pigs exhibited high clinical scores and the successful virus isolation from spleen. These results showed that the E2-specific and neutralizing antibodies induced by VN91-E2 antigen appeared at day 24 after first boost and a significant increase was observed at day 28 (p<0.01). This response reached a peak at day 35 and continued until day 63 when compared to controls. Importantly, VN91-E2 induced E2-specific and neutralizing antibodies protected experimental pigs against high virulence of CSFVs circulating in Vietnam, including genotype 1.1, 2.1, and 2.2.ConclusionThese findings also suggested that CSFV VN91-E2 subunit vaccine could be a promising vaccine candidate for the control and prevention of CSFV in Vietnam.

Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2

Classical swine fever (CSF) still threatens the swine industry in China, with genotype 2 isolates of CSFV dominating the epizootics. In 2018 the first E2 subunit marker vaccine against CSFV (Tian Wen Jing, TWJ-E2®), containing a baculovirus-expressed E2 glycoprotein of a genotype 1.1 vaccine strain, was officially licensed in China and commercialized. To evaluate the cross-protective efficacy of TWJ-E2 against different virulent genotype 2 Chinese field isolates (2.1b, 2.1c, 2.1 h, and 2.2), 4-week-old pigs were immunized with the TWJ-E2 vaccine according to the manufacturer’s instructions and then challenged with genotype 2 strains. A group vaccinated with the conventional C-strain vaccine was included for comparison. TWJ-E2 vaccinated pigs developed higher levels of E2 and neutralizing antibodies than those receiving the commercial C-strain vaccine. All TWJ-E2 and C-strain vaccinated pigs survived challenge without development of fever, clinical signs or pathological lesions. In contrast, all unvaccinated control pigs displayed severe CSF disease with 40–100% mortalities by 24 days post challenge. None of the TWJ-E2 and C-strain vaccinated pigs developed viremia, viral shedding from tonsils, Erns protein in the sera, or viral RNA loads in different tissues after challenge, all of which were detected in the challenged unvaccinated controls. We conclude that vaccination of young pigs with TWJ-E2 provides complete immune protection against genotypically heterologous CSFVs and prevents viral shedding after challenge, with an efficacy at least comparable to that elicited by the conventional C-strain vaccine.

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform.

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

Revisiting the genetic diversity of classical swine fever virus: A proposal for new genotyping and subgenotyping schemes of classification.

Classical swine fever (CSF) is a highly contagious febrile viral disease caused by CSF virus (CSFV), and it is considered one of the most important infectious diseases that affect domestic pigs and wild boar. Previous molecular epidemiology studies have revealed that the diversity of CSFV comprises three main genotypes and different subgenotypes defined using a reliable cut-off to accurately classify CSFV at genotype and subgenotype levels. However, a growing number of CSFV both complete genome and full E2 gene sequences have been submitted to GenBank (more than 500 sequences are currently available, revised on December 1, 2017). Therefore, the aim of this study was to revisit the taxonomy of CSFV at genotype and subgenotype levels, to unify nomenclature and to provide an update to the classification of CSFV. We propose here a new genotyping scheme with five well-defined CSFV genotypes (CSFV Genotypes 1-5) and 14 subgenotypes (seven for each of the CSFV Genotype 1 and CSFV Genotype 2). The findings showed in this study are relevant for molecular epidemiology approaches and will help to better understand the genetic diversity and spreading of CSFV at a global scale. The update in the classification of CSFV will allow the scientific community to establish more accurately the links among different outbreaks of the disease.

Generation and characterization of recombinant classical swine fever virus C-strain expressing the cap protein of porcine circovirus type 2

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating viral disease in swine, leading to significant economic losses to the pig husbandry. C-strain is one of the best modified live vaccines against CSF. The vaccine is highly safe and efficacious and can provide rapid and complete protection against essentially all genotypes of CSFV. Co-infections of pigs with CSFV and porcine circovirus type 2 (PCV2) occur frequently in the field, making it difficult to control the associated diseases. Here, a recombinant C-strain rHCLV-Cap expressing the Cap protein of PCV2 was constructed and evaluated in vitro and in vivo. The recombinant had comparable phenotypes to C-strain in cell cultures and rabbits. At ten days post-immunization, anti-E2, but not anti-Cap, antibodies were detected in the rabbits inoculated with the recombinant virus. Our study warrants further work to construct C-strain-based bivalent vaccines.

Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine

Modified live vaccines (MLVs) based on genotype 1 strains, particularly C-strain, have been used to prevent and control classical swine fever virus (CSFV) worldwide. Nevertheless, a shift in the predominant CSFV strains circulating in the field from genotype 1 or 3 to genotype 2 is seen. Genotype 2 is genetically distant from the vaccine strains and was recently reported during outbreaks after vaccine failure; this has raised concerns that vaccination has influenced viral evolution. In Korea in 2016, there was an unexpected CSF outbreak in a MLV-vaccinated commercial pig herd. The causative CSFV strain was genetically distinct from previously isolated Korean strains but similar to recent Chinese strains exhibiting enhanced capacity to escape neutralization; this suggests the need for global cooperative research on the evolution of CSFV. We analysed global E2 sequences, using bioinformatics tools, revealing the evolutionary pathways of CSFV. Classical swine fever virus genotypes 1 and 2 experienced different degrees and patterns of evolutionary growth. Whereas genotype 1 stayed relatively conserved over time, the genetic diversity of genotype 2 has progressively expanded, with few fluctuations. It was determined that genotype 2 evolved under lower immune pressures and at a higher evolutionary rate than genotype 1. Further, several selected codons, under diversifying selection in genotype 1 but under purifying selection in genotype 2, correspond to antigenic determinants, which could lead to evasion of vaccine-induced immunity. Our findings provide evidence that evolutionary changes in CSFV are the result of the disproportionate usage of the CSF MLVs in endemic areas; this underscores the need to develop mitigation strategies to minimize the substantial risk associated with the emergence of vaccine-escaping mutants.

An outbreak of classical swine fever in pigs in Bangladesh, 2015.

In a group of 22 healthy pigs aged between 4 and 6 months, 2 pigs became ill with high fever, complete anorexia, cough and abnormal swaying movements on 22 June 2015. One of them died on June 24 and the second died on July 3. Shortly after, the remaining pigs also fell ill and died from the same illness by 10 August 2015. We investigated the aetiology, epidemiological and clinical features of the outbreak. We recorded the clinical signs and symptoms for each pig with the date of onset of illness. Veterinarians conducted post‐mortem examinations on the 12 dead pigs, they collected tissue samples from the dead pigs and placed them in a tube containing 1 mL of nucleic acid extraction buffer (lysis buffer). We tested all the tissue samples by real‐time reverse transcription polymerase chain reaction (rRT‐PCR) to detect classical swine fever virus (CSFV) because the animals’ symptoms matched those of this disease. We also conducted a phylogentic analysis of the nucleotide sequence of the E2 gene segment of CSFV detected in a lung tissue sample. The attack rate (22/22) and the case fatality were 100%. The predominant symptoms of the disease included high fever, cough, diarrhoea and swaying movements of the hind legs prior to death. Of the 12 pigs tissue samples tested, all had evidence of the presence of CSFV RNA by rRT‐PCR. The phylogenetic analysis indicated that the virus belongs to genotype 2.2, which is closely related to CSFV genotype 2.2 reported in India. Our investigation suggests that CSF is circulating in pigs, posing a risk for communities in Bangladesh that rely on pigs for economic income and dietary protein. Future research could focus on estimating the disease and economic burden of CSFV in pig rearing areas to determine if interventions might be warranted or cost‐effective.

A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells

BackgroundClassical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells.MethodsWe designed several specific probes of CSFV RNA and reference gene β-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2).ResultsThe lowest detection threshold of the ViewRNA ISH method was 10−8, while the sensitivity of FAT and IHC were 10−5 and 10−4, respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3–9 hpi, RNA was mainly distributed in nucleus and reached a maximum at 12hpi, then RNA copy number was gradually increased around the cell nucleus during 24–48 hpi and reached the peak at 72hpi.ConclusionsTo our knowledge, this is the first to reveal the dynamic distribution of medium virulence CSFV RNA in PK15 cells using the ViewRNA ISH method. The sensitivity of the ViewRNA ISH was three to four orders of magnitude higher than that of FAT and IHC methods. The specificity experiment showed that the ViewRNA ISH was highly specific for CSFV and no cross-reaction occurred to negative control and other pig infectious virus. This assay is more suitable for studying the CSFV RNA life cycle in cell nucleus. The results proved that CSFV RNA enters into PK15 cells earlier than 0.5hpi, relative to the eclipse period of cytoplasm is 6–9 hpi and CSFV RNA has ever existed in nucleus.

Genome sequence of classical Swine Fever virus genotype 1.1 with a genetic marker of attenuation detected in a continuous porcine cell line.

The complete genome sequencing and analysis of a classical swine fever virus (CSFV) detected in a porcine kidney cell line revealed a close relationship with genotype 1.1 viruses circulating in India and China. The presence of consecutive T insertions in the 3′ untranslated region (UTR), as seen in vaccine strains of CSFV, suggested some degree of attenuation.

Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1+2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1+2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1+2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1+2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1+2.1E2 are promising candidate subunit vaccines against CSF.

The CSFV DNAChip: A novel diagnostic assay for classical swine fever virus

A novel assay, the CSFV DNAChip, was developed to clearly and rapidly discriminate three genotypes of classical swine fever virus (CSFV). Total RNA was extracted from clinical samples and then subjected to a one-step reverse-transcription polymerase chain reaction (RT-PCR) using Cy3-labeled primers from the 5′ non-coding region (NCR) of CSFV. Amplicons were hybridized to the CSFV DNAChip and fluorescence scanning was performed for detection of CSFV. A cut-off fluorescence intensity value of 5000 was determined by two-graph receiver operating curve (TG-ROC) analysis. The limit of detection values for the developed DNA chip assay were 0.313ng/μL for amplicon concentration and 1TCID50/100μL for virus titer. Using the developed DNA chip, 157 field samples (91 CSFV-positive and 66 CSFV-negative) were investigated. The genotypes determined by the CSFV DNAChip agreed completely with those determined by nucleotide sequence analysis of the viral genome. The developed CSFV DNAChip will be helpful in implementing a CSFV eradication strategy, as it provides a rapid and accurate diagnostic assay that can discriminate easily among CSFV genotypes.

Novel rope-based sampling of classical swine fever shedding in a group of wild boar showing low contagiosity upon experimental infection with a classical swine fever field strain of genotype 2.3

Several classical swine fever (CSF) epidemics in wild boar and domestic pigs in Europe during the last decades have been caused by CSF virus (CSFV) strains of genotype 2.3. This genotype is known to be virulent leading to high morbidity and mortality. We experimentally infected two eight months old wild boar with 105,5 TCID50 of CSFV genotype 2.3 and kept the animals together with five noninoculated wild boar of the same age. Our original purpose was to evaluate a non-invasive sampling method based on saliva collection using “rope-in-a-bait” sampling baits. While expecting high morbidity, high level of virus shedding and some mortality, we actually observed a subclinical course of infection with an unexpected low contagiosity. The two inoculated animals infected only three contact animals while two contact animals remained uninfected. These findings substantially add to our epidemiological understanding of CSFV circulation in wild boar populations. CSFV infected animals older than six months and in good condition may not shed sufficient virus to transmit infection to all seronegative in-contact animals. The contagiosity in relation to the animal's age is discussed. This supports the hypothesis of silent perpetuation of CSFV in wild boar populations for several months if the wild boar density is sufficiently high. The feasibility of the “rope-in-a-bait” sampling method could be proven during the short viraemic phase of infected animals during the second week of infection.

Classical swine fever virus detection

The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications. In conclusion, the ring trial showed reliability of classical swine fever diagnosis on an international level and helped to optimize CSFV-specific RT-PCR diagnostics.