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. 2009 Oct 5;4(10):e7295.
doi: 10.1371/journal.pone.0007295.

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

Maxim A Khasnatinov  1 , Katarina Ustanikova, Tatiana V Frolova, Vanda V Pogodina, Nadezshda G Bochkova, Ludmila S Levina, Mirko Slovak, Maria Kazimirova, Milan Labuda, Boris Klempa, Elena Eleckova, Ernest A Gould, Tamara S Gritsun
Affiliations

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

Maxim A Khasnatinov et al. PLoS One. .

Abstract

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic analysis of Yar strains.
MEGA version 4 was used to align E genes (between nucleotide positions 1114–2223 of Vs virus genome) of SIB TBEV strains (accession numbers are specified). Tree topology was reconstructed by Neighbor-Joining. The Tamura-Nei model was used for estimation of evolutionary distances . Bootstraps were based on 1000 replications; values below 90% are hidden. The scale bar shows the number of nucleotide substitutions per site. Geographic origins of SIB strains and clusters I, II and III are shown on the right hand side of the tree. Yar strains are highlighted using triangles. KFDV was used as the outgroup.
Figure 2
Figure 2. Identification of HA-disabling mutations.
(A). Abbreviated comparative alignment of TBEV E protein sequences (complete version is available on request). TBEV strains are specified by subtype (FE-, SIB- or WE-subtypes) and GenBank accession numbers. HA-disabling mutations are encircled. The “tick-specific” amino acids that differentiate I. persulcatus-transmitted viruses (shadowed) from the I. ricinus-transmitted viruses are highlighted in yellow; those that increase hydrophobicity are boxed and surface-faced amino acids are marked with asterisks (*). The fusion peptide is underlined. (B) Mapping of HA-disabling residues onto native dimeric conformation of E protein crystal structure (1SVB.pdb); the monomer is shown as it lies on the virion membrane. The “persulcatus” and “ricinus” residues are highlighted in orange; fusion peptide is in green and HA-deficient amino acids (arrows) are red. The position of Yar substitution D277A (red coloured) coincides with the position of “ricinus/persulcatus” substitution (orange colour is masked). (C) Residues 67, 122 and 277 (purple spheres) are mapped onto the E protein in trimeric post-fusion conformation (1URZ.pdb) . The fusion peptide is highlighted in green and can be seen protruding out of the virion membrane towards the endosomal membrane. Different subunits of the trimer are coloured in red, blue and yellow.
Figure 3
Figure 3. Effect of Yar-virus simulated mutations on HA activity of TBEV.
The engineered viruses IC-D67G, IC-E122G, IC-D277A and pGGVs (1−4×107 PFU/well) were tested for HA using chicken erythrocytes at pH 6.2.
Figure 4
Figure 4. Effect of HA-disabling mutations on growth characteristics of TBEV in mammalian models.
(A) Confluent monolayers of PS cells were infected with IC-D67G, IC-E122G, IC-D277A or pGGVsH virus at an estimated moi of 1 PFU/cell and virus yield in cell culture medium was determined at different time points post-infection by plaque assay. (B) Plaque morphology of specified Yar viruses and engineered mutants in PS cell monolayers. (C) Adult mice were inoculated intraperitoneally with 2000 PFU of IC-D67G, IC-E122G, IC-D277A or pGGVsH followed by monitoring of the morbidity rate (neuroinvasiveness test).
Figure 5
Figure 5. Effect of HA-disabling mutations on TBEV replication in ticks.
(A) Adult unfed I. ricinus females were infected with IC-D67G, IC-E122G IC-D277A or pGGVsH virus (day 0) and virus yield (lgPFU/ml; solid bars) was determined after incubation of infected fasting ticks for 2, 7, 14 and 21 days and also following the transfer of 21-day fasting ticks to feed on the mice for 3 days. (B) For tick-to-tick transmission, adult infected ticks were placed on mice in close proximity to uninfected nymphs and allowed to co-feed for 3 days. Tick-to-tick transmission rate (clear bars) is expressed as the proportion of infected nymphs to the total number of nymphs. Black triangles show the average virus titres in individually infected recipient nymphs as determined by plaque assay.
Figure 6
Figure 6. Mutagenesis of the infectious clone of TBEV.
The plasmid pGGVs660–1982H that contains the partial PrM-E gene fragment between nucleotides 660–1882 of the Vs virus genome was used as a template in PCR to synthesize megaprimers E-1171 A/G (genome positions and lengths are specified). Primers used to produce megaprimers, with targeted mutations (circles) are represented by thick arrows. Subsequently the megaprimer without the other pair of primers was used to amplify plasmid pGGVs660–1982H (solid circular line). The produced linear newly-synthesized complementary ssDNA molecules (nicked circular dotted line) with acquired mutations were annealed during the last step of PCR, randomly producing twice-nicked circular DNA. Parent Dam+ methylated DNA of the pGGVs660–1982H was removed by DpnI endonuclease digestion to facilitate clone selection.
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