Recent insights on epidemiology, diagnosis and control of Classical and African Swine Fever in pig industry

To determine serum concentrations of the selected acute-phase proteins (APPs) haptoglobin, serum amyloid A (SAA), and C-reactive protein (CRP) in pigs experimentally inoculated with classical swine fever (CSF) and African swine fever (ASF) viruses. 8 crossbred (Large White x Landrace) 10-week-old pigs. Pigs were allocated to 2 groups (4 pigs/group). One group was inoculated with the CSF virus Alfort 187 strain, whereas the other groupwas inoculated with the ASF virus Spain 70 isolate. Blood samples were collected at various time points. At the end of the study, pigs were euthanized and a complete necropsy was performed, including histologic and immunohistochemical analyses. Serum concentrations of APPs increased in pigs inoculated with CSF and ASF viruses, which suggested an acute-phase response in the course of both diseases. The most noticeable increase in concentration was recorded for SAA in both groups (up to a 300-fold increase for CSF virus and an approx 40-fold increase for ASF virus), followed by CRP and then haptoglobin, which each had only 3- to 4-fold increases. Serum concentrations of APPs increased significantly in pigs inoculated with CSF and ASF viruses. However, differences were evident in serum concentrations of the proteins evaluated in this study.

The results of the study of lymphoid organs and sera of wild boars for the presence of DNA of African swine fever (ASF) virus and RNA of classical swine fever (CSF) virus are presented, as well as the results of a serological examination for the presence of ASF and CSF virus antibodies. The study was conducted in Ukraine between 2008 and 2013. Biological samples were obtained from wild boars shot during the hunting season, and were examined by real-time PCR and ELISA. In total, 5759 sera were tested for CSF virus antibodies and 4856 for ASF virus antibodies by ELISA. Samples of lymphoid organs totalling 1129 were examined by PCR for the detection of CSF virus RNA and 8102 such samples were examined for the detection of ASF virus DNA. CSF virus antibodies were detected in 6.56% of wild boar sera. RNA of CSF virus was also identified in 1 out of 1129 samples tested. ASF virus antibodies or DNA in lymphoid organ samples were not detected.

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170–190+ kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with a genome size of approximately 12 kB, belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS) allow for the establishment of NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution, facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10 kB and 6 kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV, providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the three known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with two different genotype 1 CSF viruses. ASFV and CSFV amplicons from optimized PCR were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1,000X for ASFV, with 99% of the genome covered, and 10,000X–20,000X for CSFV, with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation, as well as genetic characterization of these high-consequence swine viruses, globally and within the United States, should an outbreak occur.

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170-190 kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with genome size of approximately 12 kB belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS), allows for the establishment of vital NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10kB and 6kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the 3 known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with 2 different genotype 1 viruses. ASFV and CSFV amplicons from optimized PCR reactions were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1000X for ASFV with 99% of the genome covered, and 10,000X-20,000X for CSFV with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation as well as genetic characterization of these high consequence swine viruses, globally and within the United States, should an outbreak occur.

Recombinase polymerase amplification (RPA) was combined with lateral flow to develop a gold nanoparticles test strip for point-of-care diagnosis of African swine fever virus (ASFV), which is called lateral flow gene assay (LFGA). Common diagnostic techniques, including polymerase chain reaction (PCR) and immunochromatography, are time-consuming and labor-intensive, and generally require costly instruments. For improvement, this assay used tailed primers to produce DNA duplexes with a single-stranded tail at one end which can hybridize with a gold nanoparticle (AuNP)-labeled oligonucleotide detection probe. And then, biotin attached to the other end of the product bound to streptavidin, which previously fixed to the test line. Therefore, there would form a sandwich structure, and gold nanoparticles labeled on the detection probe would show a red band on the test line of strip. With the low reaction temperature (37~42°C) and short reaction time (30min), LFGA can specifically identify ASFV in blood samples infected with ASFV and classical swine fever virus (CSFV), and the LOD was 102 copies/μL, which was comparable to that of agarose gel electrophoresis. In addition, blood samples infected with ASFV and CSFV were tested, and it was found that the LFGA can specifically identify ASFV DNA. In conclusion, LFGA achieves visual observation of the product after rapid RPA amplification and does not require any expensive instruments during the entire process, which is very helpful for early diagnosis of ASFV. Combined recombinase polymerase amplification (RPA) with lateral flow, we developed a gold nanoparticles test strip for point-of-care diagnosis of African swine fever virus. The upstream primers of RPA were modified with biotin, and the downstream primers were modified with a C3 spacer and an oligonucleotide tail that can be hybridized to a gold nanoparticle-labeled oligonucleotide detection probe. On the strip, the test line and control line were sprayed with streptavidin and an oligonucleotide control probe. In the presence of positive products, RPA products can form a sandwich structure on the test line. Therefore, two red lines will be displayed both on the test line and control line. When there is no positive product, only the control line is shown in red. Its low reaction temperature (37~42°C) and short time of amplification and detection (30min) make ASFV realizing point-of-care diagnosis in limited environment.

BackgroundRecently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.ResultsAfter ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.ConclusionsOur results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. Bovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

For the global pig industry, classical (CSF) and African swine fever (ASF) outbreaks are a constantly feared threat. Except for Sardinia, ASF was eradicated in Europe in the late 1990s, which led to a research focus on CSF because this disease continued to be present. However, ASF remerged in eastern Europe in 2007 and the interest in the disease, its control and epidemiology increased tremendously. The similar names and the same susceptible species suggest a similarity of the two viral diseases, a related biological behaviour and, correspondingly, similar epidemiological features. However, there are several essential differences between both diseases, which need to be considered for the design of control or preventive measures. In the present review, we aimed to collate differences and similarities of the two diseases that impact epidemiology and thus the necessary control actions. Our objective was to discuss critically, if and to which extent the current knowledge can be transferred from one disease to the other and where new findings should lead to a critical review of measures relating to the prevention, control and surveillance of ASF and CSF. Another intention was to identify research gaps, which need to be closed to increase the chances of a successful eradication of ASF and therefore for a decrease of the economic threat for pig holdings and the international trade.

The health of swine populations is increasingly threatened by diseases such as classical swine fever (CSF), African swine fever (ASF), and Japanese encephalitis (JE), which causeeconomic losses. This study investigated the seroprevalence and distribution of these pathogens across 13 districts in Chhattisgarh, Madhya Pradesh, and Telangana, India. A total of 345 serum samples and 275 whole blood samples were collected for various diagnostic testing. Samples were analyzed using reverse transcriptase polymerase chain reaction (RT-PCR), virus neutralization test (VNT), and indirect enzyme-linked immunosorbent assay (iELISA), with CSF showing the highest seroprevalence in Chhattisgarh (76.63%), while ASF waslowest (7.21%). In Madhya Pradesh, the CSF seroprevalence was 33.33%, with no ASF detected. JE positivity was 12.37% in Chhattisgarh and 44.44% in Madhya Pradesh. RT-PCR of 275 whole blood samples from Telangana revealed 11.27% positivity for CSF and 9.45% for JE, with no ASF cases. In Chhattisgarh, 7.20% of the samples showed concurrent antibody positivity for both CSF and ASF. Additionally, 2.06% were positive for CSF, ASF, and JE, indicating concurrent exposure to multiple pathogens. Age-wise analysis showed the highest CSF positivity among older pigs in Chhattisgarh and Madhya Pradesh, with higher rates observed in pigs >2 years. These findings emphasize the necessity of antigen and antibody testing for effective swine disease surveillance, revealing concurrent exposure to multiple pathogens. A combined approach using serological and molecular diagnostics strengthens surveillance, supports multi-disease control strategies, and is vital for limiting disease spread and maintaining swine health.

Detection of antibodies against classical swine fever virus in fecal samples from wild boar

Classical swine fever (CSF) also known as 'hog choleraâ' is an important viral disease of pigs in India with serious economic concern due to morbidity and mortality, and affects a vast section of the pig population in India. In present study, a structured sampling design was adopted, which covered the major regions of the Mizoram, to ascertain the economic losses due to CSF in pigs. Available estimates of the economic losses of Mizoram due to CSF are based on single values of various epidemiological and economic parameters. Overall annual morbidity, mortality and case fatality rates of CSF in Mizoram were 8.35, 5.07 and 60.70% respectively. The expected annual economic loss due to CSF in pigs in Mizoram was ` 16,69,34,465. Losses due to mortalities contributed the most to the total economic loss caused by CSF in pigs, followed by loss in body weight. This study revealed significant losses due to the incidence of CSF in pigs of Mizoram.

BackgroundAfrican swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens.ObjectivesThe aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV.MethodsThree pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5′ untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay.ResultsThe multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 100 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively.ConclusionsThe multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5' untranslated region (5'UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV.

Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT-PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay's limit of detection (LOD) was 2.89 × 102 copies/μL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay's applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study's multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.

Background: Classical swine fever (CSF) is caused by small enveloped RNA virus that belongs to the Flaviviridae family and causes high morbidity and mortality events for pig farming. In India, it is highly prevalent but with varied distribution in various geographical areas resulting in substantial economic losses to farmers. The discrepancy in prevalence estimates across studies needs to be evaluated in order to strengthen the prevention and control strategies of CSF in pigs. The aim of the present study was to provide the pooled estimate of the prevalence of classical swine fever (CSF) in India and to examine the consistency of those estimates between published studies. Methods: The data regarding prevalence of CSF in 7045 pigs were compiled from 22 published studies using comprehensive literature search for period from 1998 to 2018. The pooled prevalence of CSF was obtained under random effects model and heterogeneity between studies was determined using I² Index. Representation of meta-analysis was done using forest plot and followed by funnel plot, Baujat plot and radial plot. Result: The result of Meta-analysis revealed that pooled estimate of prevalence of CSF under random effects model was 45% (95% confidence interval: 35, 55%) in India. The heterogeneity statistic (Q) was found to be significant (p less than 0.001) with I² = 98% which indicated that prevalence of CSF was inconsistent between the studies. Likewise, results of funnel plot and Radial plot indicated heterogeneity between studies. Most influential studies for overall heterogeneity were identified using Baujat and radical plots.