Isolation, characterization, and circulation sphere of a filovirus in fruit bats.

Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.

Outbreak of piglet diarrhea associated with a new reassortant porcine rotavirus B.

Rotavirus B (RVB) is a causative agent leading to acute viral gastroenteritis diarrhea in both children and young animals, and has been commonly detected in piglets. In order to determine the causative agent of diarrheal outbreak occurring in December 2022 in piglets from a pig herd in Luoyang, Henan province of China, four common viral pathogens causing piglet diarrhea-three coronaviruses and rotavirus A (RVA) were first tested and found negative, therefore metagenomic sequencing was performed to explore other potential pathogens in the diarrheal samples. Unexpectedly, the most abundant viral reads mapped to RVB, and were de novo assembled to complete 11 viral gene segments. Sequence comparisons revealed that 5 gene segments encoding VP1, VP2, VP3, NSP3 and NSP4 of RVB strain designated as HNLY-2022 are most closely related to RVB strains derived from herbivores with low nucleotide similarities of 65.7-75.3%, and the remaining segments were relatively close to porcine RVB strains with the VP4 gene segment showing very low nucleotide identity (65.0%) with reference strains, indicating HNLY-2022 is a new reassortant RVB strain. Based on the previously proposed genotype classification criterion, the genotype constellation of RVB strain HNLY-2022 is G6-P[6]-I4-R6-C6-M6-A7-N5-T7-E5-H4 with more than half of the genotypes (P[6], R6, C6, M6, T7 and E5) newly reported. Therefore, the new reassortant RVB strain is the likely causative agent for the diarrheal outbreak of piglets occurred in China and more epidemiological studies should be conducted to monitor the spread of this newly identified porcine RVB strain.

Classical swine fever virus NS5A protein activates autophagy via the PP2A-DAPK3-Beclin 1 axis.

IMPORTANCE: Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies.

Epizootiological surveillance of porcine circoviruses in free-ranging wild boars in China.

Four species of porcine circoviruses (PCV1-4) have been reported to circulate in Chinese domestic pigs, while the epizootiology of these viruses in free-ranging wild boars in China remains unknown. In this study, tissue and serum samples collected from diseased or apparently healthy wild boars between 2018 and 2020 in 19 regions of China were tested for the prevalence of PCV1-4 infections. Positive rates of PCV1, PCV2, and PCV3 DNA in the tissue samples of Chinese wild boars were 1.6% (4/247), 58.3% (144/247), and 10.9% (27/247) respectively, with none positive for PCV4. Sequence analysis of viral genome showed that the four PCV1 strains distributed in Hunan and Inner Mongolia shared 97.5%-99.6% sequence identity with global distributed reference strains. Comparison of the ORF2 gene sequences showed that 80 PCV2 strains widely distributed in 18 regions shared 79.5%-100% sequence identity with reference strains from domestic pigs and wild boars, and were grouped into PCV2a (7), PCV2b (31) and PCV2d (42). For PCV3, 17 sequenced strains shared 97.2%-100% nucleotide identity at the genomic level and could be divided into PCV3a (3), PCV3b (2) and PCV3c (12) based on the phylogeny of ORF2 gene sequences. Serological data revealed antibody positive rates against PCV1 and PCV2 of 11.4% (19/167) and 53.9% (90/167) respectively. The data obtained in this study improved our understanding about the epidemiological situations of PCVs infection in free-ranging wild boars in China and will be valuable for the prevention and control of diseases caused by PCVs infection.

Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation, assembly of infectious virus particles, and regulation of cellular signaling pathways. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism involved has not been elucidated. Here, we reported that NS5A directly interacted with NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase (IKK) complex, to inhibit the NF-κB signaling pathway. Further investigations showed that the zinc finger domain of NEMO and the aa 126-250 segment of NS5A are essential for the interaction between NEMO and NS5A. Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation. In addition, NS5A blocked the K63-linked polyubiquitination of NEMO, thus inhibiting IKK phosphorylation, IκBα degradation, and NF-κB activation. These findings revealed a novel mechanism by which CSFV inhibits host innate immunity, which might guide the drug design against CSFV in the future.

Development of a novel bispecific antibody GR1801 for rabies.

Rabies is a zoonotic viral disease characterized by an almost 100% fatality rate once symptoms appear. However, it can be prevented through timely postexposure prophylaxis (PEP). Currently, there is a growing trend to replace polyclonal rabies immune globulin (RIG) with monoclonal antibodies (mAbs) in rabies PEP. In this study, we developed a human bispecific antibody, GR1801, by combining two mAbs, A2 and B353, which target distinct epitopes. GR1801 is an asymmetric immunoglobulin G1 molecule, with one arm (A2 targeting epitope III) in fragment antigen-binding (Fab) form and the other arm (B353 targeting epitope I) in single-chain variable fragment (scFv) form, constructed using Knobs-into-Holes technology. GR1801 demonstrated the ability to neutralize 90 naturally occurring rabies virus (RABV) glycoprotein antigenic variants, 21 pseudotyped, and 18 live street RABVs, exhibiting broad-spectrum neutralizing activity. In vivo, GR1801 provided protection equivalent to that of human RIG in golden hamsters challenged with lethal RABV. In conclusion, these findings demonstrate the neutralization potency and breadth of GR1801, which can be a promising candidate drug for rabies PEP, and a comprehensive testing against a broad spectrum of Chinese prevalent RABVs will be investigated in great detail in the future for the in vitro and in vivo studies.

IFITM3 restricts RABV infection through inhibiting viral entry and mTORC1- dependent autophagy.

Rabies, which caused by rabies virus (RABV), is a zoonotic and life-threatening disease with 100% mortality, and there is no effective treatment thus far due to the unclear pathogenesis and less of treatment targets. Interferon-induced transmembrane protein 3 (IFITM3) has recently been identified as an important anti-viral host effector induced by type I interferon. However, the role of IFITM3 in RABV infection has not been elucidated. In this study, we demonstrated that IFITM3 is a crucial restriction factor for RABV, the viral-induced IFITM3 significantly inhibited RABV replication, while knockdown of IFITM3 had the opposite effect. We then identified that IFNß induces the upregulation of IFITM3 in the absence or presence of RABV infection, meanwhile, IFITM3 positively regulates RABV-triggered production of IFNß in a feedback manner. In-depth research we found that IFITM3 not only inhibits the virus absorb and entry, but also inhibits viral replication through mTORC1-dependent autophagy. All these findings broaden our understanding of IFITM3 function and uncover a novel mechanism against RABV infection.

Virome Profiling of Chickens with Hepatomegaly Rupture Syndrome Reveals Coinfection of Multiple Viruses.

Liver diseases seriously challenge the health of chickens raised on scaled farms and cause tremendous economic losses to farm owners. The causative agents for liver diseases are still elusive, even though various pathogens, such as the hepatitis E virus, have been reported. In the winter of 2021, a liver disease was observed on a chicken farm in Dalian, China, which increased chicken mortality by up to 18%. We conducted panvirome profiling of the livers, spleens, kidneys, and recta of 20 diseased chickens. The viromic results revealed coinfection of multiple viruses, including pathogenic ones, in these organs. The viruses were highly identical to those detected in other provinces, and the vaccine and field strains of avian encephalomyelitis virus (AEV) and chicken infectious anemia virus (CIAV) cocirculated on the farm. In particular, the liver showed higher abundance of AEV and multiple fowl adenoviruses than other organs. Furthermore, the liver also contracted avian leukemia virus and CIAV. Experimental animals with infected liver samples developed minor to medium lesions of the liver and showed a virus abundance profile for AEV across internal organs similar to that in the original samples. These results suggest that coinfection with multiple pathogenic viruses influences the occurrence and development of infectious liver disease. The results also highlight that strong farm management standards with strict biosafety measures are needed to minimize the risk of pathogenic virus introduction to the farm.

Large-scale phylogenetic analysis reveals genetic diversity and geographic distribution of rabies virus in South-East and South Asia.

South-East Asia (SEA) and South Asia (SA) are two important geographic regions with the most severe enzootic rabies in the world. In these regions, phylogenetic analysis of rabies virus (RABV) has been conducted only at a country level; the results obtained from different countries are scattered and unequal, with a non-uniform system to name RABV genotypes. Therefore, it is difficult to undertake origin-tracking and compare inter-country RABV evolution and transmission. To avoid the confusion in understanding and to generate a panoramic picture of RABV genetic diversity, distribution, and transmission in SEA and SA, the present study conducted a systematic phylogenetic analysis by combining all sequences representing 2368 RABV strains submitted to GenBank by 14 rabies endemic SEA and SA countries. The results showed that RABVs circulating in two regions were classified into four major clades and many subclades: the Asia clade is circulating only in SEA, the Indian subcontinent, and Arctic-like clades only in SA, while the Cosmopolitan clade has been detected in both regions. The results also showed a wide range of hosts were infected by divergent RABV subclades, with dogs being the major transmission source. However, wildlife rabies was also found to be an important issue with 6 wild carnivore species identified as potential sources of spillover risk for sylvatic rabies to humans, domestic animals, and other wild animals. Current findings indicate that the two regions have separate virus clades circulating thus indicating the absence of cross-transmission between the regions. The study emphasizes the importance of phylogenetic analysis in the regions using uniform genotyping and naming systems for rabies surveillance, to coordinate actions of member countries to eliminate dog-mediated human rabies by 2030.

Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Virome Profiling of an Eastern Roe Deer Reveals Spillover of Viruses from Domestic Animals to Wildlife.

Eastern roe deer (Capreolus pygargus) is a small ruminant and is widespread across China. This creature plays an important role in our ecological system. Although a few studies have been conducted to investigate pathogens harbored by this species, our knowledge of the virus diversity is still very sparse. In this study, we conducted the whole virome profiling of a rescue-failed roe deer, which revealed a kobuvirus (KoV), a bocaparvovirus (BoV), and multiple circular single-stranded viruses. These viruses were mainly recovered from the rectum, but PCR detection showed systematic infection of the KoV. Particularly, the KoV and BoV exhibited closely genetic relationships with bovine and canine viruses, respectively, highly suggesting the spillover of viruses from domestic animals to wildlife. Although these viruses were unlikely to have been responsible for the death of the animal, they provide additional data to understand the virus spectrum harbored by roe deer. The transmission of viruses between domestic animals and wildlife highlights the need for extensive investigation of wildlife viruses.

DNA virome of ticks in the Northeast and Hubei provinces of China reveals diverse single-stranded circular DNA viruses.

BACKGROUND: Ticks are medically important vectors capable of transmitting a variety of pathogens to and between host species. Although the spectrum of tick-borne RNA viruses has been frequently investigated, the diversity of tick-borne DNA viruses remains largely unknown. METHODS: A total of 1571 ticks were collected from forests and infested animals, and the diversity of the viruses they harbored was profiled using a DNA-specific virome method. The viromic data were phylogenetically analyzed and validated by PCR assays. RESULTS: Although diverse and abundant prokaryotic viruses were identified in the collected ticks, only eukaryotic DNA viruses with single-stranded circular genomes covering the anelloviruses and circular replication-associated (Rep) protein-encoding single-stranded (CRESS) DNA viruses were recovered from ticks. Anelloviruses were detected only in two tick pools, but CRESS DNA viruses were prevalent across these ticks except in one pool of Dermacentor spp. ticks. Phylogenetic analyses revealed that these tick-borne CRESS DNA viruses were related to viruses recovered from animal feces, tissues and even environmental samples, suggesting that their presence may be largely explained by environmental factors rather than by tick species and host blood meals. CONCLUSIONS: Based on the results, tick-borne eukaryotic DNA viruses appear to be much less common than eukaryotic RNA viruses. Investigations involving a wider collection area and more diverse tick species are required to further support this speculation.

Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge.

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

In Vitro Effect of Flavonoids on Basophils Degranulation and Intestinal Epithelial Barrier Damage Induced by ω-5 Gliadin-Derived Peptide.

Flavonoids have antioxidant, anti-inflammatory and immunomodulatory properties, and may alleviate food allergic reactions and intestinal inflammation induced by ω-5 gliadin, a main allergen of wheat food allergy in children. In this study, a human basophil KU812 cell degranulation model and a Caco-2 monolayer cell model were constructed in vitro to evaluate the effects of four flavonoids on the allergenicity of ω-5 gliadin peptides and ω-5 gliadin peptide-induced barrier damage in Caco-2 intestinal epithelial monolayers. The results show that baicalein, luteolin, isorhamnetin and naringenin can significantly inhibit the degranulation of KU812 cells stimulated by ω-5 gliadin-derived peptide P4 and the release of IL-6 and TNF-α. In addition, the four flavonoids significantly inhibited the ω-5 gliadin-derived peptide P4 to induce the release of IL-6, IL-8 in Caco-2 cells, inhibited the release of zonulin, and significantly increase the expression of tight junction proteins Occludin and ZO-1 in the Caco-2 cell monolayer. In conclusion, baicalein, luteolin, isorhamnetin and naringenin inhibit degranulation stimulated by wheat allergen and enhance intestinal barrier functions, which supports the potential pharmaceutical application of the four flavonoids treatment for wheat food allergy.

Virome Profiling of an Amur leopard cat Reveals Multiple Anelloviruses and a Bocaparvovirus.

As a small top predator, Amur leopard cat (Prionailurus bengalensis euptilurus) is widely distributed in northeast Asia and plays an important role in the control of small rodent populations and in the maintenance of ecological equilibrium. However, the viruses harbored by this creature have been rarely investigated. Here, we report the DNA and RNA eukaryotic virome profiling of an injured Amur leopard cat followed by PCR validation, which revealed diverse anelloviruses in multiple organs and a bocaparvovirus in the lymph, but no RNA viruses. These anelloviruses have diverse genomic structures and are classified into four phylogroups with viruses of various felines, while the bocaparvovirus is extremely similar to those recovered from diarrheal domestic cats, illustrating the transmission of the virus between domestic animals and wildlife. These data provide the first insight into the genetic diversity of Amur leopard cat viruses, highlighting the need for further investigation of wild animals.

Elimination of Foreign Sequences in Eukaryotic Viral Reference Genomes Improves the Accuracy of Virome Analysis.

Widespread in public databases, foreign contaminant sequences pose a substantial obstacle in genomic analyses. Such contamination in viral genome databases is also notorious but more complicated and often causes questionable results in various applications, particularly in virome-based virus detection. Here, we conducted comprehensive screening and identification of the foreign sequences hidden in the largest eukaryotic viral genome collections of GenBank and UniProt using a scrutiny pipeline, which enables us to rigorously detect those problematic viral sequences (PVSs) with origins in hosts, vectors, and laboratory components. As a result, a total of 766 nucleotide PVSs and 276 amino acid PVSs with lengths up to 6,605 bp were determined, which were widely distributed in 39 families with many involving highly public health-concerning viruses, such as hepatitis C virus, Crimean-Congo hemorrhagic fever virus, and filovirus. The majority of these PVSs are genomic fragments of hosts including humans and bacteria. However, they cannot simply be regarded as foreign contaminants, since parts of them are results of natural occurrence or artificial engineering of viruses. Nevertheless, they severely disturb such sequence-based analyses as genome annotation, taxonomic assignment, and virome profiling. Therefore, we provide a clean version of the eukaryotic viral reference data set by the removal of these PVSs, which allows more accurate virome analysis with less time consumed than with other comprehensive databases. IMPORTANCE High-throughput sequencing-based viromics highly depends on reference databases, but foreign contamination is widespread in public databases and often leads to confusing and even wrong conclusions in genomic analysis and viromic profiling. To address this issue, we systematically detected and identified the contamination in the largest viral sequence collections of GenBank and UniProt based on a stringent scrutiny pipeline. We found hundreds of PVSs that are related to hosts, vectors, and laboratory components. By the removal of them, the resulting data set greatly improves the accuracy and efficiency of eukaryotic virome profiling. These results refresh our knowledge of the type and origin of PVSs and also have warning implications for viromic analysis. Viromic practitioners should be aware of these problems caused by PVSs and need to realize that a careful review of bioinformatic results is necessary for a reliable conclusion.

Identification and genomic characterization of a novel porcine parvovirus in China.

Porcine parvoviruses (PPVs) are a group of small non-enveloped viruses with seven species (porcine parvovirus 1-7, PPV1-7) have been identified. In this study, a novel porcine parvovirus, provisionally named porcine parvovirus 8 (PPV8), was initially identified via high-throughput sequencing (HTS) in porcine reproductive and respiratory syndrome virus-positive samples collected from swine herds in Guangdong province, 2021. The nearly full-length genome of PPV8 strain GDJM2021 is 4,380 nucleotides in length with two overlapping open ORFs encoding NS1 and VP1 respectively. Sequence analysis indicated that PPV8 shared 16.23-44.18% sequence identity at the genomic levels to PPV1-7 with the relatively highest homology to PPV1. PPV8-GDJM2021 shared 31.86-32.68% aa sequence identity of NS1 protein with those of PPV1 and porcine bufavirus (PBuV), and formed an independent branch neighboring to those formed by members of the genus Protoparvovirus. Of the 211 clinical samples collected from 1990 to 2021, 37 samples (17.5%) distributed over 12 regions in China were positive for PPV8 with time spanning 24 years (1998-2021). To our knowledge, this is the first report on the genomic characterization of the novel PPV8 and its epidemiological situations in China.

RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models.

The ongoing COVID-19 pandemic has continued to affect millions of lives worldwide, leading to the urgent need for novel therapeutic strategies. G-quadruplexes (G4s) have been demonstrated to regulate life cycle of multiple viruses. Here, we identify several highly conservative and stable G4s in SARS-CoV-2 and clarify their dual-function of inhibition of the viral replication and translation processes. Furthermore, the cationic porphyrin compound 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4) targeting SARS-CoV-2 G4s shows excellent antiviral activity, while its N-methyl-2-pyridyl positional isomer TMPyP2 with low affinity for G4 has no effects on SARS-CoV-2 infection, suggesting that the antiviral activity of TMPyP4 attributes to targeting SARS-CoV-2 G4s. In the Syrian hamster and transgenic mouse models of SARS-CoV-2 infection, administration of TMPyP4 at nontoxic doses significantly suppresses SARS-CoV-2 infection, resulting in reduced viral loads and lung lesions. Worth to note, the anti-COVID-19 activity of TMPyP4 is more potent than remdesivir evidenced by both in vitro and in vivo studies. Our findings highlight SARS-CoV-2 G4s as a novel druggable target and the compelling potential of TMPyP4 for COVID-19 therapy. Different from the existing anti-SARS-CoV-2 therapeutic strategies, our work provides another alternative therapeutic tactic for SARS-CoV-2 infection focusing on targeting the secondary structures within SARS-CoV-2 genome, and would open a new avenue for design and synthesis of drug candidates with high selectivity toward the new targets.

Detection and Characterization of a Reassortant Mammalian Orthoreovirus Isolated from Bats in Xinjiang, China.

Mammalian orthoreoviruses (MRVs) are increasingly reported to cause various diseases in humans and other animals, with many possibly originating from bats, highlighting the urgent need to investigate the diversity of bat-borne MRVs (BtMRVs). Here, we report the detection and characterization of a reassortant MRV that was isolated from a bat colony in Xinjiang, China. The BtMRV showed a wide host and organ tropism and can efficiently propagate the cell lines of different animals. It caused mild damage in the lungs of the experimentally inoculated suckling mice and was able to replicate in multiple organs for up to three weeks post-inoculation. Complete genome analyses showed that the virus was closely related to MRVs in a wide range of animals. An intricate reassortment network was revealed between the BtMRV and MRVs of human, deer, cattle, civet and other bat species. Specifically, we found a bat-specific clade of segment M1 that provides a gene source for the reassortment of human MRVs. These data provide important insights to understand the diversity of MRVs and their natural circulation between bats, humans, and other animals. Further investigation and surveillance of MRV in bats and other animals are needed to control and prevent potential MRV-related diseases.

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.