(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



Differences in neuroinvasion and protective innate immune pathways between encephalitic California Serogroup orthobunyaviruses

['Alyssa B. Evans', 'Laboratory Of Persistent Viral Diseases', 'Rocky Mountain Laboratories', 'National Institute Of Allergy', 'Infectious Diseases', 'National Institutes Of Health', 'Hamilton', 'Montana', 'United States Of America', 'Clayton W. Winkler']

Date: 2022-05

The California serogroup (CSG) of Orthobunyaviruses comprises several members capable of causing neuroinvasive disease in humans, including La Crosse orthobunyavirus (LACV), Jamestown Canyon orthobunyavirus (JCV), and Inkoo orthobunyavirus (INKV). Despite being genetically and serologically closely related, their disease incidences and pathogenesis in humans and mice differ. We have previously shown that following intraperitoneal inoculation of weanling mice, LACV was highly pathogenic while JCV and INKV were not. To determine why there were differences, we examined the ability of these viruses to invade the CNS and compared the host innate immune responses that regulated viral pathogenesis. We found that LACV was always neuroinvasive, which correlated with its high level of neuroinvasive disease. Interestingly, JCV was not neuroinvasive in any mice, while INKV was neuroinvasive in most mice. The type I interferon (IFN) response was critical for protecting mice from both JCV and INKV disease, although in the periphery JCV induced little IFN expression, while INKV induced high IFN expression. Despite their differing neuroinvasive abilities, JCV and INKV shared innate signaling components required for protection. The presence of either cytoplasmic Rig-I-Like Receptor signaling or endosomal Toll-Like Receptor signaling was sufficient to protect mice from JCV or INKV, however, inhibition of both pathways rendered mice highly susceptible to neurological disease. Comparison of IFN and IFN-stimulated gene (ISG) responses to INKV in the brains of resistant wild type (WT) mice and susceptible immune knockout mice showed similar IFN responses in the brain, but WT mice had higher ISG responses, suggesting induction of key ISGs in the brain is critical for protection of mice from INKV. Overall, these results show that the CSG viruses differ in neuroinvasiveness, which can be independent from their neuropathogenicity. The type I IFN response was crucial for protecting mice from CSG virus-induced neurological disease, however, the exact correlates of protection appear to vary between CSG viruses.

The California Serogroup (CSG) of Orthobunyaviruses contains several viruses that can cause encephalitis in humans, primarily in children, with different disease incidences. Mouse studies also showed differences between these viruses. La Crosse orthobunyavirus (LACV), the leading cause of pediatric arboviral encephalitis in the USA, is highly pathogenic in mice, while Inkoo orthobunyavirus (INKV) and Jamestown Canyon orthobunyavirus (JCV) caused only limited disease under certain conditions. In the current study, we analyzed the differences in neuroinvasion and host responses to these viruses. LACV entered the brain in all mice (highly neuroinvasive). INKV was able to invade the brain but was cleared and did not cause disease. JCV did not enter the CNS and was controlled in the periphery. Analysis of the early innate immune response showed that both INKV and JCV were controlled by type I interferon responses induced through either cytoplasmic or endosomal pattern recognition receptors. These studies show substantial differences between CSG viruses in their ability to invade the brain, cause neurological disease and the immune responses needed for the body to control these virus infections.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

To directly investigate whether the differences in CSG virus pathogenesis were mediated by differences in neuroinvasive abilities and/or the IFN response, we inoculated weanling mice with 10 5 PFU IP and conducted a time course to evaluate virus infection and IFN induction in peripheral tissues and the brain. We found that the highly pathogenic LACV invaded the brains of all mice, and the moderately pathogenic SSHV and TAHV invaded the brains of some, but not all mice. JCV did not enter the brains of any of the mice, suggesting control by peripheral immune responses. Despite not causing neuroinvasive disease, INKV gained access to the brains of most mice. Thus, INKV was neuroinvasive, but not neurovirulent, while JCV was not neuroinvasive. We further determined if there were IFN responses that correlated with neuroinvasion by examining IFN responses in the periphery to all viruses or neuropathogenesis by examining the IFN responses to INKV within the brains, as this is the only CSG virus controlled within the CNS. We also determined differences in the innate signaling pathways regulating CSG virus pathogenesis.

IFNs then bind to the IFN receptor (IFNAR) on cell surfaces to initiate a signaling cascade that results in the production of IFN stimulated genes (ISGs) with antiviral functions, including the IFN-induced proteins with tetratricopeptide repeats (IFITs) [ 11 – 14 , 16 , 19 ]. Previous studies have shown that the IFN response is important for protection during LACV infection and mediates LACV-induced age-dependent susceptibility to neuroinvasive disease in mice. Following IP inoculation, adult mice had a robust peripheral IFN response, primarily with Ifna4 and Ifnβ1, that was lacking in weanling mice, and this response mediated protection of adult mice from neuroinvasive disease [ 10 , 20 ]. Furthermore, it was determined that signaling through both cytosolic MAVS and endosomal TLR3/7/9 was required for protection of adult mice from neuroinvasive LACV disease, as adult mice deficient in either MAVS or TLR3/7/9 signaling were highly susceptible to neuroinvasive disease [ 10 ].

One of the primary mechanisms that may limit virus spread within the periphery, to the CNS, and within the CNS is the type I interferon (IFN) system. Type I IFNs include several IFN classes, including multiple IFNα subtypes, as well as IFNβ, IFNε, and IFNκ [ 11 ]. The production of these IFNs is triggered by the recognition of viral pathogen associated molecular patterns (PAMPs) by either cytoplasmic RIG-I-like (RLR) receptors or the endosomal toll-like receptors (TLR) 3, 7, and 9 [ 11 , 12 ]. Virus activated RLRs signal through the mitochondrial antiviral-signaling (MAVS) protein, then the interferon regulatory response factors (IRF)3, IRF7, and IRF5 to activate Ifn mRNA transcription and production by a number of immune and non-immune cell types [ 13 – 15 ]. Similarly, the endosomal TLRs 3, 7, and 9 signal through the adaptor proteins MYD88 (TLR7 and 9) or TRIF (TLR3) and active IRF3, IRF7, and IRF5 to stimulate IFN production [ 15 – 18 ]. The multiple pathways of activation and multiple IRF transcription factors that are capable of inducing Type I IFNs leads to large amounts of redundancy in IFN production.

We previously analyzed the pathogenesis of these five CSG viruses via intraperitoneal (IP) inoculation in C57BL/6 (B6) mice and found some similarities with the incidence and severity of disease in humans [ 8 ]. Consistent with previous studies, LACV was highly pathogenic in weanling mice [ 8 – 10 ], and SSHV and TAHV caused neuroinvasive disease in some or most weanling mice [ 8 ]. In contrast, JCV and INKV did not cause neuroinvasive disease in any weanling mice [ 8 ]. None of the viruses caused neuroinvasive disease in adult mice over the age of six weeks following IP inoculation [ 8 ]. However, when the peripheral immune system was bypassed and adult mice were inoculated intranasally (IN), LACV, SSHV, TAHV, and JCV all replicated extensively throughout the brain and caused neurological disease in nearly 100% of mice, indicating these CSG viruses are all highly neurovirulent [ 8 ]. The limiting factor for these viruses to cause neuroinvasive disease may therefore be the ability to gain access to the CNS. Interestingly, INKV only caused disease in ~25% of adult mice after IN inoculation and did not spread widely throughout the brains [ 8 ]. This indicates that INKV is only mildly neurovirulent and is likely controlled by immune responses within the brain, unlike the other CSG viruses. Determining the host immune responses involved in these differing CSG virus pathogenicities, in particular why JCV and INKV do not cause neuroinvasive disease in weanling mice, will further our understanding of CSG virus neuropathogenesis and the factors that mediate neurological disease.

The California serogroup (CSG) of Orthobunyaviruses (family Peribunyaviridae) is a serologically and genetically related group of 18 known viruses [ 1 ]. All of the CSG viruses are mosquito-borne and some have been shown to cause neuroinvasive disease in humans, which primarly occurs in children [ 1 – 7 ]. Of the neuroinvasive CSG viruses, La Crosse orthobunyavirus (LACV) is found primarily in the USA and is responsible for the most neuroinvasive cases of CSG virsues annually [ 2 , 3 ]. Snowshoe hare orthobunyavirus (SSHV) causes a handful of neuroinvasive cases annually in the USA and Canada [ 4 ]. Tahyna orthobunyavirus (TAHV) is widely distributed throughout Europe, Africa and Asia and primarily causes febrile illness, but occasionally is neuroinvasive [ 1 , 5 ]. Inkoo orthobunyavirus (INKV) is mostly restricted to Scandinavia and has only been confirmed to cause a handful of neuroinvasive cases [ 6 ]. Jamestown Canyon orthobunyavirus (JCV) is responsible for an increasing number of neuroinvasive cases in the USA and Canada, and is the only CSG virus that primarily causes severe disease in adults [ 1 , 7 ].

Results

The CSG viruses differ in neuroinvasion To determine if the differences in pathogenesis of CSG viruses correlated with differences in the ability of the viruses to invade the CNS, we inoculated weanling mice intraperitoneally (IP) with 105 PFU/mouse of the viruses and examined brain tissue at 1, 3, 5, and 7 dpi, or when a mouse showed neurological signs or other endpoint criteria. Viral RNA was evaluated via RT-qPCR with virus-specific primers, and infectious virus evaluated via plaque assay in Vero cells. LACV, the most neurovirulent of these viruses [8], had detectable viral RNA and infectious virus in the brains of all animals by 3 dpi which increased to higher levels at 5–6 dpi, the time points when mice developed clinical disease (Fig 1A and 1B). SSHV and TAHV, which were previously shown to cause neuroinvasive disease in some but not all mice at 105 PFU/mouse [8], had a range of virus levels in the brains, with the highest levels observed in mice displaying neurological signs (Fig 1C and 1F). In the brains of some SSHV- and TAHV-inoculated mice that did not have neurological signs, viral RNA and PFUs were detectable at 3–7 dpi, although in others neither viral RNA or PFUs were detected (Fig 1C and 1F). The lack of virus in some mice at 5 and 7 dpi would suggest that one reason for the lower incidence of disease with SSHV and TAHV compared to LACV is a lack of virus entering the brain in some animals. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Neuroinvasion of the CSG viruses. A, C, E, G, I) Brain tissue was evaluated for viral RNA via RT-qPCR with virus-specific primers. Dotted lines indicate mock sample average from n = 4. B, D, F, H, J) Brain tissues were evaluated for infectious virus via plaque assays of brain homogenates in Vero cells. Dotted lines represent the limit of detection. No plaques were detected in any mock controls, n = 4. Individual dots represent individual mice. For all viruses and time points, n = 6, except for: LACV n = 4 for 6 dpi, no mice survived to 7 dpi; SSHV n = 5 for 5 dpi, two of the designated 7 dpi mice developed neurological disease at 6 dpi and had to be euthanized, so n = 2 for 6 dpi, n = 4 for 7 dpi; for TAHV, one mouse in the 5 dpi group developed neurological disease at 4 dpi and had to be euthanized, so n = 1 for 4 dpi and n = 5 for 5 dpi. (G) Dunnett’s multiple comparisons tests was done for JCV viral RNA compared to mock, using Log2(%gapdh) values. Nonclinical = mice with no signs of disease; Clinical = mice displaying neurological signs or other endpoint criteria as described in the methods. https://doi.org/10.1371/journal.ppat.1010384.g001 In previous studies, neither JCV nor INKV induced neurological disease following IP inoculation in weanling mice [8]. Surprisingly, these viruses showed divergent results for their abilities to enter the CNS. Although several JCV-inoculated mice had detectable viral RNA levels in the brain, none of the levels were significantly different from mock (Fig 1G). Additionally, no infectious virus was detected in any of the JCV-inoculated mouse brains (Fig 1H). The lack of virus in the brains suggests that JCV does not cause neurological disease following IP inoculation because the virus does not gain access to the CNS. In contrast, viral RNA and infectious virus were detected in brains for INKV-inoculated mice, with two of six mice at 3 dpi, five of six mice at 5 dpi, and two of six mice at 7 dpi having detectable PFUs in brain homogenates (Fig 1I and 1J). To determine if INKV was cleared from the brains after 7 dpi, an additional set of mice were inoculated with INKV and infectious virus analyzed at 14 dpi. No infectious virus was detected in any of these brains at 14 dpi (Fig 1J). Thus, INKV entered the brains of most weanling mice from the periphery, but was either unable to replicate sufficiently or was subsequently cleared by host immune responses in the brain prior to establishing a productive infection (Fig 1I and 1J).

Virus in the periphery does not directly correlate with neuroinvasion We next determined if the differences between CSG virus levels in the CNS correlated with differences in the amount of virus in the periphery by evaluating peripheral tissues over time from the same set of B6 mice used in the neuroinvasion studies. In plasma, infectious virus was not consistently detected for any of the CSG viruses (Fig 2A). Only 25% of LACV, SSHV, and INKV-inoculated mice had detectable viremias at 1 and/or 3 dpi, with no virus detected after 3 dpi (Fig 2A). JCV and TAHV-inoculated mice did not have detectable viremia in any plasma sample (Fig 2A). Viral RNA was observed in the inguinal lymph nodes (LN) of most LACV-, SSHV-, and TAHV-inoculated mice, but in only a few JCV- and INKV-inoculated mice (Fig 2B). However, only LACV-inoculated mice had viral RNA expression significantly higher than mock on all dpi, while viral RNA was only significantly different from mock for SSHV at 3 and 6/7 dpi and TAHV at 5 dpi (Fig 2B). No significant viral RNA was detected in the LN for JCV- or INKV-inoculated mice at any dpi (Fig 2B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Virus in the periphery. A) Viremias were assessed via plaque assays of plasma samples in Vero cells. Dotted line represents the limit of detection. B-C) Viral RNA levels were analyzed via RT-qPCR with virus-specific primers in lymph nodes (B) and spleens (C). Dotted lines represent mock average from n≥3. Sample numbers are the same as described in Fig 1. Individual points represent individual mice. Dunnett’s multiple comparisons tests were done for each virus, tissue, and time point compared to mock, using Log2(%gapdh) values. Asterisks indicate sample days that had significantly higher viral RNA expression compared to mock. For mice that developed clinical signs prior to their designated time point, those values were grouped in with their designated time point (6dpi with 7dpi for SSHV, 4 dpi with 5 dpi for TAHV). Nonclinical = mice with no signs of disease; Clinical = mice displaying neurological signs or other endpoint criteria. *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009. https://doi.org/10.1371/journal.ppat.1010384.g002 Viral RNA was low to undetectable in the spleens of all mice inoculated with LACV, SSHV, JCV, or INKV (Fig 2C). TAHV-inoculated mice had detectable viral RNA over the time course, however this expression was not significantly different from mock (Fig 2C). Overall, these results suggest that none of the CSG viruses replicated extensively in the periphery. Furthermore, there was not a strong correlation between virus infection in the periphery and virus infection in the CNS. The neuroinvasive viruses (LACV, SSHV, TAHV, and INKV) were not remarkably higher in the plasma or spleens compared to the non-neuroinvasive JCV. However, the neuropathogenic viruses (LACV, SSHV, and TAHV) did appear to have higher viral RNA levels in the LNs than either of the non-neuropathogenic viruses (JCV and INKV; Fig 2B).

CSG viruses differ in peripheral interferon response From previous LACV studies we know that age-dependent resistance to neuroinvasive disease is due, in part, to a strong type I IFN response in adult mice that is lacking in weanling mice [10]. Therefore, we evaluated if there were differences in IFN responses between the CSG viruses that would help explain differences in their neuroinvasiveness and neuropathogenicity in weanling mice. To do this, we evaluated the IFN response in the periphery by performing RT-qPCR on RNA isolated from the inguinal LNs and spleens from the same time course of B6 weanling mice described above. From our analysis of viral RNA in the periphery, we determined that overall the LNs had more detectable viral RNA than spleens for the CSG viruses (Fig 2B and 2C). Most of the CSG viruses had detectable to high levels of virus in the LN at 1 dpi, therefore we analyzed mRNA expression of 10 type 1 IFNs in the 1 dpi LNs for all viruses to identify the IFN transcripts induced in response to the CSG viruses. Several IFN mRNAs were significantly increased in the LN following virus infection, although the IFN type and expression level varied between viruses. Ifnβ1 was significantly upregulated by LACV, JCV, and INKV infection and Ifnε by LACV infection (Fig 3A, 3D and 3E). Of the IFN alpha subtypes, Ifna1, Ifna4, Ifna11, and Ifa12 mRNA expression was significantly increased during INKV infection (Fig 3E), but not with the other viruses. Indeed, the only IFN mRNA with significantly different expression from mock in 1 dpi LNs from SSHV and TAHV-infected mice was Ifna9, which was lower than mock levels (Fig 3B and 3C). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. IFN response to CSG viruses in 1 dpi lymph nodes. IFN mRNA expression was evaluated via RT-qPCR for a panel of 10 Type I IFNs in lymph nodes taken at 1 dpi from mice inoculated with A) LACV; B) SSHV; C) TAHV; D) JCV; E) INKV. Expression is plotted for each samples as the fold change in %gapdh from the mock average. N = 6 for all viruses, n = 7 for mock. Each individual point represents an individual mouse. Dotted lines indicate fold change = 1. Fold change to mock is plotted on the left axis, viral RNA plotted on the right axis. One-way ANOVA analyses were performed on Log2(%gapdh) with Dunnett’s multiple comparison test performed between mock and each virus. Asterisks denote Ifn expression that was significantly different from mock for that IFN and virus (gray = significantly higher, black = significantly lower than mock): *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009. https://doi.org/10.1371/journal.ppat.1010384.g003 Expression of Ifna4, Ifna11, Ifna12, Ifnβ1, and Ifnε mRNAs was further analyzed in the LNs at 3 dpi and spleens at 1 dpi for all viruses. The full panel of 10 IFN mRNAs was also analyzed for SSHV at 3 dpi and TAHV at 4/5 dpi, because these were the days when these viruses had the highest level of viral RNA in the LNs. By 3 dpi, the IFN mRNA response in the LNs had almost entirely resolved for most mice infected with LACV, JCV, and TAHV, and the low levels were maintained for TAHV at 4/5 dpi (S1 Fig). INKV-infected mice still had increased Ifna4 and Ifnβ1 mRNA in the LN at 3 dpi, but lower than levels at 1 dpi (Figs S1 and 3E). SSHV-infected mice had increased Ifnβ1 mRNA in the LNs at 3 dpi (S1A Fig), which corresponded with the increase in viral RNA observed in the LN at 3 dpi (Fig 2). No significant upregulation in IFN mRNA expression was observed in the spleen at 1 dpi for any virus (S1C Fig). Overall, these results show that INKV had the highest and most robust IFN mRNA response in the periphery of any of the CSG viruses, with five IFN mRNAs significantly upregulated in the LNs at 1 dpi and two at 3 dpi (Figs 3E and S1A). LACV had an intermediate peripheral IFN mRNA response with two IFN mRNAs significantly upregulated in the LNs at 1 dpi, but three additional IFN mRNAs trending higher (Fig 3A). SSHV, TAHV and JCV had a low IFN mRNA response, with only one IFN mRNA significantly upregulated for SSHV (in the LN at 3 dpi) and JCV (in the LN at 1 dpi), and none significantly upregulated for TAHV (Figs 3 and S1). However, it is unclear if the increased IFN mRNA response observed in INKV was protective, as the virus appeared to be able to evade this response and gain access to the CNS.

The interferon response protects mice from JCV and INKV-induced neuroinvasive disease The results from IFN mRNA expression analysis did not yield obvious results as to which IFN may mediate protection, or if the IFN response is involved in protecting mice from JCV- and INKV-induced neuroinvasive disease at all. Therefore, to determine if the IFN response had a direct role in protecting weanling mice from JCV- and INKV-induced neuroinvasive disease, we inoculated Ifnar1-/- knockout mice, which lack a functional type I IFN receptor, with either JCV or INKV and evaluated the mice for clinical signs. INKV-inoculated Ifnar1-/- mice died at 2 dpi, while JCV-inoculated Ifnar1-/- mice all developed neurological disease or died at 3–4 dpi (Fig 6A and 6B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Disease curves and virus loads for C57BL/6 mice and Ifnar1-/- mice. Mice were inoculated IP with 105 PFU of A) INKV, n = 6 for C57BL/6, n = 9 for Ifnar1-/- or B) JCV, n = 5 for C57BL/6, n = 7 for Ifnar1-/-, and followed for neurological signs, other endpoint criteria, or death. Timepoint analysis of infectious virus via plaque assay of tissue homogenates in Vero cells of tissues from INKV-inoculated C) WT B6 mice and D) Ifnar1-/- mice. https://doi.org/10.1371/journal.ppat.1010384.g006 While the mice inoculated with JCV showed clear neurological signs of ataxia and hind limb weakness, the INKV-inoculated mice died suddenly at 2 dpi, making it impossible to determine if they had neurological signs. We therefore inoculated additional Ifnar1-/- and B6 mice with INKV and analyzed infectious virus levels in tissues from five mice each at 1 dpi for both strains, two moribund mice at 2 dpi for Ifnar1-/-, and six nonclinical mice at 3 and 5 dpi for B6. Infectious virus was measured in brains, spleens, kidneys, livers, lungs, hearts, and plasma via plaque assay in Vero cells. From all of the INKV-inoculated B6 mice, one kidney and two plasma samples at 1 dpi as well as four brains at 5 dpi had detectable plaques (Fig 6C). In contrast, at 1 dpi Ifnar1-/- mice all had high levels of virus in multiple tissues including spleen, kidney, liver, lungs, brains, and plasma (Fig 6D). The two moribund INKV-inoculated Ifnar1-/- mice at 2 dpi that tissues could be taken from both had high levels of virus in all tissues (Fig 6D). These results demonstrate that a functional type I IFN response has a crucial role in protecting mice from widespread viral dissemination and death following infection with INKV, and the development of neurological disease with JCV.

CSG viruses differ in the innate immune pathway components involved in protection of mice from neuroinvasive disease We next examined the pathways and components of the upstream innate immune system involved in generating the IFN response. Previous studies of LACV infection showed that both endosomal toll-like receptors (TLRs) and cytoplasmic Rig-I like receptors (RLRs) signaling pathways were necessary for protecting adult mice from neuroinvasive disease. Therefore, we inoculated JCV and INKV IP into mice deficient in key components of these pathways including Irf3-/-xIrf7-/- double knockout (DKO) mice, Myd88-/- mice which lack the adaptor protein for most TLRs other than TLR3, Unc93b1.3D mice that lack functional endosomal TLRs, and Mavs-/- mice that lack functional RLR signaling. Mice were followed for the development of neurological disease, which primarily consisted of ataxia and hind limb weakness/paralysis, and occasionally tremors, circling, and/or seizures. All weanling Irf3-/-xIrf7-/- DKO mice either died or developed neurological disease at 3 dpi for INKV, and at 5–6 dpi for JCV, indicating that signaling through IRF3 and/or IRF7 was critical for the protection of mice from neuroinvasive disease from JCV and INKV (Fig 7A and 7B). However, knockouts in individual pathway components resulted in surprising results in infected mice. Only 25% of Mavs-/- mice and less than 10% of Unc93b1.3D mice developed clinical signs following JCV or INKV inoculation, while none of the Myd88-/- mice showed signs of disease (Fig 7A and 7B). To determine if mice would be susceptible to JCV or INKV-induced disease without either endosomal or cytoplasmic PRRs signaling, we crossed Mavs-/- and Unc93b1.3D mice to create a Mavs-/-xUnc93b1.3D DKO mouse that lacked RLR and endosomal TLR signaling pathways. Inoculation of these mice with INKV and JCV resulted in 100% susceptibility to neuroinvasive disease with similar kinetics as the Irf3-/-xIrf7-/- DKO mice for INKV and Ifnar1-/- mice for JCV (Figs 7 and 6B). These results indicate that having either functional MAVS or TLR3/7/9 signaling pathway was sufficient for protection from neuroinvasive disease by JCV and INKV. These results contrast with previous results with LACV, where both pathways were required for protection [10]. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. Disease curves in immune deficient mouse strains. Mice were inoculated IP with 105 PFU of A) INKV or B) JCV, and followed for neurological signs, other endpoint criteria, or death. For Irf3-/-xIrf7-/- n = 8 INKV and JCV; for Myd88-/- n = 5 INKV, n = 8 JCV; for Unc93b1.3D n = 11 INKV, n = 10 JCV; for Mavs-/- n = 14 INKV, n = 10 JCV; for Mavs-/-xUnc93b1.3D n = 9 INKV, n = 10 JCV. https://doi.org/10.1371/journal.ppat.1010384.g007

IFN responses to JCV and INKV in the periphery of immune deficient mice The results from the weanling B6 mice time course studies identified several IFN mRNAs with increased expression in the LN at 1 dpi, including Ifna4, Ifna11, Ifna12, and Ifnβ1 mRNA in INKV, and Ifnβ1 mRNA in JCV. Therefore, in order to evaluate which IFNs may be responsible for the protection of JCV- and INKV-inoculated weanling mice, we compared the IFN responses to JCV and INKV between the same WT B6 mice from the previous IFN analysis that do not develop neuroinvasive disease and both DKO mouse strains (Irf3-/-xIrf7-/- mice and Mavs-/-xUnc93b.3D mice; Fig 7A and 7B). LN at 1 dpi were analyzed because they had the strongest IFN responses to JCV and INKV in the B6 mice (Fig 3A). Comparison of viral RNA levels in the LNs at 1 dpi between B6 and the two DKO strains showed that DKO mice had significantly higher viral RNA levels than the B6 mice (Fig 8A and 8C). Some differences were observed in basal IFN mRNA expression in the LN of mock-inoculated mice between WT B6 mice and the DKO strains. Mavs-/-xUnc93b1.3D mice had significantly higher expression of Ifna12 mRNA and Irf3-/-xIrf7-/- mice had significantly lower expression of Ifnβ1 mRNA compared to WT B6 mice (S4 Fig). Following virus infection Irf3-/-xIrf7-/-DKO mice had similar levels of IFN mRNA induction compared to B6 mice with either JCV or INKV infection, while the Mavs-/-xUnc93b1.3D mice had little to no IFN mRNA upregulation in response to infection (Fig 8B and 8D). The only IFN mRNA that was significantly more upregulated in B6 mice than in both DKO strains was Ifna11 in INKV-inoculated mice (Fig 8D). The significantly higher levels of induction of Ifna11 mRNA in the INKV-inoculated WT B6 mice compared to the DKO strains suggests that IFNa11 could have a role in protecting mice from INKV-induced neuroinvasive disease. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. Comparison of the IFN mRNA response in LN at 1 dpi of wild type C57BL/6 mice and immune deficient DKO mice. Viral RNA was compared in the lymph nodes (LN) at 1 dpi of mice inoculated with JCV (A) or INKV (C). B,D) IFN mRNA expression was evaluated via RT-qPCR in LN at 1 dpi of JCV-inoculated mice (B) or INKV-inoculated mice (D) in B6 and DKO mice. A,C) One-way ANOVA was performed for each tissue on Log2(%gapdh) values and Dunnett’s multiple comparisons test done between B6 and DKO mice. Asterisks represent significant ANOVA and multiple comparisons p-values. B, D) There were significantly different levels in some basal Ifn expression between mock-inoculated B6 and DKO mouse strains, therefore Log2(fold change in %gapdh from mock) values were used to normalize expression to mock for analyses. One-sample t tests were done to analyze if the expression for each mouse strain and Ifn was significantly different from 0 (equivalent to fold change = 1 = mock). One-way ANOVA with Dunnett’s multiple comparison test between mock and the DKO strains was then run. Asterisks below the lines represent expression significantly different from mock (gray = significantly higher, black = significantly lower), and asterisks above the line represent a significant difference between B6 and one (bracketed) or both (flat line) DKO strains. Asterisks denote *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009, and values for the multiple comparisons are reported as the higher value from the two DKO strains. Dotted lines indicate fold change to mock = 1. https://doi.org/10.1371/journal.ppat.1010384.g008

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010384

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.