Study and Application of Targeted Nanopore Sequencing for Swine Hepatitis E Virus Genome

Swine hepatitis E virus (sHEV) is a major zoonotic pathogen. In China, HEV genotype 4 predominates in pig herds, and the virus can transmit bidirectionally between pigs and humans, posing a threat to public health security. Current HEV detection assays have limited genome coverage and are incompatible with long-read sequencing platforms, and an efficient targeted enrichment system for the full-length sHEV genome is still lacking. In this study, we designed a set of overlapping degenerate primers based on the sequence characteristics of HEV genotypes 3 and 4 and divided them into five primer pools. A targeted multiplex RT-PCR combined with nanopore sequencing method was established for full-genome analysis of sHEV and validated using two imported swine fecal swab samples. All five primer pools successfully amplified the target viral fragments. The method achieved near-complete genome acquisition at a viral RNA concentration of 10 copies/μL, with a genome coverage rate of 84.83% at this concentration, whereas viral reads remained detectable down to 1 copy/μL, although complete genome coverage was not obtained at this level. The two tested samples shared identical viral sequences, which were identified as HEV subtype 3e by phylogenetic analysis. The established method features high sensitivity and efficient genome capture, and holds potential for full-genome sequencing, genotyping and molecular tracing of sHEV, with potential application value for samples collected at ports and on farms. This work provides technical support for surveillance, variation analysis and prevention and control of swine hepatitis E.