John K. Fazakerley

Bunyamwera Bunyavirus Nonstructural Protein NSs Counteracts the Induction of Alpha/Beta Interferon

ABSTRACT Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera virus.

Advances in dissecting mosquito innate immune responses to arbovirus infection

Arthropod-borne viruses - arboviruses - are a significant threat to public health. Whilst there is considerable knowledge about arbovirus interactions with vertebrate immunity, relatively little is known about how vectors such as mosquitoes control arbovirus infections. In this review, we discuss novel findings in the field of mosquito antiviral responses to arboviruses, in particular RNA interference, the up-and-coming field of general immune-signalling pathways, and cell death/apoptosis.

The V5A13.1 envelope glycoprotein deletion mutant of mouse hepatitis virus type-4 is neuroattenuated by its reduced rate of spread in the central nervous system

Abstract Following intracerebral inoculation of adult Balb/c Byj mice, the MHV-4 strain of mouse hepatitis virus (MHV) had an LD50 of <0.1 PFU, whereas its monoclonal antibody resistant variant V5A13.1 had an LD50 of 104.2 PFU. To determine the basis for this difference in neurovirulence we have studied the acute central nervous system (CNS) infection of these two viruses by in situ hybridization. Both viruses infected the same, specific neuroanatomical areas, predominantly neurons, and spread via the cerebrospinal fluid, along neuronal pathways and between adjacent cells. The neuronal nuclei infected and the spread of virus within the brain are described. The main difference between the parental and variant viruses was the rate at which the infection spread. MHV-4 spread rapidly, destroying large numbers of neurons and the animals died within 4 days of infection. The variant virus spread to the same areas of the brain but at a slower rate. This difference in the rate of virus spread was also apparent from the brain virus titers. The slower rate of spread of the variant virus appears to allow intervention by the immune response. Consistent with this, the variant virus spread slowly in athymic nu/nu mice, but in the absence of an intact immune response, infection and destruction of neurons eventually reached the same extent as that of the parental virus and the mice died within 6 days of infection. We conclude that the V5A13.1 variant of MHV-4 is neuroattenuated by its slower rate of spread in the CNS.

Semliki Forest Virus Nonstructural Protein 2 Is Involved in Suppression of the Type I Interferon Response

ABSTRACT The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist.

Knockdown of piRNA pathway proteins results in enhanced Semliki Forest virus production in mosquito cells

The exogenous siRNA pathway is important in restricting arbovirus infection in mosquitoes. Less is known about the role of the PIWI-interacting RNA pathway, or piRNA pathway, in antiviral responses. Viral piRNA-like molecules have recently been described following infection of mosquitoes and derived cell lines with several arboviruses. The piRNA pathway has thus been suggested to function as an additional small RNA-mediated antiviral response to the known infection-induced siRNA response. Here we show that piRNA-like molecules are produced following infection with the naturally mosquito-borne Semliki Forest virus in mosquito cell lines. We show that knockdown of piRNA pathway proteins enhances the replication of this arbovirus and defines the contribution of piRNA pathway effectors, thus characterizing the antiviral properties of the piRNA pathway. In conclusion, arbovirus infection can trigger the piRNA pathway in mosquito cells, and knockdown of piRNA proteins enhances virus production.

A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus

ABSTRACT The replicase protein nsP2 of Semliki Forest virus (SFV) has a 648RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.

Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis

The course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination.

Characterization of the cellular and cytokine response in the central nervous system following Semliki Forest virus infection

Cytokines are important mediators in the pathogenesis of central nervous system (CNS) inflammatory diseases including multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE), viral encephalitis and virus induced demyelinating diseases. We have used immunohistochemical techniques to characterize the mononuclear cell infiltrate and cytokine profiles in the CNS following infection of mice with the demyelinating A7(74) strain of Semliki Forest virus (SFV), an important viral model of MS. Mononuclear cell infiltrates in the CNS, first observed at 3 days and maximal during clearance of infectious virus, were comprised predominantly of CD8+ lymphocytes. F4/80+ macrophage/microglia and CD45/B220+ B lymphocytes were most numerous during the subsequent phase of demyelination. CD4+ T-lymphocytes were observed at low levels throughout infection. By immunostaining MHC class I, IL-1beta , IL-3 and TGF beta1 were constitutively expressed in normal mice and were upregulated following infection. MHC class II, IL-1alpha, IL-2, IL-2R, TNF-alpha and IL-6 were strongly upregulated in the CNS of SFV-infected mice and mice with chronic relapsing EAE. The spatial and temporal distribution of these cytokines during the course of disease was analysed. Whereas IL-1alpha, IL-1beta, IL-10, and TGF beta1 were observed on day 3 following infection GMCSF, IL-2 and TNF alpha were first apparent at day 7 when the cellular infiltration in the CNS was most intense. In contrast IFN gamma and IL-6 were first observed on day 10 prior to the demyelination phase of disease. Cytokines in the lesions of demyelination suggest a role in the pathogeneisis of myelin damage. Based on cytokine profiles no clear bias of either a Th1 or Th2 response was observed in the CNS during infection.

Susceptibility to a neurotropic virus and its changing distribution in the developing brain is a function of CNS maturity

Many major physiological changes occur within the rodent central nervous system (CNS) during the first few postnatal weeks. These include axonogenesis, synaptogenesis and myelination. Concomitant with CNS development over this period, there is a decrease in susceptibility to many neurotropic virus infections in that infection of suckling animals results in lethal encephalitis whereas infection of weanling animals is not lethal. The events underlying this dramatic change in susceptibility have been unclear. Here we demonstrate that age-related virulence of the neurotrophic alphavirus, Semliki Forest virus is dependent upon ability of the infection to spread in the CNS. This is not determined by maturity of interferon, or specific immune responses or the blood brain barrier, but by maturity of neuronal systems. Detailed study of the course of infection in the cortex, hippocampus and cerebellum during their postnatal development indicates that as these and other neuronal systems mature they become resistant to spread of the virus and the pattern of infection changes from widespread to focal.

Novel attenuated Chikungunya vaccine candidates elicit protective immunity in C57BL/6 mice

ABSTRACT Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that has caused severe epidemics in Africa and Asia and occasionally in Europe. As of today, there is no licensed vaccine available to prevent CHIKV infection. Here we describe the development and evaluation of novel CHIKV vaccine candidates that were attenuated by deleting a large part of the gene encoding nsP3 or the entire gene encoding 6K and were administered as viral particles or infectious genomes launched by DNA. The resulting attenuated mutants were genetically stable and elicited high magnitudes of binding and neutralizing antibodies as well as strong T cell responses after a single immunization in C57BL/6 mice. Subsequent challenge with a high dose of CHIKV demonstrated that the induced antibody responses protected the animals from viremia and joint swelling. The protective antibody response was long-lived, and a second homologous immunization further enhanced immune responses. In summary, this report demonstrates a straightforward means of constructing stable and efficient attenuated CHIKV vaccine candidates that can be administered either as viral particles or as infectious genomes launched by DNA. IMPORTANCE Similar to other infectious diseases, the best means of preventing CHIKV infection would be by vaccination using an attenuated vaccine platform which preferably raises protective immunity after a single immunization. However, the attenuated CHIKV vaccine candidates developed to date rely on a small number of attenuating point mutations and are at risk of being unstable or even sensitive to reversion. We report here the construction and preclinical evaluation of novel CHIKV vaccine candidates that have been attenuated by introducing large deletions. The resulting mutants proved to be genetically stable, attenuated, highly immunogenic, and able to confer durable immunity after a single immunization. Moreover, these mutants can be administered either as viral particles or as DNA-launched infectious genomes, enabling evaluation of the most feasible vaccine modality for a certain setting. These CHIKV mutants could represent stable and efficient vaccine candidates against CHIKV.