Infectious cDNA clone of attenuated langat tick-borne flavivirus (strain E5) and a 3 ' deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice

Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat ( LGT) strain TP21, was recovered from ticks in Malaysia. subsequently it was tested as a live attenuated vaccine for virulent tick-borne encephalitis v iruses. In a large clinical trial its attenuation was confirmed but there w as evidence of a low level of residual virulence. Thirty-five years ago fur ther attenuation of LGT TP21 was achieved by multiple passages in eggs to y ield mutant E5. To study the genetic determinants of the further attenuatio n exhibited by E5 and to allow us to manipulate the genome of this virus fo r the purpose of developing a satisfactory live attenuated tick-borne flavi virus vaccine, we recovered infectious E5 virus from a full-length cDNA clo ne. The recombinant E5 virus (clone 651) recovered from a full-length infec tious cDNA clone was more attenuated in immunodeficient mice than that of i ts biologically derived E5 parent. Increase in attenuation was associated w ith three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion i n the 3 ' -noncoding region of infectious full-length E5 cDNA. This deletio n mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3 ' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119.750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation , the E5 3 ' deletion mutant remained highly immunogenic and intraperitonea l (ip) inoculation of 10 PFU induced complete protection in Swiss mice agai nst subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.