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

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

Efficient detection of African Swine Fever Virus using minimal equipment through a LAMP PCR method.

African swine fever virus (ASFV) currently represents the biggest threat to the porcine industry worldwide, with high economic impact and severe animal health and welfare concerns. Outbreaks have occurred in Europe and Asia since ASFV was reintroduced into the continent in 2007 and, in 2021, ASFV was detected in the Caribbean, raising alarm about the reemergence of the virus in the Americas. Given the lack of vaccines against ASFV, control of the virus relies on molecular surveillance, which can be delayed due to the need for sample shipment to specialized laboratories. Isothermal PCR techniques, such as LAMP, have become increasingly attractive as point-of-care diagnostic tools given the minimal material expense, equipment, and training required. The present study aimed to develop a LAMP assay for the detection of ASFV. Four LAMP primer sets were designed, based on a consensus sequence for the ASFV p72 gene, and were tested using a synthetic plasmid containing the cloned ASFV p72 target gene as a positive control. Two primer sets, were selected for further validation, given their very short time for amplification. Both primer sets showed thermal stability, amplifying the ASFV DNA at temperatures between 60-70°C and proved to have an analytical limit of detection as low as one ASFV-plasmid DNA copy/µL, using both fluorometric and colorimetric methods. The selected primers did not yield false positive or cross reactive results with other common swine pathogens, showing high specificity. Testing of DNA-spiked samples showed that LAMP amplification was not affected by the nature of the matrices, including oral fluids, tonsils, blood, or rectal swabs. The primer sets were able to detect the two more prevalent ASFV genotypes in the field. Taken together, the results show that ASFV-LAMP-BG2 and ASFV-LAMP-BG3 would be a useful tool for rapid, highly sensitive on-site diagnostic testing.

The FlagT4G Vaccine Confers a Strong and Regulated Immunity and Early Virological Protection against Classical Swine Fever.

Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination, mostly using vaccines that do not allow for differentiation of vaccinated from infected animals (DIVA). FlagT4G vaccine is a novel candidate that confers robust immunity and shows DIVA capabilities. The present study assessed the immune response elicited by FlagT4G and its capacity to protect pigs for a short time after vaccination. Five days after a single dose of FlagT4G vaccine, animals were challenged with a highly virulent CSFV strain. A strong, but regulated, interferon-α response was found after vaccination. Vaccinated animals showed clinical and virological protection against the challenge, in the absence of antibody response at 5 days post-vaccination. Upon challenge, a rapid rise in the titers of CSFV neutralizing antibodies and an increase in the IFN-γ producing cells were noticed in all vaccinated-challenged pigs. Meanwhile, unvaccinated pigs showed severe clinical signs and high viral replication, being euthanized before the end of the trial. These animals were unable to generate neutralizing antibodies and IFN-γ responses after the CSFV challenge. The results from the present study assert the fast and efficient protection by FlagT4G, a highly promising tool for CSFV control worldwide.

Removal of the Erns RNase Activity and of the 3' Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease.

In this study, we assessed the potential synergistic effect of the Erns RNase activity and the poly-U insertion in the 3' untranslated region (UTR) of the low-virulence classical swine fever virus (CSFV) isolate Pinar de Rio (PdR) in innate and adaptive immunity regulation and its relationship with classical swine fever (CSF) pathogenesis in pigs. We knocked out the Erns RNase activity of PdR and replaced the long polyuridine sequence of the 3' UTR with 5 uridines found typically at this position, resulting in a double mutant, vPdR-H30K-5U. This mutant induced severe CSF in 5-day-old piglets and 3-week-old pigs, with higher lethality in the newborn (89.5%) than in the older (33.3%) pigs. However, the viremia and viral excretion were surprisingly low, while the virus load was high in the tonsils. Only alpha interferon (IFN-α) and interleukin 12 (IL-12) were highly and consistently elevated in the two groups. Additionally, high IL-8 levels were found in the newborn but not in the older pigs. This points toward a role of these cytokines in the CSF outcome, with age-related differences. The disproportional activation of innate immunity might limit systemic viral spread from the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms. Infection with vPdR-H30K-5U resulted in poor neutralizing antibody responses compared with results obtained previously with the parent and RNase knockout PdR. This study shows for the first time the synergistic effect of the 3' UTR and the Erns RNase function in regulating innate immunity against CSFV, favoring virus replication in target tissue and thus contributing to disease severity. IMPORTANCE CSF is one of the most relevant viral epizootic diseases of swine, with high economic and sanitary impact. Systematic stamping out of infected herds with and without vaccination has permitted regional virus eradication. However, the causative agent, CSFV, persists in certain areas of the world, leading to disease reemergence. Nowadays, low- and moderate-virulence strains that could induce unapparent CSF forms are prevalent, posing a challenge for disease eradication. Here, we show for the first time the synergistic role of lacking the Erns RNase activity and the 3' UTR polyuridine insertion from a low-virulence CSFV isolate in innate immunity disproportional activation. This might limit systemic viral spread to the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms, thus contributing to disease severity. These results highlight the role played by the Erns RNase activity and the 3' UTR in CSFV pathogenesis, providing new perspectives for novel diagnostic tools and vaccine strategies.

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

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

Development of a Dendrimeric Peptide-Based Approach for the Differentiation of Animals Vaccinated with FlagT4G against Classical Swine Fever from Infected Pigs.

Classical swine fever virus (CSFV) causes a viral disease of high epidemiological and economical significance that affects domestic and wild swine. Control of the disease in endemic countries is based on live-attenuated vaccines (LAVs) that induce an early protective immune response against highly virulent CSFV strains. The main disadvantage of these currently available LAVs is the lack of serological techniques to differentiate between vaccinated and infected animals (DIVA concept). Here, we describe the development of the FlagDIVA test, a serological diagnostic tool allowing for the differentiation between animals vaccinated with the FlagT4G candidate and those infected with CSFV field strains. The FlagDIVA test is a direct ELISA based on a dendrimeric peptide construct displaying a conserved epitope of CSFV structural protein E2. Although FlagDIVA detected anti-CSFV anti-bodies in infected animals, it did not recognize the antibody response of FlagT4G-vaccinated animals. Therefore, the FlagDIVA test constitutes a valuable accessory DIVA tool in implementing vaccination with the FlagT4G candidate.

Early and Solid Protection Afforded by the Thiverval Vaccine Provides Novel Vaccination Alternatives Against Classical Swine Fever Virus.

Classical swine fever virus (CSFV) remains a challenge for the porcine industry. Inefficient vaccination programs in some endemic areas may have contributed to the emergence of low and moderate virulence CSFV variants. This work aimed to expand and update the information about the safety and efficacy of the CSFV Thiverval-strain vaccine. Two groups of pigs were vaccinated, and a contact and control groups were also included. Animals were challenged with a highly virulent CSFV strain at 21- or 5-days post vaccination (dpv). The vaccine induced rapid and strong IFN-α response, mainly in the 5-day immunized group, and no vaccine virus transmission was detected. Vaccinated pigs showed humoral response against CSFV E2 and Erns glycoproteins, with neutralising activity, starting at 14 days post vaccination (dpv). Strong clinical protection was afforded in all the vaccinated pigs as early as 5 dpv. The vaccine controlled viral replication after challenge, showing efficient virological protection in the 21-day immunized pigs despite being housed with animals excreting high CSFV titres. These results demonstrate the high efficacy of the Thiverval strain against CSFV replication. Its early protection capacity makes it a useful alternative for emergency vaccination and a consistent tool for CSFV control worldwide.

Decrypting the Origin and Pathogenesis in Pregnant Ewes of a New Ovine Pestivirus Closely Related to Classical Swine Fever Virus.

This study shows the origin and the pathogenic role of a novel ovine pestivirus (OVPV) isolated in 2017 in Italy, as a pathogenic agent causing severe abortions after infection in pregnant ewes and high capacity for virus trans-placental transmission as well as the birth of lambs suffering OVPV-persistent infection. The OVPV infection induced early antibody response detected by the specific ELISA against classical swine fever virus (CSFV), another important virus affecting swine. The neutralizing antibody response were similar against CSFV strains from genotype 2 and the OVPV. These viruses showed high identity in the B/C domain of the E2-glycoprotein. Close molecular diagnostics cross-reactivity between CSFV and OVPV was found and a new OVPV molecular assay was developed. The phylodynamic analysis showed that CSFV seems to have emerged as the result of an inter-species jump of Tunisian sheep virus (TSV) from sheep to pigs. The OVPV and the CSFV share the TSV as a common ancestor, emerging around 300 years ago. This suggests that the differentiation of TSV into two dangerous new viruses for animal health (CSFV and OVPV) was likely favored by human intervention for the close housing of multiple species for intensive livestock production.