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Classical swine fever virus (CSFV) is the etiological agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. Systemic prophylactic immunization with the live attenuated vaccine, the C-strain vaccine, is one of the effective measures for CSF control. However, one of the limitations of the C-strain vaccine is that the field strains-infected animals cannot be differentiated from the C-strain vaccinated herds by serological tests (DIVA). This constraint hampers the practical usage of the C-strain vaccine to eradicate the CSF in China. In the current study, a novel CSF modified live marker vaccine candidate was constructed based on the attenuation of the prevalent 2.1 genotype strain by the deletion of two virulence associated functional residues in the CSFV E

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256CLIGNTTVKVHASDER271. The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever.

Classical swine fever (CSF) remains one of the most important highly contagious and fatal viral disease of swine with high morbidity and mortality. CSF is caused by classical swine fever virus (CSFV), a small, enveloped RNA virus of the genus Pestivirus. The aim of this study was to construct the a novel CSFV Fc-fusion recombinant protein and evaluate the efficacy as a vaccine against CSFV. Here, we obtained a novel subunit vaccine expressing CSFV E2 recombinant fusion protein in CHO-S cells. Functional analysis revealed that CSFV Fc-fusion recombinant protein (CSFV-E2-Fc) could bind to FcγRI on antigen-presenting cells (APCs) and significantly increase IgA levels in serum and feces, inducing stronger mucosal immune response in swine. Additionally, CSFV-E2-Fc immunization enhanced CSFV-specific T cell immune response with a Th1-like pattern of cytokine secretion, remarkably stimulated the Th1-biased cellular immune response and humoral immune response. Further, the protective effects of CSFV-E2-Fc subunit vaccines were confirmed. The data suggest that CSFV E2-Fc recombinant fusion protein may be a promising candidate subunit vaccine to elicit immune response and protect against CSFV.

The heat-stable live-attenuated classical swine fever virus (CSFV) vaccine is an urgent need in many countries of Asia, Europe and Latin America. In this study, the thermostability of lyophilized live-attenuated CSFV vaccine formulations were investigated using accelerated stability at 37 °C for 10 days. The freeze-dried heat-stable formulation ST16, containing excipient combinations of trehalose, glycine, thiourea and phosphate buffer shows the superior thermostability. Moreover, the lyophilized vaccine with formula ST16 kept loss of viral activity less than 0.5 log10 during 24 months at storage temperatures of 2–8 °C. In thermal study, ST16 stabilized the vaccine within 1.0 log10 loss after storage at up to 25 °C for 6 months and room temperature for 7 months. Even under the harshest storage conditions of 37 °C for 25 days and 45 °C for 2 weeks, the virus titer dropped less than 1.0 log10 using ST16. Besides, it is notable that ST16 excluded gelatin and exogenous proteins, which might cause allergic reactions, thus avoiding immune side effects. The vaccine formulated ST16 proved to be safe and effective when immunized to piglets in vivo. The characteristics of dried vaccines were analyzed by X-ray powder diffraction, residual water measurements, differential scanning calorimetry and it was found that vaccine antigen were preserved in an amorphous matrix with high glass transition temperature above 60 °C and low residual water content below 2%, which made the vaccine more stable during storage.

A cross-sectional study was undertaken to identify factors associated with herd-level seropositivity to Classical Swine Fever (CSF) in smallholder pig farms in West Timor, Indonesia. Questionnaires were distributed to 240 pig farmers from 8 villages in two districts to collect information about farm structure and herd information, reproductive management and performance, husbandry, pig introductions and exits, health status, farmers' knowledge and history of vaccination. Three risk factors were found to be associated with the presence of antibodies to CSF in the herd in a multivariable logistic regression model. Seropositivity was associated with the introduction of pigs in the 12-month period preceding the survey (OR 4.78, 95% CI: 1.46, 15.71). Farmers who kept goats were 3.42 (95% CI: 1.20, 9.81) times more likely to have seropositive pigs than farmers without goats. Herds that had been vaccinated against CSF were also 2.33 (95% CI: 1.10, 5.12) times more likely to be seropositive than non-vaccinated herds. This latter positive association is likely to be as a result of antibodies induced by vaccination rather than a true risk factor for infection. The results of the questionnaire highlighted the lack of implementation of biosecurity measures by smallholder farmers in West Timor, which has the potential to increase the risk of their pigs to CSF, as well as other diseases.

The classical swine fever virus (CSFV) is one of the most harmful pathogens of swine and causes considerable economic loss. Mitophagy is a selective form of autophagy that degrades damaged mitochondria by combining with lysosomes. Previous studies have been reported that CSFV infection can induce mitophagy, but which effector protein is responsible for this process remains unclear. Herein, we revealed here that the CSFV nonstructural protein 5A (NS5A) plays a critical role in inducing cellular mitophagy. Specifically, the expression of CSFV NS5A in the PK-15 cells induces membrane potential loss and mitochondrial fission, and the quantities of mitophagosomes, the expression of Parkin and PINK1 were significantly increased compared with mock cells. Intriguingly, we found that Parkin-overexpression promotes CSFV propagation. Furthermore, the expression level of reactive oxygen species (ROS) was increased by CSFV NS5A protein, while NS5A-induced mitophagy correlated with the quantity of ROS production. In summary, our results reveal a new function of NS5A in inducing cellular mitophagy and broaden our understanding of the mechanism of CSFV-induced mitophagy, which may provide a new way to develop an antiviral strategy.

Porcine circovirus associated diseases (PCVADs) are among the most important diseases affecting the worldwide swine industry. Vaccination against porcine circovirus type 2 (PCV2) infection has been utilized for disease control and effectively reduces clinical signs of PCVADs. To evaluate the efficacy of the PCV2 vaccine in field farms, we conducted a trial using conventional pigs immunized with the subunit PCV2 vaccine followed by PCV2 challenge. Immunized pigs demonstrated lower serum viral loads, less viral antigen staining in lymph nodes, and higher average daily weight gain, confirming the protective efficacy of the vaccine. However, low levels of PCV2 infection were still detected in vaccinated pigs after challenge, suggesting that the PCV2 vaccine was unable to eradicate the virus, which could lead to asymptomatic PCV2 subclinical infection (PCV2-SI) in pig farms. Additionally, PCV2 infection is a risk factor for impaired pig immune response development during the weaning to growth stages, which is a crucial period to receive vaccines against classical swine fever (CSF). Therefore, the impact of PCV2-SI or PCV2-systemic disease (PCV2-SD) on live attenuated CSF vaccine was investigated. After PCV2 challenge, there was no difference in levels of classical swine fever virus (CSFV) neutralizing antibodies (NA) between pigs with PCV2-SD and PCV2-SI, suggesting that the efficacy of CSF vaccine was compromised. Moreover, results of long-term monitoring of CSFV NA titers in PCV2-SI pigs with minimized interference by maternally-derived antibodies suggested that serum PCV2 viral loads greater than 102 copies/mL may compromise the efficacy of CSF vaccine. Overall, a conventional pig model was established to demonstrate the impaired efficacy of the subunit PCV2 vaccine and its impact on the CSF vaccine in vaccination-challenge trials. Additionally, the impaired efficacy of the PCV2 vaccine resulted in increased PCV2-SI, eventually leading to compromised the live attenuated CSF vaccine induced NA response in field farm applications.

Classical swine fever (CSF) is a highly contagious notifiable disease of pigs caused by CSF virus of Flaviviridae family. Previously, lapinized vaccines were used for the disease control, which has now been replaced with cell culture vaccines. Determination of virus titre is the key factor for development and quality control testing of classical swine fever (CSF) cell culture vaccines. Since CSFV is a non- cytopathic virus, an accurate method for the titration of this virus in cell culture has not yet been reported. Here we present a full proof method of titration of CSF cell culture viruses employing Fluorescent Antibody Technique (FAT) in 24 well plate cover slip culture of PK-15 cells. CSFV monoclonal antibodies (Mab) used in the test bind to the CSF virus particles in the cell cytoplasm of the infected cells and the immune-fluorescence signal is produced by subsequent binding of FITC conjugate with Mab. In this newly developed method, apple green fluorescence is observed in the cytoplasm of the infected cells as the virus multiplies only in the cytoplasm. The nucleus as well as the uninfected cells cytoplasm is stained red without any traces of green fluorescence. Thus, the test clearly differentiates a CSFV infected cell from the uninfected cells in the vicinity, if any, and also from the uninfected controls. The test can also quantify the accurate titres of CSF live viruses in the cell culture vaccines and hence it has wide application in routine virus titration applied for manufacturing of CSF cell culture vaccines, determination of accurate multiplicity of infection (m.o.i.) during infection and quality control of vaccines by the testing laboratories.

Porcine productive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) both are major pathogens of swine that pose a great threat to the Chinese pig industry. It has been found that PRRSV infection can lead to vaccination failure of CSFV C strain-derived modified live vaccine (CSFV-C) by interfering with the immune responses to the latter. To investigate whether PRRSV can suppress CSFV-C replication, we created a 3D4/21-based cell line PAM39 that is susceptible to both viruses by expressing PRRSV receptors CD163 and CD169, and then investigated their interplay under the condition of either sequential or simultaneous co-infection. The most significant suppressive effect came from the sequential infection when the cells were first infected by PRRSV and then followed by CSFV-C at an interval of 6 h. In addition, this effect was independent of PRRSV strains. Mechanistically, PRRSV induced an elevated level of a subset of pro-inflammatory cytokines, especially tumor necrosis factor (TNF-α), through the nuclear factor κB (NF-κB) signaling pathway to inhibit the replication of CSFV-C in vitro. Thus, our studies provide an alternative explanation on PRRSV-induced CSFV vaccination failure, and this has an important implication in CSF vaccination and control.

Here, we constructed an attenuated live marker classical swine fever (CSF) vaccine (Flc-LOM-BErns) to eradicate CSF. This was done by taking infectious clone Flc-LOM, which is based on an attenuated live CSF vaccine virus (LOM strain), and removing the full-length classical swine fever virus (CSFV) Erns sequences and the 3′ end (52 base pairs) of the CSFV capsid. These regions were substituted with the full-length bovine viral diarrhoea virus (BVDV) Erns gene sequence and the 3′ end (52 base pairs) of the BVDV capsid gene. Sows were vaccinated with the Flc-LOM-BErns vaccine 3 weeks before insemination and then challenged with virulent CSFV at the early, mid- or late stages of pregnancy. We then examined transplacental transmission to the foetuses. Piglets born to sows vaccinated with Flc-LOM-BErns did not show vertical infection, regardless of challenge time. In addition, CSFV challenge did not affect the delivery date, weight or length of the foetus. Pregnant sows inoculated with the Flc-LOM-BErns vaccine were anti-CSF Erns antibody-negative and anti-BVDV Erns antibody-positive. Challenge of pregnant sows with virulent CSFV resulted in anti-CSF Erns antibody positivity. These results strongly indicate that differential diagnosis can be conducted between the Flc-LOM-BErns vaccinated animal and virulent CSFV affected animal by detecting antibody against BVDV Erns or CSF Erns gene. Therefore, the Flc-LOM-BErns vaccine may fulfil the function of differential diagnosis which required for DIVA vaccine.

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.

Classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) are economically important swine viruses that cause reproductive failure and/or respiratory symptoms in pigs. However, the co-infection status of these viruses in Chinese swine herds is not well clarified. In this study, we evaluated the co-infection of these four viruses in 159 pigs collected from 63 herds in eight regions of China from 2016 to 2018. CSFV, PRRSV, PCV2 and PCV3 were detected in 14, 56, 43 and 4 of the pigs, respectively. The percentage of singular infections was 32.71%, while the percentages of dual infections and multiple infections were 15.72% and 3.15%, respectively. The E2 of CSFV, ORF5 of PRRSV, ORF2s of PCV2 and PCV3 from all positive samples were determined and used for phylogenetic analyses. E2-based phylogenetic tree showed that all 14 CSFVs identified in this study belong to 2.1b subtype. ORF5-based phylogenetic tree showed that PRRSV2 is predominant in China while PRRSV1 can also be detected. In addition, 35, 16, 4 and 1 of our PRRSVs are clustered with highly pathogenic PRRSV2, NADC30-like PRRSV2, classical PRRSV2 and PRRSV1, respectively. ORF2-based phylogenetic trees showed that our PCVs are grouped with 2 PCV2 subtypes (PCV2d and PCV2b) and 3 PCV3 subtypes (PCV3a, PCV3b and PCV3c), respectively. Our results provide the latest co-infection status and the diversity of four important swine viruses in Chinese swine herds, which is beneficial for understanding the epidemiology of these viruses.

Classical swine fever (CSF) continues to be a devastating infectious disease for the swine industry in China and commonly exists as wild or atypical types. From June 3rd to October 3rd, 2018, outbreaks of typical CSF cases with mortality rates of 42-86% occurred in 11 swine herds in five cities of Guangdong province, and were confirmed by RT-PCR. Phylogenetic analyses based on the nucleotide sequences of full-length E2 genes showed that the CSFV isolates collected in Guangdong, 2018 grouped into sub-subgenotype 2.1c and formed a separate clade from previously identified 2.1c isolates. Sequence comparison further confirmed the distance between the novel emergent and previously identified 2.1c isolates, with shared 94.5-98.2% and 97.8-99.7% identities at the nucleotide and amino acid levels respectively. Furthermore, 2.1c isolates collected in 2018 from Guangdong province contained a unique amino acid substitution (K174R) in the E2 protein in comparison with other 2.1c representative strains and CSFV 2.1, 2.2, 2.3 strains. Of note, the novel emergent 2.1c isolates are neutralized by sera from C-strain vaccinated sows, indicating that C-strain is still efficacious for protection against field isolates of CSFV.

Pig farming in Ecuador represents an important economic and cultural sector. Recently, the National Veterinary Service has implemented individual identification of pigs, stricter movement control and mandatory vaccination against classical swine fever (CSF), which has been prevalent since 1940. The information registered since 2017 seems valuable for applying modelling techniques assessing the risk of dissemination of animal diseases and test control strategies. Social network analysis (SNA) was used to characterise the premises into risk categories, looking for better targeting surveillance activities and identifying higher risk targets. Network modelling was used to test the disease spread and control strategies over the network, using a Susceptible, Infected, Recovered, Susceptible SIRS model to compare a target selection of high-risk nodes against the current traditional surveillance. Finally, temporal network analysis revealed general trends in the network's evolution. The network contained 751,003 movements with 6 million pigs, excluding movements to slaughterhouses. A total of 165,593 premises were involved in network movements (144,118 farms, 138 industrials, 21,337 traders and 51 markets). On an annual average, 75% of the premises (124,976) received or sent up to one movement with an annual average of 1.5 pigs. In contrast, 0.01% of the premises (166) made up to 1,372 movements, with an annual average of 11,607 pigs. Simulations on the network resulted in the dissemination of CSF, reaching a mean prevalence of 29.93% without a control strategy. The random selection strategy reduced the prevalence to 24.4%, while target selection by risk reduced to 3.3%. When comparing the static representation of the network with the temporal counterpart, causal fidelity (c = 0.62) showed an overestimation of 38% in the number of transmission paths, even though it took an average of 4.39 steps to cross the network, these steps took approximately 233 days. The characterisation of the premises acting on the Ecuadorian network, based on risk emphasis, and the application of network modelling techniques can support the redesign of the surveillance system. Minor changes could enable the implementation of risk-based surveillance, targeting specifically the nodes at higher risk and considering special attention to markets and traders.

Oral fluid sampling for the detection of classical swine fever virus infection provides a relatively inexpensive method for conducting active CSF surveillance. The purpose of this study was to detect CSFV nucleic acid and antibody in serum and oral fluid samples in a group of 10 pigs infected with the moderate CSFV strain, Paderborn. Based on clinical signs, outcome, and other results, pigs were placed into one of three disease outcome groups; Acute, Chronic and Recovered. Oral fluid and serum samples were analyzed for the presence of CSFV nucleic acid along with E2 and Erns surface protein-specific IgM, IgG and IgA responses. The results were summarized into a timeline of detection events beginning with the appearance of E2-IgM in serum (3 DPI) followed by CSFV nucleic acid in serum (6 DPI), CSFV nucleic acid in oral fluid (8 DPI), E2-IgG in serum (20 DPI), and E2-IgG in oral fluid (24 DPI). The results show that a combination of molecular and serological analyses of oral fluid can be incorporated into CSF surveillance.

Classical swine fever (CSF) is a devastating viral disease affecting pigs that causes major economic losses worldwide. Conventional assays to identify classical swine fever virus (CSFV) face challenges, such as the required molecular amplification of the target molecules via polymerase chain reaction (PCR). We designed a gold nanoflare probe to directly detect CSFV. Gold nanoparticles (AuNPs) were conjugated with a pair of complementary DNA sequences that specifically recognized and captured CSFV RNA, resulting in a fluorescence signal to indicate the existence of CSFV. The constructed nanocomposite was then utilized in a quantitative analysis to recognize the virus sequence present at amounts as low as 50 pg/μL. The CSFV-AuNP probe enabled real-time, quantitative detection of native CSFV in response to doses of the specific RNA sequence (CSFV NS2) that indicated active viral replication of CSFV Shimen in macrophages after 12, 24, and 48 h. The potential diagnostic applications of the probe were demonstrated by measuring CSFV without nucleic acid amplification in samples from seven types of tissue samples, specifically heart, spleen, kidney, liver, lymph, intestine, and muscle samples obtained from one pig confirmed to suffer CSF. The speed, sensitivity, and versatility of this CSFV-AuNP biosensor make it an ideal candidate for further application in the prevention and control of animal epidemic diseases.

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.

The development of subunit vaccines against classical swine fever is a desirable goal, because it allows discrimination between vaccinated and infected animals. In this study, humoral and cellular immune response elicited in inbred BALB/c mice by immunization with a recombinant classical swine fever virus (CSFV) E2 protein fused to porcine CD154 antigen (E2CD154) was assessed. This model was used as a predictor of immune response in swine. Mice were immunized with E2CD154 emulsified in Montanide ISA50V2 or dissolved in saline on days 1 and 21. Another group received E2His antigen, without CD154, in the same adjuvant. Montanide ISA50V2 or saline served as negative controls for each experimental group. Animals immunized with 12.5 and 2.5 μg/dose of E2CD154 developed the highest titers (>1:2000) of CSFV neutralizing antibodies. Moreover, CSFV specific splenocyte gamma-interferon production, measured after seven and twenty-eight days of immunization, was significantly higher in mice immunized with 12.5 μg of E2CD154. As a conclusion, E2CD154 emulsified in Montanide ISA50 V2 was able to induce a potent humoral and an early cellular immune response in inbred BALB/c mice. Therefore, this immunogen might be an appropriate candidate to elicit immune response in swine, control CSF disease and to eliminate CSFV in swine.

Classical swine fever (CSF) is a viral infectious disease of swine with significant economic impact in the affected countries due to the limitation of trade, culling of infected animals and production losses. In Latin America, CSF is endemic in several countries including Ecuador, Bolivia, Brazil and Peru. Since 2010, the National Veterinary Services of Peru have been working to better control and eradicate the disease with an intensive vaccination program. The aim of this study was to evaluate the effectiveness of the vaccination program and determine which factors are still contributing to the persistence of the disease in certain regions of Peru. We integrated the data from the vaccination campaign, the live pig movement network and other socioeconomic indicators into a multilevel logistic regression model to evaluate their association with CSF occurrence at district level. The results revealed that high vaccination coverage significantly reduces the risk of CSF occurrence (OR = 0.07), supporting the effectiveness of the vaccination program. Districts belonging to large and medium pig trade network communities (as identified with walktrap algorithm) had higher probability to CSF occurrence (OR = 2.83 and OR = 5.83, respectively). The human development index (HDI) and the presence of a slaughterhouse in the district was also significantly associated with an increased likelihood of CSF occurrence (OR = 1.52 and OR = 3.25, respectively). Districts receiving a high proportion of the movements from districts that were infected in the previous year were also at higher risk of CSF occurrence (OR = 3.30). These results should be useful to guide the prioritization of vaccination strategies and may help to design other intervention strategies (e.g., target education, movement restrictions, etc.) in high-risk areas to more rapidly advance in the eradication of CSF in Peru.

Classical swine fever virus (CSFV) is an ancient and economically important flavivirus that utilizes a Rab5-dependent endocytic pathway to enter host cells. Rab22a is a small GTPase that cooperates with Rab5 in the regulation of early endosome dynamics. Until now, the role of Rab22a in the flavivirus life cycle has been poorly defined. In this study, we systematically analyzed the role of Rab22a in CSFV proliferation and internalization using multiple viral replication analyses in combination with the overexpression, knockdown, and mutation of Rab22a, and found that Rab22a is involved in the entry process of CSFV. Confocal microscopy results showed that Rab22a colocalized with virus particles during the early phase of infection. Furthermore, by using glutathione S-transferase pull-down and co-immunoprecipitation assays, we verified the interaction between Rab22a and CSFV non-structural protein NS4B, and determined that NS4B can only bind to wild-type Rab22a, but not to the mutants Q64L and S19N. In addition, we explored the relationship between Rab22a, Rab5 and NS4B in CSFV internalization, and found out that these three proteins bind in early endosomes, and then through a Rab22a-Rab5-NS4B cascade allows the entry of CSFV. Taken together, our findings highlight the role of Rab proteins in CSFV internalization, and extend the understanding of the life cycle of flaviviruses.

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.

Classical swine fever virus (CSFV) C-strain was developed through hundreds of passages of a highly virulent CSFV in rabbits. To investigate the molecular basis for the adaptation of C-strain to the rabbit (ACR), a panel of chimeric viruses with the exchange of glycoproteins Erns, E1, and/or E2 between C-strain and the highly virulent Shimen strain and a number of mutant viruses with different amino acid substitutions in E2 protein were generated and evaluated in rabbits. Our results demonstrate that Shimen-based chimeras expressing Erns-E1-E2, Erns-E2 or E1-E2 but not Erns-E1, Erns, E1, or E2 of C-strain can replicate in rabbits, indicating that E2 in combination with either Erns or E1 confers the ACR. Notably, E2 and the amino acids P108 and T109 in Domain I of E2 are critical in ACR. Collectively, our data indicate that E2 is crucial in mediating the ACR, which requires synergistic contribution of Erns or E1.

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.

Early recognition and rapid elimination of infected animals is key to controlling incursions of classical swine fever virus (CSFV). In this study, the diagnostic characteristics of 10 CSFV assays were evaluated using individual serum (n = 601) and/or oral fluid (n = 1417) samples collected from -14 to 28 days post inoculation (DPI). Serum samples were assayed by virus isolation (VI), 2 commercial antigen-capture enzyme-linked immunosorbent assays (ELISA), virus neutralization (VN), and 3 antibody ELISAs. Both serum and oral fluid samples were tested with 3 commercial real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays. One or more serum samples was positive by VI from DPIs 3 to 21 and by antigen-capture ELISAs from DPIs 6 to 17. VN-positive serum samples were observed at DPIs ≥ 7 and by antibody ELISAs at DPIs ≥ 10. CSFV RNA was detected in serum samples from DPIs 2 to 28 and in oral fluid samples from DPIs 4 to 28. Significant differences in assay performance were detected, but most importantly, no single combination of sample and assay was able to dependably identify CSFV-inoculated pigs throughout the 4-week course of the study. The results show that effective surveillance for CSFV, especially low virulence strains, will require the use of PCR-based assays for the detection of early infections (