- Email: [email protected]
Astrocyte response to St. Louis encephalitis virus
Virus Research 217 (2016) 92–100
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Astrocyte response to St. Louis encephalitis virus Adriano Lara Zuza a , Heber Leão Silva Barros a , Thelma Fátima de Mattos Silva Oliveira a , Juliana Helena Chávez-Pavoni b , Renata Graciele Zanon a,∗ a b
Institute of Bioscience, Federal University of Uberlandia, Para 1720, Uberlandia, Minas Gerais CEP 38400-902, Brazil School of Medicine, Federal University of Mato Grosso, MT 270 St., Km 06, Rondonopolis, Mato Grosso, Brazil
a r t i c l e
i n f o
Article history: Received 4 January 2016 Received in revised form 7 March 2016 Accepted 8 March 2016 Available online 11 March 2016 Keywords: Flavivirus infections St. Louis encephalitis virus Astrocytes Glial fibrillary acidic protein
a b s t r a c t St. Louis encephalitis virus (SLEV), a flavivirus transmitted to humans by Culex mosquitoes, causes clinical symptoms ranging from acute febrile disorder to encephalitis. To reach the central nervous system (CNS) from circulating blood, the pathogen must cross the blood–brain barrier formed by endothelial cells and astrocytes. Because astrocytes play an essential role in CNS homeostasis, in this study these cells were infected with SLEV and investigated for astrogliosis, major histocompatibility complex (MHC)-Idependent immune response, and apoptosis by caspase-3 activation. Cultures of Vero cells were used as a positive control for the viral infection. Cytopathic effects were observed in both types of cell cultures, and the cytotoxicity levels of the two were compared. Astrocytes infected with a dilution of 1E-01 (7.7E + 08 PFU/mL) had a reduced mortality rate of more than 50% compared to the Vero cells. In addition, the astrocytes responded to the flavivirus infection with increased MHC-I expression and astrogliosis, characterized by intense glial fibrillary acidic protein expression and an increase in the number and length of cytoplasmic processes. When the astrocytes were exposed to higher viral concentrations, a proportional increase in caspase-3 expression was observed, as well as nuclear membrane destruction. SLEV immunostaining revealed a perinuclear location of the virus during the replication process. Together, these results suggest that mechanisms other than SLEV infection in astrocytes must be associated with the development of the neuroinvasive form of the disease. © 2016 Elsevier B.V. All rights reserved.
1. Introduction St. Louis encephalitis virus (SLEV) is an enveloped, positive, single-strand RNA virus that belongs to the family Flaviviridae, genus Flavivirus (Monath, 2015), and is serologically related to the Japanese encephalitis complex, which includes viruses such as Japanese encephalitis virus (JEV) and West Nile virus (WNV). SLEV is transmitted to humans mainly by Culex spp. bite (Monath and Tsai, 2002; Trent et al., 1980; Rodrigues et al., 2010). The virus is widely disseminated in the Americas (Spinsanti et al., 2008; Auguste et al., 2009; Rodrigues et al., 2010), and once inoculated, it may infect the central nervous system (CNS), causing encephalitis (Lim et al., 2008; Monath 2015). During the process of invading the CNS, the virus must cross the blood–brain barrier (BBB), formed by endotheliocytes and astrocytes, to reach the brain and spinal cord. The BBB is a highly
∗ Corresponding author at: Maria das Dores Dias 850, 601, 10, Uberlandia 38408206, Brazil. E-mail address: [email protected] (R.G. Zanon). http://dx.doi.org/10.1016/j.virusres.2016.03.005 0168-1702/© 2016 Elsevier B.V. All rights reserved.
selective, permeable membrane that regulates the passage of several substances into the brain’s extracellular fluid (Suen et al., 2014). If a virion succeeds in penetrating an endotheliocyte and crosses the basement membrane, it still has to infiltrate the astrocyte body to reach a neuron. Astrocytes, the key glial component of the CNS, are responsible for maintaining homeostasis for perfect neuronal function through the secretion of growth factors, glycogen storage, and detoxification of metals and xenobiotics (Chen et al., 2010; Furr and Marriot, 2012; Rao et al., 2012). In addition, these cells control BBB permeability, mainly due to their involvement in inflammatory processes through the expression of cytokines and nitric oxide synthase, inducing leucocyte migration and microglial activation (Bi et al., 1995). Astrocytes also express several molecules that are intimately involved in immune responses, such as major histocompatibility complex (MHC)-I and -II, antiviral interferon via pattern recognition receptors, interleukin (IL)-6, tumor necrosis factor ␣, and IL-1 (Kawai and Akira, 2008; Chauhan et al., 2010; Yoneyama and Fujita, 2010). Once a virion encounters an astrocyte, both innate and adaptive immune responses are triggered, culminating in the release of cytokines and chemotactic factors that recruit defense cells from
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
93
Fig. 1. Mortality rate in (a) Vero cells and (b) astrocytes at six days post-infection with different SLEV dilutions. Note that the viral concentration responsible for 94.03% of mortality in Vero cells caused only 38.18% of mortality in astrocytes.
the bloodstream. Thus, activated astrocytes (or astrogliosis) produce IL-6, IL-8, IL-1, IL-18, RANTES, interferon ␥-inducible protein 10, glial fibrillary acidic protein (GFAP), glutamate aspartate transporter, glutamate transporter 1, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) (Chen et al., 2010). The production of hepatocyte growth factor, ceruloplasmin, glutamine synthetase, vimentin, and reactive oxygen species by astrocytes has also been reported (Mishra et al., 2007). Astrogliosis increases the permeability of the BBB, allowing leukocytes in the bloodstream to cross into the nervous extracellular matrix. Once the BBB is disrupted, more virions can penetrate the nervous tissue coming from the blood, especially in patients with high viremia or immunologic deficiency, resulting in a massive infection of the brain and spinal cord (Dong and Benveniste, 2001; Fischer and Reichmann, 2001; Furr and Marriott, 2012). Considering that astrocytes are the maestros of BBB homeostasis, the investigation of this cell response to SLEV infection may contribute to a better comprehension of the mechanisms of viral infection of the CNS and encephalitis. To address this issue, this study aimed to investigate the astrocyte–SLEV interaction using primary astrocyte cultures compared to viral infection in the Vero cell lineage. Parameters such as astrogliosis, expression of MHCI molecules, and apoptosis were evaluated in both infected cell cultures.
2. Material and methods 2.1. Production of virus stock SLEV (BeH 355964 strain) was cultured by continuous passage in Aedes albopictus C6/36 cell linage and maintained in L15 culture medium (Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Nutricell, Campinas, Brazil), 100 U/mL penicillin, 100 L/mL streptomycin (Sigma-Aldrich) at 28 ◦ C. Cultures were harvested after 7 days or when cytopathic effect was observed. For harvesting, cells were frozen at –70 ◦ C (ultrafreezer Nuare, Plymouph, MN, USA) and immediately thawed. The suspension was transferred to a 15 mL Falcon tube and centrifuged at 1000 × g for debris removal. Supernatant was removed and the aliquots were stored at –70 ◦ C, constituting the virus stock.
2.2. Astrocyte primary culture Astrocyte primary cultures were made by dissociation of cerebral cortex of neonate Swiss mice (1–2 days postnatal). Animals were immersed in ice until reaching unconsciousness and decapitated for brain removal. All procedures in animals were submitted and approved by the Ethics Committee on animal use of the Federal University of Uberlandia (protocol number CEUA148/13).
94
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
Fig. 2. Immunofluorescence anti-SLEV (SLEV MIAF) in Vero (b, c, j, k) and astrocyte (e, f, m, n) infected cultures in different dilutions at six days post-infection. In astrocyte cultures anti-OROV (OROV MIAF) staining was used as a MIAF control (h, i, p, q). All cells were stained with DAPI, a nuclear marker. Non-infected cells served as control (a, d, g). An intense marking in the cytoplasm of Vero cells was observed in all viral dilutions, while astrocytes showed anti-SLEV marking only in undiluted dose of virus (reddish stain). Asterisks indicate apoptotic cells with vacuolated nucleus, nucleic acid dispersed in cytoplasm and viral particles in a perinuclear location. In (r), a similar characteristic was seen in OROV-infected astrocytes when stained with anti-OROV antibodies. Scale: 50 m.
Collected tissues were fragmented, washed in phosphatebuffered saline (PBS 0.1 M, pH 7.4) with no calcium or magnesium (Nutricell) and trypsinized for 10 min at 37 ◦ C. After trypsinization, DNAase (Sigma-Aldrich) was added to the solution, homogenized and then, FBS was added to block enzymatic action. Supernatant was centrifuged (250 × g for 10 min) in culture medium containing 4% bovine serum albumin (BSA; Sigma-Aldrich). DMEM was added to the cell precipitate, which was then seeded in 75 cm2 cell culture bottles (Nest Biotechnology CO., Shangai, China) and incubated at 37 ◦ C, 5% CO2. After about 10 days until reaching 80% of
cell confluence, cell cultures were purified according to McCarthy and de Vellis (1980). In short, cell cultures were gently rinsed three times with complete medium to remove floating cells. Each culture flask (with 10 mL of fresh medium) was closed and placed onto an orbital shaker for 15–18 h (37 ◦ C). Following the shaking period, the suspended cells from all flasks were removed. The culture flasks containing a nearly intact layer of astrocytes (purity ∼ 95%) were returned to incubator until reaching cell confluence. The cells were then trypsinized, centrifuged (250g for 10 min), counted (5 × 104 or 5 × 103 cells), seeded on 24 or 96-well plates (Nest
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
95
Fig. 3. Immunofluorescence of astrocyte cultures infected with SLEV in different dilutions (b–e) or OROV (f) and stained with DAPI and anti-GFAP (cytoplasmic stain) antibody at six days post-infection. Non-infected cells served as control (a). In (g), drawing represents the normal and reactive astrocytes, the dashed circle shows the location where the quantification of pixel density has been performed. The graph in (h) shows the pixel density measured in the different groups. One-way ANOVA and Bonferroni post-test. (*) p < 0.05. Scale: 50 m.
Biotechnology), respectively, and incubated for more three days at 37 ◦ C, until 80% of cell confluence to be reached, when then were infected with SLEV or Oropouche virus (OROV), one of the most common orthobunyaviruses that was used as negative control in SLEV immunostaining to evaluate the specificity of anti-SLEV antibodies. OROV was chosen because it is available in our laboratory as well as its marker for immunostaining (anti-OROV antibodies).
2.3. Astrocyte mortality rate in response to SLEV infection SLEV infection in both Vero and astrocyte cultures was evaluated from 1 to 6 days post-infection (p.i.) for cytopathic effect (CPE). In view of the differences in both growth rate and susceptibility to viruses of the two cell types, CPE was observed at 3 days p.i. in Vero cells and 6 days p.i. in astrocytes. For comparative purposes, the period of cell culture after infection used in our analyses was 6 days for both cell types. Alterations in cell cultures, such as cell morphological changes, cell disorganization, apoptosis induction and loss of cell adherence to culture plate, were considered for CPE scores, according to Norkin (2010) who defined alterations caused by viral infection with similar characteristics to the SLEV. Vero cells, known to be SLEV susceptible and permissive, were used as positive control. For this purpose, Vero cells were cultured in 96-well plates (5 × 103 cells/well, 100 L/well) for 6 days in DMEM supplemented with 10% FBS, at 37 ◦ C, 5% CO2 and humidity control. Plates were infected with seriated virus dilutions (1E-01, 1E-02, 1E-03, 1E-04, 1E-05 and 1E-06) and one row left as control group. Astrocytes were cultured under the same conditions and infected with different dilutions (1E + 01, 7.5E-01, 5E-01, 2.5E-01, 1E-01, 1E-02, 1E-03, 1E-04, 1E-05 and 1E-06). After 6 days of incubation, both cultures were submitted to MTT protocol as described below.
For MTT assay the culture medium was aspirated, and then added 50 L of MTT (Sigma Aldrich) solution (1 mg/mL) per well and incubated for more 4 h. After the incubation period, solution was completely removed and 100 L of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added in each well to solubilize formazan crystals. Plates were gently agitated at room temperature for 10 min and read at 540 nm (Elx 800, Bio-Tek, Winnosky, VT, USA). The absorbance measures were used to calculate the titer of the viral samples as described by Reed and Muench (1938). Cell mortality rate was calculated as follows: mortality rate = [(A − B)/A × 100], where A: absorbance of infected cells and B: absorbance of noninfected cells.
2.4. Immunocytochemistry To detect viral intracellular proteins in astrocyte and Vero cultures, polyclonal anti-SLEV antibodies obtained from mouse immune ascitic fluid (MIAF) and polyclonal mouse anti-OROV antibodies were used in astrocytes to confirm OROV infection and to provide a negative control of MIAF for SLEV immunostaining. Astrocytes were cultured in 96-well plates and infected for 1 h with SLEV in dilutions of 1E + 00, 5E-01, 1E-01, 1E-02, 1E-03, 1E-04, 1E-05 and 1E-06, or with OROV in the dilution of 1E-02. One plate column was left for negative control. After 6 days of incubation, culture medium was removed, cells were fixed in 4% paraformaldehyde (Synth, Diadema, Brazil) and DMEM, washed in PBS and incubated for 1 h at 37 ◦ C in 3% BSA and 0.2% Triton-X100 (Sinth,) solution. Then, astrocytes were incubated with the following polyclonal primary antibodies: goat anti-GFAP (1:100; Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-mouse cleaved caspase-3 (p11) (1:200, Abcam, Cambridge, MA, USA), mouse antiMHC-I (1:100, AbD SEROTEC, Kidlington, UK), rabbit anti-SLEV
96
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
Fig. 4. Immunofluorescence of astrocyte cultures infected with SLEV in different dilutions (b-e) or OROV (f) and stained with DAPI and anti-cleaved caspase 3 (CASP-3, asterisks) antibody at six days post-infection. Non-infected cells served as control (a). The graph (g) shows a significant increase in CASP-3 expression in all infected cells when compared to control. Mann-Whitney U test, p < 0.05 (*). Scale: 50 m.
diluted 1:100, and rabbit anti-OROV diluted 1:100 at 4 ◦ C for 24 h. All reactions were performed in duplicate. After incubation with primary antibodies, cultures were washed in PBS and incubated for 1 h with secondary antibodies (anti-goat, anti-mouse or anti-rabbit) conjugated with Alexa Fluor 488 or 594 (Jackson Lab., Bar Harbor, ME, USA). Cultures were stained with DAPI (Sigma-Aldrich) for cell nucleus visualization. Subsequently to incubation and washing, all cultures were kept in glycerol/PBS/DAPI for analysis in fluorescence inverted microscope. Four photographs of representative fields were shot from each well. Qualitative comparison was made for cultures immunostained with anti-SLEV and anti-OROV. Quantitative analysis was used for cultures immunostained with anti-GFAP, anti-caspase-3 and anti-MHC-I. Caspase-3 positive cells were counted and statistically analyzed using Mann-Whitney U test. GFAP and MHC-I immunostaining was analyzed by pixel densitometry in the photographs and statistically compared using one way ANOVA and Bonferroni post-test. All results were expressed as mean ± SD, and statistical significance was established when p < 0.05. 3. Results 3.1. Mortality rate Cell mortality rates were calculated using an MTT assay at six days p.i. for both Vero cells and astrocytes infected with different concentrations of SLEV, in order to compare virus–cell interactions.
The mortality percentage rate was calculated for each viral concentration (dilution) compared with non-infected cells (control). Cell mortality rate was 94.03% in the 1E-01Vero cell dilution (Fig. 1a). Vero cell viability was inversely proportional to decreasing concentrations of SLEV tested. The mortality rate of the astrocytes (Fig. 1b) was as high as 79.85% when exposed to the non-diluted viral solution and 38.18% in the 1E-01dilution, thus showing a reduced mortality rate of more than 50% compared to the Vero cells in the same viral concentration. As with the Vero cells, the mortality rates of the astrocytes also diminished when the cells were exposed to decreasing viral concentrations, indicating that the SLEV titer directly influenced the number of astrocytes that underwent death. 3.2. Virus invasion and astrocyte response to SLEV As shown in Fig. 2, SLEV immunostaining revealed a large amount of virions inside the astrocytes when these cells were exposed to a 7.7E + 08 PFU/mL concentration of the non-diluted virus solution (Fig. 2e, reddish stain). Immunostaining was much more intense in perinuclear locations, where the rough endoplasmic reticulum (RER) is located, and in nuclear locations in apoptotic cells, where the nucleus acquired a vacuolated aspect with dispersed chromatin in cytosol. As expected, all genetic contents were blue when stained for DAPI, a nucleic acid dye. The white asterisks, in anti-SLEV mouse immune ascitic fluids (MIAF) in a non-diluted SLEV sample, illustrate nuclear destruction during cell death, when
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
97
Fig. 5. Immunofluorescence of astrocyte cultures infected with SLEV in different dilutions (b–e) or OROV (f) and stained with DAPI and anti-MHC-I antibody (cytoplasmic stain) at six days post-infection. Non-infected cells served as control (a). The pixel densitometry analysis graph (g) shows significant increase in MHC-I expression in cells infected with non-diluted SLEV, SLEV 5E-01 and OROV 1E-02 when compared to control. One-way ANOVA and Bonferroni post-test. (*) p < 0.05. Scale: 50 m.
the nuclear envelope was scattered throughout the cytoplasm. The recently destroyed nucleus assumed a darker coloration, almost without any DAPI, and a vacuolated character. Anti-SLEV antibodies seemed to form dollops around the nucleus, and occasionally, small reddish vesicles in the cell boundary or even outside the cell. This image is characteristic of Flavivirus replication, which occurs in RER ending with vesicles budding from the Golgi apparatus, heading to the cytoplasmic membrane, when virions are released. As the dilution increased, the number of astrocytes immunostained for SLEV decreased significantly. In the 1E-06 dilution (Fig. 2n), a small decrease in the astrocyte population was observed compared to the control group, and there was almost no staining for the virus. SLEV infection in the Vero cells showed the presence of the virus in lower concentrations as well (Fig. 2k), indicating an important difference in the astrocyte response to SLEV that suggests a resistance to infection. In astrocytes infected with Oropouche virus (OROV) (Fig. 2o and r), a great number of cells were observed in apoptosis and, as expected, with no SLEV immunostaining, confirming MIAF specificity. In the 1E-02 dilution (Fig. 2r), the cells displayed an intense staining, similar to the staining described in astrocytes infected with non-diluted SLEV solution, with big perinuclear lumps and small vesicles strongly stained in the cell periphery and exterior. As mentioned previously, astrocytes are antigen-presenting cells that control BBB permeability and CNS homeostasis, and
mobilize defense cells from the bloodstream to the CNS. These actions are possible through a phenomenon called astrogliosis, which involves several structural modifications, such as an increase in the expression of GFAP and in the number and length of cytoplasmic processes, among others. In order to observe whether SLEV and OROV infection could induce astrogliosis in primary astrocyte cultures, the cells were stained for GFAP (Fig. 3). When the astrocytes were exposed to the non-diluted SLEV solution (Fig. 3b), most cells underwent apoptosis, with nuclear shrinking and/or rupture, and a decrease in the number of cytoplasmic processes, which appeared to be shorter and wider. In the 1E-06, 1E-01, and 5E-01 dilutions of SLEV, morphological alterations shown by GFAP staining were observed as an increase in the number and length of cytoplasmic processes, indicating a more intense astrogliosis reaction in the infected cells compared to the control. In the OROV-infected cells stained for GFAP (Fig. 3f), a large number of cells with a morphological aspect of cellular death were observed. The cytoplasm of the remaining cells appeared starry, with cytoplasmic processes. In addition, the number of caspase-3-positive cells increased proportionally with increasing viral concentrations (Fig. 4). The CASP-3 micrograph of non-diluted SLEV and OROV 1E-02 exhibited intense staining and a great number of cells with vacuolated nuclei due to nuclear envelope fragmentation. These images revealed the nuclear content (stained in blue), which should be restricted to the
98
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
nucleus, dispersed throughout the cytosol. A quantitative analysis was conducted to evaluate the number of apoptotic cells (cleaved caspase-3 positive), and it was found that astrocyte infection using a non-diluted SLEV solution induced cell death by apoptosis in 85.37% of cells, similar to the value observed in OROV-infected astrocytes (Fig. 4g). In increasing SLEV dilutions, the percentage of caspase-3-positive cells was less, around 12%, with no significant differences among the 5E-01, 1E-01, and 1E-06 dilutions. These data suggest that this virus strain could be well adapted to astrocytes, so that at lower concentrations, these cells would be able to coexist in a semi-permissive state of infection with the intracellular parasite, without undergoing activation or apoptosis. In other words, although astrocytes possess receptors in the plasma membrane outer layer, where the virus adsorbs and internalizes, the virus might not find an adequate environment inside the host for its replication. This finding might be one reason viral immunostaining does not increase over time. Several viruses, such as Epstein-Barr (Zuo et al., 2011) and herpes simplex virus 1 (Imai et al., 2013), hold kinase coding regions in their genome, which are generally used to avoid the immune system during infection and inhibiting MHC-I expression in the cell surfaces of the host. In contrast, Flavivirus members act by inducing the expression of MHC-I, MHC-II, and diverse adhesion molecules, using this signaling system to promote cell lysis by T cytotoxic lymphocytes, and thus increasing the host’s susceptibility to viral infection (Abraham and Manjunath, 2006). As mentioned earlier, astrocytes express MHC molecules, and, theoretically, in the adaptation process to enable a virus strain to coexist chronically with astrocytes, a signaling pathway that would need to be downregulated is the MHC. To test this hypothesis, cells in culture were checked for immune system activation via MHC-I (Fig. 5). Although it was expected that all of the astrocytes in vitro would display increased MHC-I expression after being exposed to SLEV and OROV, these cells revealed an intense increase in MHC-I signaling that was directly proportional to the viral concentration. A significant increase in MHC-I expression was noted in the SLEVinfected astrocytes in the non-diluted sample (43.9%) and the 5E-01 dilution (31.1%) (Fig. 5g). This finding suggests that even if adaption were to occur, it would not affect the ability of the astrocytes to present viral antigens via MHC-I.
4. Discussion The SLEV BeH355964 strain was first isolated in 1978 at Belem, in the state of Para, Brazil (Pinheiro et al., 1981; Rodrigues et al., 2010). Rodrigues et al. (2010), in a phylogenetic analysis based on the SLEV envelope (E) gene, suggested that the virus is widespread throughout the Americas, with approximately 30 different strains in circulation in Brazil alone. Their study also demonstrated the presence of a new viral lineage (VIII) in Brazilian Amazonia. According to their phylogenetic analysis, the strain used in the current study belongs to lineage Vb. Although no studies about the virulence of this particular strain have been described, we should consider the study of Maharaj et al. (2012), who used SLEV and WNV chimeric viruses to infect vertebrate (Vero and duck) and invertebrate (Culex) cells in vitro. The authors observed that WNV titers were 100 times greater than SLEV in both vertebrate cell lineages, and that chimeric phenotypes exhibited growth changes and cytopathogenicity. Ciota et al. (2014) evaluated the adaptive potential of SLEV to a possible new host (tick), and found that a minor change in the viral genetic consensual sequence might facilitate viral infection in ticks. However, after successive passages in the tick cell cultures, the virions lost their ability to infect the vertebrate cells. These findings arise some questions related to SLEV infection in astrocytes. Is it possible that SLEV has suffered genetic
alterations during several passages for viral stock production? Would this genetic alteration be capable of affecting viral replication in astrocytes? To discard the possibility of adaption and a false interpretation of impaired viral replication in astrocytes, viral stocks used in the present study were produced in the invertebrate C6/36 A. albopictus lineage, which is well adapted to SLEV infection and reaches high titer levels. A comparative analysis of these viral stocks was carried out in two vertebrate mammal lineages: Vero cells and astrocyte primary cultures from rodents. One secure way to evaluate astrocyte responses involves the observation of morphological alterations, such as increased wideness of intermediate filament structures and, consequently, its constitutive proteins, especially GFAP, which demonstrates robust expression when astrocytes became activated (Middeldorp and Hol, 2011; Pekny et al., 2014). Virus infection is capable of inducing astrogliosis, as reported by Mishra et al. (2007), who observed longer and wider astrocytic processes after four days of JEV infection. In a similar manner, in our study we demonstrated that astrocyte invasion by SLEV induced astrogliosis, with an increase in the density of cytoplasmic processes at a viral concentration below that of the non-diluted solution. Chen et al. (2010) reported that the morphology of astrocytes was not significantly affected after JEV infection, contradicting Mishra et al. (2007), who described cytopathic effects in astrocyte cultures four days after JEV infection. A cytopathic effect was observed in our astrocyte cultures six days after SLEV infection, but only in the non-diluted solution. At lower concentrations, no morphological changes compatible with cell damage were found. In addition, astrocyte infection induced apoptosis through the caspase-3-dependent pathway, confirmed by immunocytochemical analysis. Quantification of cleaved caspase-3-positive cells revealed that programed cell death increased with the nondiluted SLEV solution. At lower viral concentrations, the astrocytes responded with a significant increase in caspase-3 levels, but at a lower intensity, corroborating our other results that astrocytes are resistant to SLEV infection. The phenomena of astrogliosis, caspase activation, and transcription of innate immune response genes in astrocytes have also been described in Chikungunya infection, an Orthobunyavirus (Das et al., 2015), reinforcing the importance of this cell type in controlling viral infections of CNS. After WNV infection in primary astrocyte cultures, Diniz et al. (2006) described a cytopathic effect consisting of mild morphological alterations, mostly eight days after infection and stabilizing around the 30th day. According to their study, the infected astrocytes became elongated with long cytoplasmic processes before death. At the fourth day p.i., approximately 30% of the remaining cells underwent apoptosis. Given these results, the authors concluded that astrocyte infection by WNV occurred slowly, and some cells did not show signs of infection, as they were cultivated successfully for 114 days in the presence of infectious viral particles without any cytopathic effects, maintaining a persistent and permissive state of infection. These findings suggest that astrocytes might be responsible for chronic viral infections due to the maintenance of WNV in the CNS. This finding was also confirmed by Hussmann et al. (2013), who reported that reduced astrocyte susceptibility to WNV was due to a delay in the replication of the viral genome and a decrease in cell-to-cell interferon-dependent infection. As such, astrocytes might be able to regulate WNV dissemination in the CNS, constituting a potential target for WNV neuropathology research. The morphological alterations found in the current study were similar to those described by Diniz et al. (2006). After six days of infection with 7.7E + 08 PFU/mL (non-diluted SLEV solution), we found that 79.85% of astrocytes were not viable. A mortality rate of 50% of the astrocytes in culture was observed in the 1E-03 dilution, corroborating the hypothesis that, in fact, these cells are resistant
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
to SLEV. Future studies should be performed to confirm the presence of chronic SLEV infection in astrocytes, similar to what occurs in WNV, which could justify the long period of recovery postencephalitis and the sequels experienced by patients with SLEV encephalitis. In a similar manner, Diniz et al. (2006) demonstrated that the intracellular distribution of WNV in infected astrocytes would be in a perinuclear location. Uchil et al. (2006) also observed that a significant proportion (20%) of RNA-polymerase-dependent total RNA in cells infected with WNV, JEV, and dengue virus were within the nucleus, along with the most important NS3 and NS5 replicases of JEV, demonstrating the nuclear location and synthesis of vRNA. They hypothesized that the host cell nucleus would be an additional site for replicase active complexes. To better understand the perinuclear location of anti-SLEV antibodies observed in our results, further studies need to be conducted to confirm the presence and synthesis of vRNA inside the nuclei of CNS cells. The low astrocyte infectivity by SLEV might be due to the capacity of these cells to live with the intracellular virus at low titers without triggering the apoptotic pathway or in the diverse functions of these cells, especially for their neuroprotective role in the CNS through the synthesis of neurotrophic factors and neurotrophins (Villoslada and Genain, 2004). We can also suggest that astrocytes are cells that are semi- or non-permissive to SLEV (Brooks et al., 2014), as our findings revealed infected cells with a low mortality rate, demonstrated by MTT and immunostaining with cleaved caspase-3, where cell viability was only compromised in the presence of non-diluted or a low dilution of viral solution. Mishra et al. (2007) reported that astrocytes presented increased CNTF levels in acute disseminated encephalomyelitis and increased NGF levels in viral encephalitis, and that all alterations were associated with neuroimmunomodulation and neuroprotection. Flaviviruses such as WNV induce the expression of MHC-I, MHCII, and adhesion molecules such as intercellular adhesion molecule 1 (CD54), vascular cell adhesion molecule 1 (CD106), and Eselectin on the cell surface (Kesson et al., 2002). MHC-I and MHC-II molecules are involved in the activation of TCD8 and TCD4 lymphocytes, respectively, inducing an immune adaptive response (Abbas et al., 2014). Abraham and Manjunath (2006) reported that MHC-I molecules were upregulated in JEV-infected astrocytes while MHCII remained unaltered, and that the maximum expression of MHC-I on the surface of infected astrocytes occurred 48 h after infection. In our study, SLEV was incubated for six days, and we found that astrocyte infection by SLEV, in a manner similar to that of JEV, resulted in an increase in MHC-I, mostly in cultures infected with higher viral concentrations, thus confirming that astrocytes are immunocompetent cells. Our results raise new questions regarding the astrocyte–SLEV interaction. In order to better understand how SLEV invades the CNS, more research must be conducted to investigate the chronic infection of astrocytes by SLEV, as with WNV; the existence of factors that prevent viral genome replication in astrocytes, causing a reduction in interferon-dependent cell–cell infection, as observed with WNV; whether flavivirus nuclear replicase complexes of SLEV are formed as described for JEV; and whether SLEV infection raises the genic expression of neurotrophins such as NGF, CNTF, and BDNF. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We are very grateful to Professor Luiz Tadeu Moraes Figueiredo (University of São Paulo, School of Medicine, Ribeirão Preto, SP,
99
Brazil) for suppling us with the viral aliquots of SLEV (BeH 355964 strain) and OROV (BeAn 19991 strain) as well as the MIAFs used in this work, and to Professor Jonny Yokosawa (Federal University of Uberlândia, MG, Brazil) for helping us with virus replication. We gratefully acknowledge financial support from the CNPq (474050/2011-4) and Fapemig (CDS-APQ-04285-10) agencies. References Abbas, A.K., Lichtman, A.H., Pillai, S., 2014. Basic Immunology: Functions and Disorders of the Immune System, 4th ed. Elsevier, Saunders, Philadelphia. Abraham, S., Manjunath, R., 2006. Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus. Virus Res. 119, 216–220, http://dx.doi.org/10.1016/j.virusres.2006.03.006. Auguste, A.J., Pybus, O.G., Carrington, C.V.F., 2009. Evolution and dispersal of St. Louis encephalitis virus in the Americas. Infect. Genet. Evol. 9, 709–715, http:// dx.doi.org/10.1016/j.meegid.2008.07.006. Bi, Z., Barna, M., Komatsu, T., Reiss, C.S., 1995. Vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity. J. Virol. 69, 6466–6472. Brooks, G.F., Carroll, K.C., Butel, J.S., Morse, S.A., Mietzner, T.A., 2014. Jawetz, Melnick & Adelberg’s Medical Microbiology, 26th ed. McGraw-Hill Companies, Chicago. Chauhan, V.S., Furr, S.R., Sterka, D.G., Nelson, D.A., Moerdyk-Schauwecker, M., Marriott, I., Grdzelishvili, V.Z., 2010. Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses. Virology 400, 187–196, http://dx.doi.org/10.1016/j. virol.2010.01.025. Chen, C.-J., Ou, Y.-C., Lin, S.-Y., Raung, S.-L., Liao, S.-L., Lai, C.-Y., Chen, S.-Y., Chen, J.-H., 2010. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. J. Gen. Virol. 91, 1028–1037, http://dx.doi.org/10. 1099/vir.0.013565-0. Ciota, A.T., Payne, A.F., Ngo, K. a., Kramer, L.D., 2014. Consequences of in vitro host shift for St. Louis encephalitis virus. J. Gen. Virol. 95, 1281–1288, http://dx.doi. org/10.1099/vir.0.063545-0. Das, T., Hoarau, J.J., Bandjee, M.C.J., Maquart, M., Gasque, P., 2015. Multifaceted innate immune responses engaged by astrocytes, microglia and resident dendritic cells against chikungunya neuroinfection. J. Gen. Virol. 96, 294–310, http://dx.doi.org/10.1099/vir.0.071175-0. Diniz, J.A.P., Da Rosa, A.P.A.T., Guzman, H., Xu, F., Xiao, S.-Y., Popov, V.L., Vasconcelos, P.F.C., Tesh, R.B., 2006. West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection. Am. J. Trop. Med. Hyg 75 (6), 691–696. Dong, Y., Benveniste, E.N., 2001. Immune function of astrocytes. Glia 36, 180–190, http://dx.doi.org/10.1002/glia.1107. Fischer, H.-G., Reichmann, G., 2001. Brain dendritic cells and Macrophages/Microglia in central nervous system inflammation. J. Immunol. 166, 2717–2726, http://dx.doi.org/10.4049/jimmunol.166.4.2717. Furr, S.R., Marriott, I., 2012. Viral CNS infections: role of glial pattern recognition receptors in neuroinflammation. Front. Microbiol. 3, 201, http://dx.doi.org/10. 3389/fmicb.2012.00201. Hussmann, K.L., Samuel, M.a, Kim, K.S., Diamond, M.S., Fredericksen, B.L., 2013. Differential replication of pathogenic and nonpathogenic strains of West Nile virus within astrocytes. J. Virol. 87, 2814–2822, http://dx.doi.org/10.1128/ JVI. 12-02577. Imai, T., Koyanagi, N., Ogawa, R., Shindo, K., Suenaga, T., Sato, A., Arii, J., Kato, A., Kiyono, H., Arase, H., Kawaguchi, Y., 2013. Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells. PLoS One 8, e72050, http://dx.doi.org/10.1371/journal.pone.0072050. Kawai, T., Akira, S., 2008. Toll-like receptor and RIG-I-like receptor signaling. Ann. N. Y. Acad. Sci. 1143, 1–20, http://dx.doi.org/10.1196/annals.1443.020. Kesson, A.M., Cheng, Y., King, N.J.C., 2002. Regulation of immune recognition molecules by flavivirus, West Nile. Viral Immunol. 15, 273–283, http://dx.doi. org/10.1089/08828240260066224. Lim, J.K., Louie, C.Y., Glaser, C., Jean, C., Johnson, B., Johnson, H., McDermott, D.H., Murphy, P.M., 2008. Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US epidemic. J. Infect. Dis. 197, 262–265, http://dx.doi.org/10. 1086/524691. Maharaj, P.D., Anishchenko, M., Langevin, S.A., Fang, Y., Reisen, W.K., Brault, A.C., 2012. Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes. J. Gen. Virol. 93, 39–49, http://dx.doi.org/10.1099/vir.0.033159-0. McCarthy, K.D., de Vellis, J., 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85 (3), 890–902. Middeldorp, J., Hol, E.M., 2011. GFAP in health and disease. Prog. Neurobiol. 93, 421–443, http://dx.doi.org/10.1016/j.pneurobio.2011.01.005. Mishra, M.K., Koli, P., Bhowmick, S., Basu, A., 2007. Neuroprotection conferred by astrocytes is insufficient to protect animals from succumbing to Japanese encephalitis. Neurochem. Int. 50, 764–773, http://dx.doi.org/10.1016/j.neuint. 2007.01.014.
100
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
Monath, T.P., Tsai, T.F., 2002. Flaviviruses. In: Richman, D.D. (Ed.), Clinical Virology. ASM Press, Washington, D.C, pp. 1097–1151. Monath, T.P., 2015. Saint Louis Encephalitis, in: Up To Date. Waltham, MA. http:// www.uptodate.com/contents/st-louisencephalitis?source=search result&search=saint+louis+encephalitis+virus&selectedTitle=1%7E150 (acessed 31.07.15). Pekny, M., Wilhelmsson, U., Pekna, M., 2014. The dual role of astrocyte activation and reactive gliosis. Neurosci. Lett. 565, 30–38, http://dx.doi.org/10.1016/j. neulet.2013.12.071. Pinheiro, F.P., LeDuc, J.W., Travassos da Rosa, A.P., Leite, O.F., 1981. Isolation of St. Louis encephalitis virus from a patient in Belém, Brazil. Am. J. Trop. Med. Hyg. 30, 145–148. Rao, J.S., Kellom, M., Kim, H.-W., Rapoport, S.I., 2012. Neuroinflammation and synaptic loss. Neurochem. Res. 37, 903–910, http://dx.doi.org/10.1007/s11064012-0708-2. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoint. Am. J. Hyg. 27, 493–497. Rodrigues, S.G., Nunes, M.R.T., Casseb, S.M.M., Prazeres, A.S.C., Rodrigues, D.S.G., Silva, M.O., Cruz, A.C.R., Tavares-Neto, J.C., Vasconcelos, P.F.C., 2010. Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal. J. Gen. Virol. 91, 2420–2427, http://dx.doi.org/10. 1099/vir.0.019117-0. Spinsanti, L.I., Díaz, L.A., Glatstein, N., Arselán, S., Morales, M.A., Farías, A.A., Fabbri, C., Aguilar, J.J., Ré, V., Frías, M., Almirón, W.R., Hunsperger, E., Siirin, M., Da Rosa, A.T., Tesh, R.B., Enría, D., Contigiani, M., 2008. Human outbreak of St. louis encephalitis detected in Argentina, 2005. J. Clin. Virol. 42, 27, http://dx.doi.org/ 10.1016/j.jcv.2007.11.022.
Suen, W.W., Prow, N. a, Hall, R. a, Bielefeldt-Ohmann, H., 2014. Mechanism of west nile virus neuroinvasion: a critical appraisal. Viruses 6, 2796–2825, http://dx. doi.org/10.3390/v6072796. Trent, D.W., et al., 1980. Variation among strains of St. Louis encephalitis virus: basis for a genetic, pathogenetic, and epidemiologic classification. Ann. N. Y. Acad. Sci. 354, 219. Uchil, P.D., Kumar, A.V. a, Satchidanandam, V., 2006. Nuclear localization of flavivirus RNA synthesis in infected cells. J. Virol. 80, 5451–5464, http://dx.doi. org/10.1128/JVI.01982-05. Villoslada, P., Genain, C.P., 2004. Role of nerve growth factor and other trophic factors in brain inflammation. Prog. Brain Res. 146, 403–414, http://dx.doi.org/ 10.1016/S0079-6123(03)46025-1. Yoneyama, M., Fujita, T., 2010. Recognition of viral nucleic acids in innate immunity. Rev. Med. Virol. 20, 4–22, http://dx.doi.org/10.1002/rmv.633. Zuo, J., Thomas, W.A., Haigh, T.A., Fitzsimmons, L., Long, H.M., Hislop, A.D., Taylor, G.S., Rowe, M., 2011. Epstein-Barr virus evades CD4+T cell responses in lytic cycle through BZLF1-mediated downregulation of CD74 and the cooperation of vBcl-2. PLoS Pathol. 7, e1002455, http://dx.doi.org/10.1371/journal.ppat. 1002455.
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Astrocyte response to St. Louis encephalitis virus Adriano Lara Zuza a , Heber Leão Silva Barros a , Thelma Fátima de Mattos Silva Oliveira a , Juliana Helena Chávez-Pavoni b , Renata Graciele Zanon a,∗ a b
Institute of Bioscience, Federal University of Uberlandia, Para 1720, Uberlandia, Minas Gerais CEP 38400-902, Brazil School of Medicine, Federal University of Mato Grosso, MT 270 St., Km 06, Rondonopolis, Mato Grosso, Brazil
a r t i c l e
i n f o
Article history: Received 4 January 2016 Received in revised form 7 March 2016 Accepted 8 March 2016 Available online 11 March 2016 Keywords: Flavivirus infections St. Louis encephalitis virus Astrocytes Glial fibrillary acidic protein
a b s t r a c t St. Louis encephalitis virus (SLEV), a flavivirus transmitted to humans by Culex mosquitoes, causes clinical symptoms ranging from acute febrile disorder to encephalitis. To reach the central nervous system (CNS) from circulating blood, the pathogen must cross the blood–brain barrier formed by endothelial cells and astrocytes. Because astrocytes play an essential role in CNS homeostasis, in this study these cells were infected with SLEV and investigated for astrogliosis, major histocompatibility complex (MHC)-Idependent immune response, and apoptosis by caspase-3 activation. Cultures of Vero cells were used as a positive control for the viral infection. Cytopathic effects were observed in both types of cell cultures, and the cytotoxicity levels of the two were compared. Astrocytes infected with a dilution of 1E-01 (7.7E + 08 PFU/mL) had a reduced mortality rate of more than 50% compared to the Vero cells. In addition, the astrocytes responded to the flavivirus infection with increased MHC-I expression and astrogliosis, characterized by intense glial fibrillary acidic protein expression and an increase in the number and length of cytoplasmic processes. When the astrocytes were exposed to higher viral concentrations, a proportional increase in caspase-3 expression was observed, as well as nuclear membrane destruction. SLEV immunostaining revealed a perinuclear location of the virus during the replication process. Together, these results suggest that mechanisms other than SLEV infection in astrocytes must be associated with the development of the neuroinvasive form of the disease. © 2016 Elsevier B.V. All rights reserved.
1. Introduction St. Louis encephalitis virus (SLEV) is an enveloped, positive, single-strand RNA virus that belongs to the family Flaviviridae, genus Flavivirus (Monath, 2015), and is serologically related to the Japanese encephalitis complex, which includes viruses such as Japanese encephalitis virus (JEV) and West Nile virus (WNV). SLEV is transmitted to humans mainly by Culex spp. bite (Monath and Tsai, 2002; Trent et al., 1980; Rodrigues et al., 2010). The virus is widely disseminated in the Americas (Spinsanti et al., 2008; Auguste et al., 2009; Rodrigues et al., 2010), and once inoculated, it may infect the central nervous system (CNS), causing encephalitis (Lim et al., 2008; Monath 2015). During the process of invading the CNS, the virus must cross the blood–brain barrier (BBB), formed by endotheliocytes and astrocytes, to reach the brain and spinal cord. The BBB is a highly
∗ Corresponding author at: Maria das Dores Dias 850, 601, 10, Uberlandia 38408206, Brazil. E-mail address: [email protected] (R.G. Zanon). http://dx.doi.org/10.1016/j.virusres.2016.03.005 0168-1702/© 2016 Elsevier B.V. All rights reserved.
selective, permeable membrane that regulates the passage of several substances into the brain’s extracellular fluid (Suen et al., 2014). If a virion succeeds in penetrating an endotheliocyte and crosses the basement membrane, it still has to infiltrate the astrocyte body to reach a neuron. Astrocytes, the key glial component of the CNS, are responsible for maintaining homeostasis for perfect neuronal function through the secretion of growth factors, glycogen storage, and detoxification of metals and xenobiotics (Chen et al., 2010; Furr and Marriot, 2012; Rao et al., 2012). In addition, these cells control BBB permeability, mainly due to their involvement in inflammatory processes through the expression of cytokines and nitric oxide synthase, inducing leucocyte migration and microglial activation (Bi et al., 1995). Astrocytes also express several molecules that are intimately involved in immune responses, such as major histocompatibility complex (MHC)-I and -II, antiviral interferon via pattern recognition receptors, interleukin (IL)-6, tumor necrosis factor ␣, and IL-1 (Kawai and Akira, 2008; Chauhan et al., 2010; Yoneyama and Fujita, 2010). Once a virion encounters an astrocyte, both innate and adaptive immune responses are triggered, culminating in the release of cytokines and chemotactic factors that recruit defense cells from
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
93
Fig. 1. Mortality rate in (a) Vero cells and (b) astrocytes at six days post-infection with different SLEV dilutions. Note that the viral concentration responsible for 94.03% of mortality in Vero cells caused only 38.18% of mortality in astrocytes.
the bloodstream. Thus, activated astrocytes (or astrogliosis) produce IL-6, IL-8, IL-1, IL-18, RANTES, interferon ␥-inducible protein 10, glial fibrillary acidic protein (GFAP), glutamate aspartate transporter, glutamate transporter 1, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) (Chen et al., 2010). The production of hepatocyte growth factor, ceruloplasmin, glutamine synthetase, vimentin, and reactive oxygen species by astrocytes has also been reported (Mishra et al., 2007). Astrogliosis increases the permeability of the BBB, allowing leukocytes in the bloodstream to cross into the nervous extracellular matrix. Once the BBB is disrupted, more virions can penetrate the nervous tissue coming from the blood, especially in patients with high viremia or immunologic deficiency, resulting in a massive infection of the brain and spinal cord (Dong and Benveniste, 2001; Fischer and Reichmann, 2001; Furr and Marriott, 2012). Considering that astrocytes are the maestros of BBB homeostasis, the investigation of this cell response to SLEV infection may contribute to a better comprehension of the mechanisms of viral infection of the CNS and encephalitis. To address this issue, this study aimed to investigate the astrocyte–SLEV interaction using primary astrocyte cultures compared to viral infection in the Vero cell lineage. Parameters such as astrogliosis, expression of MHCI molecules, and apoptosis were evaluated in both infected cell cultures.
2. Material and methods 2.1. Production of virus stock SLEV (BeH 355964 strain) was cultured by continuous passage in Aedes albopictus C6/36 cell linage and maintained in L15 culture medium (Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Nutricell, Campinas, Brazil), 100 U/mL penicillin, 100 L/mL streptomycin (Sigma-Aldrich) at 28 ◦ C. Cultures were harvested after 7 days or when cytopathic effect was observed. For harvesting, cells were frozen at –70 ◦ C (ultrafreezer Nuare, Plymouph, MN, USA) and immediately thawed. The suspension was transferred to a 15 mL Falcon tube and centrifuged at 1000 × g for debris removal. Supernatant was removed and the aliquots were stored at –70 ◦ C, constituting the virus stock.
2.2. Astrocyte primary culture Astrocyte primary cultures were made by dissociation of cerebral cortex of neonate Swiss mice (1–2 days postnatal). Animals were immersed in ice until reaching unconsciousness and decapitated for brain removal. All procedures in animals were submitted and approved by the Ethics Committee on animal use of the Federal University of Uberlandia (protocol number CEUA148/13).
94
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
Fig. 2. Immunofluorescence anti-SLEV (SLEV MIAF) in Vero (b, c, j, k) and astrocyte (e, f, m, n) infected cultures in different dilutions at six days post-infection. In astrocyte cultures anti-OROV (OROV MIAF) staining was used as a MIAF control (h, i, p, q). All cells were stained with DAPI, a nuclear marker. Non-infected cells served as control (a, d, g). An intense marking in the cytoplasm of Vero cells was observed in all viral dilutions, while astrocytes showed anti-SLEV marking only in undiluted dose of virus (reddish stain). Asterisks indicate apoptotic cells with vacuolated nucleus, nucleic acid dispersed in cytoplasm and viral particles in a perinuclear location. In (r), a similar characteristic was seen in OROV-infected astrocytes when stained with anti-OROV antibodies. Scale: 50 m.
Collected tissues were fragmented, washed in phosphatebuffered saline (PBS 0.1 M, pH 7.4) with no calcium or magnesium (Nutricell) and trypsinized for 10 min at 37 ◦ C. After trypsinization, DNAase (Sigma-Aldrich) was added to the solution, homogenized and then, FBS was added to block enzymatic action. Supernatant was centrifuged (250 × g for 10 min) in culture medium containing 4% bovine serum albumin (BSA; Sigma-Aldrich). DMEM was added to the cell precipitate, which was then seeded in 75 cm2 cell culture bottles (Nest Biotechnology CO., Shangai, China) and incubated at 37 ◦ C, 5% CO2. After about 10 days until reaching 80% of
cell confluence, cell cultures were purified according to McCarthy and de Vellis (1980). In short, cell cultures were gently rinsed three times with complete medium to remove floating cells. Each culture flask (with 10 mL of fresh medium) was closed and placed onto an orbital shaker for 15–18 h (37 ◦ C). Following the shaking period, the suspended cells from all flasks were removed. The culture flasks containing a nearly intact layer of astrocytes (purity ∼ 95%) were returned to incubator until reaching cell confluence. The cells were then trypsinized, centrifuged (250g for 10 min), counted (5 × 104 or 5 × 103 cells), seeded on 24 or 96-well plates (Nest
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
95
Fig. 3. Immunofluorescence of astrocyte cultures infected with SLEV in different dilutions (b–e) or OROV (f) and stained with DAPI and anti-GFAP (cytoplasmic stain) antibody at six days post-infection. Non-infected cells served as control (a). In (g), drawing represents the normal and reactive astrocytes, the dashed circle shows the location where the quantification of pixel density has been performed. The graph in (h) shows the pixel density measured in the different groups. One-way ANOVA and Bonferroni post-test. (*) p < 0.05. Scale: 50 m.
Biotechnology), respectively, and incubated for more three days at 37 ◦ C, until 80% of cell confluence to be reached, when then were infected with SLEV or Oropouche virus (OROV), one of the most common orthobunyaviruses that was used as negative control in SLEV immunostaining to evaluate the specificity of anti-SLEV antibodies. OROV was chosen because it is available in our laboratory as well as its marker for immunostaining (anti-OROV antibodies).
2.3. Astrocyte mortality rate in response to SLEV infection SLEV infection in both Vero and astrocyte cultures was evaluated from 1 to 6 days post-infection (p.i.) for cytopathic effect (CPE). In view of the differences in both growth rate and susceptibility to viruses of the two cell types, CPE was observed at 3 days p.i. in Vero cells and 6 days p.i. in astrocytes. For comparative purposes, the period of cell culture after infection used in our analyses was 6 days for both cell types. Alterations in cell cultures, such as cell morphological changes, cell disorganization, apoptosis induction and loss of cell adherence to culture plate, were considered for CPE scores, according to Norkin (2010) who defined alterations caused by viral infection with similar characteristics to the SLEV. Vero cells, known to be SLEV susceptible and permissive, were used as positive control. For this purpose, Vero cells were cultured in 96-well plates (5 × 103 cells/well, 100 L/well) for 6 days in DMEM supplemented with 10% FBS, at 37 ◦ C, 5% CO2 and humidity control. Plates were infected with seriated virus dilutions (1E-01, 1E-02, 1E-03, 1E-04, 1E-05 and 1E-06) and one row left as control group. Astrocytes were cultured under the same conditions and infected with different dilutions (1E + 01, 7.5E-01, 5E-01, 2.5E-01, 1E-01, 1E-02, 1E-03, 1E-04, 1E-05 and 1E-06). After 6 days of incubation, both cultures were submitted to MTT protocol as described below.
For MTT assay the culture medium was aspirated, and then added 50 L of MTT (Sigma Aldrich) solution (1 mg/mL) per well and incubated for more 4 h. After the incubation period, solution was completely removed and 100 L of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added in each well to solubilize formazan crystals. Plates were gently agitated at room temperature for 10 min and read at 540 nm (Elx 800, Bio-Tek, Winnosky, VT, USA). The absorbance measures were used to calculate the titer of the viral samples as described by Reed and Muench (1938). Cell mortality rate was calculated as follows: mortality rate = [(A − B)/A × 100], where A: absorbance of infected cells and B: absorbance of noninfected cells.
2.4. Immunocytochemistry To detect viral intracellular proteins in astrocyte and Vero cultures, polyclonal anti-SLEV antibodies obtained from mouse immune ascitic fluid (MIAF) and polyclonal mouse anti-OROV antibodies were used in astrocytes to confirm OROV infection and to provide a negative control of MIAF for SLEV immunostaining. Astrocytes were cultured in 96-well plates and infected for 1 h with SLEV in dilutions of 1E + 00, 5E-01, 1E-01, 1E-02, 1E-03, 1E-04, 1E-05 and 1E-06, or with OROV in the dilution of 1E-02. One plate column was left for negative control. After 6 days of incubation, culture medium was removed, cells were fixed in 4% paraformaldehyde (Synth, Diadema, Brazil) and DMEM, washed in PBS and incubated for 1 h at 37 ◦ C in 3% BSA and 0.2% Triton-X100 (Sinth,) solution. Then, astrocytes were incubated with the following polyclonal primary antibodies: goat anti-GFAP (1:100; Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-mouse cleaved caspase-3 (p11) (1:200, Abcam, Cambridge, MA, USA), mouse antiMHC-I (1:100, AbD SEROTEC, Kidlington, UK), rabbit anti-SLEV
96
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
Fig. 4. Immunofluorescence of astrocyte cultures infected with SLEV in different dilutions (b-e) or OROV (f) and stained with DAPI and anti-cleaved caspase 3 (CASP-3, asterisks) antibody at six days post-infection. Non-infected cells served as control (a). The graph (g) shows a significant increase in CASP-3 expression in all infected cells when compared to control. Mann-Whitney U test, p < 0.05 (*). Scale: 50 m.
diluted 1:100, and rabbit anti-OROV diluted 1:100 at 4 ◦ C for 24 h. All reactions were performed in duplicate. After incubation with primary antibodies, cultures were washed in PBS and incubated for 1 h with secondary antibodies (anti-goat, anti-mouse or anti-rabbit) conjugated with Alexa Fluor 488 or 594 (Jackson Lab., Bar Harbor, ME, USA). Cultures were stained with DAPI (Sigma-Aldrich) for cell nucleus visualization. Subsequently to incubation and washing, all cultures were kept in glycerol/PBS/DAPI for analysis in fluorescence inverted microscope. Four photographs of representative fields were shot from each well. Qualitative comparison was made for cultures immunostained with anti-SLEV and anti-OROV. Quantitative analysis was used for cultures immunostained with anti-GFAP, anti-caspase-3 and anti-MHC-I. Caspase-3 positive cells were counted and statistically analyzed using Mann-Whitney U test. GFAP and MHC-I immunostaining was analyzed by pixel densitometry in the photographs and statistically compared using one way ANOVA and Bonferroni post-test. All results were expressed as mean ± SD, and statistical significance was established when p < 0.05. 3. Results 3.1. Mortality rate Cell mortality rates were calculated using an MTT assay at six days p.i. for both Vero cells and astrocytes infected with different concentrations of SLEV, in order to compare virus–cell interactions.
The mortality percentage rate was calculated for each viral concentration (dilution) compared with non-infected cells (control). Cell mortality rate was 94.03% in the 1E-01Vero cell dilution (Fig. 1a). Vero cell viability was inversely proportional to decreasing concentrations of SLEV tested. The mortality rate of the astrocytes (Fig. 1b) was as high as 79.85% when exposed to the non-diluted viral solution and 38.18% in the 1E-01dilution, thus showing a reduced mortality rate of more than 50% compared to the Vero cells in the same viral concentration. As with the Vero cells, the mortality rates of the astrocytes also diminished when the cells were exposed to decreasing viral concentrations, indicating that the SLEV titer directly influenced the number of astrocytes that underwent death. 3.2. Virus invasion and astrocyte response to SLEV As shown in Fig. 2, SLEV immunostaining revealed a large amount of virions inside the astrocytes when these cells were exposed to a 7.7E + 08 PFU/mL concentration of the non-diluted virus solution (Fig. 2e, reddish stain). Immunostaining was much more intense in perinuclear locations, where the rough endoplasmic reticulum (RER) is located, and in nuclear locations in apoptotic cells, where the nucleus acquired a vacuolated aspect with dispersed chromatin in cytosol. As expected, all genetic contents were blue when stained for DAPI, a nucleic acid dye. The white asterisks, in anti-SLEV mouse immune ascitic fluids (MIAF) in a non-diluted SLEV sample, illustrate nuclear destruction during cell death, when
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
97
Fig. 5. Immunofluorescence of astrocyte cultures infected with SLEV in different dilutions (b–e) or OROV (f) and stained with DAPI and anti-MHC-I antibody (cytoplasmic stain) at six days post-infection. Non-infected cells served as control (a). The pixel densitometry analysis graph (g) shows significant increase in MHC-I expression in cells infected with non-diluted SLEV, SLEV 5E-01 and OROV 1E-02 when compared to control. One-way ANOVA and Bonferroni post-test. (*) p < 0.05. Scale: 50 m.
the nuclear envelope was scattered throughout the cytoplasm. The recently destroyed nucleus assumed a darker coloration, almost without any DAPI, and a vacuolated character. Anti-SLEV antibodies seemed to form dollops around the nucleus, and occasionally, small reddish vesicles in the cell boundary or even outside the cell. This image is characteristic of Flavivirus replication, which occurs in RER ending with vesicles budding from the Golgi apparatus, heading to the cytoplasmic membrane, when virions are released. As the dilution increased, the number of astrocytes immunostained for SLEV decreased significantly. In the 1E-06 dilution (Fig. 2n), a small decrease in the astrocyte population was observed compared to the control group, and there was almost no staining for the virus. SLEV infection in the Vero cells showed the presence of the virus in lower concentrations as well (Fig. 2k), indicating an important difference in the astrocyte response to SLEV that suggests a resistance to infection. In astrocytes infected with Oropouche virus (OROV) (Fig. 2o and r), a great number of cells were observed in apoptosis and, as expected, with no SLEV immunostaining, confirming MIAF specificity. In the 1E-02 dilution (Fig. 2r), the cells displayed an intense staining, similar to the staining described in astrocytes infected with non-diluted SLEV solution, with big perinuclear lumps and small vesicles strongly stained in the cell periphery and exterior. As mentioned previously, astrocytes are antigen-presenting cells that control BBB permeability and CNS homeostasis, and
mobilize defense cells from the bloodstream to the CNS. These actions are possible through a phenomenon called astrogliosis, which involves several structural modifications, such as an increase in the expression of GFAP and in the number and length of cytoplasmic processes, among others. In order to observe whether SLEV and OROV infection could induce astrogliosis in primary astrocyte cultures, the cells were stained for GFAP (Fig. 3). When the astrocytes were exposed to the non-diluted SLEV solution (Fig. 3b), most cells underwent apoptosis, with nuclear shrinking and/or rupture, and a decrease in the number of cytoplasmic processes, which appeared to be shorter and wider. In the 1E-06, 1E-01, and 5E-01 dilutions of SLEV, morphological alterations shown by GFAP staining were observed as an increase in the number and length of cytoplasmic processes, indicating a more intense astrogliosis reaction in the infected cells compared to the control. In the OROV-infected cells stained for GFAP (Fig. 3f), a large number of cells with a morphological aspect of cellular death were observed. The cytoplasm of the remaining cells appeared starry, with cytoplasmic processes. In addition, the number of caspase-3-positive cells increased proportionally with increasing viral concentrations (Fig. 4). The CASP-3 micrograph of non-diluted SLEV and OROV 1E-02 exhibited intense staining and a great number of cells with vacuolated nuclei due to nuclear envelope fragmentation. These images revealed the nuclear content (stained in blue), which should be restricted to the
98
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
nucleus, dispersed throughout the cytosol. A quantitative analysis was conducted to evaluate the number of apoptotic cells (cleaved caspase-3 positive), and it was found that astrocyte infection using a non-diluted SLEV solution induced cell death by apoptosis in 85.37% of cells, similar to the value observed in OROV-infected astrocytes (Fig. 4g). In increasing SLEV dilutions, the percentage of caspase-3-positive cells was less, around 12%, with no significant differences among the 5E-01, 1E-01, and 1E-06 dilutions. These data suggest that this virus strain could be well adapted to astrocytes, so that at lower concentrations, these cells would be able to coexist in a semi-permissive state of infection with the intracellular parasite, without undergoing activation or apoptosis. In other words, although astrocytes possess receptors in the plasma membrane outer layer, where the virus adsorbs and internalizes, the virus might not find an adequate environment inside the host for its replication. This finding might be one reason viral immunostaining does not increase over time. Several viruses, such as Epstein-Barr (Zuo et al., 2011) and herpes simplex virus 1 (Imai et al., 2013), hold kinase coding regions in their genome, which are generally used to avoid the immune system during infection and inhibiting MHC-I expression in the cell surfaces of the host. In contrast, Flavivirus members act by inducing the expression of MHC-I, MHC-II, and diverse adhesion molecules, using this signaling system to promote cell lysis by T cytotoxic lymphocytes, and thus increasing the host’s susceptibility to viral infection (Abraham and Manjunath, 2006). As mentioned earlier, astrocytes express MHC molecules, and, theoretically, in the adaptation process to enable a virus strain to coexist chronically with astrocytes, a signaling pathway that would need to be downregulated is the MHC. To test this hypothesis, cells in culture were checked for immune system activation via MHC-I (Fig. 5). Although it was expected that all of the astrocytes in vitro would display increased MHC-I expression after being exposed to SLEV and OROV, these cells revealed an intense increase in MHC-I signaling that was directly proportional to the viral concentration. A significant increase in MHC-I expression was noted in the SLEVinfected astrocytes in the non-diluted sample (43.9%) and the 5E-01 dilution (31.1%) (Fig. 5g). This finding suggests that even if adaption were to occur, it would not affect the ability of the astrocytes to present viral antigens via MHC-I.
4. Discussion The SLEV BeH355964 strain was first isolated in 1978 at Belem, in the state of Para, Brazil (Pinheiro et al., 1981; Rodrigues et al., 2010). Rodrigues et al. (2010), in a phylogenetic analysis based on the SLEV envelope (E) gene, suggested that the virus is widespread throughout the Americas, with approximately 30 different strains in circulation in Brazil alone. Their study also demonstrated the presence of a new viral lineage (VIII) in Brazilian Amazonia. According to their phylogenetic analysis, the strain used in the current study belongs to lineage Vb. Although no studies about the virulence of this particular strain have been described, we should consider the study of Maharaj et al. (2012), who used SLEV and WNV chimeric viruses to infect vertebrate (Vero and duck) and invertebrate (Culex) cells in vitro. The authors observed that WNV titers were 100 times greater than SLEV in both vertebrate cell lineages, and that chimeric phenotypes exhibited growth changes and cytopathogenicity. Ciota et al. (2014) evaluated the adaptive potential of SLEV to a possible new host (tick), and found that a minor change in the viral genetic consensual sequence might facilitate viral infection in ticks. However, after successive passages in the tick cell cultures, the virions lost their ability to infect the vertebrate cells. These findings arise some questions related to SLEV infection in astrocytes. Is it possible that SLEV has suffered genetic
alterations during several passages for viral stock production? Would this genetic alteration be capable of affecting viral replication in astrocytes? To discard the possibility of adaption and a false interpretation of impaired viral replication in astrocytes, viral stocks used in the present study were produced in the invertebrate C6/36 A. albopictus lineage, which is well adapted to SLEV infection and reaches high titer levels. A comparative analysis of these viral stocks was carried out in two vertebrate mammal lineages: Vero cells and astrocyte primary cultures from rodents. One secure way to evaluate astrocyte responses involves the observation of morphological alterations, such as increased wideness of intermediate filament structures and, consequently, its constitutive proteins, especially GFAP, which demonstrates robust expression when astrocytes became activated (Middeldorp and Hol, 2011; Pekny et al., 2014). Virus infection is capable of inducing astrogliosis, as reported by Mishra et al. (2007), who observed longer and wider astrocytic processes after four days of JEV infection. In a similar manner, in our study we demonstrated that astrocyte invasion by SLEV induced astrogliosis, with an increase in the density of cytoplasmic processes at a viral concentration below that of the non-diluted solution. Chen et al. (2010) reported that the morphology of astrocytes was not significantly affected after JEV infection, contradicting Mishra et al. (2007), who described cytopathic effects in astrocyte cultures four days after JEV infection. A cytopathic effect was observed in our astrocyte cultures six days after SLEV infection, but only in the non-diluted solution. At lower concentrations, no morphological changes compatible with cell damage were found. In addition, astrocyte infection induced apoptosis through the caspase-3-dependent pathway, confirmed by immunocytochemical analysis. Quantification of cleaved caspase-3-positive cells revealed that programed cell death increased with the nondiluted SLEV solution. At lower viral concentrations, the astrocytes responded with a significant increase in caspase-3 levels, but at a lower intensity, corroborating our other results that astrocytes are resistant to SLEV infection. The phenomena of astrogliosis, caspase activation, and transcription of innate immune response genes in astrocytes have also been described in Chikungunya infection, an Orthobunyavirus (Das et al., 2015), reinforcing the importance of this cell type in controlling viral infections of CNS. After WNV infection in primary astrocyte cultures, Diniz et al. (2006) described a cytopathic effect consisting of mild morphological alterations, mostly eight days after infection and stabilizing around the 30th day. According to their study, the infected astrocytes became elongated with long cytoplasmic processes before death. At the fourth day p.i., approximately 30% of the remaining cells underwent apoptosis. Given these results, the authors concluded that astrocyte infection by WNV occurred slowly, and some cells did not show signs of infection, as they were cultivated successfully for 114 days in the presence of infectious viral particles without any cytopathic effects, maintaining a persistent and permissive state of infection. These findings suggest that astrocytes might be responsible for chronic viral infections due to the maintenance of WNV in the CNS. This finding was also confirmed by Hussmann et al. (2013), who reported that reduced astrocyte susceptibility to WNV was due to a delay in the replication of the viral genome and a decrease in cell-to-cell interferon-dependent infection. As such, astrocytes might be able to regulate WNV dissemination in the CNS, constituting a potential target for WNV neuropathology research. The morphological alterations found in the current study were similar to those described by Diniz et al. (2006). After six days of infection with 7.7E + 08 PFU/mL (non-diluted SLEV solution), we found that 79.85% of astrocytes were not viable. A mortality rate of 50% of the astrocytes in culture was observed in the 1E-03 dilution, corroborating the hypothesis that, in fact, these cells are resistant
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
to SLEV. Future studies should be performed to confirm the presence of chronic SLEV infection in astrocytes, similar to what occurs in WNV, which could justify the long period of recovery postencephalitis and the sequels experienced by patients with SLEV encephalitis. In a similar manner, Diniz et al. (2006) demonstrated that the intracellular distribution of WNV in infected astrocytes would be in a perinuclear location. Uchil et al. (2006) also observed that a significant proportion (20%) of RNA-polymerase-dependent total RNA in cells infected with WNV, JEV, and dengue virus were within the nucleus, along with the most important NS3 and NS5 replicases of JEV, demonstrating the nuclear location and synthesis of vRNA. They hypothesized that the host cell nucleus would be an additional site for replicase active complexes. To better understand the perinuclear location of anti-SLEV antibodies observed in our results, further studies need to be conducted to confirm the presence and synthesis of vRNA inside the nuclei of CNS cells. The low astrocyte infectivity by SLEV might be due to the capacity of these cells to live with the intracellular virus at low titers without triggering the apoptotic pathway or in the diverse functions of these cells, especially for their neuroprotective role in the CNS through the synthesis of neurotrophic factors and neurotrophins (Villoslada and Genain, 2004). We can also suggest that astrocytes are cells that are semi- or non-permissive to SLEV (Brooks et al., 2014), as our findings revealed infected cells with a low mortality rate, demonstrated by MTT and immunostaining with cleaved caspase-3, where cell viability was only compromised in the presence of non-diluted or a low dilution of viral solution. Mishra et al. (2007) reported that astrocytes presented increased CNTF levels in acute disseminated encephalomyelitis and increased NGF levels in viral encephalitis, and that all alterations were associated with neuroimmunomodulation and neuroprotection. Flaviviruses such as WNV induce the expression of MHC-I, MHCII, and adhesion molecules such as intercellular adhesion molecule 1 (CD54), vascular cell adhesion molecule 1 (CD106), and Eselectin on the cell surface (Kesson et al., 2002). MHC-I and MHC-II molecules are involved in the activation of TCD8 and TCD4 lymphocytes, respectively, inducing an immune adaptive response (Abbas et al., 2014). Abraham and Manjunath (2006) reported that MHC-I molecules were upregulated in JEV-infected astrocytes while MHCII remained unaltered, and that the maximum expression of MHC-I on the surface of infected astrocytes occurred 48 h after infection. In our study, SLEV was incubated for six days, and we found that astrocyte infection by SLEV, in a manner similar to that of JEV, resulted in an increase in MHC-I, mostly in cultures infected with higher viral concentrations, thus confirming that astrocytes are immunocompetent cells. Our results raise new questions regarding the astrocyte–SLEV interaction. In order to better understand how SLEV invades the CNS, more research must be conducted to investigate the chronic infection of astrocytes by SLEV, as with WNV; the existence of factors that prevent viral genome replication in astrocytes, causing a reduction in interferon-dependent cell–cell infection, as observed with WNV; whether flavivirus nuclear replicase complexes of SLEV are formed as described for JEV; and whether SLEV infection raises the genic expression of neurotrophins such as NGF, CNTF, and BDNF. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We are very grateful to Professor Luiz Tadeu Moraes Figueiredo (University of São Paulo, School of Medicine, Ribeirão Preto, SP,
99
Brazil) for suppling us with the viral aliquots of SLEV (BeH 355964 strain) and OROV (BeAn 19991 strain) as well as the MIAFs used in this work, and to Professor Jonny Yokosawa (Federal University of Uberlândia, MG, Brazil) for helping us with virus replication. We gratefully acknowledge financial support from the CNPq (474050/2011-4) and Fapemig (CDS-APQ-04285-10) agencies. References Abbas, A.K., Lichtman, A.H., Pillai, S., 2014. Basic Immunology: Functions and Disorders of the Immune System, 4th ed. Elsevier, Saunders, Philadelphia. Abraham, S., Manjunath, R., 2006. Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus. Virus Res. 119, 216–220, http://dx.doi.org/10.1016/j.virusres.2006.03.006. Auguste, A.J., Pybus, O.G., Carrington, C.V.F., 2009. Evolution and dispersal of St. Louis encephalitis virus in the Americas. Infect. Genet. Evol. 9, 709–715, http:// dx.doi.org/10.1016/j.meegid.2008.07.006. Bi, Z., Barna, M., Komatsu, T., Reiss, C.S., 1995. Vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity. J. Virol. 69, 6466–6472. Brooks, G.F., Carroll, K.C., Butel, J.S., Morse, S.A., Mietzner, T.A., 2014. Jawetz, Melnick & Adelberg’s Medical Microbiology, 26th ed. McGraw-Hill Companies, Chicago. Chauhan, V.S., Furr, S.R., Sterka, D.G., Nelson, D.A., Moerdyk-Schauwecker, M., Marriott, I., Grdzelishvili, V.Z., 2010. Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses. Virology 400, 187–196, http://dx.doi.org/10.1016/j. virol.2010.01.025. Chen, C.-J., Ou, Y.-C., Lin, S.-Y., Raung, S.-L., Liao, S.-L., Lai, C.-Y., Chen, S.-Y., Chen, J.-H., 2010. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. J. Gen. Virol. 91, 1028–1037, http://dx.doi.org/10. 1099/vir.0.013565-0. Ciota, A.T., Payne, A.F., Ngo, K. a., Kramer, L.D., 2014. Consequences of in vitro host shift for St. Louis encephalitis virus. J. Gen. Virol. 95, 1281–1288, http://dx.doi. org/10.1099/vir.0.063545-0. Das, T., Hoarau, J.J., Bandjee, M.C.J., Maquart, M., Gasque, P., 2015. Multifaceted innate immune responses engaged by astrocytes, microglia and resident dendritic cells against chikungunya neuroinfection. J. Gen. Virol. 96, 294–310, http://dx.doi.org/10.1099/vir.0.071175-0. Diniz, J.A.P., Da Rosa, A.P.A.T., Guzman, H., Xu, F., Xiao, S.-Y., Popov, V.L., Vasconcelos, P.F.C., Tesh, R.B., 2006. West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection. Am. J. Trop. Med. Hyg 75 (6), 691–696. Dong, Y., Benveniste, E.N., 2001. Immune function of astrocytes. Glia 36, 180–190, http://dx.doi.org/10.1002/glia.1107. Fischer, H.-G., Reichmann, G., 2001. Brain dendritic cells and Macrophages/Microglia in central nervous system inflammation. J. Immunol. 166, 2717–2726, http://dx.doi.org/10.4049/jimmunol.166.4.2717. Furr, S.R., Marriott, I., 2012. Viral CNS infections: role of glial pattern recognition receptors in neuroinflammation. Front. Microbiol. 3, 201, http://dx.doi.org/10. 3389/fmicb.2012.00201. Hussmann, K.L., Samuel, M.a, Kim, K.S., Diamond, M.S., Fredericksen, B.L., 2013. Differential replication of pathogenic and nonpathogenic strains of West Nile virus within astrocytes. J. Virol. 87, 2814–2822, http://dx.doi.org/10.1128/ JVI. 12-02577. Imai, T., Koyanagi, N., Ogawa, R., Shindo, K., Suenaga, T., Sato, A., Arii, J., Kato, A., Kiyono, H., Arase, H., Kawaguchi, Y., 2013. Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells. PLoS One 8, e72050, http://dx.doi.org/10.1371/journal.pone.0072050. Kawai, T., Akira, S., 2008. Toll-like receptor and RIG-I-like receptor signaling. Ann. N. Y. Acad. Sci. 1143, 1–20, http://dx.doi.org/10.1196/annals.1443.020. Kesson, A.M., Cheng, Y., King, N.J.C., 2002. Regulation of immune recognition molecules by flavivirus, West Nile. Viral Immunol. 15, 273–283, http://dx.doi. org/10.1089/08828240260066224. Lim, J.K., Louie, C.Y., Glaser, C., Jean, C., Johnson, B., Johnson, H., McDermott, D.H., Murphy, P.M., 2008. Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US epidemic. J. Infect. Dis. 197, 262–265, http://dx.doi.org/10. 1086/524691. Maharaj, P.D., Anishchenko, M., Langevin, S.A., Fang, Y., Reisen, W.K., Brault, A.C., 2012. Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes. J. Gen. Virol. 93, 39–49, http://dx.doi.org/10.1099/vir.0.033159-0. McCarthy, K.D., de Vellis, J., 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85 (3), 890–902. Middeldorp, J., Hol, E.M., 2011. GFAP in health and disease. Prog. Neurobiol. 93, 421–443, http://dx.doi.org/10.1016/j.pneurobio.2011.01.005. Mishra, M.K., Koli, P., Bhowmick, S., Basu, A., 2007. Neuroprotection conferred by astrocytes is insufficient to protect animals from succumbing to Japanese encephalitis. Neurochem. Int. 50, 764–773, http://dx.doi.org/10.1016/j.neuint. 2007.01.014.
100
A.L. Zuza et al. / Virus Research 217 (2016) 92–100
Monath, T.P., Tsai, T.F., 2002. Flaviviruses. In: Richman, D.D. (Ed.), Clinical Virology. ASM Press, Washington, D.C, pp. 1097–1151. Monath, T.P., 2015. Saint Louis Encephalitis, in: Up To Date. Waltham, MA. http:// www.uptodate.com/contents/st-louisencephalitis?source=search result&search=saint+louis+encephalitis+virus&selectedTitle=1%7E150 (acessed 31.07.15). Pekny, M., Wilhelmsson, U., Pekna, M., 2014. The dual role of astrocyte activation and reactive gliosis. Neurosci. Lett. 565, 30–38, http://dx.doi.org/10.1016/j. neulet.2013.12.071. Pinheiro, F.P., LeDuc, J.W., Travassos da Rosa, A.P., Leite, O.F., 1981. Isolation of St. Louis encephalitis virus from a patient in Belém, Brazil. Am. J. Trop. Med. Hyg. 30, 145–148. Rao, J.S., Kellom, M., Kim, H.-W., Rapoport, S.I., 2012. Neuroinflammation and synaptic loss. Neurochem. Res. 37, 903–910, http://dx.doi.org/10.1007/s11064012-0708-2. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoint. Am. J. Hyg. 27, 493–497. Rodrigues, S.G., Nunes, M.R.T., Casseb, S.M.M., Prazeres, A.S.C., Rodrigues, D.S.G., Silva, M.O., Cruz, A.C.R., Tavares-Neto, J.C., Vasconcelos, P.F.C., 2010. Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal. J. Gen. Virol. 91, 2420–2427, http://dx.doi.org/10. 1099/vir.0.019117-0. Spinsanti, L.I., Díaz, L.A., Glatstein, N., Arselán, S., Morales, M.A., Farías, A.A., Fabbri, C., Aguilar, J.J., Ré, V., Frías, M., Almirón, W.R., Hunsperger, E., Siirin, M., Da Rosa, A.T., Tesh, R.B., Enría, D., Contigiani, M., 2008. Human outbreak of St. louis encephalitis detected in Argentina, 2005. J. Clin. Virol. 42, 27, http://dx.doi.org/ 10.1016/j.jcv.2007.11.022.
Suen, W.W., Prow, N. a, Hall, R. a, Bielefeldt-Ohmann, H., 2014. Mechanism of west nile virus neuroinvasion: a critical appraisal. Viruses 6, 2796–2825, http://dx. doi.org/10.3390/v6072796. Trent, D.W., et al., 1980. Variation among strains of St. Louis encephalitis virus: basis for a genetic, pathogenetic, and epidemiologic classification. Ann. N. Y. Acad. Sci. 354, 219. Uchil, P.D., Kumar, A.V. a, Satchidanandam, V., 2006. Nuclear localization of flavivirus RNA synthesis in infected cells. J. Virol. 80, 5451–5464, http://dx.doi. org/10.1128/JVI.01982-05. Villoslada, P., Genain, C.P., 2004. Role of nerve growth factor and other trophic factors in brain inflammation. Prog. Brain Res. 146, 403–414, http://dx.doi.org/ 10.1016/S0079-6123(03)46025-1. Yoneyama, M., Fujita, T., 2010. Recognition of viral nucleic acids in innate immunity. Rev. Med. Virol. 20, 4–22, http://dx.doi.org/10.1002/rmv.633. Zuo, J., Thomas, W.A., Haigh, T.A., Fitzsimmons, L., Long, H.M., Hislop, A.D., Taylor, G.S., Rowe, M., 2011. Epstein-Barr virus evades CD4+T cell responses in lytic cycle through BZLF1-mediated downregulation of CD74 and the cooperation of vBcl-2. PLoS Pathol. 7, e1002455, http://dx.doi.org/10.1371/journal.ppat. 1002455.