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.

New Potential Role of European Mouflon (Ovis aries musimon) in the Epidemiology of Border Disease in the Pyrenees.

Since 2001, high-mortality outbreaks of border disease (BD) have negatively affected populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Studies in the affected areas determined that sympatric wild ruminants did not seem to have an epidemiologic role in the circulation of border disease virus (BDV). However, the recent increase in European mouflon (Ovis aries musimon) densities might enhance the risk of pathogen transmission among chamois and mouflons. We conducted a serologic and virologic investigation of BDV in European mouflon from the Spanish Pyrenees, with the aim of determining potential changes in the role of this species in BDV epidemiology. From 2018 to 2022, we detected antibodies against BDV in 31/185 (16.7%) animals but did not detect BDV RNA in any spleen sample (0/65). These results indicate that BDV infection is occurring in these mouflon populations to a greater extent than previously described, which could shift the current understanding of BD epidemiology in the Pyrenees and cause an unpredictable effect on both chamois and mouflon populations. Further studies on the molecular identification of BDV in mouflon and chamois are required to better understand the contribution of mouflon in the epidemiology of BD.

Feeding Spray-Dried Porcine Plasma to Pigs Improves the Protection Afforded by the African Swine Fever Virus (ASFV) BA71∆CD2 Vaccine Prototype against Experimental Challenge with the Pandemic ASFV-Study 2.

This study aimed to evaluate the effects of feeding spray-dried porcine plasma (SDPP) on the protection afforded by the BA71∆CD2 African swine fever virus (ASFV) vaccine prototype. Two groups of pigs acclimated to diets without or with 8% SDPP were intranasally inoculated with 105 plaque-forming units (PFU) of live attenuated ASFV strain BA71∆CD2 and, three weeks later, left in direct contact with pigs infected with the pandemic Georgia 2007/01 ASFV strain. During the post-exposure (pe) period, 2/6 from the conventional diet group showed a transient peak rectal temperature >40.5 °C before day 20 pe, and some tissue samples collected at 20 d pe from 5/6 were PCR+ for ASFV, albeit showing Ct values much higher than Trojan pigs. Interestingly, the SDPP group did not show fever, neither PCR+ in blood nor rectal swab at any time pe, and none of the postmortem collected tissue samples were PCR+ for ASFV. Differential serum cytokine profiles among groups at vaccination, and a higher number of ASFV-specific IFNϒ-secreting T cells in pigs fed with SDPP soon after the Georgia 2007/01 encounter, confirmed the relevance of Th1-like responses in ASF protection. We believe that our result shows that nutritional interventions might contribute to improving future ASF vaccination strategies.

Feeding Spray-Dried Porcine Plasma to Pigs Reduces African Swine Fever Virus Load in Infected Pigs and Delays Virus Transmission-Study 1.

The objective of this study was to evaluate the potential benefits of feeding spray-dried porcine plasma (SDPP) to pigs infected with African swine fever virus (ASFV). Two groups of twelve weaned pigs each were fed with CONVENTIONAL or 8% SDPP enriched diets. Two pigs (trojans)/group) were injected intramuscularly with the pandemic ASFV (Georgia 2007/01) and comingled with the rest of the pigs (1:5 trojan:naïve ratio) to simulate a natural route of transmission. Trojans developed ASF and died within the first week after inoculation, but contact pigs did not develop ASF, viremia, or seroconversion. Therefore, three more trojans per group were introduced to optimize the ASFV transmission (1:2 trojan:naïve ratio). Blood, nasal, and rectal swabs were weekly harvested, and at end of the study ASFV-target organs collected. After the second exposure, rectal temperature of conventionally fed contact pigs increased >40.5 °C while fever was delayed in the SDPP contact pigs. Additionally, PCR Ct values in blood, secretions, and tissue samples were significantly lower (p < 0.05) for CONVENTIONAL compared to SDPP contact pigs. Under these study conditions, contact exposed pigs fed SDPP had delayed ASFV transmission and reduced virus load, likely by enhanced specific T-cell priming after the first ASFV-exposure.

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.

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.

Effect of spray-drying and ultraviolet C radiation as biosafety steps for CSFV and ASFV inactivation in porcine plasma.

Spray-dried animal plasma (SDAP) is widely used in diets of domestic animals to improve health status and increase growth and feed efficiency. Individual steps in the SDAP manufacturing process, including spray-drying, have been validated to inactivate potential pathogens. Manufacturing standards have established a minimum exit temperature of 80°C and a minimum post-drying storage period of 14 days at 20°C for production of SDAP. Also, UV-C irradiation has been evaluated as another inactivation step that could be included in the manufacturing process. The aim of this study was to assess the inactivation effectiveness of spray-drying on Classical swine fever virus (CSFV) and African swine fever virus (ASFV) and the effect of UV-C inactivation on ASFV as redundant biosafety steps of the manufacturing process for producing spray-dried porcine plasma (SDPP). This study demonstrated that UV-C treatment of liquid porcine plasma can inactivate more than 4 Log10 TCID50/mL of ASFV at 3000 J/L. Spray-drying effectively inactivated at least 4 Log10 TCID50/mL of both CSFV and ASFV. Incorporating UV-C technology within the SDAP manufacturing process can add another biosafety step to further enhance product safety.

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.

Foetal Immune Response Activation and High Replication Rate during Generation of Classical Swine Fever Congenital Infection.

Classical swine fever virus (CSFV) induces trans-placental transmission and congenital viral persistence; however, the available information is not updated. Three groups of sows were infected at mid-gestation with either a high, moderate or low virulence CSFV strains. Foetuses from sows infected with high or low virulence strain were obtained before delivery and piglets from sows infected with the moderate virulence strain were studied for 32 days after birth. The low virulence strain generated lower CSFV RNA load and the lowest proportion of trans-placental transmission. Severe lesions and mummifications were observed in foetuses infected with the high virulence strain. Sows infected with the moderately virulence strain showed stillbirths and mummifications, one of them delivered live piglets, all CSFV persistently infected. Efficient trans-placental transmission was detected in sows infected with the high and moderate virulence strain. The trans-placental transmission occurred before the onset of antibody response, which started at 14 days after infection in these sows and was influenced by replication efficacy of the infecting strain. Fast and solid immunity after sow vaccination is required for prevention of congenital viral persistence. An increase in the CD8+ T-cell subset and IFN-alpha response was found in viremic foetuses, or in those that showed higher viral replication in tissue, showing the CSFV recognition capacity by the foetal immune system after trans-placental infection.

A Polyuridine Insertion in the 3' Untranslated Region of Classical Swine Fever Virus Activates Immunity and Reduces Viral Virulence in Piglets.

Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3' untranslated region (3' UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3' UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3' UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3' untranslated region (3' UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3' UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy.

BACKGROUND: Recent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions. RESULTS: Six litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage. CONCLUSIONS: Circulation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication.

Experimental infection with high- and low-virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease.

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free-ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high-virulence (Cadí-6) and low-virulence (Freser-5) BDV strains was performed. Pregnant and non-pregnant animals with and without antibodies against BDV were included in each group. Cadí-6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser-5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí-6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí-6 group. Chamois from both groups presented lesions in brain but the ones infected with the low-virulence Freser-5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low-virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross-protection in chamois against high-virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.

Evaluation of the effectiveness of the SurePure Turbulator ultraviolet-C irradiation equipment on inactivation of different enveloped and non-enveloped viruses inoculated in commercially collected liquid animal plasma.

The objective of this study was to evaluate the effectiveness of the SurePure Turbulator ultraviolet-C (UV-C, 254 nm wavelength) irradiation equipment on inactivation of different enveloped and non-enveloped viruses in commercially collected liquid animal plasma. Specifically, Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV), Bovine viral diarrhea virus (BVDV), Classical swine fever virus (CSFV), Swine influenza virus (SIV) as enveloped viruses and Porcine parvovirus (PPV), Swine vesicular disease virus (SVDV), Porcine circovirus type 2 (PCV-2) and Senecavirus A (SVA) as non-enveloped viruses, were inoculated in bovine or porcine plasma and subjected to different UV-C irradiation doses (0, 750, 1500, 3000, 6000 and 9000 J/L) using an UV-C device developed for opaque liquid working under turbulent flow. The enveloped viruses tested were inactivated at < 3000 J/L of UV-C, being the dose needed to inactivate 4 log TCID50 (4D) of 1612 J/L for PRV,1004 J/L for PRRSV, 1953 J/L for PEDV, 1639 J/L for SIV, 1641 J/L for CSFV and 1943 J/L for BVDV. The non-enveloped viruses tended to have higher 4D values: 2161 J/L for PPV, 3223 J/L for SVA and 3708 J/L for SVDV. Because the initial viral concentration was <4.0 Log for PCV-2, it was not possible to calculate the 4D value for this virus. In conclusion, these results demonstrated that the SurePure Turbulator UV-C treatment system is capable of inactivating significant levels of swine viruses inoculated in commercially collected porcine or bovine plasma. It was concluded that irradiation with UV-C can provide an additional redundant biosafety feature in the manufacturing process of spray-dried animal plasma.

Low CD4/CD8 ratio in classical swine fever postnatal persistent infection generated at 3 weeks after birth.

Classical swine fever virus (CSFV) is one of the most important pathogens affecting swine. After infection with a moderate virulence strain at 8 hours after birth, CSFV is able to induce viral persistence. These animals may appear clinically healthy or showed unspecific clinical signs despite the permanent viremia and high viral shedding, in absence of immune response to the virus. Given the role played by this infection in disease control, we aimed to evaluate the capacity of CSFV to induce postnatal persistent infection at 3 weeks after birth. Nine pigs were CSFV infected and sampled weekly during 6 weeks and viral, clinical, pathological and immunological tests were carried out. Also, the CD4/CD8 ratio was calculated with the purpose to relate this marker with the CSFV persistent infection. The IFN-α response was detected mainly 1 week after infection, being similar in all the infected animals. However, 44.4% of animals were CSFV persistently infected, 33.3% died and 22.2% developed specific antibody response. Interestingly, in persistently infected pigs, the T-CD8 population was increased, the T-CD4 subset was decreased and lower CD4/CD8 ratios were detected. This is the first report of CSFV capacity to confer postnatal persistent infection in pigs infected at 3 weeks after birth, an age in which the weaning could be carried out in some swine production systems. This type of infected animals shed high amounts of virus and are difficult to evaluate from the clinical and anatomopathological point of view. Therefore, the detection of this type of infection and its elimination in endemic areas will be relevant for global CSF eradication. Finally, the low CD4/CD8 ratios found in persistently infected animals may be implicated in maintaining high CSFV replication during persistence and further studies will be performed to decipher the role of these cells in CSFV immunopathogenesis.

New insights on pestivirus infections in transhumant sheep and sympatric Pyrenean chamois (Rupicapra p. pyrenaica).

Border Disease Virus (BDV) causes health and economic impact on livestock and is also of importance in wildlife conservation as it causes high mortality outbreaks in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Pastoral practices are known as a main interspecies pathogen transmission. Hence, the presence of pestivirus in transhumant sheep flocks and sympatric chamois was assessed in areas with different epidemiological scenarios of chamois BDV infections. Moreover, the present study had also the goal to identify if inter-specific infections occurred and when they happened. Five sheep flocks grazing in two alpine areas in the Pyrenees with two different BDV epidemiological scenarios in chamois populations were studied during two transhumant seasons. Sheep were sampled before and after transhumance. Pyrenean chamois sera and spleen samples from both areas where also studied during the same period. Antibodies against BDV were assessed by means of ELISA and VNT. A qRT-PCR was used in order to detect the virus. Seroprevalence in sheep ranged between 0 and 91.1% at the flock level. Chamois were found to have high seroprevalences (52.9-77.7%) in both areas, and four new BDV isolates were sequenced. One sheep farm presented persistent BDV circulation and three showed low BDV circulation. The after-transhumance period was identified as the moment when viral transmission occured in the first farm, associated to BDV strains of domestic origin, according to VNT results. However, the BDV isolate was genetical closely related to previous BDV strains from chamois origin. In another farm, antibodies in two of the three positive sera were associated to infection with a chamois-like BDV strain. Altogether indicates that occasional viral transmission from chamois to sheep may occur.

African swine fever virus infection in Classical swine fever subclinically infected wild boars.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.