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Identification of novel testing matrices for African swine fever surveillance.

ABSTRACT: African swine fever (ASF) is a devastating viral disease of pigs and wild boar, and it threatens global food security. We aimed to identify suitable sample matrices for use in ASF surveillance programs. Six pigs inoculated with ASFV were sampled at postmortem. Blood, bone marrow, ear biopsies, and oral, nasal, and rectal swabs were taken from all pigs. All samples were analyzed using 3 real-time PCR (rtPCR) assays and a LAMP assay. ASFV was detected at > 10⁷ genome copies/mL in blood; bone marrow was found to provide the highest viral load. Ct values provided by the rtPCR assays were correlated, and ASFV was detected in all oral, nasal, and rectal swabs and in all ear biopsy samples irrespective of the location from which they were taken. The LAMP assay had lower sensitivity, and detected ASFV in 54 of 66 positive samples, but delivered positive results within 17 min. We identified additional sample matrices that can be considered depending on the sampling situation: bone marrow had a high probability of detection, which could be useful for decomposed carcasses. However, ear biopsies provide an appropriate, high-throughput sample matrix to detect ASFV and may be useful during surveillance programs.

SUBMITTER: Flannery J

PROVIDER: S-EPMC7645617 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:African swine fever (ASF) has one of the highest case-fatality rates among pig diseases. Europe was considered ASF-free for about two decades until 2007, when the virus was introduced into Georgia. Since then, it has been identified throughout Eastern Europe, and reached Belgium in late 2018, increasing the risk of ASF being introduced into neighboring countries-namely Germany, Luxembourg, the Netherlands, and France. French authorities have therefore reinforced surveillance measures to improve the probability of detecting ASF rapidly if it emerges in France. Predictive modeling may help to anticipate the extent of virus spread and evaluate the efficiency of these surveillance measures. A previously published and well-documented model that simulates ASF virus spread was therefore tailored to realistically represent the French situation in terms of the geographic distribution of swine production sites and the commercial trade between them on the one hand, and the implementation of surveillance protocols on the other. The outcomes confirmed the moderate spread of ASF through the swine trade network, a situation that had been previously highlighted for the case of Denmark. However, the diversity of the French pig production landscape has revealed a huge potential for the geographic dispersal of the virus, especially should the index case occur in a low-density area, with a median source-to-case distance reaching 300 km. Free-range herds, which are more likely to have interactions with wild boars, were also identified as potential entrance gate for the virus. Transmissions from conventional herds were quasi-exclusively due to swine movement on the commercial network, representing 99% of transmission events. In contrast, 81% of transmission events occurred in the neighborhood of the index herd when the virus was introduced in free-range herds. The current surveillance measures were found relatively efficient for detecting the virus in large herds, leading to detection rates of 94%. However, infections on smaller production sites-which often have free-range herds-were more difficult to detect and would require screening protocols specifically targeting these smaller herds.

Project description:Recently 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.After 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.Our 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.

Project description:African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies.ImportanceASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.

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Identification of novel testing matrices for African swine fever surveillance.

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