Adaptation of enterovirus to vero cells and vaccine formulations thereof
An enterovirus, cell technology, applied in the direction of antiviral agents, viruses, vaccines, etc., can solve undisclosed problems
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Embodiment 1
[0089] Example 1: Virus Propagation
[0090] Vero cells were infected with a high infectious dose of enterovirus D68 strain. Initially, let the virus adsorb on T25 cm 2 The flask was shaken occasionally for about 60 to 120 min, and then DMEM was added as maintenance medium. Use the virus stock obtained from each passage at a dilution of approximately 1:3-1:10 to infect subsequent batches for the initial passage. In subsequent / subsequent passages, infection was performed with virus at higher dilutions (range 1:20-1:500) using virus stocks from previous passages. Virus was harvested when a cytopathic effect of 90% or greater was achieved or within 6 days of infection, whichever was earlier. The entire reproduction process of enterovirus D68 is carried out at a temperature not higher than 35°C and ideally 32-33°C. The titer of virus in different passages was determined by TCID50 assay and / or plaque assay, and as figure 2 shown.
[0091] At a controlled temperature in the r...
Embodiment 2
[0092] Example 2: RNA isolation and RT-PCR
[0093] RNA has been isolated from post-harvest cell-free supernatants with infected virus by the Trizol method according to standard procedures. Briefly, 750 μl Trizol was used in 250 μl virus-containing supernatant. If it was desired to obtain large amounts of viral nucleic acid material, the viral supernatant was concentrated by ultrafiltration through PEG6000 or PEG8000 (Merck, India) or using a 100 kDa cut-off membrane (Merck, India) prior to the Trizol isolation method. The isolated RNA was reverse transcribed into complementary cDNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Confirm the presence of viral RNA using virus-specific primers, such as image 3 shown. Primer sequences are provided below:
[0094] Enterovirus D68-specific primers (Calvo et al., Pediatr Infect Dis J 2016, 35:45-49):
[0095] (F)-1: GTTCYTTAATAGGRTTCRTAGCAGC
[0096] (R)-1:CTCTATTRCCAATTATGGCATTRAG
[0...
Embodiment 3
[0100] Example 3: Sequencing
[0101] For whole-genome sequencing, use an Illumina-compatible UltraTM DirectionalRNALibrary Prep Kit (New England BioLabs, MA, USA) prepared RNA sequencing library. Sequencing libraries were initially quantified by a Qubit fluorometer (Thermo Fisher Scientific, MA, U.S.A.) and their fragment size distributions were analyzed on an Agilent TapeStation. Finally, the sequencing library was accurately quantified by quantitative PCR using the Kapa Library Quantification Kit (KapaBiosystems, Wilmington, MA, U.S.A.). qPCR-quantified libraries were pooled in equimolar amounts to create the final multiplex library pool for sequencing on the Illumina NextSeq (150x 2 chemistry).
[0102] Table 1 below provides the amino acid differences and corresponding nucleotide level differences between the parental enterovirus D68 strain US / KY / 14-18953 and the virus strain US / KY / 14-18953-Vero adapted to Vero cells:
[0103]
[0104]
[0105] Table 1: Sequenci...
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