ARF1 with Sec7 Domain-Dependent GBF1 Activates Coatomer Protein I To Support Classical Swine Fever Virus Entry.

Classical swine fever virus (CSFV), a positive-sense, enveloped RNA virus that belongs to the Flaviviridae family, hijacks cell host proteins for its own replication. We previously demonstrated that Golgi-specific brefeldin A (BFA) resistance factor 1 (GBF1), a regulator of intracellular transport, mediates CSFV infection. However, the molecular mechanism by which this protein regulates CSFV proliferation remains unelucidated. In this study, we constructed a series of plasmids expressing GBF1 truncation mutants to investigate their behavior during CSFV infection and found that GBF1 truncation mutants containing the Sec7 domain could rescue CSFV replication in BFA- and GCA (golgicide A)-treated swine umbilical vein endothelial cells (SUVECs), demonstrating that the effect of GBF1 on CSFV infection depended on the activity of guanine nucleotide exchange factor (GEF). Additionally, it was found that ADP ribosylation factors (ARFs), which are known to be activated by the Sec7 domain of GBF1, also regulated CSFV proliferation. Furthermore, we demonstrated that ARF1 is more important for CSFV infection than other ARF members with Sec7 domain dependence. Subsequent experiments established the function of coatomer protein I (COP I), a downstream effector of ARF1 which is also required for CSFV infection by mediating CSFV invasion. Mechanistically, inhibition of COP I function impaired CSFV invasion by inhibiting cholesterol transport to the plasma membrane and regulating virion transport from early to late endosomes. Collectively, our results suggest that ARF1, with domain-dependent GBF1 Sec7, activates COP I to facilitate CSFV entry into SUVECs. IMPORTANCE Classical swine fever (CSF), a highly contact-infectious disease caused by classical swine fever virus (CSFV) infecting domestic pigs or wild boars, has caused huge economic losses to the pig industry. Our previous studies have revealed that GBF1 and class I and II ARFs are required for CSFV proliferation. However, a direct functional link between GBF1, ARF1, and COP I and the mechanism of the GBF1-ARF1-COP I complex in CSFV infection are still poorly understood. Here, our data support a model in which COP I supports CSFV entry into SUVECs in two different ways, depending on the GBF1-ARF1 function. On the one hand, the GBF1-ARF1-COP I complex mediates cholesterol trafficking to the plasma membrane to support CSFV entry. On the other hand, the GBF1-ARF1-COP I complex mediates CSFV transport from early to late endosomes during the entry steps.

Role of innate immunity in pathophysiology of classical swine fever virus infection.

Classical swine fever virus (CSFV) infection causes mild to severe diseases among pigs, depending on the age and immune status of the host and viral strains. CSFV targets various cells, including macrophages and conventional and plasmacytoid dendritic cells. Classical swine fever is one of the most devastating diseases of pigs which leads to high morbidity and mortality, and causes significant economic loss worldwide. In response to infection with CSFV, host innate immune system eliminates the virus by recognizing specific viral molecules via distinct cellular pattern recognition receptors. These receptors trigger downstream intracellular signaling pathways, which regulate the translocation and activation of transcription factors that control the production of cytokines and interferons (IFNs). In turn, these IFNs activate JAK-STAT signaling that governs the transcription of IFN-stimulated genes (ISGs) that play critical roles in antiviral immunity. However, CSFV has evolved different strategies to evade innate immune signaling and can establish persistent infection without being recognized by immune surveillance. In this review, we discuss the current understanding of host innate response to CSFV infection. We also summarize how CSFV evades innate immunity to establish its chronic infection.

Genetic and virulence characterization of classical swine fever viruses isolated in Mongolia from 2007 to 2015.

Classical swine fever (CSF), a highly contagious viral disease affecting domestic and wild pigs in many developing countries, is now considered endemic in Mongolia, with 14 recent outbreaks in 2007, 2008, 2011, 2012, 2014, and 2015. For the first time, CSF viruses isolated from these 14 outbreaks were analyzed to assess their molecular epidemiology and pathogenicity in pigs. Based on the nucleotide sequences of their 5'-untranslated region, isolates were phylogenetically classified as either sub-genotypes 2.1b or 2.2, and the 2014 and 2015 isolates, which were classified as 2.1b, were closely related to isolates from China and Korea. In addition, at least three different viruses classified as 2.1b circulated in Mongolia. Experimental infection of the representative isolate in 2014 demonstrated moderate pathogenicity in 4-week-old pigs, with relatively mild clinical signs. Understanding the diversity of circulating CSF viruses gleans insight into disease dynamics and evolution, and may inform the design of effective CSF control strategies in Mongolia.

Experimental infection of Bama miniature pigs with a highly virulent classical swine fever virus.

BACKGROUND: Currently, larger domestic pigs are only animals widely used in vaccine evaluation and pathogenicity study of classical swine fever virus (CSFV). This study was aimed to create an alternative animal experimental infection model of CSFV. RESULTS: Twenty specific-pathogen-free Bama miniature pigs were randomly divided into two groups and rooms, infected and non-infected, and the pigs in the infected group were inoculated intramuscularly with 104, 105 or 106 TCID50 (median tissue culture infective dose) CSFV Shimen strain (n = 5 × 3) or left uninoculated to serve as in-contact pigs (n = 3). The uninfected control pigs (n = 2) were housed in a separate room. Clinical signs, body temperature, viraemia, tissue antigen distribution, pathological changes and seroconversion were monitored. Clinical signs were observed as early as 2 days post-inoculation (dpi) in all infected pigs (though mild in contact pigs), but not non-infected control pigs. All inoculated pigs showed viraemia by 6 dpi. The in-contact pigs showed lower levels of viraemia. At 10 dpi, seroconversion was noted in five of the 15 inoculated pigs. All inoculated or one in-contact pigs died by 15 dpi. CONCLUSIONS: These results show that Bama miniature pigs support productive CSFV infection and display clinical signs and pathological changes consistent with CSFV infections observed in larger domestic pigs.

Classical swine fever virus NS2 protein promotes interleukin-8 expression and inhibits MG132-induced apoptosis.

Classical swine fever (CSF) caused by virulent strains of classical swine fever virus (CSFV) is a hemorrhagic disease of pigs and is characterized by disseminated intravascular coagulation, thrombocytopenia, and immunosuppression. Until now, the role of the NS2 protein produced by CSFV in the pathogenesis of CSF is not well understood. In this report, we investigated the function of CSFV NS2 by examining its effects on the pro-inflammatory CXC chemokine, interleukin-8 (IL-8) expression, and cell survival. Stable swine umbilical vein endothelial cell line (SUVEC) expressing CSFV NS2 were established and showed that CSFV NS2 expressing SUVEC cells express approximately 16-fold higher levels of IL-8 as compared to control vector GFP-expressing cells, GFP-E2 expressing cells, and untransfected cells. Further studies showed that CSFV NS2 induced endoplasmic reticulum stress and activated the nuclear transcription factor kappa B (NF-κB), which is responsible for the up-regulation of IL-8 and the anti-apoptotic protein, Bcl-2, expression. In addition, the GFPNS2-expressing SUVEC cells were resistant to MG132-induced apoptosis. This study suggested that CSFV NS2 plays an important role in the inflammatory response and in persistent CSFV infection. These findings provide novel information on the function of the poorly characterized CSFV NS2.

Quantification of classical swine fever virus in aerosols originating from pigs infected with strains of high, moderate or low virulence.

During epidemics of classical swine fever (CSF), the route of virus introduction into a farm is often unclear. One of the suggested routes is via the air. Under experimental conditions, airborne transmission over a short distance seems possible, but analysis of outbreak data is still inconclusive. For a better understanding of the role of airborne transmission, quantitative information is needed on concentrations of virus emitted by infected pigs. This was studied in four groups of 10 pigs in which three pigs were inoculated with either a low virulent strain (Zoelen), a low or high dose of a moderately virulent strain (Paderborn), or a highly virulent strain (Brescia). The other seven pigs in each group served as contact pigs. At several moments after infection, air samples were obtained using gelatine filters. Infectious virus and viral RNA were detected in the air of rooms housing the pigs infected with the moderately and highly virulent strains with titres of 10(1.2) to 10(3.0)TCID(50)/m(3) of infectious virus, and 10(1.6) to 10(3.8)TCID(50)equiv./m(3) of viral RNA. It was observed that the higher the dose or virulence of the virus strain used for inoculation of the pigs, the sooner virus could be detected in the air samples. This is the first study describing the quantification of (infectious) CSFV in air samples of rooms housing infected pigs, enabling to quantify the contribution of individual infected pigs to virus concentrations in aerosols. This can be used as input for quantitative models of airborne spread over large distances.

[Effect of experimental classical swine fever (CSF) infection on libido and ejaculate parameters in boars]. / Einfluss einer experimentellen Infektion mit dem Virus der klassischen Schweinepest auf Libido und Ejakulat- parameter beim Eber.

The objective of this study was to characterize the effect of an experimental infection with the classical swine fever (CSF) virus on libido and ejaculate parameters of adult boars. Four boars 10 month old were infected with a CSF field isolate (Visbek/Han95). Semen was collected every second day after infection and daily during the pyrexic phase. The only clinical signs in the boars were an increase in body temperature, but never above 39.9 degrees C and a temporally reduction of food intake. The libido was always good, so semen collection was performed in three boars without difficulty and the semen quality was always in the range of the minimum requirements for sperm that is used for artificial insemination. Although one boar had a good libido only a sperm free ejaculate could be collected on one day. The results show that a CSF virus infection of adult boars hardly causes any clinical symptoms and that sperm can be obtained despite fever. Insemination boars may thus be of special epidemiological relevance for the dissemination of the CSF virus.

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.

Quantitative assessment of clinical signs for the detection of classical swine fever outbreaks during an epidemic.

The performance of clinical signs as a diagnostic test for the detection of classical swine fever (CSF) outbreaks during the 1997-1998 CSF epidemic in The Netherlands was evaluated by constructing and analysing a receiver operating characteristic (ROC) curve. This curve assesses the discriminating ability of a diagnostic test over a range of test signals. The cut-off values for a defined diagnostic test to detect CSF outbreaks were set by different combinations of clinical signs observed. The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P<0.001) larger than the area under the random ROC curve. This indicates that clinical signs have a significantly higher performance as a diagnostic test for the detection of CSF than for flipping a coin. However, the gain in diagnostic performance compared to a random process is not as much as we would wish it to be. The optimal efficient diagnostic test combined a sensitivity of 72.7% with a specificity of 52.7%, with a combination of the following clinical signs: unsteady gait/ataxia, not eating, not reacting to antibiotic treatment, conjunctivitis, hard faecal pellets.

Epidemiological characteristics of an outbreak of classical swine fever in an area of high pig density.

The objective of this study was to analyse an outbreak of classical swine fever under a policy of non-vaccination, intensive surveillance and eradication in an area of high pig density. The virus was found in 52 herds, where some 90,000 pigs were slaughtered. The clinical signs were vague and the reports of suspect herds generally coincided with increased mortality. The interval between the first occurrence of clinical signs and the report of a suspect herd was shorter when the disease was first diagnosed in fattening pigs than when it was diagnosed in sows, boars or suckling piglets. Among fattening pigs, mortality and morbidity appeared to increase with age. The proportion of clinically ill animals was positively correlated with the proportion of serologically positive animals in a pig house during the phase when the disease was spreading. Fifty-eight per cent of pig houses containing only clinically healthy but some virologically positive pigs were serologically negative. Antigen detection was therefore critical for early disease detection. Serology was nevertheless useful to ascertain that swine fever was not endemic in the area. The secondary cases were concentrated in the close neighbourhood of the herd initially infected.

[Classical swine fever]. / Klassische Schweinepest.

The principles of aetiology, pathogenesis, diagnosis and immunology of classical swine fever as well as control measures are described. This review focuses on new diagnostic methods, immune reactions and possibilities for control of CSF in the context of the current eradication plans.

Classical swine fever in wild boar (Sus scrofa)--experimental infections and viral persistence.

A pregnant wild boar and two wild boar weaners were inoculated intranasally with a field isolate of classical swine fever virus (CSFV) recently derived from a diseased domestic pig. The clinical, pathological and haematological findings noted in the young wild boars were comparable to those in domestic weaner pigs inoculated with the same virus isolate. Both wild boars showed the acute haemorrhagic form of CSF, one animal died 18 days post inoculation (p. i.) and the second one had to be euthanized when moribund two days later. The wild boar sow did not show any signs of illness p. i. but seroconversion was noticed. Twenty-eight days p. i. birth was given to six clinically healthy offsprings. One of the newborn proved to be viraemic until death when 39 days of age. Except for poor growth no other symptoms were noticed in this piglet. The non-viraemic litter mates remained healthy, although they had close contact to the persistently infected piglet. High titres of neutralizing antibodies against CSFV were measured in the serum samples of these offsprings. All findings were more or less in accordance with observations previously made in domestic pigs when infected with CSFV around 85 to 90 days of gestation. The wild boar was calculated to have been inoculated at about 87 to 92 days of gestation.

Classical swine fever virus infection modulates serum levels of INF-α, IL-8 and TNF-α in 6-month-old pigs.

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.

Infection of mouse bone marrow-derived immature dendritic cells with classical swine fever virus C-strain promotes cells maturation and lymphocyte proliferation.

In this study, the interactions of classical swine fever virus (CSFV) C-strain and the virulent GSLZ strain with mouse bone marrow-derived immature dendritic cells (BM-imDCs) were investigated for the first time. Both the C-strain and the virulent GSLZ strain could effectively infect and replicate in mouse BM-imDCs. C-strain-infected BM-imDCs showed a greatly enhanced degree of maturation, and could effectively promote the expansion and proliferation of allogeneic naive T cells. The C-strain induced a stronger Th1 response. Infection with the virulent GSLZ strain had no obvious influence on cell maturation or lymphocyte proliferation, and failed to induce any obvious immune response. The results of this study provided initial information for research of the immunologic mechanisms of CSFV using mouse DCs as the model cells.

Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs.

Host-virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population.

Classical swine fever virus suppresses maturation and modulates functions of monocyte-derived dendritic cells without activating nuclear factor kappa B.

Classical swine fever virus (CSFV) compromises the host immune system, causing the severe disease of pigs. Dendritic cells (DCs) are the most potent inducers of immune responses. In the present study, we investigated the functional properties of porcine monocyte-derived DCs (Mo-DCs) affected by CSFV. Results showed that the expression of surface markers of DCs such as major histocompatibility complex class II (MHC-II), CD80, CD83 and CD86 were unimpaired, but an obviously increased expression of CD172a in DCs was noticed 48 h after CSFV infection. The expression profiles of cytokines were detected in cultured Mo-DCs after various treatments for 48 h by Q-RT-PCR. The findings suggested that CSFV infection significantly increased the mRNA expression of IL-10 and TNF-α, and inhibited IL-12 expression, with little effect on IFN-α and IFN-γ expression. We further demonstrated that CSFV was incapable of activating the nuclear factor kappa B (NF-κB) in infected DCs, which was characterized by an unvaried DNA binding activity of NF-κB, the lack of translocation of p65/RelA from the cytoplasm to the nucleus and the stabilization of p65/RelA expression. Furthermore, Western blot analysis indicated that the inactivation of NF-κB was due to the failure of IκBα degradation. The data demonstrated that CSFV could be replicated in DCs and CSFV infection could modulate the secretion of crucial co-stimulatory molecules and cytokines which down-regulated maturation of DCs, without activating NF-κB in DCs. Thus, the results suggested a possible mechanism for CSFV evasion of innate host defenses, providing the basis for understanding molecular pathways in CSFV pathogenesis.

Transmission of classical swine fever virus depends on the clinical course of infection which is associated with high and low levels of virus excretion.

Infection with moderately virulent strains of classical swine fever virus (CSFV) can lead to different courses of disease: either (sub)acute, resulting in death or recovery, or chronic disease. The virus excretion dynamics between these courses are quite dissimilar, but it is not known if this also results in differences in virus transmission. In this study, the excretion and transmission dynamics of the moderately virulent Paderborn strain were studied in 15 one-to-one experiments. In these experiments, a single inoculated pig was housed with a single susceptible contact pig from day 1 post-inoculation (p.i.). Each contact pig that became infected was removed and replaced by a new contact pig at day 17 p.i. and day 26 p.i. Infection of contact pigs was monitored by reverse transcription quantitative real-time PCR on oropharyngeal fluid samples. Five of the inoculated pigs developed the chronic form or died during the acute phase (high excreting pigs), while 10 pigs recovered from the infection (low excreting pigs). In the first contact period, there was no significant difference in virus excretion between the high and low excreting pigs, while in the second and third contact period, high excreting pigs excreted significantly higher quantities of virus. Over the entire study period, the reproduction ratio differed significantly between the high (143 [56.3-373]) and low excreting pigs (23.1 [11.5-45.0]). This indicates the importance of high excreting pigs in transmission of CSFV. Furthermore, this study showed the rate of CSFV infections from a contaminated environment.

Protective efficacy of a Classical swine fever virus C-strain deletion mutant and ability to differentiate infected from vaccinated animals.

Classical swine fever (CSF) continues to be the most economically damaging pig disease in the world. The disease can be effectively controlled by vaccination with the live C-strain vaccine. This vaccine, however, does not enable the serological differentiation between infected and vaccinated animals (DIVA) and its use can therefore impose severe trade restrictions. CSF-specific diagnostic ELISAs detect antibodies directed against the conserved and immunodominant A domain of the E2 structural glycoprotein. We previously reported the production of a C-strain virus in which the immunodominant TAVSPTTLR epitope of the A domain is stably mutated with the aim to render the virus suitable as a DIVA vaccine. We here report that a single vaccination with this vaccine virus protected pigs from a lethal challenge dose of the highly virulent Brescia strain. Analysis of the sera, however, demonstrated that a commercially available E2 ELISA was unsuitable as an accompanying DIVA test.