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Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line

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ABSTRACT

Tick-borne flaviviruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893A→C (29% of 5978 reads at 12 h post infection (hpi)) and 2284T→A (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells.

No MeSH data available.


Integrative Genomic Viewer alignment of LGTV TP21 sequence reads obtained at 12, 96 and 1680 hpi. The horizontal gray bars represent sequence read alignments and the colored bars were read pairs of unexpected size or orientation. Genome truncations would have appeared as clear regions interspaced between horizontal gray bars [9]. The LGTV genome map was added as a schematic above the panels.
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viruses-08-00252-f002: Integrative Genomic Viewer alignment of LGTV TP21 sequence reads obtained at 12, 96 and 1680 hpi. The horizontal gray bars represent sequence read alignments and the colored bars were read pairs of unexpected size or orientation. Genome truncations would have appeared as clear regions interspaced between horizontal gray bars [9]. The LGTV genome map was added as a schematic above the panels.

Mentions: As mentioned, our recent studies in HEK 293T cells show that DIPs were not present at the initiation of persistent infection, but were a feature once persistence was established [19]. Consequently, we were curious to see if DIPs were present in persistently infected ISE6 cells. Therefore, we deep-sequenced the LGTV genome extracted from total cellular RNA on a HiSeq 2500 sequencer (Illumina, San Diego, CA, USA) as described before [19]. The sequence reads were aligned and visualized with Integrated Genomics Viewer software (version 2.2.10, Broad Institute, Cambridge, MA, USA) [29,30], and 253,809 sequence read pairs were aligned at 12 hpi resulting in a depth of coverage of 2300-fold. 2,884,383 sequence pairs were obtained at 96 hpi to achieve a sequencing depth of 26,000-fold. At 1680 hpi, we obtained 881,289 sequence pairs to achieve an average depth of coverage of 8000-fold. Interestingly, analysis of the LGTV genome alignments failed to identify truncated genomes at any of these time points (Figure 2). This contrasted with our observations of 293T cells, in which truncations could be detected as early as 5 weeks of passaging persistently infected cells [19]. These results suggested that DIPs are not generated during persistent TBFV infection of ISE6 cells.


Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line
Integrative Genomic Viewer alignment of LGTV TP21 sequence reads obtained at 12, 96 and 1680 hpi. The horizontal gray bars represent sequence read alignments and the colored bars were read pairs of unexpected size or orientation. Genome truncations would have appeared as clear regions interspaced between horizontal gray bars [9]. The LGTV genome map was added as a schematic above the panels.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035966&req=5

viruses-08-00252-f002: Integrative Genomic Viewer alignment of LGTV TP21 sequence reads obtained at 12, 96 and 1680 hpi. The horizontal gray bars represent sequence read alignments and the colored bars were read pairs of unexpected size or orientation. Genome truncations would have appeared as clear regions interspaced between horizontal gray bars [9]. The LGTV genome map was added as a schematic above the panels.
Mentions: As mentioned, our recent studies in HEK 293T cells show that DIPs were not present at the initiation of persistent infection, but were a feature once persistence was established [19]. Consequently, we were curious to see if DIPs were present in persistently infected ISE6 cells. Therefore, we deep-sequenced the LGTV genome extracted from total cellular RNA on a HiSeq 2500 sequencer (Illumina, San Diego, CA, USA) as described before [19]. The sequence reads were aligned and visualized with Integrated Genomics Viewer software (version 2.2.10, Broad Institute, Cambridge, MA, USA) [29,30], and 253,809 sequence read pairs were aligned at 12 hpi resulting in a depth of coverage of 2300-fold. 2,884,383 sequence pairs were obtained at 96 hpi to achieve a sequencing depth of 26,000-fold. At 1680 hpi, we obtained 881,289 sequence pairs to achieve an average depth of coverage of 8000-fold. Interestingly, analysis of the LGTV genome alignments failed to identify truncated genomes at any of these time points (Figure 2). This contrasted with our observations of 293T cells, in which truncations could be detected as early as 5 weeks of passaging persistently infected cells [19]. These results suggested that DIPs are not generated during persistent TBFV infection of ISE6 cells.

View Article: PubMed Central - PubMed

ABSTRACT

Tick-borne flaviviruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893A→C (29% of 5978 reads at 12 h post infection (hpi)) and 2284T→A (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells.

No MeSH data available.