Rescue of recombinant canine distemper virus that expresses S1 subunit of SARS-CoV-2 spike protein in vitro

Front. Cell. Infect. Microbiol.

Wang

et al. 2022

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Canine distemper and canine parvoviral enteritis are infections caused by the canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2), respectively. They are two common infectious diseases that cause high morbidity and mortality in affected dogs. Combination vaccines have been broadly used to protect dogs from infections of CDV, CPV-2, and other viruses. VP2 is the most abundant protein of the CPV-2 capsid. It elicits potent immunity in animals and, therefore, is widely used for designing subunit antigen-based vaccines. In this study, we rescued a recombinant CDV (QN vaccine strain) using reverse genetics. The recombinant CDV (rCDV-VP2) was demonstrated to express stably the VP2 in cells for at least 33 serial passages in vitro. Unfortunately, a nonsense mutation was initially identified in the VP2 open reading frame (ORF) at passage-34 (P34) and gradually became predominant in rCDV-VP2 quasispecies with passaging. Neither test strip detection nor indirect immunofluorescence assay demonstrated the expression of the VP2 at P50. The P50 rCDV-VP2 was subjected to next-generation sequencing, which totally identified 17 single-nucleotide variations (SNVs), consisting of 11 transitions and 6 transversions. Out of the 17 SNVs, 1 and 9 were identified as nonsense and missense mutations, respectively. Since the nonsense mutation arose in the VP2 ORF as early as P34, an earlier rCDV-VP2 progeny should be selected for the vaccination of animals in future experiments.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recovery of Recombinant Canine Distemper Virus That Expresses CPV-2a VP2: Uncovering the Mutation Profile of Recombinant Undergoing 50 Serial Passages In Vitro

Liu

Front. Cell. Infect. Microbiol.

Wang

et al. 2022

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“…: MT236316) were optimized for codon usage bias in dogs using an online codon-optimizing tool ( https://www.vectorbuilder.cn/tool/codon-optimization.html ), followed by chemical synthesis. These two codon-optimizing sequences were independently subcloned into the Not I/ Pme I sites of another CDV cDNA clone ( 25 ) via homologous recombination using the In-Fusion® Kit (Takara, Dalian, China) according to the manufacturer's instruction. Two recombinant cDNA clones ( Figures 1C,D ) were subjected to Sanger sequencing for confirming their identities, followed by plasmid extraction using the HighPure Maxi Plasmid Kit (TIANGEN, Beijing, China) according to the manufacturer's instruction.…”

Section: Methodsmentioning

confidence: 99%

“…Unfortunately, there has been no report concerning CDV-vectored vaccines against DBV as yet. We had developed one virulence-attenuating strain (CDV QN strain) previously, and more recently, we constructed its reverse genetics platform for expressing foreign antigens ( 24 , 25 ). Considering anti-DBV vaccines unavailable for dogs, we rescued two recombinant CDVs in the present study.…”

Section: Introductionmentioning

confidence: 99%

Recovery of Two Replication-Competent Canine Distemper Viruses That Separately Express Dabie Bandavirus Gn and Gc

Lyu

et al. 2022

Front. Vet. Sci.

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Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis with a high mortality rate in humans. Additionally, dogs are frequently reported to be infected with this disease. There has been no commercially available vaccine for humans and animals as yet. The SFTS is caused by Dabie bandavirus (DBV), formerly known as SFTS virus. The DBV is now classified into the genus Bandavirus in the family Phenuiviridae. DBV Gn and Gc can induce specific immune responses in vivo. In this study, we used reverse genetics technique to construct two recombinant canine distemper viruses (rCDVs), rCDV-Gn and -Gc, which could express Gn and Gc in vitro, respectively. Both of the recombinants, derived from a common parental CDV, were independently subjected to twenty serial passages in cells for Sanger sequencing. Neither point mutation nor fragment deletion was found in the Gn open reading frame (ORF), whereas the rCDV-Gc showed a nonsynonymous mutation (A157C) in the Gc ORF, correspondingly resulting in a mutation of amino acid (T53P) in the Gc. Growth curve of the rCDV-Gc almost coincided with that of a wild-type CDV, but exhibited a significant difference from that of the rCDV-Gn. Much research remains to be performed to demonstrate whether both recombinants are able of inducing specific immune responses in vivo.

“…We have previously constructed two reverse genetics platforms for different CDV strains. Both platforms facilitate recovery of marker-tagged ( Liu et al., 2020 ) or antigen-expressing ( Liu et al., 2021 ) recombinant CDV. In this study, a recombinant eGFP-tagged CDV (rCDV-eGFP) was rescued, identified, characterized and then used as a model virus for extra forty passages separately in ribavirin- and non-treated cells.…”

Section: Introductionmentioning

confidence: 99%

Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles

Liu

Wang

Front. Cell. Infect. Microbiol.

et al. 2021

Self Cite

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.