CD22 Is Required for Protection against West Nile Virus Infection

West Nile virus (WNV) is a RNA virus of the family Flaviviridae and the leading cause of mosquito-borne encephalitis in the United States. Humoral immunity is essential for protection against WNV infection; however, the requirements for initiating effective antibody responses against WNV infection are still unclear. CD22 (Siglec-2) is expressed on B cells and regulates B cell receptor signaling, cell survival, proliferation, and antibody production. In this study, we investigated how CD22 contributes to protection against WNV infection and found that CD22 knockout (Cd22^−/−) mice were highly susceptible to WNV infection and had increased viral loads in the serum and central nervous system (CNS) compared to wild-type (WT) mice. This was not due to a defect in humoral immunity, as Cd22^−/− mice had normal WNV-specific antibody responses. However, Cd22^−/− mice had decreased WNV-specific CD8⁺ T cell responses compared to those of WT mice. These defects were not simply due to reduced cytotoxic activity or increased cell death but, rather, were associated with decreased lymphocyte migration into the draining lymph nodes (dLNs) of infected Cd22^−/− mice. Cd22^−/− mice had reduced production of the chemokine CCL3 in the dLNs after infection, suggesting that CD22 affects chemotaxis via controlling chemokine production. CD22 was not restricted to B cells but was also expressed on a subset of splenic DCIR2⁺ dendritic cells that rapidly expand early after WNV infection. Thus, CD22 plays an essential role in controlling WNV infection by governing cell migration and CD8⁺ T cell responses.     

Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.     

Small Interfering RNA-Mediated Control of Virus Replication in the CNS Is Therapeutic and Enables Natural Immunity to West Nile Virus

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Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection

Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4⁺ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection.     

Seasonality and Outbreaks in West Nile Virus Infection

In this paper we analyze the impact of seasonal variations on the dynamics of West Nile Virus infection. We are interested in the generation of new epidemic peaks starting from an endemic state. In many cases, the oscillations generated by seasonality in the dynamics of the infection are too small to be observable. The interplay of this seasonality with the epidemic oscillations can generate new outbreaks starting from the endemic state through a mechanism of parametric resonance. Using experimental data we present specific cases where this phenomenon is numerically observed.     

Surfactant Protein-D Is Essential for Immunity to Helminth Infection

Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.     

Genetic Variation in OAS1 Is a Risk Factor for Initial Infection with West Nile Virus in Man

West Nile virus (WNV) is a re-emerging pathogen that can cause fatal encephalitis. In mice, susceptibility to WNV has been reported to result from a single point mutation in oas1b, which encodes 2′–5′ oligoadenylate synthetase 1b, a member of the type I interferon-regulated OAS gene family involved in viral RNA degradation. In man, the human ortholog of oas1b appears to be OAS1. The ‘A’ allele at SNP rs10774671 of OAS1 has previously been shown to alter splicing of OAS1 and to be associated with reduced OAS activity in PBMCs. Here we show that the frequency of this hypofunctional allele is increased in both symptomatic and asymptomatic WNV seroconverters (Caucasians from five US centers; total n = 501; OR = 1.6 [95% CI 1.2–2.0], P = 0.0002 in a recessive genetic model). We then directly tested the effect of this SNP on viral replication in a novel ex vivo model of WNV infection in primary human lymphoid tissue. Virus accumulation varied markedly among donors, and was highest for individuals homozygous for the ‘A’ allele (P<0.0001). Together, these data identify OAS1 SNP rs10774671 as a host genetic risk factor for initial infection with WNV in humans.     

Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

IL-10 Signaling Blockade Controls Murine West Nile Virus Infection

West Nile virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Our data show that interleukin-10 (IL-10) is dramatically elevated both in vitro and in vivo following WNV infection. Consistent with an etiologic role of IL-10 in WNV pathogenesis, we find that WNV infection is markedly diminished in IL-10 deficient (IL-10^−/−) mice, and pharmacologic blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice. Increased production of antiviral cytokines in IL-10^−/− mice is associated with more efficient control of WNV infection. Moreover, CD4⁺ T cells produce copious amounts of IL-10, and may be an important cellular source of IL-10 during WNV infection in vivo. In conclusion, IL-10 signaling plays a negative role in immunity against WNV infection, and blockade of IL-10 signaling by genetic or pharmacologic means helps to control viral infection, suggesting a novel anti-WNV therapeutic strategy.     

Second International Diagnostic Accuracy Study for the Serological Detection of West Nile Virus Infection

Background

In recent decades, sporadic cases and outbreaks in humans of West Nile virus (WNV) infection have increased. Serological diagnosis of WNV infection can be performed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) neutralization test (NT) and by hemagglutination-inhibition assay. The aim of this study is to collect updated information regarding the performance accuracy of WNV serological diagnostics.

Methodology/Principal findings

In 2011, the European Network for the Diagnostics of Imported Viral Diseases-Collaborative Laboratory Response Network (ENIVD-CLRN) organized the second external quality assurance (EQA) study for the serological diagnosis of WNV infection. A serum panel of 13 samples (included sera reactive against WNV, plus specificity and negative controls) was sent to 48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 30 countries participated in the study. Eight laboratories achieved 100% of concurrent and correct results. The main obstacle in other laboratories to achieving similar performances was the cross-reactivity of antibodies amongst heterologous flaviviruses. No differences were observed in performances of in-house and commercial test used by the laboratories. IFA was significantly more specific compared to ELISA in detecting IgG antibodies. The overall analytical sensitivity and specificity of diagnostic tests for IgM detection were 50% and 95%, respectively. In comparison, the overall sensitivity and specificity of diagnostic tests for IgG detection were 86% and 69%, respectively.

Conclusions/Significance

This EQA study demonstrates that there is still need to improve serological tests for WNV diagnosis. The low sensitivity of IgM detection suggests that there is a risk of overlooking WNV acute infections, whereas the low specificity for IgG detection demonstrates a high level of cross-reactivity with heterologous flaviviruses.     

A Role for Ifit2 in Restricting West Nile Virus Infection in the Brain

Previous studies have demonstrated that type I interferon (IFN-I) restricts West Nile virus (WNV) replication and pathogenesis in peripheral and central nervous system (CNS) tissues. However, the in vivo role of specific antiviral genes that are induced by IFN-I against WNV infection remains less well characterized. Here, using Ifit2^−/− mice, we defined the antiviral function of the interferon-stimulated gene (ISG) Ifit2 in limiting infection and disease in vivo by a virulent North American strain of WNV. Compared to congenic wild-type controls, Ifit2^−/− mice showed enhanced WNV infection in a tissue-restricted manner, with preferential replication in the CNS of animals lacking Ifit2. Virological analysis of cultured macrophages, dendritic cells, fibroblasts, cerebellar granule cell neurons, and cortical neurons revealed cell type-specific antiviral functions of Ifit2 against WNV. In comparison, small effects of Ifit2 were observed on the induction or magnitude of innate or adaptive immune responses. Our results suggest that Ifit2 restricts WNV infection and pathogenesis in different tissues in a cell type-specific manner.     

Identification of Cellular Proteome Modifications in Response to West Nile Virus Infection

Flaviviruses are positive-stranded RNA viruses that are a public health problem because of their widespread distribution and their ability to cause a variety of diseases in humans. West Nile virus is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. Clinical manifestations of West Nile virus infection are diverse, and their pathogenic mechanisms depend on complex virus-cell interactions. In the present work, we used proteomics technology to analyze early Vero cell response to West Nile infection. The differential proteomes were resolved 24 h postinfection using two-dimensional DIGE followed by mass spectrometry identification. Quantitative analysis (at least 2-fold quantitative alteration, p < 0.05) revealed 127 differentially expressed proteins with 68 up-regulated proteins and 59 down-regulated proteins of which 93 were successfully identified. The implication for mammalian cellular responses to this neurotropic flavivirus infection was analyzed and made possible more comprehensive characterization of the virus-host interactions involved in pathogenesis. The present study thus provides large scale protein-related information that should be useful for understanding how the host metabolism is modified by West Nile infection and for identifying new potential targets for antiviral therapy.West Nile virus (WNV)¹ is a mosquito-borne flavivirus belonging to the Japanese encephalitis virus (JEV) serocomplex. The virus is maintained in nature in enzootic cycles in which it is transmitted between ornithophilic mosquitoes and avian hosts. In mammals, including humans, WNV is an encephalitic flavivirus and can cause natural infections of the central nervous system (CNS) with a neuropathogenesis involving neuroinvasiveness (ability to enter the CNS) and neurovirulence (replication within the CNS) (1). To date, no pharmacological treatment exists for WNV, and a vaccine is only available for horses.First isolated in 1937, WNV has become endemic in Africa, the Middle East, and parts of Asia and Europe (2, 3). Phylogenetics analysis groups WNV strains into two distinct lineages. Viruses in lineage 2 are found only in Africa, whereas viruses in lineage 1 are present both in Africa and in other areas, particularly Asia and Europe. Since 1999, WNV from lineage 1 (NY99) has reached North America where, in 2002, it caused the largest arboviral meningoencephalitis outbreak ever recorded in this area (4).It is known that flavivirus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to a “cytopathic effect” in various cell cultures of human, primate, rodent, and insect origin (5). Recent studies have revealed specific effects of viruses on cellular processes. It has been demonstrated that flaviviruses can induce cell death directly through viral replication and the production of proapoptotic proteins (6–11), but the mechanism of pathogenesis has not been elucidated.Although neurons are regarded as the major target of WNV in vivo (2), WNV infection has been shown to induce apoptosis in different cell lines in a similar manner in vitro (12, 13). This includes a wide range of different cell types with, in particular, the African green monkey kidney continuous cell line (Vero) recommended by the World Health Organization Collaborating Center for systematic research and isolation of arboviruses as well as a substrate to develop live attenuated and inactivated vaccines. Acute infection of Vero cells by WNV produces a lytic infection with a characteristic rounding cytopathic effect and the production of a large number of infectious particles in the culture fluid within 3 days postinfection (14). Although this permissive mammalian cell system is widely used for flavivirus isolation, propagation, and titration, to date no studies have focused on identifying Vero cellular proteins whose expression has been altered by WNV infection. We considered that Vero cells could be a good model for in vitro identification of cell protein alterations with possible implication in certain pathogenic mechanisms.In the present work, fluorescent 2D DIGE technology combined with MS analysis was used to examine the consequences of Vero cell infection by WNV. To evaluate early mammalian cell response after infection and to avoid the effect of cell death and protein degradation, the culture conditions (e.g. infectious dose and incubation time) were optimized. A total of 93 differentially expressed protein spots were identified (over ±2-fold, p < 0.05) and confirmed by fluorescent Western blot analysis. The implication for cellular responses to this flavivirus infection as well as the potential roles of certain altered identified proteins are discussed to characterize the pathophysiologic processes. This study can also provide useful clues for antiviral research.     

Threshold Conditions for West Nile Virus Outbreaks

In this paper, we study the stability and saddle-node bifurcation of a model for the West Nile virus transmission dynamics. The existence and classification of the equilibria are presented. By the theory of K-competitive dynamical systems and index theory of dynamical systems on a surface, sufficient and necessary conditions for local stability of equilibria are obtained. We also study the saddle-node bifurcation of the system. Explicit subthreshold conditions in terms of parameters are obtained beyond the basic reproduction number which provides further guidelines for accessing control of the spread of the West Nile virus. Our results suggest that the basic reproductive number itself is not enough to describe whether West Nile virus will prevail or not and suggest that we should pay more attention to the initial state of West Nile virus. The results also partially explained the mechanism of the recurrence of the small scale endemic of the virus in North America. Supported by the Chinese NSF grants 10531030 and 10671143. Supported by the Chinese NSF grants 10801074. Supported by Canada Research Chairs Program, Mathematics for Information Technology and Complex Systems (MITACS), National Microbiology Laboratory, Natural Sciences and Engineering Research Council (NSERC), Canadian Foundation of Innovation (CFI) and Ontario Innovation Trust (OIT), Ontario Ministry of Health and Long-term Care, Peel, Toronto, Chat-Kent Health Units, and Public Health Agency of Canada (PHAC). Supported by NSERC, MITACS, CFI/OIT a new opportunity fund, Early Research Award of Ministry of Research and Innovation (ERA) of Ontario, Infectious Diseases Branch of Ministry of Health and Long Term Care (MOH) of Ontario and PHAC.     

A Hamster-Derived West Nile Virus Isolate Induces Persistent Renal Infection in Mice

Background

West Nile virus (WNV) can persist long term in the brain and kidney tissues of humans, non-human primates, and hamsters. In this study, mice were infected with WNV strain H8912, previously cultured from the urine of a persistently infected hamster, to determine its pathogenesis in a murine host.

Methodology/Principal Findings

We found that WNV H8912 was highly attenuated for neuroinvasiveness in mice. Following a systemic infection, viral RNA could be detected quickly in blood and spleen and much later in kidneys. WNV H8912 induced constitutive IL-10 production, upregulation of IFN-β and IL-1β expression, and a specific IgM response on day 10 post-infection. WNV H8912 persisted preferentially in kidneys with mild renal inflammation, and less frequently in spleen for up to 2.5 months post infection. This was concurrent with detectable serum WNV-specific IgM and IgG production. There were also significantly fewer WNV- specific T cells and lower inflammatory responses in kidneys than in spleen. Previous studies have shown that systemic wild-type WNV NY99 infection induced virus persistence preferentially in spleen than in mouse kidneys. Here, we noted that splenocytes of WNV H8912-infected mice produced significantly less IL-10 than those of WNV NY99-infected mice. Finally, WNV H8912 was also attenuated in neurovirulence. Following intracranial inoculation, WNV persisted in the brain at a low frequency, concurrent with neither inflammatory responses nor neuronal damage in the brain.

Conclusions

WNV H8912 is highly attenuated in both neuroinvasiveness and neurovirulence in mice. It induces a low and delayed anti-viral response in mice and preferentially persists in the kidneys.     

Modelling the dynamics of West Nile Virus

In this work we formulate and analyze a mathematical model for the transmission of West Nile Virus (WNV) infection between vector (mosquito) and avian population. We find the Basic Reproductive Number in terms of measurable epidemiological and demographic parameters. is the threshold condition that determines the dynamics of WNV infection: if the disease fades out, and for the disease remains endemic. Using experimental and field data we estimate for several species of birds. Numerical simulations of the temporal course of the infected bird proportion show damped oscillations approaching the endemic value.