In Vitro Replication of Swine Hepatitis E Virus (HEV): Production of Cell-Adapted Strains

SIMPLE SUMMARY: The zoonotic virus of HEV-3 genotype is further classified in different subtypes whose biological role is still not clear. Most information available on the virus, both on genetic features and on replicative cycle, had been obtained by studying the genome characteristics and by performing in vitro transfection of permissive cells using replicative clones of the viruses. The lack of an efficient cell culture for HEV cultivation has hampered the study of the virus so far. In the present study, the protocol for HEV cultivation on human A549 lung cells previously established was used successfully to cultivate different subtypes of HEV-3 isolated from pig faeces. The different isolates grew similarly. Sequence analyses was performed on isolates, at different days post infection and in the following passages on cells. Analyses excluded the presence of any insertion in the hypervariable region of the genome, as observed in previous studies, and revealed a few mutations acquired in the viral genomes during the growth on cells. The protocol of HEV-3 cell cultivation was used for a quick production of a high amount of the virus in serum free medium, not requiring further purification. The obtained isolates will be used for future experiments of virus infectivity. ABSTRACT: The hepatitis E caused by the virus HEV of genotypes HEV-3 and HEV-4 is a zoonotic foodborne disease spread worldwide. HEV is currently classified into eight different genotypes (HEV-1–8). Genotypes HEV-3 and HEV-4 are zoonotic and are further divided into subtypes. Most of the information on HEV replication remains unknown due to the lack of an efficient cell cultivation system. Over the last couple of years, several protocols for HEV cultivation have been developed on different cell lines; even if they were troublesome, long, and scarcely reproducible, they offered the opportunity to study the replicative cycle of the virus. In the present study, we aimed to obtain a protocol ready to use viral stock in serum free medium that can be used with reduced time of growth and without any purification steps. The employed method allowed isolation and cell adaptation of four swine HEV-3 strains, belonging to three different subtypes. Phylogenetic analyses conducted on partial genome sequences of in vitro isolated strains did not reveal any insertion in the hypervariable region (HVR) of the genomes. A limited number of mutations was acquired in the genome during the virus growth in the partial sequences of Methyltransferase (Met) and ORF2 coding genes.