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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

Chimaeric virus growth kinetics in cell culture.Viruses are specified as shown in the text. PS cells were infected with TBEV at a multiplicity of 1 pfu per cell in at least 4 replicates. Samples of cell culture media were collected every 4 hours during the first day pi and then once a day. Virus infectivity was established by plaque assay. A-C) HyprIC and Hypr-based recombinant viruses; D-F) VsIC and Vs-based recombinant viruses. Error bars reflect 95% confidence intervals.
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pone.0158105.g003: Chimaeric virus growth kinetics in cell culture.Viruses are specified as shown in the text. PS cells were infected with TBEV at a multiplicity of 1 pfu per cell in at least 4 replicates. Samples of cell culture media were collected every 4 hours during the first day pi and then once a day. Virus infectivity was established by plaque assay. A-C) HyprIC and Hypr-based recombinant viruses; D-F) VsIC and Vs-based recombinant viruses. Error bars reflect 95% confidence intervals.

Mentions: To compare the viability and replication efficiency of the chimaeric viruses the growth cycle assays in porcine kidney cell cultures (PS) were carried out. All chimaeric viruses replicated efficiently and the kinetics of replication were comparable with the control viruses HyprIC and VsIC. However, the replacement of structural genes (plus the 5' UTR) in the HyprIC genome by the equivalent genes of VsIC, reduced virus infectivity by approximately one order of magnitude at each time point (Fig 3A). Interestingly, replacement of the Vs E protein alone or in combination with the Vs prM gene did not significantly affect the replication kinetics of Hypr-based chimaeras (Fig 3B and 3C). No significant differences in virus infectivity were observed between Vs based chimaeras and control VsIC, including viruses both with entire structural genes plus the 5’ UTR from Hypr and separate genes E or prM-E from HyprIC (Fig 3D–3F). Moreover, no differences in replication kinetics were revealed between HyprIC with long or short 3'UTR.


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)

Chimaeric virus growth kinetics in cell culture.Viruses are specified as shown in the text. PS cells were infected with TBEV at a multiplicity of 1 pfu per cell in at least 4 replicates. Samples of cell culture media were collected every 4 hours during the first day pi and then once a day. Virus infectivity was established by plaque assay. A-C) HyprIC and Hypr-based recombinant viruses; D-F) VsIC and Vs-based recombinant viruses. Error bars reflect 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920422&req=5

pone.0158105.g003: Chimaeric virus growth kinetics in cell culture.Viruses are specified as shown in the text. PS cells were infected with TBEV at a multiplicity of 1 pfu per cell in at least 4 replicates. Samples of cell culture media were collected every 4 hours during the first day pi and then once a day. Virus infectivity was established by plaque assay. A-C) HyprIC and Hypr-based recombinant viruses; D-F) VsIC and Vs-based recombinant viruses. Error bars reflect 95% confidence intervals.
Mentions: To compare the viability and replication efficiency of the chimaeric viruses the growth cycle assays in porcine kidney cell cultures (PS) were carried out. All chimaeric viruses replicated efficiently and the kinetics of replication were comparable with the control viruses HyprIC and VsIC. However, the replacement of structural genes (plus the 5' UTR) in the HyprIC genome by the equivalent genes of VsIC, reduced virus infectivity by approximately one order of magnitude at each time point (Fig 3A). Interestingly, replacement of the Vs E protein alone or in combination with the Vs prM gene did not significantly affect the replication kinetics of Hypr-based chimaeras (Fig 3B and 3C). No significant differences in virus infectivity were observed between Vs based chimaeras and control VsIC, including viruses both with entire structural genes plus the 5’ UTR from Hypr and separate genes E or prM-E from HyprIC (Fig 3D–3F). Moreover, no differences in replication kinetics were revealed between HyprIC with long or short 3'UTR.

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