Rescue of Senecavirus A to uncover mutation profiles of its progenies during 80 serial passages in vitro

Lin

Wang

et al. 2021

Microbial Pathogenesis

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The coronavirus disease 2019 (COVID-19), as an unprecedented pandemic, has rapidly spread around the globe. Its etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus Betacoronavirus in the family Coronaviridae . The viral S1 subunit has been demonstrated to have a powerful potential in inducing protective immune responses in vivo . Since April 2020, farmed minks were frequently reported to be infected with the SARS-CoV-2 in different countries. Unfortunately, there has been no available veterinary vaccine as yet. In this study, we used reverse genetics to rescue a recombinant canine distemper virus (CDV) that could express the SARS-CoV-2 S1 subunit in vitro . The S1 subunit sequence was demonstrated to be relatively stable in the genome of recombinant CDV during twenty serial viral passages in cells. However, due to introduction of the S1 subunit sequence into CDV genome, this recombinant CDV grew more slowly than the wild-type strain did. The genomic backbone of recombinant CDV was derived from a virulence-attenuating strain (QN strain). Therefore, if able to induce immune protections in minks from canine distemper and COVID-19 infections, this recombinant would be a potential vaccine candidate for veterinary use.

Section: Methodsmentioning

confidence: 99%

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

Lin

Wang

et al. 2021

Microbial Pathogenesis

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“…A total of five RNA samples were separately reverse transcribed using the 1st Strand cDNA Synthesis Kit (Takara, Dalian, China) and random hexamers, according to the manufacturer's instruction. The Illumina sequencing and library construction were performed as descried previously ( 18 ). In brief, the NEBNext® Ultra™ II RNA Library Prep Kit (NEB, Ipswich, MA, USA) was used for library construction.…”

Section: Methodsmentioning

confidence: 99%

“…Processing and analysis of NGS data were also carried out as descried previously ( 18 ). In brief, raw reads were filtered by fastp ( https://github.com/OpenGene/fastp ) to remove sequencing adapters and low-quality reads, including those reads scored < Q20.…”

Section: Methodsmentioning

confidence: 99%

“…As an alternative technique, the next-generation sequencing (NGS) has been broadly applied to analyze and to quantify the exceptionally-high diversity within viral quasispecies. By means of it, single-nucleotide polymorphisms (SNPs) in a variety of viruses can be systematically analyzed to reveal their evolutionary profiles (14)(15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

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Mutation Profiles of eGFP-Tagged Small Ruminant Morbillivirus During 45 Serial Passages in Ribavirin-Treated Cells

Zou

et al. 2021

Front. Vet. Sci.

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Small ruminant morbillivirus (SRMV), formerly known as peste-des-petits-ruminants virus, classified into the genus Morbillivirus in the family Paramyxoviridae. Its L protein functions as the RNA-dependent RNA polymerases (RdRp) during viral replication. Due to the absence of efficient proofreading activity in their RdRps, various RNA viruses reveal high mutation frequencies, making them evolve rapidly during serial passages in cells, especially treated with a certain mutagen, like ribavirin. We have previously rescued a recombinant enhanced green fluorescence protein-tagged SRMV (rSRMV-eGFP) using reverse genetics. In this study, the rSRMV-eGFP was subjected to serial passages in ribavirin-treated cells. Due to the ribavirin-exerted selective pressure, it was speculated that viral progenies would form quasispecies after dozens of passages. Viral progenies at passage-10, -20, -30, -40, and -50 were separately subjected to next-generation sequencing (NGS), consequently revealing a total of 34 single-nucleotide variations, including five synonymous, 21 missense, and one non-sense mutations. The L sequence was found to harbor eight missense mutations during serial passaging. It was speculated that at least one high-fidelity variant was present in viral quasispecies at passage-50. If purified from the population of viral progenies, this putative variant would contribute to clarifying a molecular mechanism in viral high-fidelity replication in vitro.

“…The expressions of Gn and Gc were separately analyzed by mass spectrometry (MS) at the Shanghai Biopro le Biotechnology Co., Ltd (Shanghai, China), as described previously (Liu et al 2021). In brief, culture supernatants of P10 progenies were inactivated by 0.1% formalin at 4 °C for 48 h. Protein digestion was performed with a method of filter-aided sample preparation (Wiśniewski et al 2009).…”

Section: Mass Spectrometrymentioning

confidence: 99%

Rescue of Two Recombinant Canine Distemper Viruses That Separately Express Dabie Bandavirus Gn and Gc in Vitro

Lin

Wang

et al. 2021

Preprint

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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 to construct two recombinant canine distemper viruses (rCDV), rCDV-Gn and -Gc, which could express Dabie bandavirus Gn and Gc in vitro, respectively. Two foreign sequences, Gn and Gc open reading frames, were genetically stable during twenty serial viral passages in cells. Growth curve of the rCDV-Gc basically coincided with that of a wild-type CDV, but showed a significant difference from that of the rCDV-Gn. The rCDV-Gn and -Gc were derived from a common parental CDV, the virulence-attenuating QN strain. Therefore, if proven to be efficient in resisting both canine distemper and SFTS in dogs, either or both recombinant CDVs would be potential vaccine candidates.