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Classical swine fever (CSF) is endemic in Cuba and is one of the major health problems of the Cuban swine industry. The current efforts to control the disease in Cuba include vaccination with Porvac ® , a subunit marker vaccine. Although the efficacy of Porvac against CSF virus (CSFV) subgenotype 1.4 has been extensively documented, little is known about the ability of the antibodies induced by this vaccine to neutralize other CSFV genotypes. In this study, sera collected from three pigs vaccinated with Porvac were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2, and 3. The findings from this study indicate that additional in vivo studies are warranted to confirm the ability of this vaccine to protect pigs against CSFV genotypes 2 and 3., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. Crown.)

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a severe disease that causes huge economic losses in the swine industry worldwide. In China, CSF has been under control due to extensive vaccination since 1954. However, there are still sporadic CSF outbreaks in China. Here, we isolated 27 CSFV strains from three Chinese provinces (Shaanxi, Gansu, and Ningxia) from 2011 to 2018. Phylogenetic analysis based on the full-length envelope glycoprotein E2 coding region revealed that 25 out of 27 CSFV isolates clustered within subgroups 2.1 and 2.2, while two strains from Gansu belonged to subgroup 1.1. The sequence identity among these 27 isolates varied from 79.3% to 99.8% (nucleotides) and from 83.1% to 99.7% (amino acids). Further analysis based on the E2 amino acid sequences showed that these new isolates have consistent amino acid substitutions, including R31K and N34S., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Classical swine fever virus (CSFV) reemerged in naïve pig herds on Jeju Island, South Korea, due to the accidental introduction of the LOM vaccine strain in 2014. Since this reemergence, the previously CSFV-free region has experienced numerous outbreaks, causing the virus to become endemic in provincial herds. In this study, we determined the complete genome sequences and investigated the molecular characteristics of LOM-derived field CSFV strains with unique insertion-deletion (INDEL) mutations in the 3'-untranslated region (UTR) that were responsible for ongoing sporadic outbreaks on Jeju Island in 2019. The Jeju LOM-derived variants that emerged in 2019 had their own INDEL signatures in the 3'-UTR, resulting in changes to the predicted secondary stem-loop structures. The genomes of these strains were 12,297-12,302 nucleotides in length, one nucleotide (nt) shorter or one, two, or four nt longer than the reference LOM strain. The 3'-UTR INDEL variants shared 98.8-99.0% and 98.3-98.6% identity with the LOM strain at the polyprotein and full-genome level, respectively. The total number of genetic variations between the LOM vaccine strain and the 3'-UTR INDEL isolates ranged from 161 to 202 and 37 to 45 at the nucleotide and amino acid level, respectively. These mutations were broadly dispersed throughout the genome and particularly clustered in NS2 and the 3'-UTR, possibly triggering a reversion to low virulence and allowing the virus to adapt to improve its persistence in the field. This study provides important information about the genetic evolution of LOM-derived CSFV circulating in the free region, and suggests that it arose from continuous non-lethal mutations to ensure viral fitness in host animals.

Classical swine fever (CSF) is an important viral disease of domestic pigs and wild boar. The structural proteins E2 and E rns of classical swine fever virus (CSFV), which participate in the attachment of the virion to the host cell surface and its subsequent entry, are immunogenic. The E2 and E rns proteins are used for diagnosis and the development of vaccines against CSFV infection in swine. Newcastle disease virus (NDV) has been successfully used as a viral vector to express heterologous proteins. In the present study, the E2 and E rns proteins of CSFV were expressed in cell culture as well as embryonated chicken eggs, using recombinant NDV (rNDV). Rescued rNDV expressing the E2 and E rns proteins induced the production of CSFV-neutralizing antibodies upon intranasal vaccination of pigs. Serum samples from vaccinated animals were found to neutralize both homologous and heterologous CSFV strains. Furthermore, rNDV expressing the E2 and E rns proteins of CSFV was used to develop an indirect ELISA, which was used to measure the the antibody titers of randomly collected serum samples. The results suggested that the ELISA based on rNDV-expressed E2 and E rns proteins could be used to screen for CSFV infections. This study shows that rNDV-based expression of CSFV antigens is potentially applicable for development of vaccines and diagnostic tests for CSFV infection. This approach could be an economically favorable alternative to the existing vaccine and diagnostics for CSFV in pigs.

Control of classical swine fever (CSF) in developing countries is achieved by immunization with attenuated vaccines, such as the lapinized C-strain vaccine that has been widely used in China. However, C-strain has relatively low growth rate in cell cultures, thus affecting productivity of the vaccine for the industry. In this study, eight amino acid residues were mutated on the C-strain backbone, resulting in a cell-adapted strain Cmut8. The mutant strain exhibited rapid growth with titer of about 100 fold higher than its parental C-strain. The mutation sites located at structural proteins E rns and E2 contributed more to cell adaptation than those located in non-structural proteins. Sera collected from pigs inoculated with Cmut8 and C-strain at the same dose showed similar antibody levels and neutralization titers. Pigs inoculated with different doses of Cmut8 (low, medium and high) and with C-strain offered full protection against challenge with a virulent strain, shown as absence of fever and other symptoms, marginal low levels of viral load, and no obvious gross pathological changes in major organs. Unvaccinated control pigs challenged with the virulent strain showed high fever from day 2 post-challenge and apparent clinical symptoms with two deaths. Viral load were markedly elevated in these control pigs after challenge. The pigs inoculated with high dose of Cmut8 did not show fever or other typical CSF symptoms, and no apparent pathological changes were observed in major organs. Besides, the Cmut8 strain did not induce typical fever response in rabbits. These results demonstrate that the cell-adapted Cmut8 strain remains non-pathogenic to the weaned pigs, provides full protection and could be a good candidate vaccine strain for improved yield at lower cost.

Two strains of classical swine fever virus (CSFV) (SRB/CSFV/1264/2005 and SRB/CSFV/6168/2006), producing serious clinical signs of disease during outbreaks in 2005 and 2006 in Serbia, were isolated on porcine kidney cells, and their complete genomes were determined by next-generation sequencing. This first complete genome characterization of Serbian CSFV strains provides new data about the evolution of CSFV in the Balkan region and enables further detailed phylogenetic studies of the various strains.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2 B/C/D/A ) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2 B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

In this study, 306 rectal swabs from diarrheal pigs of various ages (0-3 weeks, 3-6 weeks, and >6 weeks) were collected from 54 piggery units in different climatic zones in Haryana state, India. These samples were tested for the presence of porcine astrovirus (PAstV), porcine rotavirus group A (PRV-A), and classical swine fever virus (CSFV) by reverse transcription polymerase chain reaction (RT-PCR), and porcine circovirus 2 (PCV-2) by polymerase chain reaction (PCR). Out of the 306 samples tested, 153 (50%), 108 (35.3%), 32 (10.6%), and three (0.9%) tested positive for PAstV, PCV-2, PRV-A, and CSFV, respectively. A single infection was detected in 135 samples, while mixed infections were found in 77 samples: 70 with two viruses and seven samples with more than two. PAstV was detected most frequently (55.31%) in pigs aged 3-6 weeks. PCV-2 was more predominant in pigs aged 0-3 weeks (36.53%), whereas PRV-A was more common in pigs aged 3-6 weeks (11.3%). CSFV was observed in the age group of 0-3 weeks (1.92%). Phylogenetic analysis revealed the circulation of lineages 2 and 4 of PAstV in this region. Thus, it can be concluded that one or more than one virus is circulating in piggery units in Haryana, India., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The classical swine fever (CSF) is a highly contagious viral disease of pigs and wild boar. The CSF causes great economic losses for pork production and the occurrence of the disease is notifiable to the OIE. The objective of this work was to identify and characterize CSF virus isolates from Brazil. Seven viral isolates were obtained and the full-length E2 sequences were analyzed. Phylogenetic analysis revealed a different segregation pattern between Brazilian isolates and members of subgenotype 1.1, forming a separate group within genotype 1. Genetic distance analysis suggested the existence of two new subgenotypes, designated subgenotypes 1.5 and 1.6.

Classical swine fever (CSF) can run acute, chronic, and prenatal courses in both domestic pigs and wild boar. Although chronic infections are rare events, their epidemiological impact is very high due to the long-term shedding of virus. So far, little is known about the factors that influence disease course and outcome from either the host or virus's perspective. To elucidate the viral determinants, we analyzed the role of the viral populations for the development of chronic CSF virus (CSFV) infections. Three different animal trials that had led to both chronic and acute infections were chosen for a detailed analysis by deep sequencing. The three inocula represented sub-genogroups 2.1 and 2.3, and two viruses were wild-type CSFV, one derived from an infectious cDNA clone. These viruses and samples derived from acutely and chronically infected animals were subjected to next-generation sequencing. Subsequently, the derived full-length genomes were compared at both the consensus and the quasispecies level. At consensus level, no differences were observed between the parental viruses and the viruses obtained from chronically infected animals. Despite a considerable level of variability at the quasispecies level, no indications were found for any predictive pattern with regard to the chronicity of the CSFV infections. While there might be no direct marker for chronicity, moderate virulence of some CSFV strains in itself seems to be a crucial prerequisite for the establishment of long-term infections which does not need further genetic adaption. Thus, general host and virus factors need further investigation.

The E2 glycoprotein and 3' untranslated region (UTR) of classical swine fever virus (CSFV) are virulence determinants. To investigate the synergistic roles of E2 and 3'UTR for pathogenicity and genomic stability, a series of chimeric CSFVs were constructed by replacing the E2 gene and/or 3'UTR of virulent CSFV strain Shimen with the corresponding sequence of the lapinized 'Chinese' strain (C-strain) using a reverse genetic approach. The in vitro growth characterization and in vivo pathogenicity of the chimeric CSFVs were investigated. Our results demonstrated that the E2 glycoprotein mediates virus cell-to-cell spread and viral particle release and that the 3'UTR regulates viral RNA replication. The CSFV E2 and 3'UTR synergistically modulate infectious virus production, viral genomic stability in vitro, and attenuation in swine. This work contributes to our understanding of the structure and function of the CSFV genome and virus pathogenicity and will be useful for the development of a novel CSF vaccine.

NS5A is a multifunctional non-structural protein of classical swine fever virus (CSFV) that plays an important role in viral replication, but how it exerts its functions is unknown. Here, we report the cleavage of NS5A of the vaccine C-strain, resulting in two truncated forms (b and c). Further experiments using calpain- and caspase-family-specific inhibitors, followed by a caspase-6-specific shRNAs and inhibitor, showed that the cleavage of C-strain NS5A to produce truncated form c is mediated by caspase-6, mapping to 272 DTTD 275 , while the cleavage producing truncated form b is probably mediated by another unknown protease. shRNA-mediated downregulation of caspase-6 and blocking of enzyme activity in ST cells significantly impaired genome replication and virus production, indicating that NS5A cleavage is required for CSFV replication.

Current subgenotype 2.1 isolates of classical swine fever virus (CSFV) play a dominant role in CSF outbreaks in China, and a novel sub-subgenotype 2.1g of CSFV was recently identified, but the complete genome sequence of this new sub-subgenotype has not been reported. In this study, complete genome of 2.1g isolate GD19/2011 collected from Guangdong province of China in 2011 was sequenced. It was found to be 12,298 nucleotides (nt) in length, including a 375-nt 5'UTR, a 11,697-nt opening reading frame (ORF), and a 227-nt 3'UTR. GD19/2011 shared 91.0-93.7 % and 95.6-97.5 % nt and amino acid sequence identity, respectively, with other subgenotype 2.1 isolates. The topology of a phylogenetic tree constructed based on complete genome sequences of GD19/2011 and other CSFV isolates was identical to that obtained with full-length E2 gene sequences, but it was significantly different from those obtained with the 5'UTR and core sequences. Serial passages of GD9/2011 in PK-15 cells generated a highly cell-adapted virus stock with an infectious titer of 10(7.8) TCID50/ml at the 12(th) passage in which two amino acid substitutions, S476R and N2494S, were observed in comparison with the complete polyprotein sequence of the original isolate from kidney tissue, GD19/2011. This is the first report of the complete genome sequence of a 2.1g isolate, and the GD19/2011 isolate will be useful for further analysis of the evolution and virulence of CSFV isolates.

Recently, CP7_E2alf (SuvaxynCSF Marker), a live marker vaccine against classical swine fever virus, was licensed through the European Medicines Agency. For application of such a genetically engineered virus under field conditions, knowledge about its genetic stability is essential. Here, we report on stability studies that were conducted to assess and compare the mutation rate of CP7_E2alf in vitro and in vivo. Sequence analyses upon passaging confirmed the high stability of CP7_E2alf, and no recombination events were observed in the experimental setup. The data obtained in this study confirm the genetic stability of CP7_E2alf as an important safety component.

Classical swine fever virus (CSFV) is a noncytopathogenic virus, and the incorporation of an enhanced green fluorescent protein (EGFP) tag into the viral genome provides a means of direct monitoring of viral infection without immunostaining. It is well established that the 3' untranslated region (3'-UTR) of the CSFV plays an important role in viral RNA replication. Although CSFV carrying a reporter gene and chimeric CSFV have been generated and evaluated, a chimeric CSFV with a visible marker has not yet been reported. Here, we generated and evaluated a chimeric virus containing the EGFP tag and the 3'-UTR from vaccine strain HCLV (C-strain) in the genetic background of the highly virulent CSFV Shimen strain. The chimeric marker CSFV was fluorescent and had an approximately 100-fold lower viral titer, lower replication level of viral genome, and weaker fluorescence intensity than the recombinant CSFV with only the EGFP tag or the parental virus. Furthermore, the marker chimera was avirulent and displayed no viremia in inoculated pigs, which were completely protected from lethal CSFV challenge as early as 15 days post-inoculation. The chimeric marker virus was visible in vitro and attenuated in vitro and in vivo, which suggests that CSFV can be engineered to produce attenuated variants with a visible marker to facilitate in vitro studies of CSFV infection and replication and to develop of novel vaccines against CSF.

Classical swine fever (CSF) is endemic in Cuba and is one of the major health problems of the Cuban swine industry. The current efforts to control the disease in Cuba include vaccination with Porvac®, a subunit marker vaccine. Although the efficacy of Porvac against CSF virus (CSFV) subgenotype 1.4 has been extensively documented, little is known about the ability of the antibodies induced by this vaccine to neutralize other CSFV genotypes. In this study, sera collected from three pigs vaccinated with Porvac were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2, and 3. The findings from this study indicate that additional in vivo studies are warranted to confirm the ability of this vaccine to protect pigs against CSFV genotypes 2 and 3. [ABSTRACT FROM AUTHOR]

Classical swine fever (CSF), caused by the CSF virus (CSFV), is a highly contagious disease in pigs. In Korea, vaccination using a live-attenuated strain (LOM strain) has been used to control the disease. However, parenteral vaccination using a live-attenuated strain still faces a number of problems related to storage, cost, injection stress, and differentiation of CSFV infected and vaccinated pigs. Therefore, two kinds of new candidates for oral vaccination have been developed based on the translation of the E2 gene of the SW03 strain, which was isolated from an outbreak of CSF in 2002 in Korea, in transgenic rice calli (TRCs) from Oriza sativa L. cv. Dongjin to express a recombinant E2 protein (rE2-TRCs). The expression of the recombinant E2 protein (rE2) in rE2-TRCs was confirmed using Northern blot, SDS-PAGE, and Western blot analysis. Immune responses to the rE2-TRC in mice and pigs were investigated after oral administration. The administration of rE2-TRCs increased E2-specific antibodies titers and antibody-secreting cells when compared to animals receiving the vector alone (p < 0.05 and p < 0.01). In addition, mice receiving rE2-TRCs had a higher level of CD8+ lymphocytes and Th1 cytokine immune responses to purified rE2 (prE2) in vitro than the controls (p < 0.05 and p < 0.01). Pigs receiving rE2-TRCs also showed an increase in IL-8, CCL2, and the CD8+ subpopulation in response to stimulation with prE2. These results suggest that oral administration of rE2-TRCs can induce E2-specific immune responses.

Classical swine fever (CSF) is a major constraint to pig production worldwide, and in many developing countries, the epidemiological status is unknown. Here, for the first time, molecular identification and characterization of CSFV isolates from two recent outbreaks in Nepal are presented. Analysis of full-length E2-encoding sequences revealed that these isolates belonged to CSFV subgenotype 2.2 and had highest genetic similarity to isolates from India. Hence, for CSFV, Nepal and India should be regarded as one epidemiological unit. Both Nepalese isolates exhibited significant sequence differences, excluding a direct epidemiological connection and suggesting that CSFV is endemic in that country.

Classical swine fever (CSF) causes severe disease in pigs, characterized by hemorrhage, fever, and leucopenia. A primary target of the virus is endothelial cells, where a pro-inflammatory and pro-coagulant response occurs with downregulation of gap junctional communication; these changes establish a basis for haemostatic imbalance. The aim of this study was to gain an understanding of the effect of classical swine fever virus (CSFV) on endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability. Porcine aortic endothelial cells (PAECs) were infected with CSFV at different multiplicity of infection (M.O.I.) for 48 h. Downregulation of the transcription and translation levels of eNOS was detected by semi-quantitative RT-PCR, immunoconfocal microscopy, and western blotting. This was accompanied by a reduction in NO bioavailability and attenuation of angiogenesis. Without influence from the progeny virus titer, the decrease in eNOS protein was reversed by an ERK inhibitor (PD98059) and two PI3/Akt inhibitors (LY294002 and wortmannin). In addition, we found that the transcription factors AP1, Sp1, and GATA1/2 may be involved in the downregulation of eNOS promoter activity. In conclusion, infection of PAECs with CSFV attenuated the expression of eNOS and reduced NO bioavailability through activation of the ERK and PI3/Akt pathways.

Conventional reverse genetics for classical swine fever virus (CSFV) is based on the transfection of permissive cells with either in vitro or intracellularly synthesized RNA transcripts from a viral genomic cDNA clone. These strategies are complicated, inefficient and time-consuming. This study is aimed to develop an improved reverse genetics method for the direct, rapid and efficient recovery of CSFV from cloned cDNA. The cDNA clone pBRCISM was constructed, which harbors the full-length genomic sequence from the CSFV Shimen strain flanked by the cytomegalovirus promoter (an RNA polymerase II promoter), a chimeric intron, and hammerhead ribozyme sequences at the 5'-end and the hepatitis delta virus ribozyme and SV40 polyadenylation signal sequences at the 3'-end. Infectious progeny virus was rescued from PK-15 cells directly transfected with pBRCISM, and its morphology, one-step growth characteristics and pathogenicity were indistinguishable from the parent virus and virus rescued from classical reverse genetics. The reverse genetics based on RNA polymerase II yielded a 120-fold increase in the titer of nascent virus in 12-h less time than a reverse genetics method based on in vitro transcription. The full-length cDNA clone remained stable and infectious after 20 passages in bacterial cells, in contrast to the instability of the full-length clone without the intron after 9 passages. The improved reverse genetics method developed in the present study is efficient, stable, convenient and cost-effective and will be valuable for the rapid recovery of CSFV mutants.

Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever. [ABSTRACT FROM AUTHOR]

The sequence TAVSPTTLR is a conserved and linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus. In this study, TAVSPTTLR-directed antibodies, induced either by virions or by an epitope-focused immunogen, were characterized. The results revealed that despite the same epitope specificity, the antibodies induced by different immunogens varied significantly both in the neutralizing test and in binding inhibition assays. This suggests that the protective immunity induced by this epitope is due to more than simply the epitope specificity and that this epitope might need essential contributions from its flanking context to induce functional epitope-specific antibodies.

The Chinese hog cholera lapinized virus (HCLV), also called the "Chinese strain" or "C-strain" of classical swine fever virus (CSFV), was developed in China in the 1950s. There are uncertainties about the genetic heterogeneity and origin of this vaccine virus. The objectives of this study were to investigate the genetic heterogeneity of the C-strain, for which nucleotide sequences have been submitted to GenBank from different laboratories, and to determine whether there is any evidence to support the hypothesis that the C-strain originated from the Shimen strain. Analysis of 150 nearly complete E2 gene sequences revealed that the C-strain clade includes several HCLV vaccine strains, cell-culture-adapted Riems strains, and viruses isolated from diseased pigs. The whole-genome phylogeny indicated that the ancestor of the C-strain was only distantly related to the Shimen strain. Therefore, there was no phylogenetic evidence to support the Shimen-origin hypothesis.

The pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.

The immune response to CSFV and the strategies of this virus to evade and suppress the pigs' immune system are still poorly understood. Therefore, we investigated the transcriptional response in the tonsils, median retropharyngeal lymph node (MRLN), and spleen of pigs infected with CSFV strains of similar origin with high, moderate, and low virulence. Using a porcine spleen/intestinal cDNA microarray, expression levels in RNA pools prepared from infected tissue at 3 dpi (three pigs per virus strain) were compared to levels in pools prepared from uninfected homologue tissues (nine pigs). A total of 44 genes were found to be differentially expressed. The genes were functionally clustered in six groups: innate and adaptive immune response, interferon-regulated genes, apoptosis, ubiquitin-mediated proteolysis, oxidative phosphorylation and cytoskeleton. Significant up-regulation of three IFN-gamma-induced genes in the MRLNs of pigs infected with the low virulence strain was the only clear qualitative difference in gene expression observed between the strains with high, moderate and low virulence. Real-time PCR analysis of four response genes in all individual samples largely confirmed the microarray data at 3 dpi. Additional PCR analysis of infected tonsil, MRLN, and spleen samples collected at 7 and 10 dpi indicated that the strong induction of expression of the antiviral response genes chemokine CXCL10 and 2'-5' oligoadenylate synthetase 2, and of the TNF-related apoptosis-inducing ligand (TRAIL) gene at 3 dpi, decreased to lower levels at 7 and 10 dpi. For the highly and moderately virulent strains, this decrease in antiviral and apoptotic gene expression coincided with higher levels of virus in these immune tissues.

To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E(+) strain), WB82 and WB82/E(+) strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E(+) strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E(+) strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV.

The nonstructural protein 3 (NS3) of members of the family Flaviviridae possesses multiple enzyme activities that are likely to be essential for viral replication. Here, we cloned and expressed full-length CSFV NS3 protein (NS3FL) and its N-terminal truncated version (ntNS3) in E. coli. NTPase activities of the purified NS3FL and ntNS3 proteins and their reaction conditions were investigated. The results showed that CSFV NS3FL and ntNS3 proteins contained a specific polynucleotide-stimulated NTPase acitivity. Characterization of ntNS3 NTPase activity showed that optimal reaction conditions with respect to pH, MgCl2 and monovalent cations were similar to those of bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV). Site-directed mutagenesis analysis demonstrated that the GxGK(232)T to GxGAT mutation in the conserved motif I abolished the NTPase activity of ntNS3, whereas substitution of TATPA(354) for TATPV in the motif III had no effect on the enzyme activity. Moreover, the kinetic properties (K(m) and k(cat)) of CSFV NS3 were more similar to those of BVDV. Our results provide insight into the structure-function relationship of CSFV NS3 and facilitate our understanding of its role in the replication cycle of CSFV.

African swine fever virus (ASF) has circulated in Vietnam since 2018, causing significant losses to the pig industry. Quick, accurate diagnosis of African swine fever virus (ASFV) infection is crucial for controlling the disease. The detection of the virus in piglets with congenital tremors is described in this paper. ASFV was detected in brain tissues by polymerase chain reaction (PCR) and immunohistochemistry. Classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and pseudorabies virus were not detected by PCR, suggesting that the ASFV was the cause of these neurological signs., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Two new classical swine fever virus (CSFV) isolates obtained from naturally infected swine were found to exhibit a cytopathogenic (cp) phenotype. According to their reactivity with monoclonal antibodies (mabs) the isolates cpBW1 and cpMVP1 were classified as antigenic types "Lothringen'92" and "Flandern'90", respectively. In Northern blot analyses and PCR assays CSFV RNA of subgenomic length was detected in infected cells indicating the presence of defective interfering particles. Nucleotide sequencing of cDNA fragments obtained by reverse transcription (RT)-PCR allowed to localize in the genomes of the cpMVP1 and cpBW1 DIs deletions of 4,764 and 4,746, respectively. From the latter isolate a noncytopathogenic (noncp) biotype was obtained by cloning in cell culture. CSFV cpBW1 and a noncp isolate were used separately for infection of pigs. Viremia occurred much quicker after infection with cp virus and clinical signs were more prominent compared to animals infected with noncp virus.

RNA-dependent RNA polymerase (RdRp) is the replicase of positive-strand RNA viruses. Expression and characterization of the replicase are the first steps in the elucidation of the virus replication mechanism. We expressed nonstructural protein 5B (NS5B) of classical swine fever virus (CSFV) as a fusion protein with green fluorescent protein (GFP) in porcine kidney cells (PK-15 cells), natural host cells of CSFV. The expressed CSFV NS5B-GFP fusion protein possessed RdRp activity. By fluorescence microscope it was observed that the density of the fusion protein near cytoplasmic membranes was higher than that in other parts of cells. This was in contrast to the distribution of the GFP alone which was uniformly distributed throughout the cytoplasm. The GFP is a signal for the location of NS5B in a host cell that allows in vitro and in vivo investigation of the distribution of plus-strand RNA virus RdRp.

Phylogenetic analyses of large numbers of classical swine fever strains have revealed a high degree of sequence conservation in the genomic regions examined, suggesting either a recent common ancestor or a low evolution rate. This low variability is in contrast to findings with other RNA viruses. To investigate the consequence of this apparent genetic stability on phylogenetic examinations, the Belgian field isolate Wingene'93 was passaged in pigs as well as in cell culture by various methods. Sequence analyses of viruses collected after various passages in three target regions proposed for phylogenetic studies (5' NTR, E2, and NS5B) revealed a complete sequence conservation. Only when the amount of passaged virus was lowered, mimicking a genetic bottleneck, a single point mutation was observed in the E2 gene. Additionally, only four nucleotide substitutions were observed when the genome of a virus obtained after 96 cell passages in persistently infected cells was compared with its parental virus, the recombinant virus derived from an infectious cDNA clone of CSFV strain Alfort/187. This low mutation frequency observed both in vitro and in vivo demonstrates that classical swine fever virus is genetically stable. Hence, even minor mutations can be considered significant in molecular epidemiological studies.

We describe an alternative reverse genetics system for generating classical swine fever virus (CSFV) based on swine RNA polymerase I promoter (pSPI)-mediated vRNA transcription. The recombinant plasmid pSPT/SM harboring a full-length CSFV Shimen strain cDNA, flanked by a swine RNA polymerase I (pol I) promoter sequence at the 5′ end and a murine pol I terminator sequence at the 3′ end, was constructed. When the plasmid pSPT/SM was introduced into PK-15 cells by transfection, an infectious CSFV with termini identical to those of the parental virus was generated directly. CSFV rescued from this reverse genetics system exhibited similar growth kinetics and plaque formation compared with the parental CSFV. When the novel reverse genetics system was used to generate the CSFV vaccine C-strain, infectious virus was detected in the supernatant of PK-15 cells transfected with the recombinant plasmid pSPT/C. This novel reverse genetics system is a simple and efficient tool for the investigation of the structure and function of the viral genome, for molecular pathogenicity studies, and for the development of genetically engineered vaccines for CSFV. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction was utilized to determine the sequence of a 280 base pair fragment from cDNAs of the 5' noncoding region of 29 isolates of classical swine fever virus. Phylogenetic analysis of the sequences revealed low level genomic variation that correlated with the geographic origins of the isolates.

Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.

Classical swine fever is a contagious disease of pigs characterized by fatal hemorrhagic fever. Classical swine fever virus (CSFV) induces the expression of pro-inflammatory and pro-coagulant factors of vascular endothelial cells and establishes a long-term infection. This study aimed to understand the effect of CSFV on endothelial connexin 43 (Cx43) expression and gap junctional intercellular coupling (GJIC). Porcine aortic endothelial cells were infected with CSFV at different multiplicity of infection for 48 h. Semi-quantitative RT-PCR, immunoconfocal microscopy, and Western blotting showed that the transcription and translation of Cx43 were reduced, and this was associated with an attenuation of GJIC. This decrease occurred in a time-dependent manner. An ERK inhibitor (PD98059), a JNK inhibitor (SP600125), and proteasome/lysosome inhibitors all significantly reversed the reduction in Cx43 protein levels without any influence on the titer of progeny virus. In addition, CSFV activated ERK and JNK in a time-dependent manner and down-regulated Cx43 promoter activity, mainly through decreased AP2 binding. This effect was primarily caused by the replication of CSFV rather than a consequence of cytokines being induced by CSFV infection of endothelial cells. [ABSTRACT FROM AUTHOR]

Infection of pigs with CSFV can lead to either acute disease, resulting in death or recovery, or chronic disease. The mechanisms by which CSFV manipulates the pig's first line of defence to establish a chronic infection are poorly understood. Therefore, pigs were infected with moderately virulent CSFV, and whole blood was collected on a regular basis during a period of 18 days. Using whole-genome microarrays, time-dependent changes in gene expression were recorded in blood cells of chronically diseased pigs and pigs that recovered. Bioinformatics analysis of regulated genes indicated that different immunological pathways were regulated in chronically diseased pigs compared to recovered pigs. In recovered pigs, antiviral defence mechanisms were rapidly activated, whereas in chronically diseased pigs, several genes with the potential to inhibit NF-κB- and IRF3/7-mediated transcription of type I interferons were up-regulated. Compared to recovered pigs, chronically diseased pigs failed to activate NK or cytotoxic T-cell pathways, and they showed decreased gene activity in antigen-presenting monocytes/macrophages. Remarkably, in chronically diseased pigs, genes related to the human autoimmune disease systemic lupus erythematosus (SLE) were up-regulated during the whole period of 18 days. CSFV pathology in kidney and skin resembles that of SLE. Furthermore, enzymes involved in the degradation of 1,25-dihydroxyvitamin D3 and of tryptophan to kynurenines were expressed at different levels in chronically diseased and recovered pigs. Both of these chemical processes may affect the functions of T helper/regulatory cells that are crucial for tempering the inflammatory response after a viral infection.

Classical swine fever virus (CSFV) infection results in highly significant economic losses. Previous studies have suggested that CSFV can be recognized by RIG-I-like receptors (RLRs) to trigger innate defenses. However, the role of mitochondrial antiviral signaling protein (MAVS), the adaptor of RLRs, is still unknown during CSFV infection. Here, we showed that CSFV infection increased MAVS expression in porcine alveolar macrophages (PAMs). Additionally, intracellular reactive oxygen species (ROS) were involved in MAVS expression in CSFV-infected PAMs. Moreover, MAVS enhanced the induction of antiviral and pro-inflammatory cytokines and apoptosis, and inhibited CSFV replication. However, CSFV still establishes a persistent infection in the host. Thus, how CSFV antagonises MAVS-mediated host cell defense was investigated. Importantly, CSFV N pro inhibited MAVS-induced interferons and pro-inflammatory cytokines and apoptosis. Furthermore, IRF3-knockdown also suppressed MAVS-induced host cell defense. Taken together, these results demonstrate that intracellular ROS is involved in CSFV-induced MAVS expression and MAVS induces antiviral cytokines and apoptosis to inhibit CSFV replication while CSFV N pro inhibits MAVS-mediated host cell defenses possibly through degradation of IRF3. These data offer novel insights into the immunomodulatory effects of CSFV infection on the host innate response.

Archival swine DNA samples from Indonesia and Mongolia, some of which were previously shown to be positive for African swine fever virus, were screened for the presence of porcine circovirus 2 (PCV-2) and porcine circovirus 3 (PCV-3) by PCR. Samples from both countries were positive for PCV-2 (three from Mongolia and two from Indonesia), while none were positive for PCV-3. The PCV-2 amplicons were sequenced, and phylogenetic analysis revealed that the PCV-2 strains belonged to four different genotypes: PCV-2a (Mongolia), PCV-2b (Mongolia and Indonesia), PCV-2d (Indonesia), and PCV-2g (Mongolia). This is the first report of ASFV/PCV-2 coinfection in pigs and the first report of the presence of PCV-2 in Mongolia. [ABSTRACT FROM AUTHOR]

Infectious bronchitis (IB) is a highly contagious respiratory disease caused by a gammacoronavirus that has been circulating for many years in chickens in Bangladesh, resulting in significant economic losses. The aim of this study was to detect and characterize infectious bronchitis virus (IBV) from clinical outbreaks and surveillance samples. Real-time RT-PCR was used to detect IBV in pooled lung and tracheal tissue samples (n = 78), oropharyngeal swabs (n = 19), and pooled fecal samples (n = 13) from live-bird markets. Both respiratory and nephropathogenic forms of IB were suspected at necropsy (n = 7) from clinical outbreaks. Sequencing of hypervariable regions (HVR1-2 and HVR3) of the region of the spike gene (S) encoding the S1 subunit of five isolates revealed circulation of the Mass-like, QX-like, and 4/91-like genotypes of IBV in Bangladesh. Each genotype was extremely variable, as shown by separate clustering of the viruses in a phylogenetic tree and high nucleotide (nt) sequence divergence (38.8–41.2% and 25.7–37.4% in the HVR1-2 and HVR3 sequence, respectively). The unique mutation G65E was observed in each Mass-like isolate, and Y328S was observed in each 4/91-like Bangladeshi isolate. Three neutralizing epitope sites were predicted within the HVRs that differed significantly among the three genotypes. In addition, one Bangladeshi isolate carried fixed mutations at 294F and 306Y, like other pathogenic QX-like IBVs, which could affect epitopes involved in neutralization, facilitating virus circulation among vaccinated flocks. Therefore, continuous screening and genotype characterization will be necessary to track the epidemiology of IBV and control IB infection in Bangladesh. [ABSTRACT FROM AUTHOR]

In 2007, African swine fever virus (ASFV) was introduced into the Transcaucasian countries and Russia. Since then, it has spread alarmingly and reached the European Union. ASFV strains are highly virulent and lead to almost 100% mortality under experimental conditions. However, the possibility of dose-dependent disease courses has been discussed. For this reason, a study was undertaken to assess the risk of chronic disease and the establishment of carriers upon low-dose oronasal infection of domestic pigs and European wild boar. It was demonstrated that very low doses of ASFV are sufficient to infect especially weak or runted animals by the oronasal route. Some of these animals did not show clinical signs indicative of ASF, and they developed almost no fever. However, no changes were observed in individual animal regarding the onset, course and outcome of infection as assessed by diagnostic tests. After amplification of ASFV by these animals, pen- and stablemates became infected and developed acute lethal disease with similar characteristics in all animals. Thus, we found no indication of prolonged or chronic individual courses upon low-dose infection in either species. The scattered onset of clinical signs and pathogen detection within and among groups confirms moderate contagiosity that is strongly linked with blood contact. In conclusion, the prolonged course at the "herd level" together with the exceptionally low dose that proved to be sufficient to infect a runted wild boar could be important for disease dynamics in wild-boar populations and in backyard settings.

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 invitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity invitro and CSFV production. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field. [ABSTRACT FROM AUTHOR]

Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 "Margarita" and the recent field isolate CSF1058 "Pinar del Rio". Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.

Summary. The expression of tumor necrosis factor (TNF)-a and apoptosis was studied in lymph nodes from pigs infected with Classical swine fever virus (CSFV). Pigs were inoculated with CSFV and euthanized at 3, 5, 7, and 10 days postinoculation. An increase in TNF-a expression was detected in CSFV-infected lymph nodes using a reverse transcription-polymerase chain reaction, and TNF-a protein was detected in lymph nodes by immunohistochemistry. The majority of TNF-a-positive cells also expressed the SWC3a antigen, a specific marker for porcine leukocytes. By combined use of in situ hybridization and immunohistochemistry, CSFV infection was detected in lymph nodes macrophage. Lymphocytes death occurred by apoptosis that was characterized by condensed shrunken cells and the formation of apoptotic bodies, some of them contained pyknotic nuclear remnants. Apoptosis was detected in situ by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) reaction. A double-labeling experiment using immunohistochemistry and TUNEL reaction for the detection of CSFV and apoptosis demonstrated that the majority of labeled cells were positive for CSFV or apoptosis. This suggestes that CSFV can induce apoptosis directly and indirectly. Apoptotic cells induced by viral infection were more abundant than CSFV-infected cells in all lymph nodes tested. A double-labeling experiment using immunohistochemistry and TUNEL reaction for the detection of TNF-a and apoptosis demonstrated that labeled cells were positive for either TNF-a or apoptosis, and both. The present study addressed two important issues regarding CSFV-induced apoptosis: (i) viral infection and apoptosis colocalize at the cell level; and (ii) one or more factors (e.g., TNF-a) released from macrophages may induce apoptosis in uninfected bystander cells. [ABSTRACT FROM AUTHOR]