First report of the novel atypical porcine pestivirus in Spain and a retrospective study.

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.

Post-Natal Persistent Infection With Classical Swine Fever Virus in Wild Boar: A Strategy for Viral Maintenance?

In this study, fifteen wild boar piglets were intranasally inoculated <10 h after birth with the moderately virulent classical swine fever virus (CSFV) strain Catalonia 01. At 5 days post-inoculation, seven other animals within 48 h of birth were put in contact with them. Viral replication and innate and specific immune responses were evaluated. Of the inoculated animals, 46.67% remained post-natally persistently infected and were apparently healthy with neither humoral nor cellular immunological responses specific to CSFV and with high viral loads in their blood, organs and body secretions. Moreover, the present data extend the time period to 48 h after birth when a moderately virulent CSFV strain could lead to post-natal persistent infection given the generation of persistently infected wild boars in the contact group (33.33%). The innate immune response to the virus, as measured by type I IFN-α in serum, was mostly not impaired in the persistently infected wild boars. Interestingly, a decrease and lack of IFN-γ-producing cells against CSFV and PHA was observed. In endemic countries where wild swine species are increasing and low and moderate virulence CSFV strains are prevalent, the possible generation of this form of disease cannot be ruled out.

Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses.

African swine fever (ASF) and classical swine fever (CSF) are two highly infectious transboundary animal diseases (TADs) that are serious threats to the pig industry worldwide, including in China, the world's largest pork producer. In this study, a duplex real-time PCR assay was developed for the rapid detection and differentiation of African swine fever virus (ASFV) and classical swine fever virus (CSFV). The assay was performed on a portable, battery-powered PCR thermocycler with a low sample throughput (termed as 'T-COR4 assay'). The feasibility and reliability of the T-COR4 assay as a possible field method was investigated by testing clinical samples collected in China. When evaluated with reference materials or samples from experimental infections, the assay performed in a reliable manner, producing results comparable to those obtained from stationary PCR platforms. Of 59 clinical samples, 41 had results identical to a two-step CSFV real-time PCR assay. No ASFV was detected in these samples. The T-COR4 assay was technically easy to perform and produced results within 3 h, including sample preparation. In combination with a simple sample preparation method, the T-COR4 assay provides a new tool for the field diagnosis and differentiation of ASF and CSF, which could be of particular value in remote areas.

Increased viral load and prevalence of Torque teno sus virus 2 (TTSuV2) in pigs experimentally infected with classical swine fever virus (CSFV).

Torque teno sus viruses (TTSuVs) are considered non-pathogenic viruses, although lately they have been linked to porcine circovirus diseases, mainly with post weaning multisystemic wasting syndrome (PMWS). These associations point out a possible pathogenic role of TTSuVs or, alternatively, that TTSuV replication is up-regulated under disease conditions. In order to further explore the association of TTSuVs with disease occurrence, TTSuVs prevalence and viral load were assessed before and after an experimental infection with a highly pathogenic classical swine fever (CSF) virus (CSFV) isolate. Serum samples from 56 animals were analyzed by means of a real time quantitative PCR (qPCR) for TTSuV1 and TTSuV2 before and after (between 6 and 13 days post-inoculation) the CSFV challenge. Based on the post-infection clinical evolution and immune response against CSFV, the animals were divided into two groups: group I, with protecting immunity against CSFV and no clinical signs at the day of necropsy, and group II, with no detectable immune response against CSFV and moderate to severe clinical signs. TTSuVs qPCR results indicated that TTSuV2 and not TTSuV1 load in serum increased significantly after challenge with CSFV in the group of pigs with clinical signs, specifically in those with a moderate course of the disease. Therefore, this study emphasizes the different behaviour of both TTSuVs, as already found in the PMWS background, and further supports the association of TTSuV2 with disease occurrence.

Chimeric calicivirus-like particles elicit specific immune responses in pigs.

Virus-like particles (VLPs) have received considerable attention due to their potential application in veterinary vaccines and, in particular, VLPs from rabbit haemorrhagic disease virus (RHDV) have successfully shown to be good platforms for inducing immune responses against an inserted foreign epitope in mice. The aim of this study was to assess the immunogenicity of chimeric RHDV-VLPs as vaccine vectors in pigs. For this purpose, we have generated chimeric VLPs containing a well-known T epitope of 3A protein of foot-and-mouth disease virus (FMDV). Firstly, RHDV-VLPs were able to activate immature porcine bone marrow-derived dendritic cells (poBMDCs) in vitro. Secondly, pigs were inoculated twice in a two-week interval with chimeric RHDV-VLPs at different doses intranasally or intramuscularly. One intramuscularly treated group was also inoculated with adjuvant Montanide™ ISA 206 at the same time. Specific IgG and IgA antibodies against RHDV-VLPs were induced and such levels were higher in the adjuvanted group compared with other groups. Interestingly, anti-RHDV-VLP IgA responses were higher in groups inoculated intramuscularly than those that received the VLPs intranasally. Two weeks after the last immunisation, specific IFN-γ-secreting cells against 3A epitope and against RHDV-VLPs were detected in PBMCs by ELISPOT. The adjuvanted group exhibited the highest IFN-γ-secreting cell numbers and lymphoproliferative specific T cell responses against 3A epitope and RHDV-VLP. This is the first immunological report on the potential use of chimeric RHDV-VLPs as antigen carriers in pigs.

Interferon-gamma induction correlates with protection by DNA vaccine expressing E2 glycoprotein against classical swine fever virus infection in domestic pigs.

Classical swine fever (CSF) is a highly contagious viral infection affecting domestic and wild pigs. For classical swine fever virus (CSFV), immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T cell responses in protection against CSFV is largely unknown. Here we have extended these studies to deeply characterize the role of T cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies. Constant levels of CSFV-specific IFN-gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed. Severe clinical signs of the disease, including high titers of viremia, pyrexia and virus spread to different organs, were recorded in the non-vaccinated challenged animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short peak of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vaccinated animals. Our results support the relevance of the induction of a strong T cellular response to confer a solid protection upon DNA vaccination against CSFV. Further experiments are needed to be done in order to clarify the key cytokines playing a role in CSFV-protection and to obtain emergency vaccines capable to confer robust and fast protection.

Molecular epidemiology of classical swine fever in Cuba.

The origin and evolution of the classical swine fever (CSF) epizootic that occurred in Cuba from 1993 to 1997 has been investigated by the analysis of E2 gene sequences from 15 representative viral isolates as well as the vaccine and the challenge strains used in this country. In the phylogenetic tree derived from these sequences, the Cuban isolates were located in a defined cluster within the previously reported genomic subgroup 1.2. This cluster was related, although distinguishable, from the live vaccine used in Cuba since 1965. Two further groups were identified. One of them included the early viruses isolated in the western part of Cuba until 1996 and the strain Margarita, used for vaccine potency tests since 1965. These results are consistent with the strain Margarita being the origin of the western outbreaks. The viruses isolated from 1996 in eastern Cuba defined a related, but independent group. The level of sequence variation observed in this group does not exclude an independent origin for the eastern isolates.

An RT-PCR assay for the specific detection of classical swine fever virus in clinical samples.

A simple reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been developed for the specific amplification of DNA after reverse transcription of RNA from the classical swine fever virus (CSFV). A pair of oligonucleotides was selected from an area of high homology in the genome of CSFV strains, but which differed from the corresponding sequences in the genome of bovine viral diarrhea virus (BVDV) strains. Using these primers (CSFV1-CSFV2), a CSFV specific DNA band of 174 bp was amplified from the CSFV RNA extracted from four reference strains and 14 field isolates, as well as from 25 organ extracts and eight buffy coats and serum samples of experimentally infected animals. No amplification was observed with the RNA from four BVDV reference and vaccine strains and seven field isolates. This RT-PCR assay made it possible, in a one-step reaction, to detect CSFV rapidly, sensitively and specifically in cell culture supernatants and in clinical specimens.