Long-term passage of duck Tembusu virus in BHK-21 cells generates a completely attenuated and immunogenic population with increased genetic diversity

Zhang, Lijiao; Sun, Mengxu; Zhang, Qingshui; Wang, Jun; Cao, Yunyun; Cui, Shangjin; Su, Jingliang

doi:10.1016/j.vaccine.2019.10.080

Cited by 9 publications

(25 citation statements)

References 46 publications

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“…We also assessed the pathogenicity by infecting a group of 4-day-old ducklings in each group, as described above. Consistent with our previous study, ducklings infected with WT virus exhibited depression, loss of food uptake, and ataxia from 3 dpi, resulting in a mortality rate of 70% (36), whereas none of the ducklings infected with ChimE virus showed evident signs of illness throughout the test period. These results suggested that mutations in the E protein impaired virus replication in the blood, brain, and heart of ducklings, leading to virulence attenuation.…”

Section: Resultssupporting

confidence: 91%

“…In our previous study, a chimeric virus (ChimE) was generated by introducing the mutated E genes of the attenuated P310-4C virus (a clone of the 310th passaged TMUV) into the backbone of a virulent TMUV strain (wild type [WT]). When subcutaneously (s.c.) injected into ducklings, this chimeric virus showed significant virulence attenuation compared with WT virus (36). However, it was unclear which stage(s) was affected by the mutated E gene during the TMUV infection process.…”

Section: Resultsmentioning

confidence: 99%

“…We recently developed a highly attenuated TMUV vaccine candidate, P310, by sequential passaging in BHK-21 cells, resulting in a total of 23 amino acid substitutions during the adaptive process (36). The chimeric virus with the mutated E gene showed increased growth in BHK cells and significantly reduced pathogenicity in subcutaneously (s.c.) inoculated ducklings, highlighting the importance of these mutations to the phenotypic changes of attenuated TMUV.…”

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confidence: 99%

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Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis

Sun

Zhang

Cao

et al. 2020

J Virol

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Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV. IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis

Sun

Zhang

Cao

et al. 2020

J Virol

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“…Different variations may occur when producing large quantities (bulk) in the laboratory. Due to the low sensitivity of RNA-bound RNA polymerase, RNA viruses always produce a pool of variants during replication (Zhang et al, 2020). This phenomenon provides a potential for the rapid evolution of the virus, but it also makes up the majority of mutations that have detrimental effects on virus stability.…”

Section: Discussionmentioning

confidence: 99%

Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates

Karakuş

Taştan

Kançağı

et al. 2020

Preprint

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The COVID-19 outbreak caused by SARS-CoV-2 has created an unprecedented health crisis since there is no coronavirus vaccine in the market due to the novelty of this virus. Therefore, SARS-CoV-2 vaccines have become very important to reduce morbidity and mortality. At this point, inactivated vaccines are important because the straightforward process of existing infrastructure used for several licensed human vaccines can be used for SARS-CoV-2. Inactive vaccines provide an antigenic presentation similar to that when they encounter invasive virus particles of the immune system. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. Our candidate OZG-3861 version 1 (V1) is an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1) is the GM-CSF adjuvant added vaccine candidate. We applied the candidates intradermal to BALB/c mice to assess the toxicity and immunogenicity of the OZG-3861 V1 and SK-01 V1. Here, we report our preliminary results in vaccinated mice. When considered in terms of T and B cell responses, it was observed that especially the vaccine models containing GM-CSF as an adjuvant caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature. Another finding showed that the presence of adjuvant is more important in T cell response rather than B cell. The vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study encouraged us to start the challenge test using infective SARS-CoV-2 viruses and our second version of gamma-irradiated inactivated vaccine candidates in humanized ACE2+ mice.

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“…Subsequently, it was reported in Malaysia (Homonnay et al, 2014) and Thailand (Thontiravong et al, 2015). Since the emergence of the disease, different types of TMUV vaccine candidates have been developed in China, such as live-attenuated vaccines (Li et al, 2014;Wang et al, 2016;He et al, 2019;Huang et al, 2019;Zhang et al, 2020), inactivated vaccines (Lin et al, 2015;Zhang et al, 2017;Liu et al, 2018), subunit vaccines (Zhao et al, 2015;Ma et al, 2016), recombinant duck enteritis virus-, Newcastle disease virus-, and adenovirusvectored vaccines (Chen et al, 2014;Zou et al, 2014Zou et al, , 2017Sun et al, 2018;Tang et al, 2019), and DNA vaccines (Huang et al, 2018a,b;Tang et al, 2018). Among them, live-attenuated TMUV WFG36 (Wang et al, 2016) and FX2010 (Li et al, 2014) vaccines and inactivated TMUV HB vaccine (Liu et al, 2018) have been licensed to use in ducks in China.…”

Section: Introductionmentioning

confidence: 99%

The Neutralizing Antibody Response Elicited by Tembusu Virus Is Affected Dramatically by a Single Mutation in the Stem Region of the Envelope Protein

Liu

Cui

et al. 2020

Front. Microbiol.

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Long-term passage of duck Tembusu virus in BHK-21 cells generates a completely attenuated and immunogenic population with increased genetic diversity

Cited by 9 publications

References 46 publications

Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis

Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis

Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates

The Neutralizing Antibody Response Elicited by Tembusu Virus Is Affected Dramatically by a Single Mutation in the Stem Region of the Envelope Protein

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