P108 and T109 on E2 Glycoprotein Domain I Are Critical for the Adaptation of Classical Swine Fever Virus to Rabbits but Not for 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 demonstrated that P108 and T109 on the E2 glycoprotein (E2^P108-T109) in domain I (E2^DomainI) rather than R132, S133, and D191 in domain II (E2^DomainII) determine C-strain's adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197-206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2^P108-T109, E2^DomainI, or E2^DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVE^rns carrying the E^rns glycoprotein of C-strain were generated and evaluated. We found that E2^P108-T109 or E2^DomainI but not E2^DomainII of C-strain renders vSM-HCLVE^rns adaptive to rabbits, suggesting that E2^P108-T109 in combination with the E^rns glycoprotein (E2^P108-T109-E^rns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2^P108-T109-E^rns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2^P108-T109-E^rns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2^DomainII and E^rns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the E^rns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.