Safety and DIVA Capability of Novel Live Attenuated Classical Swine Fever Marker Vaccine Candidates in Pregnant Sows.

Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in Erns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in Erns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in Erns combined with a 5-168 amino acids deletion of Npro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates.

Assessment of the Safety Profile of Chimeric Marker Vaccine against Classical Swine Fever: Reversion to Virulence Study.

Chimeric marker vaccine candidates, vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE-/PAPeV Erns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE-/PhoPeV Erns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE- strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE-/PAPeV Erns was significantly higher than that of vGPE-/PhoPeV Erns and vGPE-. In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.

Investigation of the safety and protective efficacy of an attenuated and marker M. bovis-BoHV-1 combined vaccine in bovines.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry worldwide; it is caused by multiple bacterial or viral coinfections, of which Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens. Although live vaccines have demonstrated better efficacy against BRD induced by both pathogens, there are no combined live and marker vaccines. Therefore, we developed an attenuated and marker M. bovis-BoHV-1 combined vaccine based on the M. bovis HB150 and BoHV-1 gG-/tk- strain previously constructed in our lab and evaluated in rabbits. This study aimed to further evaluate its safety and protective efficacy in cattle using different antigen ratios. After immunization, all vaccinated cattle had a normal rectal temperature and mental status without respiratory symptoms. CD4+, CD8+, and CD19+ cells significantly increased in immunized cattle and induced higher humoral and cellular immune responses, and the expression of key cytokines such as IL-4, IL-12, TNF-α, and IFN-γ can be promoted after vaccination. The 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- combined strain elicited the most antibodies while significantly increasing IgG and cellular immunity after challenge. In conclusion, the M. bovis HB150 and BoHV-1 gG-/tk- combined strain was clinically safe and protective in calves; the mix of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its low amount of shedding and highest humoral and cellular immune responses compared with others. This study introduces an M. bovis-BoHV-1 combined vaccine for application in the cattle industry.

Preliminary molecular study for DIVA trial of antigenically characterized circulating bovine herpesvirus subtype 1.1 in Egypt.

Using marker vaccines to control bovine alphaherpesvirus-1 (BoHV-1) is a novel strategy for differentiation between infected and vaccinated animals (DIVA). In this study, multiplex real-time PCR targeting gD and gE genes was applied for BoHV-1 screening on 60 clinical samples from cattle with a history of vaccination, in some cases by US2-deleted marker vaccines, that were suffering from severe respiratory symptoms. Conventional PCR targeting the gC and US2 flanking region was done for molecular characterization and identification of the US2-deleted vaccine strain. Six samples were positive for BoHV-1 by both RT-PCR and conventional PCR. Surprisingly, a conventional PCR DIVA trial based on the US2 gene revealed that only one sample that exhibited the US2 gene was a wild virus, while others that did not exhibit the US2 gene were vaccine viruses. Phylogenetic characterization classifies the samples as BoHV-1.1. This finding reveals the circulation of vaccine virus in field-diseased animals, which threatens the eradication program.

Efficacy of an orally administered classical swine fever live marker vaccine (Flc-LOM-BErns strain) in pigs.

In several countries, classical swine fever (CSF) has not been detected in domestic pigs, but has been detected in wild boars, making the disease difficult to control. To overcome this problem, we inoculated pigs with a CSF live marker vaccine (Flc-LOM-BErns strain), which has "distinguish infection from vaccinated animals (DIVA)" function, to determine whether it is suitable as an oral vaccine specifically for wild boars. Pigs inoculated intramuscularly or orally with the Flc-LOM-BErns vaccine were challenged 2 or 4 weeks later, respectively, with virulent CSFV. Pigs administered the oral Flc-LOM-BErns strain (105.0 and 6.0 TCID50/dose), and those vaccinated intramuscularly (103.0 TCID50/dose), had normal numbers of leukocytes and normal body temperature. Also, they generated protective neutralizing antibodies and anti-BVDV Erns antibodies. In addition, all pigs in these groups survived, with no CSFV RNA detected in feces, spleen, or other organs. Thus, the Flc-LOM-BErns vaccine shows excellent safety and efficacy, while having DIVA function and suitability for oral inoculation.

Generation and Efficacy of Two Chimeric Viruses Derived from GPE- Vaccine Strain as Classical Swine Fever Vaccine Candidates.

A previous study proved that vGPE- mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE- vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE- vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE- vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE- vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE-/PAPeV Erns or vGPE-/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns affirmed their properties, as the vGPE- vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Safety, efficacy, and DIVA feasibility on a novel live attenuated classical swine fever marker vaccine candidate.

Classical swine fever virus (CSFV) is the etiological agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. Systemic prophylactic immunization with the live attenuated vaccine, the C-strain vaccine, is one of the effective measures for CSF control. However, one of the limitations of the C-strain vaccine is that the field strains-infected animals cannot be differentiated from the C-strain vaccinated herds by serological tests (DIVA). This constraint hampers the practical usage of the C-strain vaccine to eradicate the CSF in China. In the current study, a novel CSF modified live marker vaccine candidate was constructed based on the attenuation of the prevalent 2.1 genotype strain by the deletion of two virulence associated functional residues in the CSFV Erns, H79, and C171. Meanwhile, four residues S14, G22, E24, and E25 were identified specifically for the 6B8 mAb binding to the CSFV E2 as the novel conformational epitope. Then four substitutions of S14K, G22A, E24R, and G25D were further incorporated in the double deletion construct as a negative serological marker. Finally, the double-deletion marker MLV candidate GD18-ddErnHC-KARD was rescued, and its safety and efficacy profiles were evaluated in piglets. The safety study results indicated that the candidate did not induce fever, clinical signs, or pathological lesions with a high dose of 105.0 TCID50, and in addition, no virus shedding was detected until 21 days post-inoculation. Meanwhile, the efficacy study results demonstrated that at a low dose of 103.0 TCID50, it conferred complete clinical protection and no virus shedding was detected after the challenge with a highly virulent Shimen strain. Importantly, the infected animals were differentiated using the accompanied DIVA ELISA. These results constitute a proof-of-concept for rationally designing a CSF antigenically marked modified live vaccine candidate.

Disease control tools to secure animal and public health in a densely populated world.

Animal health is a prerequisite for global health, economic development, food security, food quality, and poverty reduction, while mitigating against climate change and biodiversity loss. We did a qualitative review of 53 infectious diseases in terrestrial animals with data from DISCONTOOLS, a specialist database and prioritisation model focusing on research gaps for improving infectious disease control in animals. Many diseases do not have any appropriate control tools, but the prioritisation model suggests that we should focus international efforts on Nipah virus infection, African swine fever, contagious bovine pleuropneumonia, peste des petits ruminants, sheeppox and goatpox, avian influenza, Rift Valley fever, foot and mouth disease, and bovine tuberculosis, for the greatest impact on the UN's Sustainable Development Goals. Easy to use and accurate diagnostics are available for many animal diseases. However, there is an urgent need for the development of stable and durable diagnostics that can differentiate infected animals from vaccinated animals, to exploit rapid technological advances, and to make diagnostics widely available and affordable. Veterinary vaccines are important for dealing with endemic, new, and emerging diseases. However, fundamental research is needed to improve the convenience of use and duration of immunity, and to establish performant marker vaccines. The largest gap in animal pharmaceuticals is the threat of pathogens developing resistance to available drugs, in particular for bacterial and parasitic (protozoal, helminth, and arthropod) pathogens. We propose and discuss five research priorities for animal health that will help to deliver a sustainable and healthy planet: vaccinology, antimicrobial resistance, climate mitigation and adaptation, digital health, and epidemic preparedness.

Genetically distinct pestiviruses pave the way to improved classical swine fever marker vaccine candidates based on the chimeric pestivirus concept.

Classical swine fever (CSF) is one of the most important viral diseases of pigs. In many countries, the use of vaccines is restricted due to limitations of subunit vaccines with regard to efficacy and onset of protection as well as failure of live vaccines to differentiate infected from vaccinated animals (DIVA principle). Chimeric pestiviruses based on CSF virus (CSFV) and the related bovine viral diarrhea virus (BVDV) have been licensed as live marker vaccines in Europe and Asia, but cross-reactive antibodies can cause problems in DIVA application due to close antigenic relationship. To develop marker vaccine candidates with improved DIVA properties, three chimeric viruses were generated by replacing Erns of CSFV Alfort-Tübingen with homologue proteins of only distantly related pestiviruses. The chimeric viruses "Ra", "Pro", and "RaPro" contained Erns sequences of Norway rat and Pronghorn pestiviruses or a combination of both, respectively. In porcine cells, the "Pro" chimera replicated to high titers, while replication of the "Ra" chimera was limited. The "RaPro" chimera showed an intermediate phenotype. All vaccine candidates were attenuated in a vaccination/ challenge trial in pigs, but to different extents. Inoculation induced moderate to high levels of neutralizing antibodies that protected against infection with a genetically heterologous, highly virulent CSFV. Importantly, serum samples of vaccinated animals did not show any cross-reactivity in a CSFV Erns antibody ELISA. In conclusion, the Erns antigen from distantly related pestiviruses can provide a robust serological negative marker for a new generation of improved CSFV marker vaccines based on the chimeric pestivirus concept.

Evaluation of novel inactivated vaccines for the SAT 1, SAT 2 and SAT 3 serotypes of foot-and-mouth disease in pigs.

BACKGROUND: The foot-and-mouth disease (FMD) virus is classified into seven serotypes, of which the South African types have South African Territories (SAT)1, SAT2, and SAT3 that are prevalent in Africa. Especially SAT2 have spread to Arabian Peninsula and the Palestinian Autonomous Territories. Of these viruses, the incidence of SAT2 is the highest. It is important to prepare for the spread of the virus to other continents, even though most FMD viruses are bovine-derived. In particular, due to the high breeding density of pigs in Asia, more attention is usually paid to the immunity and protection of pigs than cattle. For this reason, this study investigated the immunity and protection of pigs against the SAT viruses. METHODS: Specific vaccines were developed for SAT1, SAT2, and SAT3 serotypes. These vaccine viruses were designed to be distinguished from the wild-type strain. An immunogenicity test was conducted using these vaccines in both cattle (n = 5/group) and pigs (n = 20/group). RESULTS: High virus-neutralizing titer of antibodies (> 1:100) was induced in only 2 weeks after the immunization of cattle with the individual vaccine for SAT1, SAT2 or SAT3, and a clear immune response was induced after the second immunization in pigs. When the vaccinated pigs (n = 4-5/group) were challenged by the homologous wild-type virus strain 4 weeks after immunization, all the pigs were protected from the challenge. CONCLUSIONS: This study confirmed that these vaccines can be used against SAT1, SAT2, and SAT3 viruses in cattle and pigs. The vaccine strains developed in this study are expected to be used as vaccines that can protect against FMD in the event of a future FMD outbreak in pigs in consideration of the situation in Asia.

Assessment of the efficacy of an attenuated live marker classical swine fever vaccine (Flc-LOM-BErns) in pregnant sows.

Here, we constructed an attenuated live marker classical swine fever (CSF) vaccine (Flc-LOM-BErns) to eradicate CSF. This was done by taking infectious clone Flc-LOM, which is based on an attenuated live CSF vaccine virus (LOM strain), and removing the full-length classical swine fever virus (CSFV) Erns sequences and the 3' end (52 base pairs) of the CSFV capsid. These regions were substituted with the full-length bovine viral diarrhoea virus (BVDV) Erns gene sequence and the 3' end (52 base pairs) of the BVDV capsid gene. Sows were vaccinated with the Flc-LOM-BErns vaccine 3 weeks before insemination and then challenged with virulent CSFV at the early, mid- or late stages of pregnancy. We then examined transplacental transmission to the foetuses. Piglets born to sows vaccinated with Flc-LOM-BErns did not show vertical infection, regardless of challenge time. In addition, CSFV challenge did not affect the delivery date, weight or length of the foetus. Pregnant sows inoculated with the Flc-LOM-BErns vaccine were anti-CSF Erns antibody-negative and anti-BVDV Erns antibody-positive. Challenge of pregnant sows with virulent CSFV resulted in anti-CSF Erns antibody positivity. These results strongly indicate that differential diagnosis can be conducted between the Flc-LOM-BErns vaccinated animal and virulent CSFV affected animal by detecting antibody against BVDV Erns or CSF Erns gene. Therefore, the Flc-LOM-BErns vaccine may fulfil the function of differential diagnosis which required for DIVA vaccine.

Protection against transplacental transmission of moderately virulent classical swine fever virus using live marker vaccine "CP7_E2alf".

Classical swine fever (CSF) remains as one of the most important infectious diseases of swine. While prophylactic vaccination is usually prohibited in free countries with industrialized pig production, emergency vaccination is still foreseen. In this context, marker vaccines are preferred as they can reduce the impact on trade. The live-attenuated Suvaxyn® CSF Marker vaccine by Zoetis (based on pestivirus chimera "CP7_E2alf"), was recently licensed by the European Medicines Agency. Its efficacy for the individual animal had been shown in prior studies, but questions remained regarding protection against transplacental transmission. To answer this question, a trial with eight pregnant sows and their offspring was performed as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Six of the sows were intramuscularly vaccinated on day 44 of gestation, while the other two remained as unvaccinated controls. All sows were challenged with the moderately virulent CSFV strain "Roesrath" and euthanized shortly before the calculated farrowing date. Sows and piglets were grossly examined and necropsied. Organs (spleen, tonsil, lymph node, and kidney), EDTA-blood and serum were collected from all animals. All samples were tested for antibodies against CSFV glycoproteins E2 and Erns as well as CSFV (virus, antigen and genome). It could be demonstrated that the vaccine complies with all requirements, i.e. no virus was found in the blood of vaccinated sows and their fetuses, and no antibodies were found in the serum of the fetuses from the vaccinated sows. All controls were valid. Thus, it was demonstrated that a single dose vaccination in the sows efficiently protected the offspring against transplacental infection with a moderately virulent CSFV strain.

Reduced specificity of Erns antibody ELISAs for samples from piglets with maternally derived antibodies induced by vaccination of sows with classical swine fever marker vaccine CP7_E2alf.

Successful implementation of marker vaccines against classical swine fever virus is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA) as well as the development of a testing scheme during emergency vaccination. In this context, special attention needs to be paid to breeding farms, because the offspring of marker vaccinated sows possess maternally derived antibodies (MDAs). So far, limited information is available on the influence of MDAs on serological testing in the context of a DIVA strategy. Therefore, two commercially available Erns antibody ELISAs were compared, using serum samples of piglets with a high-to-moderate titre of MDAs against marker vaccine CP7_E2alf. False-positive results were detected by both Erns antibody ELISAs for serum samples of piglets with an age of up to 4 weeks. Interestingly, most samples tested false-positive in the first Erns antibody ELISA were identified correctly by the other Erns antibody ELISA and vice versa. In conclusion, in case of emergency vaccination of sows, the specificity of both ELISAs in newborn piglets younger than 4 weeks may be relatively low. This could be addressed in a testing strategy by either not sampling piglets up to the age of 4 weeks or using both ELISAs in a screening-confirmation set-up.

Long-term study (2005-2010) on the vaccination with BoHV-1 glycoprotein E-deleted marker vaccine in selected two dairy herds in Turkey.

A follow-up study from 2005 to 2010 was carried out in two herds where eradication programme for the bovine herpes virus-1 (BoHV-1) infection depends on the vaccination with inactivated glycoprotein E-deleted vaccine that was started in 2001 following the vaccination with inactivated conventional vaccine between 1999 and 2001. For serological screening, a total of 12,976 sera sampled over several sampling times approximately 6 months of interval during 5 years (2005-2010) were tested for glycoprotein E (gE)- and glycoprotein B-specific antibodies using ELISA. According to the serological evidence, the long-term persistence of BoHV-1 antibodies, success of marker vaccine, first vaccination time of the calves in herds regularly vaccinated, etc. were discussed in this paper. In conclusion, the vaccination programme using gE (-) marker vaccines, with making efforts to prevent the other factors about transmission of infection, was suggested for the eradication of BoHV-1 infection in Turkey as many EU countries. This is the first report on the BoHV-1 eradication programme in some dairy cattle in Turkey.

Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges.

Classical swine fever (CSF) represents a major health and trade problem for the pig industry. In endemic countries or those with a wild boar reservoir, CSF remains a priority for Veterinary Services. Surveillance as well as stamping out and/or vaccination are the principle tools of prevention and control, depending on the context. In the past decades, marker vaccines and accompanying diagnostic tests allowing the discrimination of infected from vaccinated animals have been developed. In the European Union, an E2 subunit and a chimeric live vaccine have been licensed and are available for the use in future disease outbreak scenarios. The implementation of commonly accepted and globally harmonized concepts could pave the way to replace the ethically questionable stamping out policy by a vaccination-to-live strategy and thereby avoid culling of a large number of healthy animals and save food resources. Although a number of vaccines and diagnostic tests are available worldwide, technological advancement in both domains is desirable. This work provides a summary of an analysis undertaken by the DISCONTOOLS group of experts on CSF. Details of the analysis can be downloaded from the web site at http://www.discontools.eu/.

Early protection events in swine immunized with an experimental live attenuated classical swine fever marker vaccine, FlagT4G.

Prophylactic vaccination using live attenuated classical swine fever (CSF) vaccines has been a very effective method to control the disease in endemic regions and during outbreaks in previously disease-free areas. These vaccines confer effective protection against the disease at early times post-vaccination although the mechanisms mediating the protection are poorly characterized. Here we present the events occurring after the administration of our in-house developed live attenuated marker vaccine, FlagT4Gv. We previously reported that FlagT4Gv intramuscular (IM) administered conferred effective protection against intranasal challenge with virulent CSFV (BICv) as early as 7 days post-vaccination. Here we report that FlagT4Gv is able to induce protection against disease as early as three days post-vaccination. Immunohistochemical testing of tissues from FlagT4Gv-inoculated animals showed that tonsils were colonized by the vaccine virus by day 3 post-inoculation. There was a complete absence of BICv in tonsils of FlagT4Gv-inoculated animals which had been intranasal (IN) challenged with BICv 3 days after FlagT4Gv infection, confirming that FlagT4Gv inoculation confers sterile immunity. Analysis of systemic levels of 19 different cytokines in vaccinated animals demonstrated an increase of IFN-α three days after FlagT4Gv inoculation compared with mock infected controls.

Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status.

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle. The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA. The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated. Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples. The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples.

The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine.

Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved "AEKNPLE" epitope spanning amino acids 109-115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant "AEKNPLE" epitope in NSP 3A of FMDV.