Abstract
BackgroundCongenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood–brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported.MethodsPrimary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated ‘SBCMV’. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry.ResultsHCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6 compared to both mock-infected as well as heat-killed virus controls. A 6.6-fold induction of IL-6 and no induction TNF-alpha was observed in SBCMV-infected cell supernatants at 24 hours postinfection. Using archival brain tissue from a patient coinfected with HCMV and HIV, we also found evidence of HCMV infection of pericytes using dual-label immunohistochemistry, as monitored by NG2 proteoglycan staining.ConclusionHCMV lytic infection of primary human brain pericytes suggests that pericytes contribute to both virus dissemination in the CNS as well as neuroinflammation.
Highlights
Congenital human cytomegalovirus (HCMV) infections can result in central nervous system (CNS) abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss
Cytomegalovirus protein expression in pericytes and upregulation of proinflammatory cytokine proteins and messenger ribonucleic acid (mRNA) Using Western blots, we observed HCMV major immediate early gene (MIE) 1, 2 and the pp65 late virion protein expression in pericytes infected with primary HCMV isolate from a patient (SBCMV) compared to mock-infected control cells (Figure 2A)
Temporal expression of the gene encoding HCMV pp65 virion protein was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in pericytes infected with either the SBCMV isolate or the Towne lab HCMV strain after 12, 24, 48, 72, or 96 hours of incubation
Summary
Congenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Human cytomegalovirus (HCMV) is a ubiquitous pathogen and is the most common infectious cause of congenital disease [1,2,3,4]. Congenital infections caused by HCMV represent the leading infectious cause of mental retardation and deafness in children [5,6]. Other abnormalities in newborns include vision loss, motor deficits, seizures, and sensorineural hearing loss [7,8,9]. HCMV, a common opportunistic infection in patients with HIV/AIDS [10], is often found in brain tissue from these patients [11] where it can be associated with cytomegalovirus-induced encephalitis [12,13]
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