The pellet was resuspended in 10 mM Tris (pH 7.5) containing 5 mM MgCl 2 and 180 units of benzonase and incubated for 30 min at room temperature. When 90 to 100% cytopathic effect was observed, the supernatant and cells were collected and centrifuged at 300 × g for 10 min the cell pellet was washed and resuspended in 10 mM Tris (pH 7.5) with protease inhibitor cocktail, homogenized, and centrifuged at 3000 × g. Briefly, Vero cells were infected at a multiplicity of infection (MOI) of 10 −2 PFU/cell for 1 h at 37☌. Genomic DNA from HSV-1 clinical strain (B 3x 1.1) ( 18) was isolated following the protocol optimized earlier ( 19). We recently developed a method to extract intact full-length genomic DNA from HSV-1 and HSV-2, followed by sequencing and capturing their genome as a single read by ONT. In this observation, we performed a hybrid assembly to obtain the genome sequence of an HSV-1 clinical strain using both ONT long reads and Illumina short reads. Furthermore, obtaining a full-length accurate HSV genome will be valuable for studying determinants of drug resistance, virulence, pathogenesis, and viral evolution ( 16). Because some of the replication- and virulence-related genes are present in repeat regions, capturing both copies of repeat elements is important to understand the process, but this is possible only with the full-length genome sequence ( 8). ( 17) identified the DNA isomers of HSV-2 strain G by ONT but reported them as partial genome fragments. In addition, short reads would need extensive coverage and computational processing and thus, cannot be reliably assembled to generate a full-length HSV-1 genome. Capturing HSV variants with their nucleotide sequence has been a challenge using short-read shotgun sequencing platforms due to extremely high GC content (70 to 85%) in some regions, such as the inverted repeats of the genome genetic variations and complexities associated with isolating intact viral genomic DNA ( 12 – 16). The presence of these isomers was identified earlier by restriction digestion, southern blotting, and probe-based methods ( 7 – 11). Although HSV-1 has been reported to form genome isomers because of recombination events during replication, the significance of these isomers in virulence or viral fitness is not completely understood ( 6). The termini of unique long (U L) and short (U S) regions are flanked by the terminal and internal repeat of the long region (TRL and IRL) and the terminal and internal repeat of the short region (IRS and TRS) ( Fig. 1A) ( 4, 5). The genome of HSV-1 is complex and comprised of unique long (U L) and unique short (U S) regions flanked by repeat elements on both ends. HSV-1 is widespread in the human population, causing mild to severe skin and neurological infections ( 1 – 3). Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus that belongs to the Alphaherpesvirinae subfamily within the Herpesviridae family.
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