Transcriptome Analysis Reveals a Signature Profile for Tick-Borne Flavivirus Persistence in HEK 293T Cells

Abstract

Tick-borne flaviviruses (TBFVs) cause febrile illnesses, which may progress to severe encephalitis and/or death in humans globally. Most people who recover from severe acute disease suffer from debilitating neurological sequelae, which may be due to viral persistence, infection-induced neurological cell damage, host response, or some combination of these. Acute TBFV infection of human embryonic kidney (HEK) 293T cells in vitro results in the death of >95% of infected cells by day 5. However, replacing cell growth medium allows surviving cells to repopulate and become persistently infected for extended periods of time. The mechanisms responsible for initiation and maintenance of viral persistence remain vague. We subjected the HEK 293T cell transcriptome to deep sequencing to identify genes differentially expressed during acute infection and persistent infection. A total of 451 genes showed unique significant differential expression levels in persistently infected cells relative to the acute phase of infection. Ingenuity Pathway Analysis results suggested that the expression of prosurvival oncogenes AKT2 and ERBB2 was upregulated in persistently infected cells, whereas proapoptotic genes, such as Bad and the beta interferon 1 (IFN-␤1) gene, were downregulated. Genes encoding antiviral cytokines such as the CCL5, tumor necrosis factor alpha (TNF-␣), and CXCL10 genes were upregulated during the acute phase, but the same genes were relatively quiescent in persistently infected cells. Exogenous induction of apoptosis demonstrated that persistently infected cells were resistant to apoptosis in a dosedependent manner. In summary, the differential transcriptome profiles of acute-phase compared to persistently infected HEK 293T cells demonstrated an evasion of apoptosis, which may be critical for a chronic TBFV infection state. These results provide a basis for further study of the mechanisms of TBFV persistence.IMPORTANCE Tick-borne flaviviruses (TBFVs) cause life-threatening encephalitic disease in humans worldwide. Some people who recover from severe disease may suffer prolonged neurological symptoms due to either virus-or host response-induced cell damage or a combination of the two that are linked to viral persistence. By examining the genes that are significantly differentially expressed in acute TBFV infection versus persistent TBFV infection, we may be able to find the molecular basis of viral persistence. Here we used deep sequencing of the host cell transcriptome to discover that the expression levels of prosurvival genes were upregulated in persistently infected cells relative to acute TBFV infections whereas the expression levels of genes that promote programmed cell death were downregulated. In addition, persistently infected cells were also resistant to exogenous chemical induction of cell death, in a dose-dependent manner, compared to uninfected cells. Our results pave the way for further studies aimed at understanding the precise mechanisms of TBFV persistence.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

“…Furthermore, clear evidence of caspase mediated programmed cell death was apparent in the neuroblastoma cell line, leading to an acute lytic crisis. This is very similar to observations from our lab with a tick-borne flavivirus (32, 39), where the lytic crisis is followed by the initiation and long-term maintenance of a persistent infection. In the light of long-term presence of ZIKV in human semen (6), it will be important to determine if ZIKV persistence can also be established in SK-N-SH cells and to characterize that state.…”

“…Immunofluorescence was performed as above, using 1:1000 diluted rabbit monoclonal anti-active caspase 3 (Abcam) and 1:1000 diluted Alexa Fluor 594 anti-rabbit specific IgG (Life Technologies). TUNEL staining was performed as in (32) using in situ cell death detection kit, TMR red (Roche).…”

Analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection

“…The unique pattern of IFN signalling in DAOY cells might be virus-and cell-type-specific, since previous work characterized IFN-l as an inducer of IFN-a expression in HSV1-infected human neurons [17]. Furthermore, high up-regulation of IFN-b in LGTV-infected HEK 293T cells was also described [48]. However, DAOY cells express the type I IFN receptor and are responsive to IFN-b treatment.…”

“…This correlates with TBEV-associated immunopathogenesis in the brain [8,9]. Transcriptome analysis of LGTV-infected HEK 293 T cells revealed enhanced expression of CCL5/RANTES, CXCL10/IP-10 and TNF-a cytokines [48]. CCR5 (specifically the CCR5Delta32 allele) has been associated with the severity of TBEV-induced disease, suggesting that differential regulation of CCL5/ RANTES, etc.…”

Flavivirus Infection ofIxodes scapularis(Black-Legged Tick)Ex VivoOrganotypic Cultures and Applications for Disease Control

“…LGTV and POWV stock titers were determined in immunofocus assays as described by Offerdahl et al and Mlera et al (57)(58)(59). The same method was used to determine infectious LGTV and POWV titers in experimental samples, with two technical replicates per sample.…”