Removal of the Erns RNase Activity and of the 3' Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease.

In this study, we assessed the potential synergistic effect of the Erns RNase activity and the poly-U insertion in the 3' untranslated region (UTR) of the low-virulence classical swine fever virus (CSFV) isolate Pinar de Rio (PdR) in innate and adaptive immunity regulation and its relationship with classical swine fever (CSF) pathogenesis in pigs. We knocked out the Erns RNase activity of PdR and replaced the long polyuridine sequence of the 3' UTR with 5 uridines found typically at this position, resulting in a double mutant, vPdR-H30K-5U. This mutant induced severe CSF in 5-day-old piglets and 3-week-old pigs, with higher lethality in the newborn (89.5%) than in the older (33.3%) pigs. However, the viremia and viral excretion were surprisingly low, while the virus load was high in the tonsils. Only alpha interferon (IFN-α) and interleukin 12 (IL-12) were highly and consistently elevated in the two groups. Additionally, high IL-8 levels were found in the newborn but not in the older pigs. This points toward a role of these cytokines in the CSF outcome, with age-related differences. The disproportional activation of innate immunity might limit systemic viral spread from the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms. Infection with vPdR-H30K-5U resulted in poor neutralizing antibody responses compared with results obtained previously with the parent and RNase knockout PdR. This study shows for the first time the synergistic effect of the 3' UTR and the Erns RNase function in regulating innate immunity against CSFV, favoring virus replication in target tissue and thus contributing to disease severity. IMPORTANCE CSF is one of the most relevant viral epizootic diseases of swine, with high economic and sanitary impact. Systematic stamping out of infected herds with and without vaccination has permitted regional virus eradication. However, the causative agent, CSFV, persists in certain areas of the world, leading to disease reemergence. Nowadays, low- and moderate-virulence strains that could induce unapparent CSF forms are prevalent, posing a challenge for disease eradication. Here, we show for the first time the synergistic role of lacking the Erns RNase activity and the 3' UTR polyuridine insertion from a low-virulence CSFV isolate in innate immunity disproportional activation. This might limit systemic viral spread to the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms, thus contributing to disease severity. These results highlight the role played by the Erns RNase activity and the 3' UTR in CSFV pathogenesis, providing new perspectives for novel diagnostic tools and vaccine strategies.

Pathology of the outbreak of subgenotype 2.5 classical swine fever virus in northern Vietnam.

Classical swine fever (CSF) is an endemic disease in southeastern Asia and is one of the most important swine diseases in Vietnam. This study was conducted to characterize the pathology of natural cases of CSF in northern Vietnam in 2018 and their genetic prevalence. A total of 10 representative pigs were collected from four provinces (Hung Yen, Ha Noi, Quang Ninh and Thai Binh) during five outbreaks and examined pathologically. The gross and histopathological findings showed the disease was expressed as the acute or the subacute to chronic form of CSF, depending on the age of the animals. The most consistently observed lesions associated with infection by the classical swine fever virus (CSFV) included lymphoid depletions in tonsils, lymph node and spleen; histiocytic hyperplasia in spleen; cerebral haemorrhage; perivascular cuffing in the brain; renal erythrodiapedesis; urothelial vacuolation and degeneration and interstitial pneumonia. The immunohistochemical findings showed a ubiquitous CSFV antigen mainly in the monocytes/macrophages and in the epithelial and endothelial cells in various organs. CSFV neurotropism was also found in the small neurons of the cerebrum and the ganglia of the myenteric plexus. Analysis of the full-length envelope protein (E2) genome sequence showed that all strains were genetically clustered into subgenotype 2.5, sharing a nucleotide identity of 94.0%-100.00%. Based on the results of this study, the strain was categorized as a moderately virulent CSFV.

Isolation and Characterization of Porcine Astrovirus 5 from a Classical Swine Fever Virus-Infected Specimen.

Many new astroviruses have been identified in humans and other animals in recent years, but only a few have been successfully isolated for extensive biological study. Here, we report an unusual isolation of a porcine astrovirus 5 (PAstV5) strain from a clinical classical swine fever virus (CSFV)-infected tissue sample. Incubation of porcine PK-15 cells with an extract of the CSFV-positive tissue resulted in unexpected cytopathic effects (CPEs), and high-throughput viromic sequencing identified PAstV5 and porcine circovirus type 2 (PCV2) as well as CSFV in the culture. After clearance of CSFV and PCV2, a pure PAstV5 strain, named PAstV5-AH29-2014, was obtained. Analysis revealed virus of typical astroviral morphology with a genome of 6,448 nucleotides, sharing 84.3 to 88.9% nucleotide identity with previously published PAstV5 strains. A mechanistic study showed that CSFV coinfection was likely an important factor for successful isolation by significantly enhancing PAstV5 replication in PK-15 cells via suppression of a type I interferon response. Altogether, PAstV5-AH29-2014, as the first isolated PAstV5 strain, will provide critical material for the investigation of the biological and pathogenic properties of this virus as well as for future development of relevant biological and diagnostic reagents.IMPORTANCE Porcine astroviruses are mainly associated with gastroenteritis and neurological diseases in pigs, and five genotypes have been identified (PAstV1-5). However, the clinical manifestations of genotypes other than PAstV1 have not yet been determined because of the failure of in vitro virus isolation. Here, we report a surprising isolation of a PAstV5 strain from a clinical classical swine fever virus (CSFV)-infected tissue sample, which can stably passage in PK-15 cells, and coinfection with CSFV significantly enhanced the replication of PAstV5, possibly through suppression of beta interferon production. Thus, the first isolated PAstV5 strain will be useful for investigating the biological and pathogenic properties of this virus, and the findings obtained in this study provide new insights into defining the interaction mechanism between CSFV and PAstV5.

Swine Host Protein Coiled-Coil Domain-Containing 115 (CCDC115) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2 during Virus Replication.

Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2-CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2-CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.

Recombinant pseudorabies virus expressing E2 of classical swine fever virus (CSFV) protects against both virulent pseudorabies virus and CSFV.

Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.

A Polyuridine Insertion in the 3' Untranslated Region of Classical Swine Fever Virus Activates Immunity and Reduces Viral Virulence in Piglets.

Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3' untranslated region (3' UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3' UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3' UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3' untranslated region (3' UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3' UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle.

The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence.IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

Experimental infection of pigs with a classical swine fever virus isolated in Japan for the first time in 26 years.

Following an outbreak of classical swine fever (CSF) in Japan, 2018, CSFV JPN/1/2018 was isolated from an infected pig sample. In this study, we carried out a comparative experimental infection in pigs using this strain and the highly virulent ALD strain and compared outcomes, including clinical manifestation, virus shedding patterns and antibody responses. Although pigs inoculated orally or intramuscularly with JPN/1/2018 developed hyperthermia and had decreased leucocyte numbers, they survived for the whole experimental period and showed less severe clinical signs than those infected with the ALD strain. We confirmed the presence of characteristic multifocal infarction of the margin of the spleen that arises following infection with JPN/1/2018, albeit that this finding was not observed in all infected pigs. Both viruses efficiently spread to contact pigs in a similar manner, suggesting in transmissibility between the two strains. Viral RNAs were detected in all clinical samples, especially whole blood samples, before the pigs developed hyperthermia until at least approximately 2 weeks after inoculation. Our findings will be valuable for the investigations into epidemic events occurring in Japan and for establishing diagnostic strategies and control measures against CSF.

Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy.

BACKGROUND: Recent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions. RESULTS: Six litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage. CONCLUSIONS: Circulation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication.

Fluorophore labelled BVDV: a novel tool for the analysis of infection dynamics.

Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.

Virulence evaluation of classical swine fever virus subgenotype 2.1 and 2.2 isolates circulating in China.

Classical swine fever (CSF) remains an important pig disease in China, where it usually presents with mild or atypical clinical manifestations, with large scale outbreaks rarely seen. This has led to speculation about the possible circulation of viral strains of low virulence. To investigate this possibility, five field isolates within the predominant genotype 2 (2.1b, 2.1c, 2.1 h and 2.2) were evaluated and compared by experimental infection of naturally farrowed but colostrum-deprived piglets. All infected piglets displayed clinical signs, including persistent high fever, depression, anorexia, dyspnea, conjunctivitis, constipation, and hesitant gait. Typical pathological lesions, including pulmonary edema, hemorrhagic or cellulosic exudation, and swelling and hemorrhage of lymph nodes, were observed. Viremia and Erns protein expression in the blood of all infected animals were detectable from 3 to 5 days post infection (DPI), their presence correlating with the onset of fever, clinical signs and leukopenia. E2 antibody did not develop in any of the field CSFV-infected piglets during the disease course, while Erns antibody was detectable in 4-56% of infected animals at various time points. Mortalities ranged from 20 to 80% within 21 DPI, progressing to 100% by 43 DPI. Based on clinical scores and fatalities within 21 DPI, 2 of the 5 field isolates were classified as of moderate virulence and 3 of high virulence; i.e., no field isolates of low virulence were identified. The study has provided data supporting the use of these isolates as challenge viruses to evaluate the efficacy of current CSF vaccines.

Pulmonary intravascular macrophages regulate the pathogenetic mechanisms of pulmonary lesions during acute courses of classical swine fever.

Classical swine fever (CSF) is a highly contagious and often fatal viral disease of domestic pigs and wild boar. Pulmonary oedema and haemorrhages in lung parenchyma are common lesions in the acute forms of CSF that may compromise pig survival and whose pathogenetic mechanisms remain unclear. The appearance of pulmonary lesions in pigs infected with Alfort/187 strain of classical swine fever virus (CSFV) euthanized between 2 and 17 days postinfection (dpi) and the role played by cytokines secreted by different pulmonary macrophage populations in the evolution of lesions was evaluated in this study. Microscopic changes of alveolar septal thickening along with oedema and haemorrhages became more severe at middle-late stages of the experiment. A significant increase in the number of pulmonary macrophages, mainly pulmonary intravascular macrophages (PIMs), was observed coinciding with the onset of alveolar septal thickening from Day 4 pi. PIMs were the main target of CSFV from initial stages of infection while the presence of infected pulmonary alveolar macrophages (PAMs) was scarce and late. Initial infection of PIMs induced phagocytic and biosynthetic activation with subsequent release of chemotactic cytokines. TNFα and, to a lesser extent, IL-1α secreted by PIMs were the major cytokines involved, while IL-6 played only a minor role. On the contrary, results suggested only a secondary role of PAMs as source of cytokines. The presence of vascular changes from Day 9 pi coincided with the highest levels of infected PIMs and the highest number of pro-inflammatory cytokines secreting PIMs. Activation and phagocytosis of platelets were observed in the lungs of infected pigs from early stages, also coinciding with the expression of cytokines with a proven procoagulant activity. The existence of intravascular coagulation phenomena in lung was ruled out.

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV.

Protection against transplacental transmission of moderately virulent classical swine fever virus using live marker vaccine "CP7_E2alf".

Classical swine fever (CSF) remains as one of the most important infectious diseases of swine. While prophylactic vaccination is usually prohibited in free countries with industrialized pig production, emergency vaccination is still foreseen. In this context, marker vaccines are preferred as they can reduce the impact on trade. The live-attenuated Suvaxyn® CSF Marker vaccine by Zoetis (based on pestivirus chimera "CP7_E2alf"), was recently licensed by the European Medicines Agency. Its efficacy for the individual animal had been shown in prior studies, but questions remained regarding protection against transplacental transmission. To answer this question, a trial with eight pregnant sows and their offspring was performed as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Six of the sows were intramuscularly vaccinated on day 44 of gestation, while the other two remained as unvaccinated controls. All sows were challenged with the moderately virulent CSFV strain "Roesrath" and euthanized shortly before the calculated farrowing date. Sows and piglets were grossly examined and necropsied. Organs (spleen, tonsil, lymph node, and kidney), EDTA-blood and serum were collected from all animals. All samples were tested for antibodies against CSFV glycoproteins E2 and Erns as well as CSFV (virus, antigen and genome). It could be demonstrated that the vaccine complies with all requirements, i.e. no virus was found in the blood of vaccinated sows and their fetuses, and no antibodies were found in the serum of the fetuses from the vaccinated sows. All controls were valid. Thus, it was demonstrated that a single dose vaccination in the sows efficiently protected the offspring against transplacental infection with a moderately virulent CSFV strain.

Complete genomic characteristics and pathogenic analysis of the newly emerged classical swine fever virus in China.

BACKGROUND: Classical swine fever (CSF) is one of the most devastating and highly contagious viral diseases in the world. Since late 2014, outbreaks of a new sub-genotype 2.1d CSF virus (CSFV) had caused substantial economic losses in numbers of C-strain vaccinated swine farms in China. The objective of the present study was to explore the genomic characteristics and pathogenicity of the newly emerged CSFV isolates in China during 2014-2015. RESULTS: All the new 8 CSFV isolates belonged to genetic sub-genotype 2.1d. Some genomic variations or deletions were found in the UTRs and E2 of these new isolates. In addition, the pathogenicity of HLJ1 was less than Shimen, suggesting the HLJ1 of sub-genotype 2.1d may be a moderated pathogenic isolate and the C-strain vaccine can supply complete protection. CONCLUSIONS: The new CSFV isolates with unique genomic characteristics and moderate pathogenicity can be epidemic in many large-scale C-strain vaccinated swine farms. This study provides the information should be merited special attention on establishing prevention and control policies for CSF.

Classical swine fever virus induces pyroptosis in the peripheral lymphoid organs of infected pigs.

Classical swine fever virus (CSFV) causes a highly lethal disease in pigs, which is characterized by immunosuppression. Leukopenia is known to be a possible mechanism of immunosuppression during CSFV infection. As a new and specialized form of cell death, pyroptosis is the key response of the innate immune system to pathogens, and is widely involved in the occurrence and development of infectious diseases. However, the relationship between CSFV and pyroptosis has not been explored. In this study, we investigated the occurrence of pyroptosis in pigs following CSFV infection. According to qRT-PCR assay results, the prevalence of this virus in peripheral lymphoid organs (tonsils, lymph nodes, and spleen) was much higher than that in other organs. Severe bleeding, necrosis, and a significant reduction in lymphocytes were found in the peripheral lymphoid organs of CSFV-infected pigs based on histological examination. In-depth studies showed that an increased ratio of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were present in the peripheral lymphoid organs of the CSFV-infected group according to immunohistochemistry. Meanwhile, the p10 subunit and activity of caspase-1, which is a regulator of pyroptosis, the N-terminal domain of gasdermin D, which is an executor of pyroptosis, and the cleavage and secretion of IL-1b, which is a product of pyroptosis were increased in the peripheral lymphoid organs of the CSFV-infected group. Together, these results demonstrated that pyroptosis is involved in CSFV-induced cell death in vivo, which provides a new understanding of the mechanism associated with lymphocyte depletion and immunosuppression in pigs infected with this virus.

Classical Swine Fever Outbreak after Modified Live LOM Strain Vaccination in Naive Pigs, South Korea.

We report classical swine fever outbreaks occurring in naive pig herds on Jeju Island, South Korea, after the introduction of the LOM vaccine strain. Two isolates from sick pigs had >99% identity with the vaccine stain. LOM strain does not appear safe; its use in the vaccine should be reconsidered.

Tissue expression of Toll-like receptors 2, 3, 4 and 7 in swine in response to the Shimen strain of classical swine fever virus.

The Toll-like receptors (TLRs) of the innate immune system provide the host with the ability to detect and respond to viral infections. The present study aimed to investigate the mRNA and protein expression levels of TLR2, 3, 4 and 7 in porcine tissues upon infection with the highly virulent Shimen strain of classical swine fever virus (CSFV). Reverse transcription­quantitative polymerase chain reaction was used to detect the mRNA expression levels of CSFV and TLR, whereas western blotting was used to detect the expression levels of TLR proteins. In addition, tissues underwent histological examination and immunohistochemistry to reveal the histopathological alterations associated with highly virulent CSFV infection and to detect TLR antigens. Furthermore, porcine monocyte­derived macrophages (pMDMs) were prestimulated with peptidoglycan from Staphylococcus aureus (PGN­SA), polyinosinic­polycytidylic acid [poly (I:C)], lipopolysaccharide from Escherichia coli 055:B5 (LPS­B5) or imiquimod (R837) in order to analyze the association between TLR expression and CSFV replication. Following stimulation for 12 h (with TLR­specific ligands), cells were infected with CSFV Shimen strain. The results revealed that the expression levels of TLR2 and TLR4 were increased in the lung and kidney, but were decreased in the spleen and lymph nodes in response to CSFV. TLR3 was strongly expressed in the heart and slightly upregulated in the spleen in response to CSFV Shimen strain infection, and TLR7 was increased in all examined tissues in the presence of CSFV. Furthermore, R837 and LPS­B5 exerted inhibitory effects on CSFV replication in pMDMs, whereas PGN­SA and poly(I:C) had no significant effect. These findings highlight the potential role of TLR expression in the context of CSFV infection.

Restoration of glycoprotein Erns dimerization via pseudoreversion partially restores virulence of classical swine fever virus.

The classical swine fever virus (CSFV) represents one of the most important pathogens of swine. The CSFV glycoprotein Erns is an essential structural protein and an important virulence factor. The latter is dependent on the RNase activity of this envelope protein and, most likely, its secretion from the infected cell. A further important feature with regard to its function as a virulence factor is the formation of disulfide-linked Erns homodimers that are found in virus-infected cells and virions. Mutant CSFV lacking cysteine (Cys) 171, the residue responsible for intermolecular disulfide bond formation, were found to be attenuated in pigs (Tews BA, Schürmann EM, Meyers G. J Virol 2009;83:4823-4834). In the course of an animal experiment with such a dimerization-negative CSFV mutant, viruses were reisolated from pigs that contained a mutation of serine (Ser) 209 to Cys. This mutation restored the ability to form disulphide-linked Erns homodimers. In transient expression studies Erns mutants carrying the S209C change were found to form homodimers with about wt efficiency. Also the secretion level of the mutated proteins was equivalent to that of wt Erns. Virus mutants containing the Cys171Ser/Ser209Cys configuration exhibited wt growth rates and increased virulence when compared with the Cys171Ser mutant. These results provide further support for the connection between CSFV virulence and Erns dimerization.

Autophagy induces apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV.

Lymphocyte depletion and immunosuppression are typical clinical characteristics of pigs infected with classical swine fever virus (CSFV). The apoptosis of virus-infected and bystander cells plays a role in the immunopathology of classical swine fever (CSF). Here, we offer the first evidence that autophagy is involved in apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV. Using immunohistochemical assays, we observed that more LC3II-positive cells appear in the T-cell zone of spleens. Spleen cell apoptosis was demonstrated using flow cytometry and TUNEL staining. Confocal immunofluorescence revealed that partial LC3II-positive cells were simultaneously TUNEL-positive. By cultivating spleen cells ex vivo, we demonstrated that the inhibition of autophagy by 3-MA treatment inhibited apoptosis and death of T lymphocytes caused by CSFV infection but did not have this effect  on B lymphocytes. Further observations demonstrated that uninfected cells in the spleen were also undergoing autophagy in vivo. In summary, these results linked autophagy with the apoptosis and cell death of splenic T cells, providing a new outlook to understand the mechanism of T lymphocyte depletion and immunosuppression during CSF.