This study aimed to provide information regarding viral pathogenesis and molecular epidemiology linked with recently reported atypical porcine pestivirus (APPV) strains and to determine the circulation of APPV in Spain from 1997 to 2016. Two-day-old piglets with moderate-severe congenital tremor (CT) from a Spanish farm were received for diagnostic purposes. Sera, nasal and rectal swabs and tissue samples were collected. qRT-PCR was performed in these samples, and a retrospective study to detect APPV RNA was carried out using a serum collection from 1997 to 2016. APPV genome was identified with high and moderate RNA loads in different tissues of the CT affected pigs. High APPV RNA load was detected in lymphoid organs, suggesting that these constitute a target for APPV replication. In 89 of the 642 retrospectively analysed samples (13.9%), APPV genome was detected. CT cases were related to the presence of APPV in viraemic piglets below 1 week of age, in which the viral RNA load was the highest. A considerable number of animals between 4 and 14 weeks of age and some 1-week-old piglets were viraemic in the absence of CT, which can act as carriers of the virus. The relative risk of APPV and CT was 8.5 (CI 95% 5.8-12.5). Thus, our data show that APPV infection is epidemiologically related to CT. Phylogenetic analysis from 1615 NS2-3 nucleotides showed only one defined APPV clade, grouping the most phylogenetically related strains from Europe and China. Of this clade, there are other strains from Europe, USA and China. This data confirm the high APPV genetic diversity, not being able to cluster this virus according to the geographic area. Our result showed that APPV has been circulating in Spain at least since 1997, being the earliest date of detection of this virus worldwide and suggesting that APPV may be widespread.

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.

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 E, 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 (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.

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.

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

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.

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