A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID₅₀. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.     

Molecular detection of Torque teno sus virus in lymphoid tissues in concomitant infections with other porcine viral pathogens

In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screened by PCR for the presence of TTSuV1, TTSuV2, PCV-2, PPV and CSFV in spleen samples. The variability of the porcine TTSuV sequences obtained was investigated by phylogenetic analysis. This study showed for the first time that TTSuV1 and TTSuV2 were present in Cuban swine herds. The investigation revealed the following infection rates: TTSuV1 40%, TTSuV2 37.5%, PCV-2 70%, PPV 37.5% and CSFV in 52.5%. The presence of two or more of these viruses at different rates in the same spleen samples was revealed. Also, a higher genetic diversity of TTSuV2 sequences was observed regarding TTSuV1 sequences.     

Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.     

Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV

The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation, that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). Our results confirm the role of CCL20 to increase antibody-mediated responses. At the same time suggest the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs previously reported. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN. Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge. The levels of IFN-alpha detected in pigs immunized with pE2 and principally in non-vaccinated challenged animals can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed. Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV. To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the immune induction and protection against virus challenge in swine infection model.     

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.     

Recent advances in the development of recombinant vaccines against classical swine fever virus: cellular responses also play a role in protection

Classical swine fever virus (CSFV) is the causative agent of one of the most devastating porcine haemorrhagic viral diseases, classical swine fever (CSF). CSFV mainly infects endothelial cells and macrophages and at the same time promotes bystander apoptosis of the surrounding T cells, causing strong immune suppression and high mortality rates. Most animals experience acute infection, during which they either die or survive by producing neutralising antibodies to the virus. However, in a few cases, the impaired immune system cannot control viral progression, leading to chronic infection. Efficient live attenuated vaccines against CSFV exist and are routinely used only in endemic countries. The ability of these vaccines to replicate in the host, even at very low rates, makes it extremely difficult to distinguish vaccinated from infected animals, favouring a restricted policy regarding vaccination against CSFV in non-endemic countries. There is a clear need for efficient and safer marker vaccines to assist in the control of future CSF outbreaks. In this review article, some of the most recent advances in the field of recombinant vaccines against CSFV are presented and the nature of the protective immune responses they induce is discussed.