Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota

Bi, Youkun; Jin, Zhongyuan; Wang, Yanhong; Mou, Sujing; Wang, Wenbin; Wei, Qiaolin; Huo, Na; Liu, Siqi; Wang, Xinglong; Yang, Zhicong; Chen, Hongjun; Xiao, Sa

doi:10.1089/vim.2019.0007

Cited by 9 publications

(6 citation statements)

References 41 publications

(41 reference statements)

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“…The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed using the following primary antibodies: an anti-CARD11 mouse PAb, a guinea pig anti-LaSota P, an anti-NP guinea pig PAb (25), an anti-HN guinea pig PAb, an anti-M synthetic peptide (KLEKGHTLAK YNPFK) mouse PAb (49), an anti-L mouse PAb, a DYKDDDK tag (3B9) mouse monoclonal antibody (MAb) (Abmart), an anti-DDDDK tag rabbit MAb (Abcam), a hemagglutinin (HA) tag (C29F4) rabbit MAb (Cell Signaling Technology), a GST tag mouse (3B10) MAb (Sungene Biotech), or ␤-tubulin (3G7) mouse MAb (Sungene Biotech). The secondary antibodies included goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sungene Biotech) and goat anti-rabbit IgG (H&L) HRP antibody (ZETA).…”

Section: Fig 12 Legend (Continued)mentioning

confidence: 99%

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Wang

Chang

Yao

et al. 2019

J Virol

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Host factors play multiple essential roles in the replication and pathogenesis of mammalian neurotropic viruses. However, the cellular proteins of the central nervous system (CNS) involved in avian neurotropic virus infection have not been completely elucidated. Here, we employed a gene microarray to identify caspase recruitment domain-containing protein 11 (CARD11), a lymphoma-associated scaffold protein presenting brain-specific upregulated expression in a virulent neurotropic Newcastle disease virus (NDV)-infected natural host. Chicken primary neuronal cells infected with NDV appeared slightly syncytial and died quickly. CARD11 overexpression inhibited viral replication and delayed cytopathic effects; conversely, depletion of CARD11 enhanced viral replication and cytopathic effects in chicken primary neuronal cells. The inhibition of viral replication by CARD11 could not be blocked with CARD11-Bcl10-MALT1 (CBM) signalosome and NF-κB signaling inhibitors. CARD11 was found to interact directly with the viral phosphoprotein (P) through its CC1 domain and the X domain of P; this X domain also mediated the interaction between P and the viral large polymerase protein (L). The CARD11 CC1 domain and L competitively bound to P via the X domain that hindered the P-L interaction of the viral ribonucleoprotein (RNP) complex, resulting in a reduction of viral polymerase activity in a minigenome assay and inhibition of viral replication. Animal experiments further revealed that CARD11 contributed to viral replication inhibition and neuropathology in infected chicken brains. Taken together, our findings identify CARD11 as a brain-specific antiviral factor of NDV infection in avian species. IMPORTANCE Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.

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Section: Fig 12 Legend (Continued)mentioning

confidence: 99%

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Wang

Chang

Yao

et al. 2019

J Virol

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“…The pepscan analysis was performed by the Novasnow Science & Technology China with anti-LaSota hyperimmune chicken serum as described previously (Bi et al 2019). The HN protein sequences were linked and elongated with neutral GSGSGSG linkers at the C-and N-termini to avoid truncated peptides.…”

Section: Pepscan Analysismentioning

confidence: 99%

“…Recently, this technology was also successfully used for a comprehensive antigenic analysis of viral proteins (21,22). In NDV, two immunodominant B-cell linear epitopes of the viral M protein were identi ed by using this pepscan approach (23).…”

Section: Introductionmentioning

confidence: 99%

“…Then, the chickens were immunized with 0.2 ml per chicken of inactivated vaccine through subcutaneous injection on days 14 and 28. Blood samples were collected on day 35(23).Six-week-old SPF female BALB/c mice (purchased from DaShuo, China) were subcutaneously immunized with 0.2 ml per mouse with inactivated LaSota vaccine (Yebio Qingdao, China) at days 1, 14 and 28. The sera were collected at day 35 and kept at -80 °C.…”

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confidence: 99%

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Identification of a Potential Neutralizing Linear Epitope of Hemagglutinin-neuraminidase in Newcastle Disease Virus

Jin

Wei

et al. 2020

Preprint

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BackgroundThe hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization.MethodsIn this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmid and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test.ResultsFive IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328-342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242-256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope.ConclusionsThe HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242-256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.

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“…Current strategies for epitope identification depend upon detection of epitopes within an individual full-length protein, a low-throughput approach that requires prior knowledge of the antigenic protein, its sequence, and its conformational structure. Technologies to screen for epitopes at the whole proteome level have been developed (e.g., proteomic microarrays, phage and yeast display); however, these technologies require extensive use of synthetic biology and other time-consuming methodologies (e.g., library construction, peptide/protein array preparation, heterologous protein expression) (3,(5)(6)(7)(8)(9)(10)(11). Another major disadvantage of display technologies and use of non-native expression systems is that these methods do not reliably replicate the native properties of the antigenic proteins, including their post-translational modifications, which can lead to inaccurate identification of epitopes.…”

Section: Introductionmentioning

confidence: 99%

Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

et al. 2021

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Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

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Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota

Cited by 9 publications

References 41 publications

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons

Identification of a Potential Neutralizing Linear Epitope of Hemagglutinin-neuraminidase in Newcastle Disease Virus

Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

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