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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.


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Langat virus (LGTV) replication kinetics in ISE6 cells. (A) Detection of the expression of LGTV E protein by confocal microscopy. Few cells were infected at 12 h post infection (hpi) as indicated by viral E protein staining in a low number of cells, but almost all of the cells were infected at 96 and 1680 hpi. The scale bar represents 10 µm; (B) LGTV TP21 titer obtained by an immunofocus assay using an anti-E antibody; (C) LGTV RNA genome copy numbers measured by quantitative PCR (qPCR). DAPI: 4',6-diamidino-2-phenylindole.
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viruses-08-00252-f001: Langat virus (LGTV) replication kinetics in ISE6 cells. (A) Detection of the expression of LGTV E protein by confocal microscopy. Few cells were infected at 12 h post infection (hpi) as indicated by viral E protein staining in a low number of cells, but almost all of the cells were infected at 96 and 1680 hpi. The scale bar represents 10 µm; (B) LGTV TP21 titer obtained by an immunofocus assay using an anti-E antibody; (C) LGTV RNA genome copy numbers measured by quantitative PCR (qPCR). DAPI: 4',6-diamidino-2-phenylindole.

Mentions: Immunofluorescence was used to evaluate the extent of Langat virus (LGTV) infection in the ISE6 cultures. 105 ISE6 cells in 4-well Labtek chamber slides (Nunc®, Sigma-Aldrich, Atlanta, GA, USA) were infected at a MOI of 5, and prepared for immunofluorescent microscopy at 12, 96, and 1680 h post infection (hpi). At each time point, cells were washed twice with PBS, fixed with 4% paraformaldehyde, probed with a mouse monoclonal anti-E (11H12) antibody (a kind gift from Dr. Connie Schmaljohn, USAMRID, Fort Detrick, Frederick, MD, USA) and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Examination of these preparations revealed that few cells were infected at 12 hpi. However, a higher number of cells were infected at 96 hpi as shown by positive staining for the LGTV E protein (Figure 1A). Furthermore, the fraction of ISE6 cells positive for E appeared to remain stable out to 1680 h (Figure 1A). These results indicated that most cells in the cultures were expressing LGTV proteins by 96 hpi and maintained expression for an extended period.


Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line
Langat virus (LGTV) replication kinetics in ISE6 cells. (A) Detection of the expression of LGTV E protein by confocal microscopy. Few cells were infected at 12 h post infection (hpi) as indicated by viral E protein staining in a low number of cells, but almost all of the cells were infected at 96 and 1680 hpi. The scale bar represents 10 µm; (B) LGTV TP21 titer obtained by an immunofocus assay using an anti-E antibody; (C) LGTV RNA genome copy numbers measured by quantitative PCR (qPCR). DAPI: 4',6-diamidino-2-phenylindole.
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Related In: Results  -  Collection

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viruses-08-00252-f001: Langat virus (LGTV) replication kinetics in ISE6 cells. (A) Detection of the expression of LGTV E protein by confocal microscopy. Few cells were infected at 12 h post infection (hpi) as indicated by viral E protein staining in a low number of cells, but almost all of the cells were infected at 96 and 1680 hpi. The scale bar represents 10 µm; (B) LGTV TP21 titer obtained by an immunofocus assay using an anti-E antibody; (C) LGTV RNA genome copy numbers measured by quantitative PCR (qPCR). DAPI: 4',6-diamidino-2-phenylindole.
Mentions: Immunofluorescence was used to evaluate the extent of Langat virus (LGTV) infection in the ISE6 cultures. 105 ISE6 cells in 4-well Labtek chamber slides (Nunc®, Sigma-Aldrich, Atlanta, GA, USA) were infected at a MOI of 5, and prepared for immunofluorescent microscopy at 12, 96, and 1680 h post infection (hpi). At each time point, cells were washed twice with PBS, fixed with 4% paraformaldehyde, probed with a mouse monoclonal anti-E (11H12) antibody (a kind gift from Dr. Connie Schmaljohn, USAMRID, Fort Detrick, Frederick, MD, USA) and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Examination of these preparations revealed that few cells were infected at 12 hpi. However, a higher number of cells were infected at 96 hpi as shown by positive staining for the LGTV E protein (Figure 1A). Furthermore, the fraction of ISE6 cells positive for E appeared to remain stable out to 1680 h (Figure 1A). These results indicated that most cells in the cultures were expressing LGTV proteins by 96 hpi and maintained expression for an extended period.

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.


Related in: MedlinePlus