Classical Swine Fever Virus Strain Research Articles

Emergence of classical swine fever virus in Israel in 2009

Classical swine fever (CSF) re-emerged in Israel in February 2009 after an absence of 62 years. The outbreak occurred on a domestic pig farm in northern Israel and affected domestic pigs and wild boar. On the basis of phylogenetic analysis of a 190 base pair fragment of the E2 glycoprotein gene, the Israeli CSF virus strain belonged to genotype 2.1 and was genetically most similar to a Chinese CSF virus strain.

Substitution of Specific Cysteine Residues in the E1 Glycoprotein of Classical Swine Fever Virus Strain Brescia Affects Formation of E1-E2 Heterodimers and Alters Virulence in Swine

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.

Molecular Characterization of Lapinized Classical Swine Fever Vaccine Strain by Full-Length Genome Sequencing and Analysis

The complete genome of a lapinized classical swine fever virus (CSFV) vaccine strain was amplified into nine overlapping fragments by RT-PCR, and nucleotide sequences were determined. Complete genome sequence alignment and phylogenetic analysis indicated 92.6–98.6% identities at the nucleotide level with other reported CSFV strains and could be grouped into subgroup 1.1 along with other attenuated strains of CSFV. The 5′-UTR demonstrated >97.0% nucleotide similarity with most of vaccine CSFV strains from China. Further, its 3′-UTR sequence indicated a length similar to all the CSFV strains from China with >98.0% nucleotide similarity, although high length heterogeneity of 3′-UTR was reported among different CSFV strains. There was 12 nt (TTTTCTTTTTTT) insertion in 3′-UTR similar to other reported attenuated vaccine strains. However, secondary structure of 3′-UTR indicated that Indian CSFV strain requires further passage to obtain a 3′-UTR structure similar to most of the attenuated strains.

Identification of an NTPase motif in classical swine fever virus NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.

PS3-46 Differential inflammatory response to herpes simplex virus type 1 variants in a murine model

Several studies have highlighted the important role of cytokines in disease development of classical swine fever virus (CSFV) infection. In the present study, we examined the kinetics of 7 porcine cytokines in serum from pigs infected with 3 different CSFV strains. Based on the clinical picture in 6-month-old Danish pigs, the strains used for inoculation were classified as being of low (Bergen), low to moderate (Eystrup) and moderate to high (Lithuania) virulence. The cytokines interferon-alpha (INF-α), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) showed increased levels after CSFV infection with more or less comparable course in the 3 groups. However, the cytokine level peaked with a 2–3 days delay in pigs infected with the low virulent strain compared to those infected with a moderately or highly virulent strain. These findings may indicate that INF-α, IL-8 and TNF-α are involved in the immune response during CSFV infection with strains of different virulence.

High-resolution epitope mapping for monoclonal antibodies to the structural protein Erns of classical swine fever virus using peptide array and random peptide phage display approaches

The structural glycoprotein E(rns) (an envelope protein with RNase activity) of classical swine fever virus (CSFV) is not well characterized with respect to its antigenic structure and organization. Here, we investigated the antigenic sites on E(rns) by raising mAbs against the Escherichia coli expressed E(rns) of CSFV strain Alfort/187 and defined the B-cell epitopes recognized by these antibodies. Eighteen mAbs to E(rns) were identified and they were classified as either immunoglobulin subclass G1 or G2b. Using an array of overlapping 12-mer peptides, spanning aa 27-227 of E(rns), the epitopes for 12 mAbs were mapped to a high resolution of six to eight residues, which cluster in five discrete locations, ¹³GIWPEKIC³⁸ (group I), ⁶⁵NYTCCKLQ⁷² (group II), ¹²⁷QARNRPTT¹³⁴ (group III), ¹⁴⁵SFAGTVIE¹⁵² (group IV) and ¹⁶¹VEDILY¹⁶⁶ (group V). Two mAbs recognize two or more antigenic determinants, including the group II epitope. The epitopes for four other mAbs could not be mapped using the overlapping 12-mer peptides. Random peptide phage display with one mAb from each of all the groups except group V further identified some conserved residues that may be critical for binding antibodies, i.e. Trp³³ in the epitope of group I, Leu⁷¹ in the epitope of group II, Gln¹²⁷ and Apn¹³⁰ in the epitope of group III, and Ser¹⁴⁵ and Gly¹⁴⁸ in the epitope of group IV. This study has provided new insights into the structure and organization of epitopes on the CSFV E(rns) and valuable epitope information for the rational design of vaccines, drugs and diagnostic immunoassays for CSFV.

Characterization of Essential Domains and Plasticity of the Classical Swine Fever Virus Core Protein

Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins Erns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal deletions, were introduced into the Core coding sequence in the context of an infectious cDNA clone of classical swine fever virus strain Alfort. Amino acids 179 to 180, 194 to 198, and 208 to 212 proved to be of special importance for the generation of progeny virus. The results of transcomplementation of a series of C-terminally truncated Core molecules indicate the importance of Ala255 at the C terminus. The plasticity of Core protein was examined by the construction of concatemeric arrays of Core coding regions and the insertion of up to three yellow fluorescent protein (YFP) genes between two Core genes. Even a Core fusion protein with more than 10-fold-increased molecular mass was integrated into the viral particle and supported the production of infectious progeny virus. The unexpected plasticity of Core protein brings into question the formation of a regular icosahedric particle and supports the idea of a histone-like protein-RNA interaction. All viruses with a duplicated Core gene were unstable and reverted to the wild-type sequence. Interestingly, a nonviable YFP-Core construct was rescued by a mutation within the C-terminal domain of the nonstructural protein NS3.

Molecular epidemiology of current classical swine fever virus isolates of wild boar in Germany

Classical swine fever (CSF) has caused significant economic losses in industrialized pig production, and is still present in some European countries. Recent CSF outbreaks in Europe were mainly associated with strains of genogroup 2 (subgroup 2.3). Although there are extensive datasets regarding 2.3 strains, there is very little information available on longer fragments or whole classical swine fever virus (CSFV) genomes. Furthermore, there are no detailed analyses of the molecular epidemiology of CSFV wild boar isolates available. Nevertheless, complete genome sequences are supportive in phylogenetic analyses, especially in affected wild boar populations. Here, German CSFV strains of subgroup 2.3 were fully sequenced using two different approaches: (i) a universal panel of CSFV primers that were developed to amplify the complete genome in overlapping fragments for chain-terminator sequencing; and (ii) generation of a single full-length amplicon of the CSFV genome obtained by long-range RT-PCR for deep sequencing with next-generation sequencing technology. In total, five different strains of CSFV subgroup 2.3 were completely sequenced using these newly developed protocols. The approach was used to study virus spread and evolutionary history in German wild boar. For the first time, the results of our study clearly argue for the possibility of a long-term persistence of genotype 2.3 CSFV strains in affected regions at an almost undetectable level, even after long-term oral vaccination campaigns with intensive monitoring. Hence, regional persistence in wild boar populations has to be taken into account as an important factor in the continual outbreaks in affected areas.

Classical swine fever in 6- and 11-week-old pigs: Haematological and immunological parameters are modulated in pigs with mild clinical disease

The severity of classical swine fever virus (CSFV) infection is believed to be determined by different factors, including the virulence of the strain as well as factors related to the host. In the present study, we infected 6- and 11-week-old pigs of unique sanitary status with CSFV strain Eystrup to elucidate the influence of age on virulence. In both age-groups, a mild clinical course correlated well with the gross-pathological findings at necropsy. The minor variations of clinical, pathological, haematological and immunological parameters between the various age-groups demonstrated that a time-span of approximately 1 month of age did not play a significant role for the severity of CSF disease in young, weaned pigs. The detailed analysis of various haematological and cellular immunological parameters proved to provide a valuable set of objective reference values for healthy control pigs and for pigs with mild clinical CSF disease. Despite that only mild disease occurred in the infected pigs, modulations of haematological and immunological parameters were observed. Depletion of B cell and a number of T cell populations in peripheral blood was observed in both age-groups, however, the changes being most pronounced in the 6-week-old pigs. In the infected pigs, but not in any of the controls, a population of large granulocytes (LG) developed in peripheral blood. The LG, which were demonstrated to be identical to low-density granulocytes, appeared before the development of viraemia. Therefore, we suggest detection of LDG to be used as an additional tool in early CSF diagnosis. The observation that pigs with a unique, high sanitary status only developed mild disease after infection with CSFV strain Eystrup emphasizes the important role of the host in the CSFV virulence puzzle.

The classical swine fever virus N-terminal protease Npro binds to cellular HAX-1

The positive-stranded RNA genome of classical swine fever virus (CSFV) encodes 12 known proteins. The first protein to be translated is the N-terminal protease (N(pro)). N(pro) helps evade the innate interferon response by targeting interferon regulatory factor-3 for proteasomal degradation and also participates in the evasion of dsRNA-induced apoptosis. To elucidate the mechanisms by which N(pro) functions, we performed a yeast two-hybrid screen in which the anti-apoptotic protein HAX-1 was identified. The N(pro)-HAX-1 interaction was confirmed using co-precipitation assays. A dramatic redistribution of both N(pro) and HAX-1 was observed in co-transfected cells, as well as in transfected cells infected with wild-type CSFV, but not in cells infected with an N(pro)-deleted CSFV strain.

Improved sero-monitoring assay for classical swine fever (CSF) using the recombinant E2 protein of a recent Korean isolate

Classical swine fever (CSF) is a highly contagious disease of pigs that causes fever, diarrhea and paralysis, often resulting in death. E2 is the major structural protein of the CSF virus (CSFV) and mediates the entrance of the virus, subsequently inducing a neutralizing immune response. In this study, the E2 gene of a recent Korean isolate of CSF, SW03, was cloned and the DNA sequence was compared to other strains via phylogenetic analysis. With the purified E2 protein, an enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of CSFV infection. The sensitivity and specificity of the E2-ELISA were 96.1% and 94.8%, respectively. A total of 17 out of 485 field-collected pig sera tested demonstrated conflicting results between two ELISA methods, a commercial kit and the E2-ELISA. Of these sera, 60% were determined to be CSFV positive by a virus neutralization test (VNT), suggesting involvement of different immune responses in the cases of CSFV infection. As the E2-ELISA was developed using a recent Korean isolate, SW03, this assay is capable of rapidly identifying newly emerging CSFV strains.

POTENCY TESTING OF ATTENUATED LIVE VACCINE LAVIR- K® AGAINST CLASSICAL SWINE FEVER

In the paper are presented the results of investigating protective effect of attenuated live vaccine against classical swine fever (CSF) under experimental conditions. The vaccine contains C-strain of CSF. In the Republic of Serbia it is used for systemic immunoprophylaxis. Potency testing was performed according to the guidelines of the European Pharmacopoeia, 5th Edition, 2005 (01/2005:0065) and the guidelines published by the World Organisation for Animal Health (OIE) (Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th Edition, 2008, Vol. 1, Chapter 2.8.3). The experiments were carried out on twelve weaner pigs seven weeks of age randomly assigned to two groups consisting of five, and one group consisting of two pigs. Before introducing the experimental protocol of vaccination and artificial infection, the presence of specific antibodies against CSF was controlled by ELISA test, i.e. by serum neutralisation test that proved no presence of antibodies against bovine virus diarrhea (BVD). The pigs in the fist group were vaccinated with attenuated live vaccine Lavir-K® (Veterinary Institute Zemun a.d. Belgrade) in a 1:40 dilution, and the pigs from second group were vaccinated in a 1:160 dilution. The group three served as a control group that was not vaccinated. Fourteen days post vaccination all animals were challenged with highly virulent CSF virus strain Beker, and fourteen days after artificial infection animals were daily observed and their body temperature was recorded. In the group of animals vaccinated with vaccine Lavir-K® in a 1:40 dilution there were no clinical characteristic symptoms of CSF, while in the group vaccinated in a 1:160 dilution two pigs became ill with obvious CSF symptoms. One pig died on day 7 p.v. In the control group, i.e. two non-vaccinated pigs, there was an acute form of CSF and they died seven days after the infection. Calculation of the Protective Dose 50 (PD50) of Lavir-K® was done using the equation by Spearman-Kaerber and it was calculated to be 184. Having in mind that one vaccine doses should have protective value of > 100, it may be concluded that tested vaccine fulfils the requirements for vaccine potency as laid down in the European Pharmacopoeia and the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals.

Patterns of gene expression in swine macrophages infected with classical swine fever virus detected by microarray

Infection of domestic swine with highly virulent, classical swine fever virus (CSFV) strain Brescia, causes lethal disease in all infected animals. However, the molecular mechanisms involved in modulating the host cellular processes and evasion of the immune response have not been clearly established. To gain insight into, the early host response to CSFV, we analyzed the pattern of gene expression in infected swine macrophages, using custom designed swine microarrays. Macrophages, the target cell for CSFV infection, were isolated from primary cultures of peripheral blood mononuclear cells, allowing us to utilize identical uninfected macrophages at the same time points as CSFV-infected macrophages, allowing only genes induced by CSFV to be identified. First, microarray probes were optimized by screening 244,000 probes for hybridization with RNA from infected and uninfected macrophages. Probes that hybridized and passed quality control standards were used to design a 44,000 probe microarray for this study. Changes in expression levels of 79 genes (48 up- and 31 down-regulated) during the first 48 h post-infection were observed. As expected many of the genes with an altered pattern of expression are involved in the development of an innate immune response. Several of these genes had differential expression in an attenuated strain NS4B.VGIv, suggesting that some of these differences are responsible for virulence. The observed gene expression profile might help to explain the immunological and pathological changes associated with infection of pigs with CSFV Brescia.

Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003-2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.

Antigenic domains analysis of classical swine fever virus E2 glycoprotein by mutagenesis and conformation-dependent monoclonal antibodies

Glycoprotein E2 of classical swine fever virus (CSFV) is the major antigenic protein exposed on the outer surface of the virion that induces main neutralizing antibodies during infection in pigs. This study displays the differences in antigenicity of E2 between vaccine and field strains of CSFV by their variable reaction patterns between expressed proteins and monoclonal antibodies (mAbs). The D/A domains of various CSFVs were relatively conserved and recognized by all mAbs against the A domain. However, mAbs against B/C domains were able to differentiate field viruses TD/96/TWN (subgroup 2.1) and 94.4/IL/94/TWN (subgroup 3.4) from the vaccine virus LPC/AHRI (subgroup 1.1). By analysis of expressed truncated proteins, the epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779. Site-directed mutagenesis further showed that residues 693C, 737C, 771L, 772L, 773F and 774D were critical for the reactivity of E2 protein with mAbs. Thus, the B/C domains are responsible for antigen specificity among various CSFVs, and the disulfide bond and motif 771LLFD 774 are essential for the structural integrity of its conformational recognition. These data significantly increase our understanding of the antigenic structure of E2 for antibody binding.

A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus

A simple and rapid assay for the detection of Classical swine fever virus (CSFV) was established using reverse transcription loop-mediated isothermal amplification (RT-LAMP). This study describes the amplification of the genomic RNA of CSFV under isothermal conditions (63 °C) within one hour, using a set of six primers (two outer primers, two inner primers and two loop primers). This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR. This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV. PRRSV. SIV. PRV-PCV, thus showed a good specificity. Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition, either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye. Because RT-LAMP is low-cost and produces rapid results, it has the potential to be an excellent tool for CSFV surveillance in the field, especially in developing countries.

5'-UTR-based phylogenetic analysis of Classical swine fever virus isolates from India

Classical swine fever (CSF) caused by Classical swine fever virus (CSFV) is a globally significant disease of pigs. Genetic typing of CSFV isolates can help in understanding the epidemiology of disease and trace down the source of outbreak. 5'-UTR sequence analysis and subsequent genetic classification of nine CSFV field isolates from India indicated that 3 isolates belonged to genotype 2.1 and were closely related to European CSFV strains. The remaining 6 isolates belonged to genotype 1 that contained old and new strains. However, the genotype 2.1 group consisted of recent field isolates only. The study showed circulation of both genotypes 1 and 2.1 in north-eastern part of India.

Global transcriptional profiles in peripheral blood mononuclear cell during classical swine fever virus infection

Classical swine fever virus (CSFV) is an etiologic agent that causes a highly contagious disease in pigs. Laying a foundation to solve problems in its pathogenic mechanism, microarray analysis was performed to detect the gene transcriptional profiles in peripheral blood mononuclear cells (PBMC) following infection with a Chinese highly virulent CSFV strain Shimen. Three susceptible pigs were inoculated intramuscularly with a lethal dose (1.0 × 10 6 TCID 50) of CSFV. Pigs showed classical CSF signs, depletion of lymphocytes and monocytes consistent with CSFV infection, and the CSFV genome was also confirmed in the PBMC. The PBMC were isolated at 1, 3, 6 and 9 days post-inoculation (dpi). Total RNA were extracted and subjected to microarray analysis. Data showed that expression of 847 genes wherein 467 genes were known function and the remaining 380 genes were unknown function, and 541 up- and 306 down-regulation, altered after infection. There were 54, 181, 438 and 354 up- and 61, 120, 218 and 145 down-regulated genes presented on 1, 3, 6 and 9 dpi, respectively. These genes were involved in immune response (14.5%), apoptosis (3.3%), signal transduction (7.6%), transcription (4.4%), metabolism (11%), transport (3.9%), development (6.8%) and cell cycle (3.7%). Results demonstrated its usefulness in exploring the pathogenic mechanisms of CSFV.

Mutations in Classical Swine Fever Virus NS4B Affect Virulence in Swine

NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.

Acute induction of cell death-related IFN stimulated genes (ISG) differentiates highly from moderately virulent CSFV strains

Classical swine fever (CSF) severity is dependent on the virulence of the CSF virus (CSFV) strain. The earliest event detected following CSFV infection is a decrease in lymphocytes number. With some CSFV strains this leads to lymphopenia, the severity varying according to strain virulence. This lymphocyte depletion is attributed to an induction of apoptosis in non-infected bystander cells. We collected peripheral blood mononuclear cells (PBMC) before and during 3 days post-infection with either a highly or moderately virulent CSFV strain and subjected them to comparative microarray analysis to decipher the transcriptomic modulations induced in these cells in relation to strain virulence. The results revealed that the main difference between strains resided in the kinetics of host response to the infection: strong and immediate with the highly virulent strain, progressive and delayed with the moderately virulent one. Also although cell death/apoptosis-related IFN stimulated genes (ISG) were strongly up-regulated by both strains, significant differences in their regulation were apparent from the observed differences in onset and extent of lymphopenia induced by the two strains. Furthermore, the death receptors apoptotic pathways (TRAIL-DR4, FASL-FAS and TNFa-TNFR1) were also differently regulated. Our results suggest that CSFV strains might exacerbate the interferon alpha response, leading to bystander killing of lymphocytes and lymphopenia, the severity of which might be due to the host’s loss of control of IFN production and downstream effectors regulation.