Lymphocytes have a role in protection, but not in pathogenesis, during La Crosse Virus infection in mice
© The Author(s). 2017
Received: 14 October 2016
Accepted: 7 March 2017
Published: 24 March 2017
La Crosse Virus (LACV) is a primary cause of pediatric viral encephalitis in the USA and can result in severe clinical outcomes. Almost all cases of LACV encephalitis occur in children 16 years or younger, indicating an age-related susceptibility. This susceptibility is recapitulated in a mouse model where weanling (3 weeks old or younger) mice are susceptible to LACV-induced disease, and adults (greater than 6 weeks) are resistant. Disease in mice and humans is associated with infiltrating leukocytes to the CNS. However, what cell types are infiltrating into the brain during virus infection and how these cells influence pathogenesis remain unknown.
In the current study, we analyzed lymphocytes recruited to the CNS during LACV-infection in clinical mice, using flow cytometry. We analyzed the contribution of these lymphocytes to LACV pathogenesis in weanling mice using knockout mice or antibody depletion. Additionally, we studied at the potential role of these lymphocytes in preventing LACV neurological disease in resistant adult mice.
In susceptible weanling mice, disease was associated with infiltrating lymphocytes in the CNS, including NK cells, CD4 T cells, and CD8 T cells. Surprisingly, depletion of these cells did not impact neurological disease, suggesting these cells do not contribute to virus-mediated damage. In contrast, in disease-resistant adult animals, depletion of both CD4 T cells and CD8 T cells or depletion of B cells increased neurological disease, with higher levels of virus in the brain.
Our current results indicate that lymphocytes do not influence neurological disease in young mice, but they have a critical role protecting adult animals from LACV pathogenesis. Although LACV is an acute virus infection, these studies indicate that the innate immune response in adults is not sufficient for protection and that components of the adaptive immune response are necessary to prevent virus from invading the CNS.
KeywordsLymphocytes La Crosse Virus Brain Central nervous system Encephalitis
La Crosse Virus (LACV) is a tri-segmented, single-stranded, negative-sense RNA virus of the genus Orthobunyavirus, in the Bunyaviridae family. The virus is primarily transmitted by the Eastern Tree Hole mosquito (Ochlerotatus triseriatus) and was first shown to cause human neurological disease when isolated from the brain of a 4-year girl who died from viral encephalitis in La Crosse, Wisconsin in 1960 . Cases of LACV encephalitis have been reported in 29 states, and the virus has been found in other invasive mosquito species, suggesting this is an emerging disease [2, 3]. LACV is a primary cause of pediatric viral encephalitis in the USA, with almost all cases of LACV-induced neurological disease observed in children under the age of 16. Some of these cases can be severe, with symptoms such as paralysis or coma and in a few cases per year, even death. In contrast, in adults and older teens LACV infection is generally either asymptomatic or causes a mild febrile disease. It does not cause encephalitis, suggesting that in adults virus is effectively cleared following infection.
Currently, there is no vaccine available to inhibit LACV infection [4, 5] or therapy to treat virus-induced encephalitis . Thus, a better understanding of the mediators of viral pathogenesis in the central nervous system (CNS) as well as the mechanisms by which adults are protected from the development of encephalitis is necessary to mitigate disease impact. In patient brain biopsies and animal models of LACV-induced encephalitis, a common feature of disease pathology is infiltration of leukocytes in the brain [6–8]. However, the make-up of this infiltrating population and their potential role in LACV pathogenesis remains unknown. We have previously established that circulating leukocytes do not contribute to virus neuroinvasion  as has been suggested for other encephalitic viruses [10, 11]. However, these cells may have other roles in influencing viral pathogenesis. Infiltrating lymphocytes including natural killer (NK) cells, CD8+ T cells, CD4+ T cells, and B cells are often necessary for viral clearance from the brain, mediate recovery from multiple encephalitic virus infections [12–14] and may protect against LACV-induced neuronal damage. However, these cells can also contribute to neuronal damage, as observed with West Nile encephalitis [15–17]. Determining which cell populations gain access to the CNS during LACV infection, as well as the influence of these cells on disease, is essential for developing therapeutics to inhibit this disease.
The murine model of LACV infection is ideal for studying neuropathogenesis and disease resistance, as it recapitulates the age-dependent susceptibility observed in humans [18, 19]. Following peripheral infection, adult mice are resistant to neurological disease and have low CNS titers , while weanlings are susceptible and have high CNS titers [18, 20]. This age-related resistance in adults appears to be due to the inability of LACV to replicate in the periphery and/or invade the CNS, as administration of LACV to the CNS by intracerebral  or intranasal inoculation  results in neurological disease in adult mice. Suppression of type I interferon (IFN) signaling in the periphery or depletion of myeloid dendritic cells (DCs) in adult animals can result in neurological disease [21, 22], suggesting that an efficient anti-viral type I IFN response is necessary for preventing disease in adult animals. However, it remains unknown whether the innate immune response is sufficient to control LACV infection in adult animals, or if adaptive immune responses, including the production of neutralizing antibodies, are also necessary in preventing the development of neurological disease.
In the current study, we examined the role of different lymphocyte populations in mediating neurological disease following LACV infection in weanling mice. Additionally, we determined if these lymphocytes influenced viral clearance and protection in adult mice. Lymphocyte-deficient transgenic mice and depletion studies demonstrated that lymphocytes did not significantly influence LACV-induced neurological disease in young animals. However, both B and T cells were necessary for efficient virus clearance and the prevention of neurological disease in adult animals. These findings demonstrate that while lymphocytes are not mediators of disease in young susceptible animals, they do provide protection in resistant adult animals. Furthermore, this study indicates that both innate and adaptive immune responses are essential for efficient virus clearance and the prevention of neurological disease following LACV infection.
Infection of mice with LACV and neurological disease criteria
All animal studies were conducted using the animal protocol RML2014-011, which was approved by the NIH/NIAID/RML Institutional Animal Care and Use Committee. Founder wildtype (C57BL/6), Rag1 -/-, and μMT -/- mice on the C57BL/6 background were purchased from Jackson Laboratories and maintained in a breeding colony at RML. LACV 1978 stock, a human isolate, was a kind gift from Richard Bennett (NIAID, NIH) and has been previously described . Mice at 3 (weanling) or 6–8 (adult) weeks of age were inoculated with 103 plaque forming units (PFU) of LACV in phosphate-buffered saline (PBS) intraperitoneally (i.p.) in a volume of 200 μl/mouse. Mock infections consisted of an equal volume of Vero cell culture supernatant diluted into PBS. Mice were observed daily for signs of neurological disease that included hunched posture, seizures, reluctance, or inability to move normally or paralysis. Animals with clear clinical signs of neurological disease were scored as clinical and euthanized immediately.
Treatment of mice with cell-depleting antibodies
For the depletion of T cells, anti-CD8 clone 169.4 and anti-CD4 clone 191.1 hybridomas were grown in RPMI media containing 10% FBS to a concentration of 2 mg/ml as measured by 260/280 absorbance. Supernatants (gift of Dr. Kim Hasenkrug, NIAID, NIH) were harvested and spun at 500 × g for 10 min to remove any cellular debris and then stored at −20 °C until use. Weanling mice were injected i.p. with 0.5 ml of the supernatant a total of three times (1, 3, and 5 days post infection (dpi)). Dual CD8 T cell- and CD4 T cell-depleted mice received two injections (a total of 1 ml of supernatant) at each indicated time point. Adult LACV-infected mice followed the same injection schedule with two additional injection days at 12 and 19 dpi. Control mice were injected on the same schedules with 10% FBS in RPMI. T cell depletion was confirmed by flow cytometry using CD3, CD4, CD8a, and CD8b.2 antibodies.
LACV-infected weanling mice were depleted of natural killer (NK)-cells by the i.p. administration of 50 μl of rabbit anti-Asialo-GM1 (Wako) at 1, 3, and 5 dpi. Adult LACV-infected mice received the same injections with an additional injection at 9 dpi. NK cell depletion was confirmed by flow cytometry using NK1.1 and CD49b (clone DX5) antibodies.
Evans Blue dye treatment
LACV-infected mice were given Evans Blue dye (200 μl of 20 mg/ml intravenously) in PBS at 6 dpi, just prior to the onset of clinical disease. Thirty minutes following dye infusion, mice were perfused transcardially with 5 ml of heparinized saline (100 U/ml) and the brains removed and processed for immunohistochemistry as indicated below. Dye leakage was visualized using epifluorescence microscopy in the TRITC channel.
Tissues processing for flow cytometry
For phenotypic profiling, verification of T cell depletion studies and lymphocyte activation/proliferation analysis, whole brains from mock and LACV-infected weanling mice were isolated at specific time points and a single-cell suspension made by homogenization and passage through a 70 μm filter. Individual mice were compared to allow determination of variation between animals. Cells were pelleted and resuspended in 70% Percoll/PBS and underlayed on a 0–30% step Percoll gradient which was centrifuged at 500g for 20 min at 4 °C. CNS immune cells were recovered at the 30–70% interface, rinsed in PBS, and placed on ice to await fixing or staining. For verification of antibody-mediated cell depletions and lymphocyte-activation/proliferation analysis, the spleens from weanling and adult mice were homogenized through a 70 μm filter to generate a single-cell suspension and red blood cells were removed using 2% dextran T500–PBS and/or lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 M EDTA).
Phenotyping CNS-infiltrating immune cells and splenocytes by flow cytometry
Cells were isolated as described above and then processed for flow cytometry as previously published . Briefly, cells were fixed in 2% paraformaldehyde and then permeabilized with 0.1% saponin–2% bovine serum albumin (BSA) in PBS. Fc receptors were blocked using CD16/CD32 Fcγ III/II (BD Biosciences, clone 2.4G2). Cells were stained using the following panel of antibodies (all antibodies used for flow cytometry were purchased from BD Pharmigen, BioLegend, Miltenyi, eBiosciences, or Molecular Probes) to establish a lymphocyte phenotype: CD45-PE (30-F11), CD4-APC/Cy7 (GK1.5), CD8a-PB (53-6.7), CD8b.2-FITC (53-5.8), CD3-PerCP/Cy5.5 (UCHT1), CD19-PE-CF594 (1D3), NK1.1-AF700 (PK136), and CD49b (DX5)-PE (DX5). The following antibodies were used in various combinations with the antibodies from the lymphocyte panel to exclude non-lymphocytic cells: CD11c-PE/Cy7 (HL3), pDCA1-APC (JF05-1C2.4.1), CD11b-APC (M1/70), Ly6G-PB (1A8), Ly6C-AF700 (HK1.4), and F480-BV510 (BM8). All flow cytometry data was obtained using an LSRII (BD Biosciences) and analyzed using either FlowJo software (version 10.2; TreeStar, Inc) or FCS Express software (version 3, De Novo). Live cells were retained and doublets excluded using SSC-A and FSC-A gating and then live cells were gated by time to exclude any artifact caused by erratic sample flow.
Analysis of CNS-infiltrating and splenic lymphocyte activation and proliferation by flow cytometry
To determine lymphocyte activation and proliferation state, CNS-infiltrating cells and splenocytes were isolated as described above and surface labeled for ~30 min at 4 °C. Antibodies used for surface staining were as follows: Pacific Blue-anti-CD8 (53-6.7) and CD4 (RM4-5), APC/Cy7-anti-CD3e (17A2), PerCP-Cy5.5-anti-CD43 (1B11), AF488-anti-CD25 (PC61), FITC-anti-CD107a (1D4B), PE-anti-CD11a (2D7), BV605-anti-KLRG1 (2F1), PE-CF594-anti-PD1 (J43), and PE-Cy7-anti-CD62L (MEL-14). Cells were then fixed overnight using reagents and following recommendations from the eBioscience Foxp3 kit, permeabilized, and then stained intracellularly with APC-anti-Foxp3 (FJK-16s) or APC-anti-Granzyme B (GRB05) and Alexa700-anti-Ki-67 (B56). Cells were then washed and fixed for 30 min at 4 °C with 2% paraformaldehyde. Flow cytometry data was collected as described above. T cells were positively gated by either CD4+ or CD8+ and CD3+ co-expression, and in the case of CD4+ T helper cells, Foxp3 exclusion. T cells were further analyzed for the expression of the phenotyping markers listed above. Splenocytes from the naïve weanlings and adults (and non-specific Ig or FMO controls where appropriate) were used to determine gating placement.
Viremia and neutralizing antibody detection
For detection of viremia, serially diluted plasma was plated directly to Vero cells as described below. For quantification of neutralizing antibody, serially diluted plasma was mixed with 102 PFU of LACV in a final volume of 200 μl in DMEM/2% FBS/1% Pen Strep. The mixture was incubated for 1 h at 37 °C for neutralization. After neutralization, the 200 μl mixture was added to confluent Vero cells in a 24-well plate and incubated again for 1 h at 37 °C. After incubation, 500 μl of 1.5% carboxymethyl cellulose in MEM was overlaid onto the cells and the cells were incubated undisturbed at 37 °C for 5 days. Cells were then fixed by adding 10% formaldehyde to each well until full and allowed to sit for 1 h at room temperature. After fixation, plates were rinsed gently with deionized water and stained with 0.35% crystal violet for 15 min. Plates were rinsed and allowed to air dry inverted. Viral titer was calculated by dividing the number of plaques per given sample by the plasma dilution factor multiplied by the volume of each well. Neutralizing antibody titer was determined by the dilution that inhibited at least 50% of plaque formation when compared to cells infected with the 102 LACV.
Real-time PCR analysis of mRNA expression was completed as previously described . The primers used include Gapdh.2-152F (TGCACCACCAACTGCTTAGC), Gapdh.2-342R (TGGATGCAGGGATGATGTTC), LACVs.2-552F (ATTCTACCCGCTGACCATTG), and LACVs.2-650R (GTGAGAGTGCCATAGCGTTG). Primers were subjected to BLAST analysis (NCBI) to ensure detection of only the specified gene and were tested on positive controls to ensure amplification of a single product. Data for each sample were calculated as the percent difference in threshold cycle (C T ) value (ΔC T = C T for glyceraldehyde-3-phosphate dehydrogenase [GAPDH] gene − C T for specified gene). Gene expression was plotted as the percentage of gene expression relative to that of the GAPDH gene.
All statistical analyses were performed using Prism software Version 7.01 (GraphPad) and are described in the figure legends.
Lymphocyte infiltration into the CNS is associated with areas of LACV infection in weanling mice
Encephalitis associated with LACV infection in both mice and humans is in part characterized by perivascular infiltration of leukocytes into the CNS [7, 8], although the makeup of the infiltrate is not known. Immunohistochemical analysis of brain tissue sections from LACV-infected weanling mice showed CD3+ T cells in areas of LACV-infected neurons at the time of clinical disease (Fig. 1a). To determine lymphocyte subtypes and when they first entered the CNS, we analyzed cellular infiltrate in the CNS by flow cytometry at pre- and clinical time points using a lymphocyte-specific antibody panel described in the “Methods”. Although the majority of infiltrating CD45hi cells in clinical mice were CD11b+ positive myeloid cells (data not shown), CD4+ T cells, CD8+ T cells, and NK1.1+ lymphocytes were consistently found in the CNS (Fig. 1b, c). CD8+ and CD4+ T cells were increased in the CNS relative to mock controls at the onset (~7 dpi) of LACV-induced neurological disease (Fig. 1d, e) while CD19+ B cells were not detected in the CNS at any point during LACV infection (Fig. 1g). NK1.1+ NK cells were significantly increased in the CNS at the pre-clinical 5 dpi time point and remained elevated through to clinical disease (Fig. 1f). Thus, lymphocyte infiltration of the weanling brain occurs during LACV encephalitis and could play a critical role in neurological disease.
NK cells are minor contributors to LACV-induced pathogenesis in weanling mice
Infiltrating CD4+ and CD8+ T cells are proliferative and express effector cell markers but do not contribute to LACV-induced neurological disease
To determine a role for these infiltrating, activated T cells in viral pathogenesis, we depleted each cell type with monoclonal antibodies in weanling mice prior to LACV infection. Flow analysis of infiltrating cells in the brain demonstrated efficient depletion of each cell type with their respective antibodies (Fig. 3c). A slight decrease in CD8+ T cells in the CNS was observed in anti-CD4-treated mice, suggesting that CD4 depletion may affect the CD8+ T cell recruitment to the CNS (Fig. 3c). However, depletion of either T cell subtype in weanling mice did not significantly alter the occurrence or onset of neurological disease following LACV infection (Fig. 3d), suggesting that T cells are not involved in either the development of neurological disease or in inhibiting its progression.
IgG antibody is present in the CNS during LACV-induced neurological disease in weanling mice but does not alter neurological disease
To directly determine whether antibody responses were important for LACV pathogenesis, B cell deficient weanling μMT -/- mice  were infected with LACV and followed for neurological disease. No difference was observed in either clinical disease development (Fig. 4d) or virus infection in the brain (not shown) in weanling μMT -/- mice compared to age-matched wildtype controls. Thus, despite the detection of IgG in the CNS of LACV-infected mice, the antibody response did not appear to influence LACV pathogenesis in susceptible weanling mice.
Combined B cell and T cell deficiency does not influence LACV-induced neurological disease in weanling mice
Despite the fact that B cells (Fig. 4d) and T cells (Fig. 3d) were not individually necessary for the development of LACV-induced neurological disease, it remained possible that the loss of both cellular and humoral adaptive immune responses could influence the development of LACV-induced neurological disease. Therefore, weanling Rag1 -/- mice, which are deficient in both mature B and T cells , were infected with LACV and monitored for neurological symptoms. No difference was observed in disease onset or occurrence between wildtype and Rag1 -/- mice (Fig. 4d) and virus infection was evident in all brain regions in both groups of mice as demonstrated by immunohistochemistry (Fig 4e, f). Also, cellular apoptosis was evident in similar regions of the brain in both wildtype and Rag1 -/- mice, suggesting wide-spread neuronal death and neuropathology (Fig. 4g, h). Collectively, these findings demonstrate that the adaptive immune response does not appear to play a significant role in mediating LACV-induced neurological damage in young mice despite the presence of lymphocytes within the CNS.
Adaptive immune responses are necessary for peripheral control of virus infection in adult mice
Neutralizing antibody producing B cells are essential for controlling LACV infection
Analysis of wildtype mice indicated the production of anti-LACV neutralizing antibody occurred in most mice by 7–10 dpi (Fig. 5d). As expected, uMT -/- and Rag1 -/- mice did not produce neutralizing antibody (Fig. 5d). To determine whether B cells and neutralizing antibodies were essential for protection from LACV-induced neurological disease, we infected μMT -/- mice with 103 PFU of LACV. Most, although not all, μMT -/- mice developed neurological disease compared to controls indicating B cells are required for efficient control of LACV infection (Fig. 5a). Susceptibility correlated with detectable viral RNA in the brain and the spleen of uMT -/- mice, which were, comparable to viral RNA levels observed in Rag1 -/- mice (Fig. 5b). Thus, humoral immune responses are necessary for protection against LACV-induced neurological disease in adult animals.
Both CD4 and CD8 T cells are necessary for neurological disease resistance
Since CD4+ and CD8+ T cells suppressed neurological disease in adult mice, we examined these cells for activation and proliferation markers. Mice were analyzed at 7 dpi, the earliest time point of disease in anti-CD4/anti-CD8-treated mice (Fig. 6b). In three of eight mice, an increase in CD4+ helper T cells expressing Ki-67, CD43, and CD11a was observed (Fig. 6c, gray circles). These same mice had increased percentages of CD8+ T cells expressing Ki67, Granzyme B, and CD107a and a decrease in expression of CD62L (Fig. 6d). Thus, LACV infection in adult mice did not induce a consistently robust CD4+ or CD8+ T cell response by 7 dpi, with only a subset of animals showing a measurable increase in T cell proliferation and activation. However, these cells are still essential for protection as depletion of both cell types resulted in all animals developing neurological disease (Fig. 6b), suggesting that a more robust response occurs later in infection or that a modest response is sufficient to clear virus.
NK cells do not contribute to LACV clearance
Anti-viral type I interferon (IFN) signaling is important for acute virus infections. Our previous studies have found that weanling mice that produce low levels of type I IFN do not efficiently control LACV replication early during peripheral infection and go on to develop neurological disease. By contrast, disease-resistant adult mice produce a robust type I IFN response that suppresses measurable viral RNA out to 4 dpi . However, by 5 dpi, viral RNA is significantly increased in the spleens of adult mice, suggesting that a low level of viral replication is taking place  and that other mechanisms of protection are needed. In the current study, we show that lymphocytes are necessary for efficient control of peripheral viral infection and the prevention of neurological disease. In contrast, lymphocytes do not appear to have a role in weanling mice, either in preventing disease development or contributing to pathogenesis.
Our current findings demonstrate that LACV infection is not completely controlled by the type I IFN response and that the adaptive immune response is necessary, through both T cell and B cell responses, to inhibit virus replication and prevent neurological disease. Interestingly, the onset of disease in Rag1 -/-, μMT -/-, or anti-CD4/anti-CD8-treated adult mice generally occurred after 10 dpi and ranged from 7–20 dpi (Figs. 5 and 6). In contrast, disease induced in weanling mice (Figs. 3 and 4) or in adult mice with deficient innate immune responses  had a general onset between 5 and 10 days. This delay in disease onset in Rag1 -/-, μMT -/-, or anti-CD4/anti-CD8-treated adult mice may be due to the ability of the type I IFN response to suppress early virus replication for a short time, resulting in a longer time period for the virus to reach the CNS and induce damage. This low-level prolonged virus replication appears to be controlled by the development of the adaptive immune response, requiring both neutralizing antibodies and T cell responses. How long LACV infection can persist in resistant adult mice without the development of CNS disease and what cell type is infected in the periphery are questions that remain to be addressed more fully to understand the role of the adaptive immune response in LACV pathogenesis.
In contrast to the clear role of lymphocytes in protection against LACV infection in adult animals, there was no substantial effect of depletion or deficiency in lymphocyte function on the development of neurological disease in weanling mice. This finding was surprising, as infiltrating lymphocytes influence pathogenesis in several other cases of viral encephalitis by either clearing infection [15–17] or exacerbating disease [13, 14]. Furthermore, analysis of these cells in LACV-infected weanling mice shows a strong activation phenotype, both in the brain and in the spleen. This contrasts with the results observed in adult mice infected with LACV, where CD4+ and CD8+ T cells are important for protection, but only a subset of mice had detectable T cell responses at 7 dpi. Thus, the inability of CD4+ and CD8+ T cells to affect LACV-induced neurological disease in weanling mice is not due to an inability of these cells to respond to virus infection. Indeed, the strong T cell response in weanling mice may be due to the higher level of virus replication, and resulting viral antigen, in the younger mice compared to the adults.
An unanticipated result of treating adult mice with vehicle control reagents containing normal fetal calf or rabbit serum was a trend toward increased incidence of LACV neurological disease (Figs. 6b and 7c). This suggests that heterologous serum elements are in some way increasing overall viral infection. Possible mechanisms include enhanced viral replication or infection, prolonged viral half-life, suppression of host immune response, or enhanced viral entry into the brain. Little published evidence exists to support the idea that heterologous serum facilitates viral replication or infection; however, this possibility cannot be discounted. By contrast, it is unlikely that serum elements are enhancing viral neuroinvasion due to the fact that weanling mice treated with the same serum-rich reagents did not have faster disease onset than untreated LACV-infected weanling mice (Figs. 2 and 3 and ). Instead, it is more likely that injection of mice with a low dose of heterologous serum is inducing a modest “immunological paralysis” through a mechanism involving suppression of lymphocyte reactivity . This effect, however, is minor as most control-treated adult mice remain resistant to LACV neurological disease.
In conclusion, the adaptive immune response, including the cellular response of CD4+ and CD8+ T cells as well as the humoral B cell response, does not appear to have a critical role in LACV-mediated damage in the CNS in disease-susceptible weanling animals. However, both humoral and cellular adaptive immune responses are critical for disease protection in adult animals. It is possible the more mature and presumptively more efficient adaptive response  in resistant adult animals compared to susceptible weanling mice is responsible for this disparity in disease resistance. Furthermore, the adaptive immune response may be more suited to controlling LACV infection in a minor cell population in the periphery, rather than a massive infection of neurons in the CNS. Indeed, when adult mice are infected either through the intranasal route or by direct intracranial injection, they develop neurological disease with similar speed and frequency as weanling mice despite having a fully developed adaptive immune response [9, 22]. This insufficiency of response may be related in part to lymphocyte accessibility to the brain compared to the peripheral tissues as well as to the extremely responsiveness of the brain to injury . Thus, enhancing the peripheral adaptive immune response against LACV in susceptible individuals may be protective against the development of neurological disease by clearing infection prior to the virus entering the CNS where infection cannot be efficiently controlled.
We would like to thank Drs. Suzette Priola, Shelly Robertson, Paul Policastro, and Stefano Boi for the critical readings of the manuscript. We also thank Donna Norton, Shelby Malingo, and Maarit von Kutzleben for the outstanding care and husbandry of the immunocompromised mice used in these studies.
This work was supported by the Division of Intramural Research, National Institutes of Health, National Institute of Allergy and Infectious Diseases.
Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
CWW, LMM, KGT, and TAW completed all animal studies and processed cells for flow cytometry. ABC analyzed cells by flow cytometry and helped design panels for flow cytometry. KEP, LMM, KGT, and CWW designed the experiments. CWW and KEP wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animal studies were conducted using the animal protocol RML2014-011, which was approved by the NIH/NIAID/RML Institutional Animal Care and Use Committee.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Thompson WH, Kalfayan B, Anslow RO. Isolation of California encephalitis group virus from a fatal human illness. Am J Epidemiol. 1965;81:245–53.View ArticlePubMedGoogle Scholar
- McJunkin JE, Nahata MC, De Los Reyes EC, Hunt WG, Caceres M, Khan RR, Chebib MG, Taravath S, Minnich LL, Carr R, et al. Safety and pharmacokinetics of ribavirin for the treatment of la crosse encephalitis. Pediatr Infect Dis J. 2011;30:860–5.View ArticlePubMedGoogle Scholar
- Westby KM, Fritzen C, Paulsen D, Poindexter S, Moncayo AC. La Crosse encephalitis virus infection in field-collected Aedes albopictus, Aedes japonicus, and Aedes triseriatus in Tennessee. J Am Mosq Control Assoc. 2015;31:233–41.View ArticlePubMedGoogle Scholar
- Schuh T, Schultz J, Moelling K, Pavlovic J. DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells. Hum Gene Ther. 1999;10:1649–58.View ArticlePubMedGoogle Scholar
- Bennett RS, Gresko AK, Nelson JT, Murphy BR, Whitehead SS. A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys. J Virol. 2012;86:420–6.View ArticlePubMedPubMed CentralGoogle Scholar
- McJunkin JE, de los Reyes EC, Irazuzta JE, Caceres MJ, Khan RR, Minnich LL, Fu KD, Lovett GD, Tsai T, Thompson A. La Crosse encephalitis in children. N Engl J Med. 2001;344:801–7.View ArticlePubMedGoogle Scholar
- McJunkin JE, Khan R, de los Reyes EC, Parsons DL, Minnich LL, Ashley RG, Tsai TF. Treatment of severe La Crosse encephalitis with intravenous ribavirin following diagnosis by brain biopsy. Pediatrics. 1997;99:261–7.View ArticlePubMedGoogle Scholar
- Bennett RS, Cress CM, Ward JM, Firestone CY, Murphy BR, Whitehead SS. La Crosse virus infectivity, pathogenesis, and immunogenicity in mice and monkeys. Virol J. 2008;5:25.View ArticlePubMedPubMed CentralGoogle Scholar
- Winkler CW, Race B, Phillips K, Peterson KE. Capillaries in the olfactory bulb but not the cortex are highly susceptible to virus-induced vascular leak and promote viral neuroinvasion. Acta Neuropathol. 2015;130:233–45.View ArticlePubMedGoogle Scholar
- Miller F, Afonso PV, Gessain A, Ceccaldi PE. Blood-brain barrier and retroviral infections. Virulence. 2012;3:222–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Suen WW, Prow NA, Hall RA, Bielefeldt-Ohmann H. Mechanism of West Nile virus neuroinvasion: a critical appraisal. Viruses. 2014;6:2796–825.View ArticlePubMedPubMed CentralGoogle Scholar
- Griffin DE. Immune responses to RNA-virus infections of the CNS. Nat Rev Immunol. 2003;3:493–502.View ArticlePubMedGoogle Scholar
- Poli A, Kmiecik J, Domingues O, Hentges F, Blery M, Chekenya M, Boucraut J, Zimmer J. NK cells in central nervous system disorders. J Immunol. 2013;190:5355–62.View ArticlePubMedGoogle Scholar
- Liu T, Chambers TJ. Yellow fever virus encephalitis: properties of the brain-associated T-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for CD4+ lymphocytes. J Virol. 2001;75:2107–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang Y, Lobigs M, Lee E, Mullbacher A. CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis. J Virol. 2003;77:13323–34.View ArticlePubMedPubMed CentralGoogle Scholar
- McCandless EE, Zhang B, Diamond MS, Klein RS. CXCR4 antagonism increases T cell trafficking in the central nervous system and improves survival from West Nile virus encephalitis. Proc Natl Acad Sci U S A. 2008;105:11270–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Teo TH, Lum FM, Claser C, Lulla V, Lulla A, Merits A, Renia L, Ng LF. A pathogenic role for CD4+ T cells during Chikungunya virus infection in mice. J Immunol. 2013;190:259–69.View ArticlePubMedGoogle Scholar
- Johnson RT. Pathogenesis of La Crosse virus in mice. Prog Clin Biol Res. 1983;123:139–44.PubMedGoogle Scholar
- Janssen R, Gonzalez-Scarano F, Nathanson N. Mechanisms of bunyavirus virulence. Comparative pathogenesis of a virulent strain of La Crosse and an avirulent strain of Tahyna virus. Lab Invest. 1984;50:447–55.PubMedGoogle Scholar
- Pekosz A, Griot C, Stillmock K, Nathanson N, Gonzalez-Scarano F. Protection from La Crosse virus encephalitis with recombinant glycoproteins: role of neutralizing anti-G1 antibodies. J Virol. 1995;69:3475–81.PubMedPubMed CentralGoogle Scholar
- Blakqori G, Delhaye S, Habjan M, Blair CD, Sanchez-Vargas I, Olson KE, Attarzadeh-Yazdi G, Fragkoudis R, Kohl A, Kalinke U, et al. La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts. J Virol. 2007;81:4991–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Taylor KG, Woods TA, Winkler CW, Carmody AB, Peterson KE. Age-dependent myeloid dendritic cell responses mediate resistance to la crosse virus-induced neurological disease. J Virol. 2014;88:11070–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Butchi NB, Woods T, Du M, Morgan TW, Peterson KE. TLR7 and TLR9 trigger distinct neuroinflammatory responses in the CNS. Am J Pathol. 2011;179:783–94.View ArticlePubMedPubMed CentralGoogle Scholar
- Gangappa S, Deshpande SP, Rouse BT. Bystander activation of CD4(+) T cells can represent an exclusive means of immunopathology in a virus infection. Eur J Immunol. 1999;29:3674–82.View ArticlePubMedGoogle Scholar
- Shrestha B, Diamond MS. Role of CD8+ T cells in control of West Nile virus infection. J Virol. 2004;78:8312–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Bien CG, Bauer J. T-cells in human encephalitis. Neuromolecular Med. 2005;7:243–53.View ArticlePubMedGoogle Scholar
- Burdeinick-Kerr R, Wind J, Griffin DE. Synergistic roles of antibody and interferon in noncytolytic clearance of Sindbis virus from different regions of the central nervous system. J Virol. 2007;81:5628–36.View ArticlePubMedPubMed CentralGoogle Scholar
- Hooper DC, Morimoto K, Bette M, Weihe E, Koprowski H, Dietzschold B. Collaboration of antibody and inflammation in clearance of rabies virus from the central nervous system. J Virol. 1998;72:3711–9.PubMedPubMed CentralGoogle Scholar
- Kitamura D, Roes J, Kuhn R, Rajewsky K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 1991;350:423–6.View ArticlePubMedGoogle Scholar
- Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S, Papaioannou VE. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 1992;68:869–77.View ArticlePubMedGoogle Scholar
- Brandstadter JD, Yang Y. Natural killer cell responses to viral infection. J Innate Immun. 2011;3:274–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Dresser DW, Mitchison NA. The mechanism of immunological paralysis. Adv Immunol. 1968;8:129–81.View ArticlePubMedGoogle Scholar
- PrabhuDas M, Adkins B, Gans H, King C, Levy O, Ramilo O, Siegrist CA. Challenges in infant immunity: implications for responses to infection and vaccines. Nat Immunol. 2011;12:189–94.View ArticlePubMedGoogle Scholar
- Wekerle H. Immune protection of the brain—efficient and delicate. J Infect Dis. 2002;186 Suppl 2:S140–4.View ArticlePubMedGoogle Scholar