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Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Khasnatinov MA, Tuplin A, Gritsun DJ, Slovak M, Kazimirova M, Lickova M, Havlikova S, Klempa B, Labuda M, Gould EA, Gritsun TS - PLoS ONE (2016)

Bottom Line: The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks.In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively.Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

View Article: PubMed Central - PubMed

Affiliation: Federal State Public Science Institution «Scientific Centre of Family Health and Human Reproduction Problems», Irkutsk, Russian Federation.

ABSTRACT
Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

No MeSH data available.


Related in: MedlinePlus

Impact of genetic background on NVT of TBEV.A) Correlation of non-viratemic transmission (NVT) rates of different TBEV chimaeras with reproduction rate in different life stages of ticks. The trend lines are estimated by the polynomial method. The Pearson`s correlation coefficients between NVT rate (solid trend line) and titres in ticks (dashed lines) are shown by brackets with indicated r values. B) Correlation of TBEV gene replacements with NVT rate. The solid and dashed trend-lines reflect the changes in Hypr and Vs NVT rate efficiency respectively, in correspondence with size of exchanged gene fragments. Trend-lines were drawn using order 3 polynomial method with R-square value shown at the right end of the corresponding trend line. C) Correlation between NVT rate and the amino acid substitutions. The amino acid differences between C-prM-E region of polyprotein of control and chimaeric viruses are plotted on the X-axis as number of amino acid substitutions per site in comparison to VsIC sequence. The NVT rates are plotted on the Y-axis. The linear regression is calculated using equation [Y = 914.53*X + 4.77] and shown as solid line (goodness of fit R2 = 0.58). The Pearson’s correlation coefficient (r) is shown in top left corner of the panel.
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pone.0158105.g008: Impact of genetic background on NVT of TBEV.A) Correlation of non-viratemic transmission (NVT) rates of different TBEV chimaeras with reproduction rate in different life stages of ticks. The trend lines are estimated by the polynomial method. The Pearson`s correlation coefficients between NVT rate (solid trend line) and titres in ticks (dashed lines) are shown by brackets with indicated r values. B) Correlation of TBEV gene replacements with NVT rate. The solid and dashed trend-lines reflect the changes in Hypr and Vs NVT rate efficiency respectively, in correspondence with size of exchanged gene fragments. Trend-lines were drawn using order 3 polynomial method with R-square value shown at the right end of the corresponding trend line. C) Correlation between NVT rate and the amino acid substitutions. The amino acid differences between C-prM-E region of polyprotein of control and chimaeric viruses are plotted on the X-axis as number of amino acid substitutions per site in comparison to VsIC sequence. The NVT rates are plotted on the Y-axis. The linear regression is calculated using equation [Y = 914.53*X + 4.77] and shown as solid line (goodness of fit R2 = 0.58). The Pearson’s correlation coefficient (r) is shown in top left corner of the panel.

Mentions: To evaluate further whether or not the observed changes in NVT rate were caused by modifications of the chimaeric viral genomes rather then titre, we applied three independent methods of regression and correlation analysis. Firstly, analysis of correlations between the NVT, titres of TBEV in salivary glands of females and in nymphs of different chimaeric viruses; secondly, regression analysis of correlations between TBEV genome fragment replacements with NVT rate and thirdly, regression analysis of correlation between NVT rate and amino acid similarity of the structural proteins of studied viruses (Fig 8A, 8B and 8C respectively). When we arranged the viruses in order of decreasing NVT rate, a strong association between TBEV NVT rate, virus titre in the salivary glands of adult ticks and nymphs and the specific TBEV genetic background was revealed. There was a strong correlation (Fig 8A) between virus titre in female ticks and NVT rate (r = 0.88), in nymphs the correlation with NVT rate was also strong, albeit less robust (r = 0.78). In summary, the pattern of change in recovered infectivity of the different viruses was very similar in nymphs and female ticks (Fig 8A) indicating equivalent decrease of virus fitness for the invertebrate host.


Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Khasnatinov MA, Tuplin A, Gritsun DJ, Slovak M, Kazimirova M, Lickova M, Havlikova S, Klempa B, Labuda M, Gould EA, Gritsun TS - PLoS ONE (2016)

Impact of genetic background on NVT of TBEV.A) Correlation of non-viratemic transmission (NVT) rates of different TBEV chimaeras with reproduction rate in different life stages of ticks. The trend lines are estimated by the polynomial method. The Pearson`s correlation coefficients between NVT rate (solid trend line) and titres in ticks (dashed lines) are shown by brackets with indicated r values. B) Correlation of TBEV gene replacements with NVT rate. The solid and dashed trend-lines reflect the changes in Hypr and Vs NVT rate efficiency respectively, in correspondence with size of exchanged gene fragments. Trend-lines were drawn using order 3 polynomial method with R-square value shown at the right end of the corresponding trend line. C) Correlation between NVT rate and the amino acid substitutions. The amino acid differences between C-prM-E region of polyprotein of control and chimaeric viruses are plotted on the X-axis as number of amino acid substitutions per site in comparison to VsIC sequence. The NVT rates are plotted on the Y-axis. The linear regression is calculated using equation [Y = 914.53*X + 4.77] and shown as solid line (goodness of fit R2 = 0.58). The Pearson’s correlation coefficient (r) is shown in top left corner of the panel.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920422&req=5

pone.0158105.g008: Impact of genetic background on NVT of TBEV.A) Correlation of non-viratemic transmission (NVT) rates of different TBEV chimaeras with reproduction rate in different life stages of ticks. The trend lines are estimated by the polynomial method. The Pearson`s correlation coefficients between NVT rate (solid trend line) and titres in ticks (dashed lines) are shown by brackets with indicated r values. B) Correlation of TBEV gene replacements with NVT rate. The solid and dashed trend-lines reflect the changes in Hypr and Vs NVT rate efficiency respectively, in correspondence with size of exchanged gene fragments. Trend-lines were drawn using order 3 polynomial method with R-square value shown at the right end of the corresponding trend line. C) Correlation between NVT rate and the amino acid substitutions. The amino acid differences between C-prM-E region of polyprotein of control and chimaeric viruses are plotted on the X-axis as number of amino acid substitutions per site in comparison to VsIC sequence. The NVT rates are plotted on the Y-axis. The linear regression is calculated using equation [Y = 914.53*X + 4.77] and shown as solid line (goodness of fit R2 = 0.58). The Pearson’s correlation coefficient (r) is shown in top left corner of the panel.
Mentions: To evaluate further whether or not the observed changes in NVT rate were caused by modifications of the chimaeric viral genomes rather then titre, we applied three independent methods of regression and correlation analysis. Firstly, analysis of correlations between the NVT, titres of TBEV in salivary glands of females and in nymphs of different chimaeric viruses; secondly, regression analysis of correlations between TBEV genome fragment replacements with NVT rate and thirdly, regression analysis of correlation between NVT rate and amino acid similarity of the structural proteins of studied viruses (Fig 8A, 8B and 8C respectively). When we arranged the viruses in order of decreasing NVT rate, a strong association between TBEV NVT rate, virus titre in the salivary glands of adult ticks and nymphs and the specific TBEV genetic background was revealed. There was a strong correlation (Fig 8A) between virus titre in female ticks and NVT rate (r = 0.88), in nymphs the correlation with NVT rate was also strong, albeit less robust (r = 0.78). In summary, the pattern of change in recovered infectivity of the different viruses was very similar in nymphs and female ticks (Fig 8A) indicating equivalent decrease of virus fitness for the invertebrate host.

Bottom Line: The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks.In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively.Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

View Article: PubMed Central - PubMed

Affiliation: Federal State Public Science Institution «Scientific Centre of Family Health and Human Reproduction Problems», Irkutsk, Russian Federation.

ABSTRACT
Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

No MeSH data available.


Related in: MedlinePlus