Function investigation of p11.5 in ASFV infection.

Virus replication relies on complex interactions between proteins. A comprehensive understanding of the protein interactions may provide clues for developing new antiviral strategies. In the case of African swine fever virus (ASFV), only a few protein interactions have been identified so far. In this study, we demonstrate that ASFV protein p72 interacts with p11.5 using co-immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS). It was found that protein p72 interacts specifically with p11.5 at sites amino acids (aa) 1-216 of p72 and aa 1-68 of p11.5. To assess the importance of p11.5 in ASFV infection, we developed a recombinant virus (ASFVGZΔA137R) by deleting the A137R gene from the ASFVGZ genome. Compared with ASFVGZ, the infectious progeny virus titers of ASFVGZΔA137R were reduced by approximately 1.0 logs. In addition, we demonstrated that the growth defect was partially attributable to a higher genome copies-to-infectious virus titer ratios produced in ASFVGZΔA137R-infected MA104 cells than in those infected with ASFVGZ. This finding suggests that MA104 cells infected with ASFVGZΔA137R may generate larger quantities of noninfectious particles. Importantly, we found that p11.5 did not affect virus-cell binding or endocytosis. Collectively, we show for the first time that the interaction between ASFV p72 and p11.5. Our results effectively provide the relevant information of the p11.5 protein. These results extend our understanding of complex interactions between viral proteins, paving the way for further studies of the potential mechanism and pathogenesis of ASFV infection.

Regulation of Wnt/ß-catenin signaling by Marek's disease virus in vitro and in vivo.

Marek's disease virus (MDV) infection causes immunosuppression in the host, ultimately inducing tumor formation and causing significant economic losses to the poultry industry. While the abnormal activation of the Wnt/ß-catenin signaling pathway is closely associated with the occurrence and development of tumors. However, the relationship between MDV and the Wnt/ß-catenin pathway remains unclear. In this study, we found that the MDV RB1B strain, but not the MDV vaccine strain CVI988, activated the Wnt/ß-catenin signaling pathway by increasing the phosphorylation level of GSK-3ß in chicken embryo fibroblast (CEF). In vivo infection experiments in SPF chickens also confirmed that the RB1B strain activated the Wnt/ß-catenin signaling pathway, while the CVI988 strain did not lead to its activation. Moreover, unlike the Meq protein encoded by the CVI988 strain, the Meq protein encoded by the RB1B strain specifically activated the Wnt/ß-catenin signaling pathway in CEF cells. The findings from these studies extend our understanding of the regulation of Wnt/ß-catenin signaling by MDV, which make a new contribution to understanding the virus-host interactions of MDV.

Metabolomics analysis of CEF cells infected with avian leukosis virus subgroup J based on UHPLC-QE-MS.

Avian leukosis virus subgroup J (ALV-J) is a retrovirus that can cause immunosuppression and tumors in chicken. However, relative pathogenesis is still not clear. At present, metabolomics has shown great potential in the screening of tumor metabolic markers, prognostic evaluation, and drug target design. In this study, we utilize an untargeted metabolomics approach based on ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) to analyze the metabolic changes in chicken embryo fibroblast (CEF) cells infected by ALV-J. We found that ALV-J infection significantly altered a wealth of metabolites compared with control group. Additionally, most of the differentially expressed metabolites belonged to lipid metabolism, purine nucleotide metabolism and amino acid metabolism. Among them, the proportion of lipid metabolites account for the highest proportion (around 31%). Results suggest that these changes may be conductive to the formation of virion, thereby promoting the replication of ALV-J. These data provided metabolic evidence and potential biomarkers for the cellular metabolic changes induced by ALV-J, and provided important insight for further understanding the replication needs and pathogenesis of ALV-J.

Isolation and genomic characterization of chicken infectious anemia virus in Jiangsu province of China during 2020-2022.

As an immunosuppressive disease virus, chicken infectious anemia virus (CIAV) mainly infects chickens, causing aplastic anemia and systemic lymphoid tissue atrophy. In recent years, the prevalence of CIAV in the poultry industry globally has caused huge economic losses. In this study, a total of 223 clinical samples, including anal swabs, tissues, blood, and vaccines, were collected from 19 broiler farms or breeding companies in Jiangsu province, with symptoms of significant anemia and immunosuppression during 2020-2022. Among them, 75 samples (75/223, 33.6%) were positive for CIAV in polymerase chain reaction (PCR) test, and 20 CIAV strains were successfully isolated. The phylogenetic trees of the 20 isolates and 42 CIAV strains deposited in GenBank formed four distinct groups (A-D). And the isolates mainly belonged to Group A but with high genetic diversity. Analysis for VP1 indicated that these isolates possess key characteristics of highly pathogenic strains. Meanwhile, VP2 and VP3 were much conserved with much fewer mutations compare to VP1. The above epidemiological study of CIAV provides novel insights into molecular characterization of CIAV and lays the foundation for developing efficient strategies for control of CIAV in China.

Single Cell Analysis of Lung Lymphatic Endothelial Cells and Lymphatic Responses during Influenza Infection.

Tissue lymphatic vessels network plays critical roles in immune surveillance and tissue homeostasis in response to pathogen invasion, but how lymphatic system per se is remolded during infection is less understood. Here, we observed that influenza infection induces a significant increase of lymphatic vessel numbers in the lung, accompanied with extensive proliferation of lymphatic endothelial cells (LECs). Single-cell RNA sequencing illustrated the heterogeneity of LECs, identifying a novel PD-L1+ subpopulation that is present during viral infection but not at steady state. Specific deletion of Pd-l1 in LECs elevated the expansion of lymphatic vessel numbers during viral infection. Together these findings elucidate a dramatic expansion of lung lymphatic network in response to viral infection, and reveal a PD-L1+ LEC subpopulation that potentially modulates lymphatic vessel remolding.

Establishment and application of a VP3 antigenic domain-based peptide ELISA for the detection of antibody against goose plague virus infection.

The detection of antibody against goose plague virus (GPV) infection has never had a commercialized test kit, which has posed challenges to the prevention and control of this disease. In this study, bioinformatics software was used to analyze and predict the dominant antigenic regions of the main protective antigen VP3 of GPV. Three segments of bovine serum albumin (BSA) vector-coupled peptides were synthesized as ELISA coating antigens. Experimental results showed that the VP3-1 (358-392aa) peptide had the best reactivity and specificity. By using the BSA-VP3-1 peptide, a detection method for antibody against GPV infection was established, demonstrating excellent specificity with no cross-reactivity with common infectious goose pathogen antibodies. The intra-batch coefficient of variation and inter-batch coefficient of variation were both less than 7%, indicating good stability and repeatability. The dynamic antibody detection results of gosling vaccines and the testing of 120 clinical immune goose serum samples collectively demonstrate that BSA-VP3-1 peptide ELISA can be used to detect antibody against GPV in the immunized goose population and has higher sensitivity than traditional agar gel precipitation methods. Taken together, the developed peptide-ELISA based on VP3 358-392aa could be useful in laboratory viral diagnosis, routine surveillance in goose farms. The main application of the peptide-ELISA is to monitor the antibody level and vaccine efficacy for GPV, which will help the prevention and control of gosling plague.

Efficient cross-protection against serotype 4/8a fowl adenoviruses (FAdVs): recombinant FAdV-4 with FAdV-8a Fiber.

IMPORTANCE: Epidemiological data reveal that FAdV-4 and FAdV-8a are the dominant serotypes of FAdVs in the poultry industry in China. Although three commercial inactivated vaccines against FAdV-4 have been licensed in China, the bivalent vaccine against both FAdV-4 and FAdV-8a is not available. Here, we used CRISPR-Cas9 and Cre-LoxP system to generate a recombinant virus FAdV4-F/8a-rF2 expressing the Fiber of FAdV-8a. Notably, FAdV4-F/8a-rF2 was highly attenuated and could provide efficient protection against both FAdV-4 and FAdV-8a in the chicken infection model, highlighting the applaudable application of FAdV4-F/8a-rF2 as a novel live-attenuated bivalent vaccine against the diseases caused by the infection of FAdV-4 and FAdV-8a.

Metabolomic profiling of Marek's disease virus infection in host cell based on untargeted LC-MS.

Marek's disease (MD) caused by Marek's disease virus (MDV), poses a serious threat to the poultry industry by inducing neurological disease and malignant lymphoma in infected chickens. However, the underlying mechanisms how MDV disrupts host cells and causes damage still remain elusive. Recently, the application of metabolomics has shown great potential for uncovering the complex mechanisms during virus-host interactions. In this study, chicken embryo fibroblasts (CEFs) infected with MDV were subjected to ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) and multivariate statistical analysis. The results showed that 261 metabolites were significantly altered upon MDV infection, with most changes occurring in amino acid metabolism, energy metabolism, nucleotide metabolism, and lipid metabolism. Notably, MDV infection induces an up-regulation of amino acids in host cells during the early stages of infection to provide the energy and intermediary metabolites necessary for efficient multiplication of its own replication. Taken together, these data not only hold promise in identifying the biochemical molecules utilized by MDV replication in host cells, but also provides a new insight into understanding MDV-host interactions.

Identification of key residues of B cell epitopes in hemagglutinin of H6 influenza A virus.

IMPORTANCE: Since the escape immunity of influenza A viruses (IAVs) is mainly caused by the continuous antigenic variations in HA, the identification of key antigenic epitopes is crucial for better understanding of the escape immunity and vaccine development for IAVs. The antigenic sites of several HA subtypes, including H1, H3, H5, and H9, have been well characterized, whereas those of H6 subtype are poorly understood. Here, we mapped nine key residues of antigenic epitopes in H6 through escape mutants using a panel of MAbs. Moreover, MAbs 4C2 and 6E3, targeting 140 and 89 residues, respectively, could protect mice against lethal challenge of MA E-Teal/417. These key residues of antigenic epitopes identified here provide the molecular targets for further elucidating the antigenic evolution of H6 and better preparing the vaccine against H6 IAV.

3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

IMPORTANCE: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

Vector-delivered artificial miRNA effectively inhibits Porcine epidemic diarrhea virus replication.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea, dehydration, vomiting, and high mortality to suckling piglets. As a strategy for antiviral therapy, artificial microRNA (amiRNA) mediated suppression of viral replication has recently become increasingly important. In this study, we evaluated the advantages of using an amiRNA vector against PEDV. METHODS: In this study, we evaluated the advantages of using an amiRNA vector against PEDV. We designed two single amiRNA sequences for different conserved sequences of the PEDV S and N genes, and tested their inhibitory effects on PEDV in Vero cells. RESULTS: It was obvious from the CCK-8 results that the transient transfection of amiRNA was non-toxic to the cells. In addition, our results showed that the transient expression of two amiRNAs (amiRNA-349 and amiRNA-1447) significantly reduced the expression of viral RNA and protein in the cells. The TCID50 results showed that the release of virus particles into the culture supernatant was significantly reduced, with an effect as high as 90%. To avoid virus mutation escape, the above two single amiRNA sequences were tandem in this study (amiRNA-349 + 1447), enabling a single microRNA to be expressed simultaneously. The real-time PCR and Western blot results showed that the inhibitory effect was significantly enhanced in each of the different time periods. The TCID50 results showed that the release of virus particles in the culture supernatant was significantly reduced at the different time periods. CONCLUSIONS: In summary, these results suggest that an RNAi based on amiRNA targeting the conserved region of the virus is an effective method to improve PEDV nucleic acid inhibitors and provide a novel treatment strategy for PEDV infection.

Identification of novel B cell epitopes in Fiber-2 protein of duck adenovirus 3 and their application.

Duck adenovirus 3 (DAdV-3), a newly emerged duck adenovirus, has resulted in significant economic losses to the duck industry across China since 2014. However, little is known about the B cell epitopes in major antigen of DAdV-3 and the serological approach for detection of DAdV-3 is not available. In this study, four monoclonal antibodies (mAbs) specific to Fiber-2 protein of DAdV-3 were first generated and designated as 2G10, 3D9, 5E6, and 6B12. Indirect immunofluorescence assay (IFA) showed that all of the mAbs reacted with the Fiber-2. Moreover, mAbs 2G10, 5E6, and 6B12 demonstrated good activity with Fiber-2 in Western blot. Notably, the Fiber-2 could be immunoprecipitated efficiently by mAb 3D9. Epitope mapping revealed that mAbs 2G10, 3D9, 5E6, and 6B12 recognized 397-429aa, 463-481aa, 67-99aa, and 1-66aa of Fiber-2, respectively. Besides, a novel sandwich ELISA for efficient detection of DAdV-3 was developed based on mAb 3D9 and horseradish peroxidase (HRP) conjugated mAb 3D9. The sandwich ELISA only reacted with DAdV-3 but not with other duck-associated viruses. The limit of detection of the ELISA was 6.25 × 103 TCID50/mL. Overall, the mAbs generated laid the foundation for elucidating the critical role of Fiber-2 in mediating infection and pathogenesis, and the sandwich ELISA approach established here provided efficient and rapid serological diagnostic tool for DAdV-3.

A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek's disease virus vaccines and field strains.

To evaluate the effect of the vaccine and differentiate vaccine from virulent MDV, a new quadruplex real-time PCR assay based on TaqMan probes was developed to differentiate and accurately quantify HVT, CVI988 and virulent MDV-1. The results showed that the limit of detection (LOD) of the new assay was 10 copies with correlation coefficients >0.994 of CVI988, HVT and virulent MDV DNA molecules without cross-reactivity with other avian disease viruses. The intra-assay and inter-assay coefficients of variation (CVs) of Ct values for the new assay were less than 3%. Analysis of replication kinetics of CVI988 and virulent MDV of collected feathers between 7 and 60 days post-infection (dpi) showed MD5 had no significant effect on the genomic load of CVI988 (p > 0.05), while vaccination with CVI988 could significantly reduce the viral load of MD5 (p < 0.05). Combined with meq gene PCR, this method can effectively identify virulent MDV infections in immunized chickens. These results demonstrated that this assay could distinguish between the vaccine and virulent MDV strains and had the advantages of being reliable, sensitive and specific to confirm the immunization status and monitor the circulation of virulent MDV strains.

An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene.

Recently, the infection of serotype 4 fowl adenovirus (FAdV-4) in chicken flocks has become endemic in China, which greatly threatens the sustainable development of poultry industry. The development of recombinant FAdV-4 expressing foreign genes is an efficient strategy for controlling both FAdV-4 and other important poultry pathogens. Previous reverse genetic technique for generating the recombinant fowl adenovirus is generally inefficient. In this study, a recombinant FAdV-4 expressing enhanced green fluorescence protein (EGFP), FA4-EGFP, was used as a template virus and directly edited fiber-2 gene to develop an efficient double-fluorescence approach to generate recombinant FAdV-4 through CRISPR/Cas9 and Cre-Loxp system. Moreover, using this strategy, a recombinant virus FAdV4-HA(H9) stably expressing the HA gene of H9N2 influenza virus was generated. Chicken infection study revealed that the recombinant virus FAdV4-HA(H9) was attenuated, and could induce haemagglutination inhibition (HI) titer against H9N2 influenza virus at early time points and inhibit the viral replication in oropharynx. All these demonstrate that the novel strategy for constructing recombinant FAdV-4 expressing foreign genes developed here paves the way for rapidly developing attenuated FAdV-4-based recombinant vaccines for fighting the diseases caused by both FAdV-4 and other pathogens.

A novel recombinant serotype 4 fowl adenovirus expressing fiber-2 protein of duck adenovirus 3.

Recently, the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and duck adenovirus 3 (DAdV-3) were outbroken and widespread, causing substantial economic losses to the duck industry. Therefore, there is an urgent need to generate a recombinant genetic engineering vaccine candidate against both FAdV-4 and DAdV-3. In this study, a novel recombinant FAdV-4 expressing the Fiber-2 protein of DAdV-3, designated as rFAdV-4-Fiber-2/DAdV-3, was generated based on CRISPR/Cas9 and Cre-LoxP systems. Indirect immunofluorescence assay (IFA) and western blot (WB) showed that the Fiber-2 protein of DAdV-3 in rFAdV-4-Fiber-2/DAdV-3 was expressed successfully. Moreover, the growth curve revealed that rFAdV-4-Fiber-2/DAdV-3 replicated efficiently in LMH cells and even showed a stronger replication ability compared to the wild type FAdV-4. The generation of the recombinant rFAdV-4-Fiber-2/DAdV-3 provides a potential vaccine candidate against both FAdV-4 and DAdV-3.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

A novel mAb broadly neutralizes SARS-CoV-2 VOCs in vitro and in vivo, including the Omicron variants.

Novel immune escape variants have emerged as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Many of the variants cause breakthrough infections in vaccinated populations, posing great challenges to current antiviral strategies targeting the immunodominance of the receptor-binding domain within the spike protein. Here, we found that a novel broadly neutralizing monoclonal antibody (mAb), G5, provided efficient protection against SARS-CoV-2 variants of concern (VOCs) in vitro and in vivo. A single dose of mAb G5 could significantly inhibit the viral burden in mice challenged with the mouse-adapted SARS-CoV-2 or SARS-CoV-2 Omicron BA.1 variant, as well as the body weight loss and cytokine release induced by mouse-adapted SARS-CoV-2. The refined epitope recognized by mAb G5 was identified as 1148 FKEELDKYF1156 in the stem helix of subunit S2. In addition, a human-mouse chimeric mAb was generated based on the variable region of heavy chain and VL genes of mAb G5. Our study provides a broad antibody drug candidate against SARS-CoV-2 VOCs and reveals a novel target for developing pan-SARS-CoV-2 vaccines.

Chicken Glycogen Synthase Kinase 3ß Suppresses Innate Immune Responses and Enhances Avian Leukosis Virus Replication in DF-1 Cells.

Glycogen synthase kinase 3ß (GSK3ß) is a widely distributed multifunctional serine/threonine kinase. In mammals, GSK3ß regulates important life activities such as proinflammatory response, anti-inflammatory response, immunity, and cancer development. However, the biological functions of chicken GSK3ß (chGSK3ß) are still unknown. In the present study, the full-length cDNA of chGSK3ß was first cloned and analyzed. Absolute quantification of chicken chGSK3ß in 1-day-old specific-pathogen-free birds has shown that it is widely expressed in all tissues, with the highest level in brain and the lowest level in pancreas. Overexpression of chGSK3ß in DF-1 cells significantly decreased the gene expression levels of interferon beta (IFN-ß), IFN regulatory factor 7 (IRF7), Toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5), MX-1, protein kinase R (PKR), and oligoadenylate synthase-like (OASL), while promoting the replication of avian leukosis virus subgroup J (ALV-J). Conversely, levels of most of the genes detected in this study were increased when chGSK3ß expression was knocked down using small interfering RNA (siRNA), which also inhibited the replication of ALV-J. These results suggest that chGSK3ß plays an important role in the antiviral innate immune response in DF-1 cells, and it will be valuable to carry out further studies on the biological functions of chGSK3ß. IMPORTANCE GSK3ß regulates many life activities in mammals. Recent studies revealed that chGSK3ß was involved in regulating antiviral innate immunity in DF-1 cells and also could positively regulate ALV-J replication. These results provide new insights into the biofunction of chGSK3ß and the virus-host interactions of ALV-J. In addition, this study provides a basis for further research on the function of GSK3 in poultry.

Amino Acid Variation at Hemagglutinin Position 193 Impacts the Properties of H9N2 Avian Influenza Virus.

Despite active control strategies, including the vaccination program in poultry, H9N2 avian influenza viruses possessing mutations in hemagglutinin (HA) were frequently isolated. In this study, we analyzed the substitutions at HA residue 193 (H3 numbering) of H9N2 and investigated the impact of these mutations on viral properties. Our study indicated that H9N2 circulating in the Chinese poultry have experienced frequent mutations at HA residue 193 since 2013, with viruses that carried asparagine (N) being replaced by those with alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), and serine (S), etc. Our results showed the N193G mutation impeded the multiple cycles of growth of H9N2, and although most of the variant HAs retained the preference for human-like receptors as did the wild-type N193 HA, the N193E mutation altered the preference for both human and avian-like receptors. Furthermore, these mutations substantially altered the antigenicity of H9N2 as measured by both monoclonal antibodies and antisera. In vivo studies further demonstrated that these mutations showed profound impact on viral replication and transmission of H9N2 in chicken. Viruses with D, E, or S at residue 193 acquired the ability to replicate in lungs of the infected chickens, whereas virus with G193 reduced its transmissibility in infected chickens to those in direct contact. Our findings demonstrated that variations at HA residue 193 altered various properties of H9N2, highlighting the significance of the continued surveillance of HA for better understanding of the etiology and effective control of H9N2 in poultry. IMPORTANCE H9N2 are widespread and have sporadically caused clinical diseases in humans. Extensive vaccinations in poultry helped constrain H9N2; however, they might have facilitated the evolution of the virus. It is therefore of importance to monitor the variation of the circulating H9N2 and evaluate its risk to both veterinary and public health. Here, we found substitutions at position 193 of HA from H9N2 circulated since 2013 and assessed the impact of several mutations on viral properties. Our data showed these mutations resulted in substantial antigenic change. N193E altered the binding preference of HA for human-like to both avian and human-like receptors. More importantly, N193G impaired the growth of H9N2 and its transmission in chickens, whereas mutations from N to D, E, and S enhanced the viral replication in lungs of chickens. Our study enriched the knowledge about H9N2 and may help implement an effective control strategy for H9N2.

A novel monoclonal antibody efficiently blocks the infection of duck adenovirus 3 by targeting Fiber-2.

Duck adenovirus 3 (DAdV-3), identified as the causative agent of a disease characterized by swelling and hemorrhage of liver and kidney, has caused substantial economic losses to duck industry in China. However, the neutralizing epitopes and the infection mechanism of DAdV-3 have not been extensively elucidated. In this study, a novel monoclonal antibody (mAb) targeting Fiber-2 protein of DAdV-3 was generated and designated as mAb 3E7. Indirect immunofluorescence assay showed that mAb 3E7 specifically reacted with the Fiber-2 in LMH cells transfected with pcDNA3.1-Fiber-2 or infected with DAdV-3. Moreover, mAb 3E7 could immunoprecipitate the Fiber-2 and efficiently inhibit the infection of DAdV-3 in vitro. Further epitope mapping revealed mAb 3E7 recognized the epitope 108LALGDGLE115 in Fiber-2, which was highly conserved among DAdV-3 strains. These findings not only identified a novel neutralizing epitope in Fiber-2, but also paved the way for further elucidating the vital roles of Fiber-2 in the infection and pathogenesis of DAdV-3.