A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post-vaccination monitoring.

BACKGROUND: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post-vaccination monitoring. RESULTS: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). CONCLUSIONS: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

Plant-made E2 glycoprotein single-dose vaccine protects pigs against classical swine fever.

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs.

Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which has received much attention due to their potential applications in lignin degradation. A new DyP from Thermomonospora curvata (TcDyP) was identified and characterized. Unlike other A-type enzymes, TcDyP is highly active toward a wide range of substrates including model lignin compounds, in which the catalytic efficiency with ABTS (kcat(app)/Km(app) = (1.7 × 10(7)) m(-1) s(-1)) is close to that of fungal DyPs. Stopped-flow spectroscopy was employed to elucidate the transient intermediates as well as the catalytic cycle involving wild-type (wt) and mutant TcDyPs. Although residues Asp(220) and Arg(327) are found necessary for compound I formation, His(312) is proposed to play roles in compound II reduction. Transient kinetics of hydroquinone (HQ) oxidation by wt-TcDyP showed that conversion of the compound II to resting state is a rate-limiting step, which will explain the contradictory observation made with the aspartate mutants of A-type DyPs. Moreover, replacement of His(312) and Arg(327) has significant effects on the oligomerization and redox potential (E°') of the enzyme. Both mutants were found to promote the formation of dimeric state and to shift E°' to a more negative potential. Not only do these results reveal the unique catalytic property of the A-type DyPs, but they will also facilitate the development of these enzymes as lignin degraders.

Complete genome sequence of a novel sub-subgenotype 2.1g isolate of classical swine fever virus from China.

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.

In vitro adaptation and genome analysis of a sub-subgenotype 2.1c isolate of classical swine fever virus.

Classical swine fever (CSF) still causes substantial economic losses in the pig industry in China. This study reports the isolation and characterization of a field CSF virus named GD53/2011 from pig kidney tissue collected during a CSF outbreak in Guangdong province, China. Phylogenetic analysis based on the full-length E2 gene sequence revealed that this isolate belongs to CSFV sub-subgenotype 2.1c. To further understand the replication characteristics, GD53/2011 was subsequently adapted in PK-15 cells, and its full-length genome was sequenced. After adaptation in PK-15 cells, the titer of GD53/2011 was significantly increased from 10(3.39) TCID50/ml at passage 6 (F6) to 10(8.50) TCID50/ml at passage 46 (F46) with the peak titer obtained at 48 h post-inoculation. Sequence comparison revealed that the E(rns) gene at passages 6, 15, and 25 of GD53/2011 was identical to that in the original tissue, but one amino acid substitution (S476R) was detected at passages 35 and 46. Furthermore, E2 gene sequences at passages 6, 15, 25, 35, and 46 was found identical to that in the original tissue, indicating that the E2 gene was stable during CSF virus adaptation in PK-15 cells. Full-length protein sequence comparison of GD53/2011 with other 2.1 sub-subgenotype isolates showed that Core and NS5A, rather than E2, are more genetically variable. Taken together, a field CSFV strain GD53/2011 was isolated, fully sequenced, and adapted to high growth titer in PK-15 cells, which might be suitable for future studies on CSFV infection, replication, and vaccine development.

Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge.

BACKGROUND: Classical swine fever (CSF) or hog cholera is a highly contagious swine viral disease. CSF endemic countries have to use routine vaccination with modified live virus (MLV) vaccines to prevent and control CSF. However, it is impossible to serologically differentiate MLV vaccinated pigs from those infected with CSF virus (CSFV). The aim of this study is to develop a one-dose E2-subunit vaccine that can provide protection against CSFV challenge. We hypothesize that a vaccine consisting of a suitable adjuvant and recombinant E2 with natural conformation may induce a similar level of protection as the MLV vaccine. RESULTS: Our experimental vaccine KNB-E2 was formulated with the recombinant E2 protein (Genotype 1.1) expressed by insect cells and an oil-in-water emulsion based adjuvant. 10 pigs (3 weeks old, 5 pigs/group) were immunized intramuscularly with one dose or two doses (3 weeks apart) KNB-E2, and 10 more control pigs were administered normal saline solution only. Two weeks after the second vaccination, all KNB-E2 vaccinated pigs and 5 control pigs were challenged with 5 × 10(5) TCID50 CSFV Honduras/1997 (Genotype 1.3, 1 ml intramuscular, 1 ml intranasal). It was found that while control pigs infected with CSFV stopped growing and developed high fever (>40 °C), high level CSFV load in blood and nasal fluid, and severe leukopenia 3-14 days post challenge, all KNB-E2 vaccinated pigs continued to grow as control pigs without CSFV exposure, did not show any fever, had low or undetectable level of CSFV in blood and nasal fluid. At the time of CSFV challenge, only pigs immunized with KNB-E2 developed high levels of E2-specific antibodies and anti-CSFV neutralizing antibodies. CONCLUSIONS: Our studies provide direct evidence that pigs immunized with one dose KNB-E2 can be protected clinically from CSFV challenge. This protection is likely mediated by high levels of E2-specific and anti-CSFV neutralizing antibodies.

Iron status of channel catfish Ictalurus punctatus affected by channel catfish anemia and response to parenteral iron.

Originally reported in 1983, channel catfish anemia (CCA), also 'white lip' or 'no blood,' is a major idiopathic disease affecting commercial production in the Mississippi Delta region of the USA. Affected individuals are characterized by lethargy, anorexia, extreme pallor, and packed cell volumes often below 5%, but a definitive cause for CCA remains elusive. Records from the National Warmwater Aquaculture Center (NWAC) reveal that, on average, CCA accounted for 4.7% of case submissions from 1994 to 2012. Known infectious agents, parasites, and perturbations in commonly measured water quality variables have been largely excluded, and research has focused on potential feed-related etiologies, particularly folic acid deficiency. No natural or anthropogenic contaminants have been found in feeds, and no associations have been made to any particular feed brand or formulation, or to the age or condition of the feed itself. Contrary to reports indicating a short clinical course, NWAC records indicate an insidious condition where certain ponds have contained fish diagnosed with CCA for up to 4 consecutive years and individual outbreaks have persisted for at least 5 mo. Investigation into the iron status of CCA-affected fish revealed values consistent with iron deficiency anemia, including low-packed cell volume (mean ± SE, 5.6 ± 1.0 vs. 24.8 ± 2.4%), serum iron (35.2 ± 3.5 vs. 104.4 ± 18.5 µg dl-1), liver iron (12.2 ± 2.6 vs. 23.3 ± 4.6 µg g-1), and percent transferrin saturation (14.5 ± 2.7 vs. 26.9 ± 3.1%) in anemic and healthy controls, respectively. Administration of parenteral iron produced complete recovery and returned iron indices to within the ranges of normal controls. Despite these findings, factors predisposing a state of hypoferremia remain unknown.

Structural and functional analysis of the pro-domain of human cathelicidin, LL-37.

Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.

Complete genome sequence of an overattenuated highly pathogenic porcine reproductive and respiratory syndrome virus.

JXA1-P170 is an overattenuated highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that has been passaged in vitro 170 times. Vaccination with JXA1-P170 cannot protect pigs against JXA1 challenge. Compared with the parental virus JXA1, JXA1-P170 contains 1 nucleotide (nt) deletion and 113 nt mutations leading to 59 amino acid substitutions. Here we announce the first complete genome sequence of the overattenuated HP-PRRSV.

Complete genome sequence of two novel Chinese virulent porcine reproductive and respiratory syndrome virus variants.

A highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), characterized by a discontinuous 30-amino-acid deletion in its Nsp2-coding region, has been emerging in China since 2006. Here, we report the complete genomic sequence of two novel Chinese virulent PRRSV variants with additional NSP2-gene deletions, which will help us understand the molecular and evolutionary characteristics of PRRSV in Asia.

An easy method to generate recombinant pseudorabies virus expressing the capsid protein of Porcine circovirus type 2d.

Introduction: Homologous recombination is an effective way to generate recombinant viruses for vaccine research such as pseudorabies virus (PRV) and adenovirus. Its efficiency can be affected by the integrity of viral genome and the linearization sites. Methods: In the study, we described a simple approach to isolate the viral DNA with high genomic integrity for large DNA viruses and a time-saving method to generate recombinant PRVs. Several cleavage sites in the PRV genome were investigated by using the EGFP as a reporter gene for identification of PRV recombination. Results: Our study showed that cleavage sites of XbaI and AvrII are ideal for PRV recombination which showed higher recombinant efficiency than others. The recombinant PRV-EGFP virus can be easily plaque purified in 1-2 weeks after the transfection. By using PRV-EGFP virus as the template and XbaI as the linearizing enzyme, we successfully constructed the PRV-PCV2d_ORF2 recombiant virus within a short period by simply transfecting the linearized PRV-EGFP genome and PCV2d_ORF2 donor vector into BHK-21 cells. This easy and efficient method for producing recombinant PRV might be adapted in other DNA viruses for the generation of recombinant viruses.

Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.

A Cell-Adapted Live-Attenuated Vaccine Candidate Protects Pigs against the Homologous Strain VNUA-ASFV-05L1, a Representative Strain of the Contemporary Pandemic African Swine Fever Virus.

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. Currently, there is no ASF vaccine commercially available. All infected animals must be isolated and culled immediately upon the confirmation of the presence of the virus. Studies leading to the rational development of protective ASF vaccines are urgently needed. Here, we generated a safe and efficacious live-attenuated vaccine (LAV) VNUA-ASFV-LAVL2 by serially passaging a field isolate (VNUA-ASFV-05L1, genotype II) in porcine alveolar macrophages (PAMs, 65 passages) and an immortalized porcine alveolar macrophage cell line (3D4/21, 55 passages). VNUA-ASFV-LAVL2 can efficiently replicate in both PAMs and 3D4/21 cells. It provides 100% protection, even with the low dose of 102 HAD50, to the vaccinated pigs against the challenge of contemporary pandemic ASFV field isolate. Pigs vaccinated with this LAV in a dose range of 102 to 105 HAD50 remained clinically healthy during both the 28-day observation period of immunization and the 28-day observation period of challenge. VNUA-ASFV-LAVL2 was eliminated from blood by 28 days post-inoculation (DPI), and from feces or oral fluids by 17 DPI. Although the vaccine strain in serum remained a safe and attenuated phenotype after five passages in swine, a reversion-to-virulence study using blood or tissue homogenates at peak viremia will be conducted in the future. ASFV-specific IgG antibodies and significant cellular immunity were detected in vaccinated pigs before the ASFV challenge. These results indicate that the VNUA-ASFV-LAVL2 strain is a safe and efficacious LAV against the genotype II ASFV strain responsible for current ASF outbreaks in Asia.

2023 International African Swine Fever Workshop: Critical Issues That Need to Be Addressed for ASF Control.

The 2023 International African Swine Fever Workshop (IASFW) took place in Beijing, China, on 18-20 September 2023. It was jointly organized by the U.S.-China Center for Animal Health (USCCAH) at Kansas State University (KSU) and the Chinese Veterinary Drug Association (CVDA) and sponsored by the United States Department of Agriculture Foreign Agricultural Service (USDA-FAS), Harbin Veterinary Research Institute, and Zoetis Inc. The objective of this workshop was to provide a platform for ASF researchers around the world to unite and share their knowledge and expertise on ASF control and prevention. A total of 24 outstanding ASF research scientists and experts from 10 countries attended this meeting. The workshop included presentations on current ASF research, opportunities for scientific collaboration, and discussions of lessons and experiences learned from China/Asia, Africa, and Europe. This article summarizes the meeting highlights and presents some critical issues that need to be addressed for ASF control and prevention in the future.

IL-1ß promotes stemness and invasiveness of colon cancer cells through Zeb1 activation.

BACKGROUND: IL-1ß is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1ß may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1ß in colon cancer stem cell (CSC) development. METHODS: Using self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1ß on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116. RESULTS: We found that IL-1ß can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1ß-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1ß-induced spheres, indicating that there might be a close association between EMT and IL-1ß-induced CSC self-renewal. Indeed, IL-1ß treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1ß-induced EMT and stem cell formation. CONCLUSION: Our findings indicate that IL-1ß may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1ß and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Pro-inflammatory cytokine interleukin-1ß promotes the development of intestinal stem cells.

OBJECTIVE: We investigated the effect of IL-1ß on the development of intestinal epithelial stem cells. MATERIALS AND METHODS: Normal intestinal epithelial cell line IEC-18 cells were cultured in the presence or absence of 200 pM of IL-1ß in serum-free medium (SFM) for various time periods. The effects of IL-1ß on intestinal stem cell self-renewal and IEC-18 cell proliferation were evaluated by a colony formation assay, MTT assay, and a focus formation assay. The expression of stemness genes including Bmi-1, Lgr-5, c-myc, Nanog, and ß-catenin in IEC-18 cells were measured by quantitative PCR and western blot analysis. RESULTS: IEC-18 cells grew as a monolayer in SFM in the absence of IL-1ß. Cellular spheres were formed when IEC-18 cells were grown in SFM in the presence of IL-1ß. IL-1ß induced the development of large colonies in the soft-agar as well as the formation of foci when IEC-18 cells were cultured in type-I collagen-coated plates. The expression of Bmi-1, Lgr-5, c-myc, Nanog, and ß-catenin were significantly increased in IEC-18 cells treated with IL-1ß. CONCLUSION: Our studies provide direct evidence the IL-1ß may play an important role in the self-renewal of intestinal epithelial stem cells and the development of cancer stem cells.

Emulsion Adjuvants for Use in Veterinary Vaccines.

The use of emulsion as adjuvants is widely used in veterinary vaccines. Emulsion adjuvants are inexpensive, stable, and relatively easy to prepare into vaccine formulations. Here we describe the preparation of oil-in-water emulsion adjuvant that has been shown to enhance immune responses and protect against diseases in pigs. This emulsion adjuvant and its variations could potentially be used alone or in combination with other adjuvants in veterinary vaccine formulations.

A Novel Competitive ELISA for Specifically Measuring and Differentiating Immune Responses to Classical Swine Fever C-Strain Vaccine in Pigs.

Classical swine fever can be controlled effectively by vaccination with C-strain vaccine. In this study, we developed a novel competitive enzyme-linked immunosorbent assay (cELISA) based on a C-strain Erns specific monoclonal antibody (mAb 1504), aiming to serologically measure immune responses to C-strain vaccine in pigs, and finally to make the C-strain become a DIVA-compatible vaccine. The cELISA system was established based on the strategy that mAb 1504 will compete with the C-strain induced antibodies in the pig serum to bind the C-strain Erns protein. The cELISA was optimized and was further evaluated by testing different categories of pig sera. It can efficiently differentiate C-strain immunized from wild-type CSFV-infected pigs and lacks cross-reaction with other common swine viruses and viruses in genus Pestivirus such as Bovine viral diarrhea virus (BVDV). The C-strain antibody can be tested in pigs 7-14 days post vaccination with this cELISA. The sensitivity and specificity of the established cELISA were 100% (95% confidence interval: 95.60 to 100%) and 100% (95% confidence interval: 98.30 to 100%), respectively. This novel cELISA is a reliable tool for specifically measuring and differentiating immune responses to C-strain vaccine in pigs. By combining with the wild-type CSFV-specific infection tests, it can make the C-strain have DIVA capability.

Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus.

Classical swine fever virus (CSFV) is a major animal pathogen threatening the global pork industry. To date, numerous anti-CSFV monoclonal antibodies (mAbs) and their recognizing epitopes have been reported. However, few mAbs were systematically characterized for the capacity to differentiate field CSFV isolates from CSF vaccine strains, and the molecular basis associated with antigenic differences between vaccines and field isolates is still largely unknown. In the present study, recombinant CSFV structural glycoproteins E2 of both virulent and vaccine strains and Erns of vaccine strain were expressed using eukaryotic cells and murine mAbs generated against E2 and Erns. After serial screening and cloning of the hybridomas, the viral spectra of mAbs were respectively determined by indirect fluorescent antibody assay (IFA) using 108 CSFVs, followed by Western blot analysis using expressed glycoproteins of all CSFV sub-genotypes including vaccine strains. The antigenic structures recognized by these mAbs were characterized by epitope mapping using truncated, chimeric, and site-directed mutated E2 and Erns proteins. We have identified two vaccine-specific, one field isolate-specific, and two universal CSFV-specific mAbs and five novel conformational epitopes with critical amino acid (aa) motifs that are associated with these five mAbs: 213EPD215, 271RXGP274, and 37LXLNDG42 on E2 and 38CKGVP42, W81, and D100/V107 on Erns. Particularly, E213 of E2 is field isolate-specific, while N40 of E2 and D100/V107 of Erns are vaccine strain-specific. Results from our study further indicate that N40D of E2 mutation in field strains was likely produced under positive selection associated with long-term mass vaccination, leading to CSFV evasion of host immune response. Taking together, this study provides new insights into the antigenic structure of CSFV E2 and Erns and the differentiating mAbs will contribute to the development of a diagnostic strategy to differentiate C-strain vaccination from natural infection (DIVA) of CSFV in terms of elimination of CSF in China.

Genome Sequence of a Virulent African Swine Fever Virus Isolated in 2020 from a Domestic Pig in Northern Vietnam.

This study reports the genome sequence of an isolated African swine fever (ASF) virus (VNUA-ASFV-05L1/HaNam) obtained at the fourth passage on pulmonary alveolar macrophages. The virus was isolated during a typical acute ASF outbreak in pigs in a northern province of Vietnam in 2020.