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. 1998 Feb 17;95(4):1746-51.
doi: 10.1073/pnas.95.4.1746.

Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4

A G Pletnev  1 , R Men
Affiliations

Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4

A G Pletnev et al. Proc Natl Acad Sci U S A. .

Abstract

Langat virus (LGT) strain TP21 is the most attenuated of the tick-borne flaviviruses for humans. Even though LGT has low-level neurovirulence for humans, it, and its more attenuated egg-passage derivative, strain E5, exhibit significant neurovirulence and neuroinvasiveness in normal mice, albeit less than that associated with tick-borne encephalitis virus (TBEV), the most virulent of the tick-borne flaviviruses. We sought to reduce or ablate these viral phenotypes of TP21 and E5 by using a strategy that had been used successfully in the past to reduce neurovirulence and abolish neuroinvasiveness of TBEV, namely substitution of structural protein genes of the tick-borne flavivirus for the corresponding genes of dengue type 4 virus (DEN4). In pursuit of these objectives different combinations of LGT genes were substituted into the DEN4 genome but only chimeras containing LGT structural proteins premembrane (preM) and envelope glycoprotein (E) were viable. The infectious LGT(preM-E)/DEN4 chimeras were restricted in replication in simian cell cultures but grew to moderately high titer in mosquito cell culture. Also, the chimeras were at least 5,000 times less neurovirulent than their parental LGT virus in suckling mice. Significantly, the chimeras lacked detectable evidence of neuroinvasiveness after i.p. inoculation of Swiss mice or the more permissive SCID mice with 10(5) or 10(7) plaque-forming units (PFU), respectively. Nonetheless, i.p. inoculation of Swiss mice with 10 or 10(3) PFU of either chimeric virus induced LGT neutralizing antibodies and resistance to fatal encephalitis caused by i.p. challenge with LGT TP21. The implications of these observations for development of a live attenuated TBEV vaccine are discussed.

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Figures

Figure 1
Figure 1
Growth of parental and chimeric viruses in simian and mosquito cells. Chimeric virus inocula were grown in mosquito cells, parental LGT TP21 and E5 virus inocula were grown in simian Vero cells, whereas DEN4 parental virus inoculum for mosquito cells was grown in mosquito cells and DEN4 inoculum for simian cells was grown in simian Vero cells. Simian LLCMK2 or Vero cells were infected with: (i) DEN4, TP21, or E5 virus at MOI of 0.01 or (ii) chimeric TP21/DEN4 or E5/DEN4 virus at MOI of 0.5. Mosquito C6/36 cells were infected with: (i) DEN4, TP21/DEN4, or E5/DEN4 virus at MOI of 0.01 or (ii) with TP21 or E5 virus at MOI of 1,000. Cells were harvested at indicated time (day after infection), and the virus titer was determined by a plaque assay on the same cells used for study of virus replication. Plaques were enumerated after 7 or 8 days of infection. In other experiments complete restriction of LGT TP21 and E5 in mosquito cells also was observed when MOI ranging from 0.01 to 1,000 were used.
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