Natalie A. Prow

Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling

ABSTRACT Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms to circumvent the hosts antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2 (P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral pathogenesis.

Characterization of Virulent West Nile Virus Kunjin Strain, Australia, 2011

An encephalitis outbreak among horses was caused by a pathogenic variant of Kunjin virus.

A new insect - specific flavivirus from northern Australia suppresses replication of West Nile virus and Murray Valley encephalitis virus in co-infected mosquito cells

Recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. To date, there is no information on the presence or prevalence of these insect-specific flaviviruses (ISFs) in Australian mosquito species. To assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (RT-PCR) and flavivirus universal primers that are specific to the NS5 gene to detect these viruses in mosquito pools collected from the Northern Territory. Of 94 pools of mosquitoes, 13 were RT-PCR positive, and of these, 6 flavivirus isolates were obtained by inoculation of mosquito cell culture. Sequence analysis of the NS5 gene revealed that these isolates are genetically and phylogenetically similar to ISFs reported from other parts of the world. The entire coding region of one isolate (designated 56) was sequenced and shown to have approximately 63.7% nucleotide identity and 66.6% amino acid identity with its closest known relative (Nakiwogo virus) indicating that the prototype Australian ISF represents a new species. All isolates were obtained from Coquillettidia xanthogaster mosquitoes. The new virus is tentatively named Palm Creek virus (PCV) after its place of isolation. We also demonstrated that prior infection of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10–43 fold (1 to 1.63 log) at 48 hr post-infection, suggesting that superinfection exclusion can occur between ISFs and vertebrate-infecting flaviviruses despite their high level of genetic diversity. We also generated several monoclonal antibodies (mAbs) that are specific to the NS1 protein of PCV, and these represent the first ISF-specific mAbs reported to date.

The Inflammatory Cytokine, Interleukin-1 Beta, Mediates Loss of Astroglial Glutamate Transport and Drives Excitotoxic Motor Neuron Injury in the Spinal Cord During Acute Viral Encephalomyelitis

Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT‐1, was examined. GLT‐1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL‐1β release also limited paralysis and prevented the loss of GLT‐1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT‐1 levels as well as lower production of IL‐1β and relatively preserved GLT‐1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT‐1 expression was largely maintained following infection of IL‐1β‐deficient animals. Together, these data show that IL‐1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

ABSTRACT West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8+ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.

Mechanism of West Nile Virus Neuroinvasion: A Critical Appraisal

West Nile virus (WNV) is an important emerging neurotropic virus, responsible for increasingly severe encephalitis outbreaks in humans and horses worldwide. However, the mechanism by which the virus gains entry to the brain (neuroinvasion) remains poorly understood. Hypotheses of hematogenous and transneural entry have been proposed for WNV neuroinvasion, which revolve mainly around the concepts of blood-brain barrier (BBB) disruption and retrograde axonal transport, respectively. However, an over‑representation of in vitro studies without adequate in vivo validation continues to obscure our understanding of the mechanism(s). Furthermore, WNV infection in the current rodent models does not generate a similar viremia and character of CNS infection, as seen in the common target hosts, humans and horses. These differences ultimately question the applicability of rodent models for pathogenesis investigations. Finally, the role of several barriers against CNS insults, such as the blood-cerebrospinal fluid (CSF), the CSF-brain and the blood-spinal cord barriers, remain largely unexplored, highlighting the infancy of this field. In this review, a systematic and critical appraisal of the current evidence relevant to the possible mechanism(s) of WNV neuroinvasion is conducted.

JE-ADVAX Vaccine Protection against Japanese Encephalitis Virus Mediated by Memory B Cells in the Absence of CD8+ T Cells and Pre-Exposure Neutralizing Antibody

ABSTRACT JE-ADVAX is a new, delta inulin-adjuvanted, Japanese encephalitis (JE) candidate vaccine with a strong safety profile and potent immunogenicity that confers efficient immune protection not only against JE virus but also against related neurotropic flaviviruses such as West Nile virus. In this study, we investigated the immunological mechanism of protection by JE-ADVAX vaccine using knockout mice deficient in B cells or CD8+ T cells and poor persistence of neutralizing antibody or by adoptive transfer of immune splenocyte subpopulations. We show that memory B cells induced by JE-ADVAX provide long-lived protection against JE even in the absence of detectable pre-exposure serum neutralizing antibodies and without the requirement of CD8+ T cells. Upon virus encounter, these vaccine-induced memory B cells were rapidly triggered to produce neutralizing antibodies that then protected immunized mice from morbidity and mortality. The findings suggest that the extent of the B-cell memory compartment might be a better immunological correlate for clinical efficacy of JE vaccines than the currently recommended measure of serum neutralizing antibody. This may explain the paradox where JE protection is observed in some subjects even in the absence of detectable serum neutralizing antibody. Our investigation also established the suitability of a novel flavivirus challenge model (β2-microglobulin-knockout mice) for studies of the role of B-cell memory responses in vaccine protection.

Neutralizing monoclonal antibodies to the E2 protein of chikungunya virus protects against disease in a mouse model.

Chikungunya virus (CHIKV) recently caused the largest epidemic ever recorded for this virus involving an estimated 1.4-6.5million cases, with imported cased reported in over 40 countries. The number of monoclonal antibodies specific for this re-emerging alphavirus is currently limited. Herein we describe the generation and characterisation of five monoclonal antibodies specific for the E2 glycoprotein of CHIKV. The antibodies detected a range of CHIKV isolates in several assays including ELISA, Western blot, immunofluorescence assay (IFA) and immunohistochemistry (IHC) without evidence of cross-reactivity with other alphaviruses. Four antibodies also neutralised CHIKV in vitro, two of which provided complete protection against arthritis in a CHIKV mouse model when administered prior to infection. Given the current shortage of widely available reagents for CHIKV, these specific antibodies will be useful not only in research, but may also provide the basis for new diagnostics and treatments.

Protection from fatal viral encephalomyelitis: AMPA receptor antagonists have a direct effect on the inflammatory response to infection

Neuronal cell death during fatal acute viral encephalomyelitis can result from damage caused by virus replication, glutamate excitotoxicity, and the immune response. A neurovirulent strain of the alphavirus Sindbis virus (NSV) causes fatal encephalomyelitis associated with motor neuron death in adult C57BL/6 mice that can be prevented by treatment with the prototypic noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor antagonist GYKI 52466 [Nargi-Aizenman J, et al. (2004) Ann Neurol 55:541–549]. To determine the mechanism of protection, NSV-infected mice were treated with 7-acetyl-5-(4-aminophenyl)-8(R)-methyl-8,9-dihydro-7H-1,3-dioxolo-(4,5-h)-benzodiazepine (talampanel), a potent, orally available member of the 2,3 benzodiazepine class of noncompetitive AMPA glutamate receptor antagonists. Talampanel-treated mice were protected from NSV-induced paralysis and death. Examination of the brain during infection showed significantly less mononuclear cell infiltration and no increase in astrocyte expression of glial fibrillary acidic protein in treated mice compared with untreated mice. Lack of CNS inflammation was attributable to failure of treated mice to induce activation and proliferation of lymphocytes in secondary lymphoid tissue in response to infection. Antibody responses to NSV were also suppressed by talampanel treatment, and virus clearance was delayed. These studies reveal a previously unrecognized effect of AMPA receptor antagonists on the immune response and suggest that prevention of immune-mediated damage, in addition to inhibition of excitotoxicity, is a mechanism by which these drugs protect from death of motor neurons caused by viral infection.

Viral RNA Intermediates as Targets for Detection and Discovery of Novel and Emerging Mosquito-Borne Viruses

Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents a rapid, sequence-independent, and cost-effective approach to virus discovery.