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.

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.

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.

Identification of an Immunosuppressive Cell Population during Classical Swine Fever Virus Infection and Its Role in Viral Persistence in the Host.

Classical swine fever virus (CSFV) remains a highly important pathogen, causing major losses in the swine industry. Persistent infection is highly relevant for CSFV maintenance in the field; however, this form of infection is not fully understood. An increase in the granulocyte population has been detected in CSFV persistently infected animals. The aim of this work was to evaluate the possible immunosuppressive role of these cells in CSFV persistent infection. The phenotype of peripheral blood and bone marrow cells from persistently infected and naïve animals was evaluated by flow cytometry, and the capacity of specific cell subsets to reduce the interferon gamma (IFN-γ) response against unspecific and specific antigen was determined using co-culture assays. The frequency of granulocytic cells was increased in cells from CSFV persistently infected pigs and they showed a phenotype similar to immunosuppressive cell populations found in persistent infection in humans. These cells from persistently infected animals were able to reduce the IFN-γ response against unspecific and specific antigen. Our results suggest that immature immunosuppressive cell populations play a role in CSFV persistent infection in swine. The information obtained by studying the role of myeloid derived suppressor cells (MDSC) during CSFV persistent infection may extrapolate to other viral persistent infections in mammals.

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.

Corrigendum to "Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows".

Here we evaluated the effect of double vaccination with a novel subunit marker vaccine candidate based in the CSFV E2 glycoprotein fused to the porcine CD154 to prevent CSFV vertical transmission. A lentivirus-based gene delivery system was used to obtain a stable recombinant HEK 293 cell line for the expression of E2 fused to porcine CD154 molecule. Six pregnant sows were distributed in two groups and at 64days of gestation animals numbered 1-4 (group 1) were vaccinated via intramuscular inoculation with 50µg of E2-CD154 subunit vaccine. Animals from group 2 (numbered 5 and 6, control animals) were injected with PBS. Seventeen days later sows from group 1 were boosted with the same vaccine dose. Twenty-seven days after the first immunization, the sows were challenged with a virulent CSFV Margarita strain and clinical signs were registered. Samples were collected during the experiment and at necropsy to evaluate immune response and virological protection. Between 14 and 18days after challenge, the sows were euthanized, the foetuses were obtained and samples of sera and tissues were collected. E2-CD154 vaccinated animals remained clinically healthy until the end of the study; also, no adverse reaction was shown after vaccination. An effective boost effect in the neutralizing antibody response after the second immunization and viral challenge was observed and supports the virological protection detected in these animals after vaccination. Protection against CSFV vertical transmission was found in the 100% of serums samples from foetus of vaccinated sows. Only two out of 208 samples (0.96%) were positive with Ct value about 36 corresponding to one tonsil and one thymus, which may be non-infective viral particles. Besides, its DIVA potential and protection from vertical transmission, the novel CSFV E2 bound to CD154 subunit vaccine, is a promising alternative to the live-attenuated vaccine for developing countries.

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.

A bivalent dendrimeric peptide bearing a T-cell epitope from foot-and-mouth disease virus protein 3A improves humoral response against classical swine fever virus.

Three dendrimeric peptides were synthesized in order to evaluate their immunogenicity and their potential protection against classical swine fever virus (CSFV) in domestic pigs. Construct 1, an optimized version of a previously used dendrimer, had four copies of a B-cell epitope derived from CSFV E2 glycoprotein connected to an also CSFV-derived T-cell epitope through maleimide instead of thioether linkages. Construct 2 was similarly built but included only two copies of the B-cell epitope, and in also bivalent construct 3 the CSFV T-cell epitope was replaced by a previously described one from the 3A protein of foot-and-mouth disease virus (FMDV). Animals were inoculated twice with a 21-day interval and challenged 15days after the second immunization. Clinical signs were recorded daily and ELISA tests were performed to detect antibodies against specific peptide and E2. The neutralising antibody response was assessed 13days after challenge. Despite the change to maleimide connectivity, only partial protection against CSFV was again observed. The best clinical protection was observed in group 3. Animals inoculated with constructs 2 and 3 showed higher anti-peptide humoral response, suggesting that two copies of the B-cell epitope are sufficient or even better than four copies for swine immune recognition. In addition, for construct 3 higher neutralizing antibody titres against CSFV were detected. Our results support the immunogenicity of the CSFV B-cell epitope and the cooperative role of the FMDV 3A T-cell epitope in inducing a neutralising response against CSFV in domestic pigs. This is also the first time that the FMDV T-cell epitope shows effectivity in improving swine immune response against a different virus. Our findings highlight the relevance of dendrimeric peptides as a powerful tool for epitope characterization and antiviral strategies development.

Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows.

Here we evaluated the effect of double vaccination with a novel subunit marker vaccine candidate based in the CSFV E2 glycoprotein fused to the porcine CD154 to prevent CSFV vertical transmission. A lentivirus-based gene delivery system was used to obtain a stable recombinant HEK 293 cell line for the expression of E2 fused to porcine CD154 molecule. Six pregnant sows were distributed in two groups and at 64days of gestation animals numbered 1-4 (group 1) were vaccinated via intramuscular inoculation with 50µg of E2-CD154 subunit vaccine. Animals from group 2 (numbered 5 and 6, control animals) were injected with PBS. Seventeen days later sows from group 1 were boosted with the same vaccine dose. Twenty-seven days after the first immunization, the sows were challenged with a virulent CSFV Margarita strain and clinical signs were registered. Samples were collected during the experiment and at necropsy to evaluate immune response and virological protection. Between 14 and 18days after challenge, the sows were euthanized, the foetuses were obtained and samples of sera and tissues were collected. E2-CD154 vaccinated animals remained clinically healthy until the end of the study; also, no adverse reaction was shown after vaccination. An effective boost effect in the neutralizing antibody response after the second immunization and viral challenge was observed and support the virological protection detected in these animals after vaccination. Protection against CSFV vertical transmission was found in the 100% of serums samples from foetus of vaccinated sows. Only two out of 208 samples (0.96%) were positive with Ct value about 36 corresponding to one tonsil and one thymus, which may be non-infective viral particles. Besides, its DIVA potential and protection from vertical transmission, the novel CSFV E2 bound to CD154 subunit vaccine, is a promising alternative to the live-attenuated vaccine for developing countries.

Novel poly-uridine insertion in the 3'UTR and E2 amino acid substitutions in a low virulent classical swine fever virus.

In this study, we compared the virulence in weaner pigs of the Pinar del Rio isolate and the virulent Margarita strain. The latter caused the Cuban classical swine fever (CSF) outbreak of 1993. Our results showed that the Pinar del Rio virus isolated during an endemic phase is clearly of low virulence. We analysed the complete nucleotide sequence of the Pinar del Rio virus isolated after persistence in newborn piglets, as well as the genome sequence of the inoculum. The consensus genome sequence of the Pinar del Rio virus remained completely unchanged after 28days of persistent infection in swine. More importantly, a unique poly-uridine tract was discovered in the 3'UTR of the Pinar del Rio virus, which was not found in the Margarita virus or any other known CSFV sequences. Based on RNA secondary structure prediction, the poly-uridine tract results in a long single-stranded intervening sequence (SS) between the stem-loops I and II of the 3'UTR, without major changes in the stem- loop structures when compared to the Margarita virus. The possible implications of this novel insertion on persistence and attenuation remain to be investigated. In addition, comparison of the amino acid sequence of the viral proteins Erns, E1, E2 and p7 of the Margarita and Pinar del Rio viruses showed that all non-conservative amino acid substitutions acquired by the Pinar del Rio isolate clustered in E2, with two of them being located within the B/C domain. Immunisation and cross-neutralisation experiments in pigs and rabbits suggest differences between these two viruses, which may be attributable to the amino acid differences observed in E2. Altogether, these data provide fresh insights into viral molecular features which might be associated with the attenuation and adaptation of CSFV for persistence in the field.

Classical Swine Fever Virus vs. Classical Swine Fever Virus: The Superinfection Exclusion Phenomenon in Experimentally Infected Wild Boar.

Two groups with three wild boars each were used: Group A (animals 1 to 3) served as the control, and Group B (animals 4 to 6) was postnatally persistently infected with the Cat01 strain of CSFV (primary virus). The animals, six weeks old and clinically healthy, were inoculated with the virulent strain Margarita (secondary virus). For exclusive detection of the Margarita strain, a specific qRT-PCR assay was designed, which proved not to have cross-reactivity with the Cat01 strain. The wild boars persistently infected with CSFV were protected from superinfection by the virulent CSFV Margarita strain, as evidenced by the absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in ST cells, demonstrating SIE at the tissue level in vivo. Furthermore, the data analysis of the Margarita qRT-PCR, by means of calculated ΔCt values, supported that PBMCs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the Margarita virus strain, while this virus was able to infect naive PBMCs efficiently. In parallel, IFN-α values were undetectable in the sera from animals in Group B after inoculation with the CSFV Margarita strain. Furthermore, these animals were unable to elicit adaptive humoral (no E2-specific or neutralising antibodies) or cellular immune responses (in terms of IFN-γ-producing cells) after inoculation with the second virus. Finally, a sequence analysis could not detect CSFV Margarita RNA in the samples tested from Group B. Our results suggested that the SIE phenomenon might be involved in the evolution and phylogeny of the virus, as well as in CSFV control by vaccination. To the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of IFN-α, which might be associated with the lack of innate immune mechanisms.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs.

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

Serum cross-reaction among virulence-associated trimeric autotransporters (VtaA) of Haemophilus parasuis.

Glässer's disease is a fibrinous polyserositis and polyarthritis of swine caused by the bacterium Haemophilus parasuis. Control by vaccination has been limited for years due to lack of cross-protection among strains. However, 6 trimeric autotransporters (VtaA) of the Nagasaki strain were shown to be antigenic and gave partial protection to a lethal challenge. The antigenic relationship among the VtaAs was examined by immunizing mice with individual VtaA showing that they cross-reacted by ELISA mainly with VtaA from the same group. When sera from protected and non-protected vaccinated piglets were examined no differences in VtaA cross-reactivity profiles were found. In addition, sera from commercial pigs immunized with a single VtaA (VtaA9) showed a wider range of VtaA cross-reaction, probably due to the previous colonization by H. parasuis. These results can help the development of new vaccine formulations against H. parasuis by allowing a rational VtaA selection.

Genomic and antigenic characterization of monomeric autotransporters of Haemophilus parasuis: an ongoing process of reductive evolution.

The genome of the highly pathogenic Haemophilus parasuis Nagasaki strain (serovar 5) was sequenced to 99 % completion. A genomic comparison with two other pathogenic serovar 5 H. parasuis strains identified six genes per genome (bmaA1-bmaA6) encoding ß-barrel monomeric autotransporters, bmaA2 and bmaA3 being pseudogenes in at least one strain. The remaining encoded proteins were predicted to belong to the subtilisin (BmaA1 and BmaA4) and cysteine (BmaA5 and BmaA6) protease families. Allelic polymorphism was detected in other H. parasuis strains by comparative genomic hybridization using microarrays. Recombination events were observed, some of them leading to gene disruption in one of the three strains, although synteny around bmaA genes was conserved. These results suggest that bmaA genes are undergoing a process of reductive evolution. To evaluate their use as potential vaccine antigens, the products of the passenger domains of bmaA1, bmaA4, bmaA5 and bmaA6 were produced in Escherichia coli as recombinant proteins. They were detected by immunoblotting using sera of colostrum-deprived piglets recovering from a sublethal infection with H. parasuis (Nagasaki). The existence of specific antibodies after infection with H. parasuis also demonstrated in vivo expression. Using proteomics, only BmaA6 was detected in the in vitro-grown Nagasaki strain. Interestingly, the translocator domain was found in the outer membrane, while the passenger domain was located in supernatants. These results indicate that BmaA proteins could be considered as immunogen candidates to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be studied further.

Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA).

Haemophilus parasuis is the etiological agent of Glässer's disease in swine, characterized by fibrinous polyserositis, polyarthritis and meningitis. The lack of a vaccine against a broad spectrum of strains has limited the control of the disease. Recently, virulence associated trimeric autotransporters (VtaA) were described as antigenic proteins of H. parasuis. In this study 6 VtaA were produced as recombinant proteins and used to immunize snatch-farrowed, colostrum-deprived piglets. Immunized animals developed specific systemic and mucosal antibodies. The protective capacity of the anti-VtaA antibodies was evaluated by the inoculation of 3 × 10(8) or 6 × 10(6) colony forming units (CFU) of the highly virulent strain Nagasaki. Vaccinated animals had a delayed course of disease and 33 or 57%, respectively, of the animals survived the lethal challenge. The partial protection achieved with the recombinant VtaA supports their potential as candidates to be included in future vaccine formulations against H. parasuis.

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus.

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo.

Glässer's disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki's VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.