Identification of two novel ephemeroviruses in pigs infected by classical swine fever virus

Ephemeroviruses are arthropod-borne rhabdoviruses within Ephemerovirus genus and have been isolated exclusively from cattle and haematophagous arthropods (mosquitoes and biting midges) without any member detected or isolated up to date from pigs, although some serological surveys have indicated that pigs may be a silent host for ephemerovirus infection. Here, many viral reads annotated to, but genetically distinct from, the existing members within the Ephemerovirus genus have been identified in the meta-transcriptomic data of two clinical classical swine fever virus (CSFV)-infected samples (HeN10 and GDMM7). The nearly complete genome sequences of the two novel ephemeroviruses have been obtained through contig assembly, specific RT-PCR and sequencing, therefore named as porcine ephemeroviruses (PoEVs). Genome nucleotide sequence analysis showed that PoEV strains HeN10 and GDMM7 have similar genome organization and 66.5% genomic identity to each other, but both are genetically distant from all members of the Ephemerovirus genus with identity being only 51.1–59.6%. Furthermore, comparison of the most conserved ephemeroviral proteins N and L indicated that PoEV strains HeN10 and GDMM7 share a high sequence identity to each other (N: 78.1%; L: 70.7%), but are diverged from the known ephemeroviruses (N: 43.4–60.7%; L: 47.6–58.5%). The genetic distance is significantly beyond the criteria for demarcation of viruses assigned to different ephemerovirus species. Thereby, two novel viruses named as PoEV1 (strain HeN10) and PoEV2 (strain GDMM7) are identified and these appear to represent two new species within the Ephemerovirus genus. The present study showed the first genome evidence of pig ephemeroviruses, likely expanding the known host range of ephemerovirus.