PRRSV-Vaccinated, Seronegative Sows and Maternally Derived Antibodies (II): Impact on PRRSV-1 Vaccine Effectiveness and Challenge Outcomes in Piglets.

Vaccination against the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is widely practiced in both sows and piglets. However, it has been shown that multivaccinated sows sometimes lack a detectable antibody response, testing seronegative in ELISA (non-responders). Moreover, PRRSV-vaccinated piglets can remain seronegative as well, which is mainly attributed to the interference of maternally derived antibodies (MDAs). The current study investigated the impact of the sow's immune status on the PRRSV vaccine effectiveness in the progeny. The experimental trial included forty-eight piglets (n = 48) originating from a commercial Belgian breeding herd, with twenty-four piglets born from PRRSV vaccinated responder sows (E+ piglets) and twenty-four piglets born from PRRSV vaccinated non-responder sows (E- piglets). Eight piglets in each group were either non-vaccinated (NoVac piglets; n = 8), intramuscularly vaccinated (IM piglets; n = 8), or intradermally vaccinated (ID piglets; n = 8), with the same PRRSV-1 vaccine as used in the sow population. Vaccination was performed at weaning at three weeks of age, and all study piglets were challenged with a high dose of the PRRSV-1 07V063 strain at 6 weeks of age. A clear interference of MDAs was observed in the E+ piglets: 66.7% of the vaccinated E+ piglets lacked an antibody response at 3 weeks post-vaccination (non-responders). Consequently, post-challenge, only the responding E+ piglets had a significantly reduced serum viremia compared to the E+ NoVac piglets. The observed viremia in the non-responding E+ piglets was similar to the viremia of the E+ NoVac piglets. In the vaccinated E- piglets, a lack of antibody response at 3 weeks post-vaccination was observed in 18.8% of the piglets. Interestingly, despite the lack of a vaccine antibody response, the non-responding E- piglets had a significantly reduced serum viremia compared to the NoVac E- piglets. In contrast, the viremia of the responding E- piglets was only numerically reduced compared to the NoVac E- piglets. Finally, some clear differences were observed in both the kinetics of infection and the immune responses post-challenge between the E+ and E- piglets. The results of this study confirm the consequences of the MDA interference on the induced partial protection of PRRSV vaccination in experimentally challenged piglets. More research is warranted to understand the immunological mechanisms behind MDA interference in PRRSV vaccination and to explain the observed differences between E+ and E- piglets.

Differential infection behavior of African swine fever virus (ASFV) genotype I and II in the upper respiratory tract.

African swine fever virus (ASFV) is a substantial threat to pig populations worldwide, contributing to economic disruption and food security challenges. Its spread is attributed to the oronasal transmission route, particularly in animals with acute ASF. Our study addresses the understudied role of nasal mucosa in ASFV infection, using a nasal explant model. The explants remained viable and revealed a discernible ASFV infection in nasal septum and turbinates post-inoculation. Interestingly, more infected cells were found in the turbinates despite its thinner structure. Further analyses showed (i) a higher replication of genotype II strain BEL18 than genotype I strain E70 in the epithelial cell layer, (ii) a preference of ASFV infection for the lamina propria and a tropism of ASFV for various susceptible cell types in different areas in the nasal mucosa, including epithelial cells, macrophages, and endothelial cells. Using porcine respiratory epithelial cells (PoRECs), isolated from nasal tissue, we found a difference in infection mechanism between the two genotypes, with genotype I favoring the basolateral surface and genotype II preferring the apical surface. Moreover, disruption of intercellular junctions enhanced infection for genotype I. This study demonstrated that ASFV may use the respiratory mucosa for entry using different cell types for replication with a genotype difference in their infection of respiratory epithelial cells.

PRRSV-Vaccinated, Seronegative Sows and Maternally Derived Antibodies (I): Impact on PRRSV-1 Challenge Outcomes in Piglets.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains an infectious agent with high importance in the swine industry. In this study, the influence of maternally derived antibodies (MDAs) on an experimental PRRSV-1 challenge is investigated. Piglets included in the study (n = 36) originated from a Belgian farrow-to-finish herd in which the sow population was routinely vaccinated with a modified live vaccine against PRRSV. Eighteen piglets were born from three PRRSV-seropositive sows (responders to vaccination) and had a clear presence of PRRSV-specific MDAs (E+ piglets). The other eighteen piglets were born from three PRRSV-seronegative sows (non-responders to vaccination) and did not have PRRSV-specific MDAs (E- piglets). In each group, twelve piglets were intranasally challenged with a high dose of the heterologous PRRSV-1 07V063 strain, the remaining piglets were mock-challenged (PBS) and served as controls. During the first days after infection, higher serum viremia and nasal shedding were observed in the challenged E- piglets compared to the challenged E+ piglets. However, at 10 days post-infection, the peak serum viremia was significantly higher in the E+ piglets in comparison to the E- piglets and serum viremia remained slightly higher in this group until the end of the study. Additionally, the two challenged groups had a different immune response to the PRRSV infection. The E- challenged piglets showed an earlier and more intense seroconversion, leading to significantly higher antibody titers at 10 dpi compared to the E+ challenged piglets. Furthermore, a trend towards both higher induction of serum IFN-γ and higher induction of IFN-γ secreting cells was observed in the E- challenged piglets. In contrast, a significantly higher induction of serum TNF-α at 7 dpi was seen in the E+ challenged piglets compared to the E- challenged piglets. The results gathered in this study suggest that PRRSV-specific MDAs induce partial protection during the early stages of infection but are not sufficient to protect against a high challenge dose. The presence of piglets lacking PRRSV-specific MDAs might pose a risk for PRRSV infection and enhanced transmission in pig farms in young piglets.

Follow-Up of PRRSv-Vaccinated Piglets Born from PRRSv-Vaccinated, ELISA-Seropositive and ELISA-Seronegative Sows.

Vaccination against the porcine reproductive and respiratory syndrome virus (PRRSv) is widely used to prevent production losses in the swine industry. In this study, piglets born from both PRRSv-vaccinated ELISA-seropositive sows (E+ piglets) and PRRSv-vaccinated ELISA-seronegative sows (E- piglets) were followed-up pre-vaccination, 3 weeks post-vaccination (wpv) and 8 wpv in two Belgian farrow-to-finish herds. The aim of the study was to analyze the presence of PRRSv-specific maternally-derived antibodies (MDAs) and the PRRSv vaccine response in both groups of piglets. The E- piglets lacked the presence of PRRSv-specific MDAs (0% seropositive), while these were present in the E+ piglets (97% seropositive). Due to this, the E- piglets showed a strong initial vaccine response (72-80% seroconversion) and vaccine viremia (65-75% PCR positive) at 3 wpv. In contrast, the E+ piglets showed only limited initial vaccine responses (25-61% with increased ELISA values) and vaccine viremia (30-31% PCR positive) at 3 wpv. By 8 wpv, the proportion of seropositive E- piglets (78-100%) and seropositive E+ piglets (55-90%) increased in both herds. However, a difference in vaccine viremia duration was observed between both herds at 8 wpv, with a decrease in the proportion of PCR positive piglets in herd 1 (E-: 47%; E+: 25%) and an increase in the proportion of PCR positive piglets in herd 2 (E-: 85%; E+: 92%). This study identified clear differences in the presence of PRRSv-specific maternally-derived antibodies and PRRSv vaccine responses between E- and E+ piglets. Further research is warranted to elicit the biological relevance of these observed differences.

Porcine Reproductive and Respiratory Syndrome virus (PRRSv): A Cross-Sectional Study on ELISA Seronegative, Multivaccinated Sows.

Vaccination against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) is widely used to control clinical disease, but the effectiveness appears in some cases to be suboptimal. Field reports have stated the presence of routinely PRRSv-vaccinated but ELISA seronegative sows: the ELISA non-responders. The real extent of this phenomenon (prevalence-origin-consequences) was not yet investigated. In this study, the prevalence of ELISA non-responders was assessed by measuring PRRSv-specific antibodies in 1400 sows, originating from 70 PRRSv-vaccinating sow herds, using IDEXX ELISA (ELISA 1) and CIVTEST E/S ELISA (ELISA 2). Neutralizing antibodies (NAbs) were quantified in a virus neutralization assay. Univariable logistic regression was used to identify herd risk factors for the presence of ELISA non-responders. The global prevalence of non-responders varied from 3.5% (ELISA 1) to 4.1% (ELISA 2), the herd-level prevalence was 40% and the within-herd prevalence ranged from 5% to 20% (ELISA 1) and from 5% to 30% (ELISA 2). The ELISA non-responders had significantly lower NAbs than the ELISA responders. Herds using the combination of one modified live vaccine and one killed vaccine had a significantly reduced risk of having ELISA non-responders. A first assessment of the prevalence and possible consequences of ELISA non-responders has been provided by this study. The clinical importance, origin and underlying immunological mechanisms warrant further research.

WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015-2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

Towards Efficient Early Warning: Pathobiology of African Swine Fever Virus "Belgium 2018/1" in Domestic Pigs of Different Age Classes.

African swine fever (ASF) is one of the most important and devastating viral diseases in wild boar and domestic pigs worldwide. In the absence of vaccines or treatment options, early clinical detection is crucial and requires a sound knowledge of disease characteristics. To provide practitioners and state veterinarians with detailed information, the objective of the present study was to characterize the ASF virus (ASFV) isolate "Belgium 2018/1" in subadult and weaning domestic pigs. To this end, two animal trials were performed. Trial A included eight subadult domestic pigs and trial B five weaner pigs. In general, clinical signs and pathological lesions were in line with previous studies utilizing highly virulent ASF genotype II viruses. However, in trial A, four subadult domestic pigs survived and recovered, pointing to an age-dependent outcome. The long-term fate of these survivors remains under discussion and would need further investigation.

Evaluation of seven commercial African swine fever virus detection kits and three Taq polymerases on 300 well-characterized field samples.

African swine fever virus (ASFV) is a complex double stranded DNA virus, responsible for a highly infectious and fatal disease in pigs and boars and for important deterioration of animal welfare. Over the last decade, the disease spread to several European and Asian countries causing unprecedented dramatic economic losses in pig industry. In the absence of a vaccine, affected countries rely on trustful diagnostic tests and adapted testing policies to set up control programs to fight against the disease. In this study, we evaluated the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases on 300 well-characterized wild boar samples collected in Belgium during the 2018-2019 outbreak. This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results.

The African swine fever virus isolate Belgium 2018/1 shows high virulence in European wild boar.

African swine fever (ASF) is one of the most important and complex viral diseases in domestic pigs and wild boar. Over the last decade, the disease has spread to several European and Asian countries and is now one of the major threats to profitable pig production worldwide. One of the more recently affected western countries is Belgium. To date, only wild boar are affected in a rather defined area in the Luxembourg region close to France, Luxembourg and Germany. While detailed sequence analyses were recently performed, biological characterization was still pending. Here, we report on the experimental inoculation of four sub-adult wild boar to further characterize the virus and its distribution in different tissues. After oronasal inoculation with the virus strain 'Belgium 2018/1', all animals developed an acute and severe disease course with typical pathomorphological and histopathological lesions. Organs and blood samples were positive in qPCR, haemadsorption test and antigen lateral flow devices (LFD). Virus and viral genome were also detected in genitals and accessory sex glands of two boars. There were no antibodies detectable in commercial antibody ELISAs, antibody LFDs and indirect immunoperoxidase tests. Thus, the genotype II ASF virus isolate 'Belgium 2018/1' showed a highly virulent phenotype in European wild boar similar to parental viruses like Armenia 2007 and other previously characterized ASFV strains. The study also provided a large set of well-characterized sample materials for test validation and assay harmonization.

Comparative Analysis of Whole-Genome Sequence of African Swine Fever Virus Belgium 2018/1.

We analyzed the whole-genome sequence of African swine fever virus Belgium 2018/1. The strain fits into the European genotype II (>99.98% identity). The high-coverage sequence revealed 15 differences compared with an improved African swine fever virus Georgia 2007/1 sequence. However, in the absence of genetic markers, no spatial or temporal correlations could be defined.

Phylogeographic Analysis of African Swine Fever Virus, Western Europe, 2018.

In September 2018, African swine fever in wild boars was detected in Belgium. We used African swine fever-infected spleen samples to perform a phylogenetic analysis of the virus. The causative strain belongs to genotype II, and its closest relatives are viruses previously isolated in Ukraine, Belarus, Estonia, and European Russia.

Seroprevalence of border disease virus and other pestiviruses in sheep in Algeria and associated risk factors.

BACKGROUND: Border disease virus (BDV) is a pestivirus responsible for significant economic losses in sheep industry. The present study was conducted between 2015 and 2016 to determine the flock seroprevalence of the disease in Algeria and to identify associated risk factors. 56 flocks from nine departments were visited and 689 blood samples were collected from adult sheep between 6 and 24 months of age (n = 576) and from lambs younger than 6 months (n = 113). All samples were tested by RT-PCR as well as by Ag-ELISA, to detect Persistently Infected (PI) animals. Serum samples from adults were tested by Ab-ELISA (Enzyme Linked Immuno-Sorbent Assay), to detect specific antibodies against pestivirus and 197 of them were further characterized by VNT (virus neutralization test) for the detection of neutralizing antibodies specific for BDV and for Bovine virus diarrhea virus (BVDV-1 and BVDV-2). RESULTS: No PI animals were found among the 689 sheep tested. 144/197 sera were positive in VNT for BDV, and 2 sera were strongly positive BVDV-2. Fifty-five flocks (98%) had at least one seropositive animal and the apparent within-flock seroprevalence was estimated to be 60.17% (95% C.I.: 52.96-66.96). The true seroprevalence based on estimated sensitivity and specificity of the Ab-ELISA was 68.20% (95% C.I.; 60.2-76.3). Several risk factors were identified as linked to BDV such as climate, landscape, flock management and presence of other ruminant species in the farm. CONCLUSION: These high seroprevalence rates suggest that BDV is widespread and is probably endemic all over the country. Further studies are needed to detect and isolate the virus strains circulating in the country and understand the distribution and impact of pestiviruses in the Algerian livestock.

Reproductive Disorders and Leptospirosis: A Case Study in a Mixed-Species Farm (Cattle and Swine).

Animal leptospirosis, exempt in rodents, manifests as peculiar biology where the animal can function, simultaneously or not, as a susceptible host or reservoir. In the first case, clinical symptoms are likely. In the second case, infection is subclinical and manifestations are mild or absent. Mild clinical symptoms encompass reproductive failure in production animals for host-adapted Leptospira sp. serovars. This work presents a study on Leptospira sp. infection in a mixed-species (bovine and swine) farm with documented reproductive disorders in the cattle unit. A long calving interval (above 450 days) was the hallmark observed in cows. Some cows (2/26 tested) presented a high titre of antibodies against Leptospira sp. serogroup Sejroe, but the overall within-herd prevalence was low (11.5% and 7.7% for cut-off titres of 1:30 and 1:100, respectively). The in-herd prevalence of leptospirosis in the sow unit (determined for 113/140 animals) was high when using a lowered cut-off threshold (32.7% vs. 1.8% for cut-off titre of 1:30 and 1:100, respectively). In this unit, the most prevalent serogroup was Autumnalis. The final diagnostic confirmation of Leptospira sp. maintenance within the farm was obtained through detection by PCR of Leptospira sp. DNA in an aborted swine litter. Despite the fact that a common causative infective agent was diagnosed in both species, the direct link between the two animal units was not found. Factors such as drinking from the same water source and the use of manure prepared with the swine slurry might raise suspicion of a possible cross-contamination between the two units. In conclusion, this work suggests that leptospirosis be included in the differential diagnosis of reproductive disorders and spontaneous abortions in production animals and provides data that justify the use of a lowered threshold cut-off for herd diagnosis.

Encephalomyocarditis virus in a captive Malayan tapir (Tapirus indicus).

A 5-month-old female captive Malayan tapir (Tapirus indicus) died suddenly without preceding symptoms. Gross necropsy revealed numerous white circular and linear foci in the myocard. Differential diagnosis all turned out negative, except for encephalomyocarditis virus. Histopathology revealed mineralisation of myocardial cells and interstitial infiltration of lymphocytes, plasma cells and less neutrophils. Encephalomyocarditis virus was detected by PCR. Although encephalomyocarditis virus occurs in many mammals, this is the first published description of this virus in a Malayan tapir.

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.

First TBEV serological screening in Flemish wild boar.

In the frame of a Flemish wildlife surveillance in 2013, a serological screening was performed on sera from wild boar (Sus scrofa; n=238) in order to detect tick-borne encephalitis virus (TBEV)-specific antibodies. Neutralising antibodies were titrated with a seroneutralisation test (SNT), using two cut-off titres (1/10-1/15). Seven wild boars were found TBEV-seropositive and showed moderate (>1/15) to high (>1/125) SNT-titres; three individuals had borderline results (1/10-1/15). This study demonstrated the presence of TBEV-specific antibodies in wild boar and highlighted potential TBEV-foci in Flanders. Additional surveillance including direct virus testing is now recommended.

Clinical problems due to encephalomyocarditis virus infections in two pig herds.

BACKGROUND: Infections with encephalomyocarditis virus may cause myocarditis and sudden death in young pigs and reproduction disorders in sows. The presence of encephalomyocarditis virus infected rodents is considered a major risk factor for transmission of the virus to pigs. There is currently no effective treatment. Tightening up biosecurity, applying effective rodent control and reducing stress are the main control measures. CASE PRESENTATION: Two farrow-to-finish herds suffering from problems with sudden death are presented. In herd A, suckling piglets from 3 to 12 days old were dying acutely whereas in herd B, piglets at the end of the nursery period (8-10 weeks) were showing identical problems. A presumptive diagnosis of encephalomyocarditis virus infection was made because typical lesions were observed in some of the affected pigs. These lesions were not always present in pigs dying acutely or in some cases the lesions were very subtle. Therefore other causes had to be ruled out based upon clinical history, clinical signs and diagnostic tests. A conclusive diagnosis was finally established by showing encephalomyocarditis virus in heart tissue using conventional gel-based polymerase chain reaction tests. The real-time PCR test that gave initially negative result was further optimized to avoid false negative results. CONCLUSIONS: Typical lesions are not always present in piglets infected with encephalomyocarditis virus, indicating the importance of examining multiple animals. Problems in suckling piglets may occur in affected herds without reproductive problems in sows. Transmission routes of EMCV in swine are not fully understood. A stand-empty period following thorough cleaning and disinfection is recommended for controlling EMC virus infections.

Study of the virulence of serotypes 4 and 9 of African horse sickness virus in IFNAR(-/-), Balb/C and 129 Sv/Ev mice.

African horse sickness virus (AHSV) is a double-stranded RNA virus which belongs to the family Reoviridae, genus Orbivirus. Recent studies have focused on the interferon-α/ß receptor knock-out mice (IFNAR(-/-)) as a small animal laboratory for the development of AHSV vaccines. The aim of this work was to study in vivo the virulence of two strains of AHSV and to compare the outcome of the infection of three mouse strains. To address this, AHSV serotypes 4 (AHSV-4) and 9 (AHSV-9) were inoculated subcutaneously (SC) and intranasally (IN) in two immunocompetent mouse strains (Balb/C and 129 Sv/Ev (129 WT)) as well as IFNAR(-/-) mice (on 129 Sv/Ev genetic background). In IFNAR(-/-) mice, fatality up to 50% was measured and significantly more clinical signs were observed in comparison with SC inoculated immunocompetent mice. The observed clinical signs were significantly more severe after AHSV-4 infection, in particular in immunocompetent mice inoculated by IN route. Considering RNAemia, significantly higher viral loads were measured following AHSV-4 infection. In the organs of 129 WT inoculated by IN route, significantly higher viral loads were detected after AHSV-4 infection. Together the results support a higher virulence for AHSV-4 compared to AHSV-9 and a higher clinical impact following infections in IN inoculated mice, at least in the investigated strains. The study also brought indirect evidences for type I IFN involvement in the control of AHSV infection.

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus.

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas.