Molecular Mechanism Of The E2-mediated Adaptation Of Classical Swine Fever Virus C-strain To Rabbits

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?HCLV?developed by hundreds of passages in rabbits in the 1950s.In contrast to the highly virulent Shimen strain,C-strain is adaptive to rabbits?ATR?and attenuated in pigs.Previously,we demonstrated that the E2 glycoprotein is crucial for C-strain ATR.However,the mechanism of C-strain ATR mediated by the E2 glycoprotein retatins elusive.And whether the amino acids associated with ATR of CSFV affect the virulence in pigs?To investigate the mechanism of C-strain ATR mediated by the E2 glycoprotein,chimeric viruses based on C-strain were generated for determining the exact amino acids on the E2 glycoprotein.Rabbit experiments showed that the Domain I on the E2 is the determinant for C-strain ATR.Next,we focused on the different amino acids on the Domain I and found that three amino acids?K105/P108/T109?determined the ATR of C-strain.To further investigate the effects of these amino acids on the ATR of C-strain,we constructed a series of mutant viruses.According to the rabbit experiment results,we demonstrated that P108 and T109 are crucial for the ATR of C-strain.Then,three chimeric viruses containing the E2^P108-T109,E2^DomainI or E2^DomainII of C-strain in the backbone of the non-rabbit-adaptive Shimen mutant vSM-HCLVE^rns were generated and evaluated for CSFV ATR.Rabbit experiments show that the E2^P108-T109 or E2^DomainIomainI but not E2^DomainII of C-strain could render vSM-HCLVE^rns to ATR,suggesting that the E2^P108-T109-E^rns of C-strain confer the Shimen strain ATR.Meanwhile,rabbit spleen lymphocytes were identified as target cells of C-strain.Next,primary rabbit spleen lymphocytes were infected with parental viruses?the Shimen strain and C-strain?and chimeric viruses(vSM-HCLVE^rnsE2^DomainI,vSM-HCLVE^rnsE2^DomainIIomainII and vSME2^L108P-I109T-HCLVE^rns).The results showed that viral genome copy numbers of rabbit-adaptive CSFV mutants(vSME2^L108P-I109T-HCLVE^rns,vSM-HCLVE^rnsE2^DomainI)or the parental virus C-strain increased as time went on.In contrast,the viral genome copy numbers of non-rabbit-adaptive CSFVs(vSM-HCLVE^rnsE2^DomainII or the Shimen strain)decreased as time went on,further demonstrating that chimeric viruses vSME2^L108P-I109T-HCLVE^rnsns or vSM-HCLVE^rnsE2^DomainI but not vSM-HCLVE^rnsE2^DomainII are adaptive to primary rabbit spleen lymphocytes.Furthermore,primary swine macrophages were inoculated with a series of chimeric viruses to analyze the growth profiles.The viral genome copy numbers of the Shimen strain and three chimeric viruses but not C-strain were indistinguishable in primary swine macrophages.However,the viral titers in the supernatants of the macrophages infected with the Shimen strain were higher than those infected with chimeric viruses.Since the E2 glycoprotein has been reported to be responsible for viral entry and tropism,a series of pseudotyped viruses?pps?bearing the E^rns,E1 and E2 glycoproteins from the Shimen strain?SMpps?,C-strain?HCLVpps?,vSM-HCLVE^rnsE2^DomainI(SME1-HCLVE^rnsE2^DomainIpps),vSM-HCLVE^rnsE2^DomainII(SME1-HCLVE^rnsE2^DomainIIpps)and vSME2^L108P-I109T-HCLVE^rns(SME1E2^L108P-I109T-HCLVE^rnspps)were generated to investigate whether their differences occur in viral entry.The results showed that pseudotyped viruses based on rabbit-adaptive but not non-rabbit-adaptive can enter rabbit spleen lymphocytes,indicating that C-strain ATR results from the improved entry efficiency.However,these pseudotyped viruses could enter swine SK6 cells with similar efficiency.Finally,whether the key amino acids contributing to the viral ATR affect viral virulence in pigs was investigated.The pigs experiment showed that E2^P108-T109-E^rns of C-strain contribute to the viral ATR but do not alter the virulence of the Shimen strain in pigs,whereas the E2^DomainII-E^rnsns of C-strain attenuate the Shimen strain.Our results provide vital information on the different molecular basis of CSFV adaptation to rabbits and attenuation in pigs.This study also implies that novel live attenuated vaccines against CSF may be developed by targeting genetic modifications instead of random evolution through blind cell passage.