Adaptation of Tick-Borne Encephalitis Virus to BHK-21 Cells Results in the Formation of Multiple Heparan Sulfate Binding Sites in the Envelope Protein and Attenuation In Vivo

Abstract

Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E. The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)-dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.

Evaluation of Genetically Engineered Derivatives of a Chinese Strain of Foot-and-Mouth Disease Virus Reveals a Novel Cell-Binding Site Which Functions in Cell Culture and in Animals

“…4) that facilitates removal of viruses from the animal's circulation, preventing interaction with the specific cell types needed to produce vesicular disease. Taken together, our results support an expanding body of literature on the acquisition of HS binding by experimentally propagated viruses and the role of this altered receptor specificity in attenuation (10,12,13,25,30,33,34,38). In these cases, the ability to bind to HS appears to correlate with the finding that attenuated viruses are rapidly cleared from the circulation, preventing systemic spread and disease.…”

Clusteron structure of tick-borne encephalitis virus populations

“…This minimizes genetic artefacts caused by adaptation and/or replication by RNA polymerase. It is known that TBEV can change genetic and phenotypic properties relatively quickly, within 4-8 passages, during adaptation to model systems, either cell cultures, or laboratory animals (Mandl et al, 2001;Romanova et al, 2007). That's why the ''tick'' sequences of their genomes have the greatest interest for studying TBEV evolution.…”

“…Glycoprotein E homodimers is located parallel to the lipid membrane on the outer surface of the virus particles (Rey et al, 1995). It was shown that the majority of mutations that alter the properties of a pathogenic virus are grouped within domains of the envelope glycoprotein E (Mandl et al, 2001;Ternovoi et al, 2003;Hayasaka et al, 2004;Romanova et al, 2007). The nucleotide sequence corresponding to the N-terminus of the glycoprotein E, is variable and is therefore often used for genotyping TBEV isolates, for differential genetic diagnosis of TBE and in phylogenetic studies (Hayasaka et al, 1999;Ternovoi et al, 2003;Kovalev et al, 2009Kovalev et al, , 2010.…”

Molecular Basis for Adaptation of a Chimeric Dengue Type‐4/Japanese Encephalitis Virus to Vero Cells

“…Detailed information is shown for 5' NTR, E, and NS4B in Table 1, and the information of the remaining silent mutations is shown in Table 2. Compared with conservative amino acid substitutions, the nonconservative mutations are of major importance, because they confer a change in charge or side-chain polarity (15,18). Amino acid substitutions were designated as conservative or nonconservative mutations as previously described (30).…”