32nd International Dynamics & Evolution of Human Viruses

NOVEL STRATEGY FOR WHOLE-GENOME SEQUENCING OF HEPATITIS A VIRUS USING NGS-ILLUMINA TECHNOLOGY AND PHYLOGENETIC COMPARISON WITH PARTIAL VP1/2A GENOMIC REGION

Not scheduled

20m

Abbaye de Royaumont, Asnières-sur-Oise, France

Poster Software, tools & methods Virtual posters

Speaker

Maria Belen Pisano (Instituto de Virologia Dr. Vanella, Facultad de Ciencias Medicas, Universidad Nacional de Cordoba)

Description

Molecular epidemiology of hepatitis A virus (HAV) plays a critical role in identifying outbreak origin and conducting surveillance. Although it is mostly carried out using short partial VP1/2A genomic sequences, utilizing whole-genome sequences (WGS) provides more accurate and robust information. In Argentina, where HAV vaccination is mandatory since 2005, the local sequence information is scarce and only one complete genome is available to date.
We aimed to develop a strategy for HAV whole-genome sequencing by adapting the NGS Illumina COVIDSeq test, to obtain HAV WGS from central Argentina, and to compare phylogenetic analyses of WGS with partial VP1/2A genomic region.
Twenty-five primer pairs were designed utilizing the Primal Scheme program (https://primalscheme.com/), and used to amplify partial genomic fragments (400bp) that comprise the entire HAV genome sequence. 16 previously HAV positive plasma (n=11) and stool samples (n=5) from Argentina were subjected to nucleic acid extraction with the High Pure Viral Nucleic Acid kit (Roche), DNA library preparation using the Illumina COVIDSeq Test and sequencing in a MiSeq equipment. Bioinformatic analysis were performed with BWA, SAMtools e iVar, and phylogenetic analyses with IQ-Tree and MEGA using WGS obtained and VP1/2A partial sequences of 1084pb and 422pb.
Eleven samples could be amplified and sequenced, with coverage between 79.3%-100% (>90% in 9 samples). The likelihood tree topology tests showed that the partial sequences trees had significantly lower likelihoods than the WGS tree (mainly the shorter sequences). However, phylogenetic analyses of partial sequences allowed genotype identification and an overview of phylogenetic relationships (with reduced phylogenetic support of clades).
The amplicon-based NGS WGS tool developed by adapting the COVIDSeq test to HAV proved to be efficient in generating new complete and near-complete viral sequences. The study of partial 1084bp VP1/2A region would constitute a useful alternative for outbreak investigation and surveillance when WGS is not possible.