P108 and T109 on the E2 glycoprotein Domain I are critical for the adaptation of classical swine fever virus to rabbits but not the virulence in pigs.

The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs in contrast with the highly virulent CSFV Shimen strain. Previously, we have demonstrated that P108 and T109 on the E2 glycoprotein Domain I (E2P108-T109 on the E2DomainI) rather than R132, S133 and D191 on the Domain II (E2DomainII) determine C-strain's adaptation to rabbits (ATR). However, it remains elusive that whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring the E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that the E2P108-T109 or E2DomainI but not E2DomainII of C-strain render vSM-HCLVErns to be adaptive to rabbits, suggesting that the E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confer the Shimen strain ATR, creating new rabbit-adaptive CSFVs. Mechanistically, the E2P108-T109-Erns of C-strain mediate viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that the E2P108-T109-Erns of C-strain do not affect viral virulence compared with the Shimen strain. Conversely, the substitution of the E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and virulence in pigs are not overlapping. Our findings provide new insights into the adaptation mechanism of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually lead to adaptation in cell cultures or non-susceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to non-susceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that the P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of rabbit-adaptive C-strain confer the adaptation of the highly virulent CSFV Shimen strain to rabbits by affecting viral entry during infection, but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular basis of CSFV adaptation to rabbits and attenuation in pigs.