Dr Sareen Galbraith

A Semliki Forest virus (SFV) recombinant particle vaccine vector was constructed expressing the viral E1 and E2 envelope proteins of the RA27/3 vaccine strain of rubella virus. This vector induced high titres of antibody after intramuscular administration to Balb/C mice, both following initial vaccination and a boost 4 weeks later. This occurred for antibody as measured by ELISA and as measured by a latex agglutination test. However, co-administration of similar particles expressing the measles virus H protein and the mumps virus HN protein with the rubella protein expressing vector resulted in reduction of the anti-rubella immune response. © 2007 Elsevier Ltd. All rights reserved.

Major virulence determinants of Semliki Forest virus (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5′ conserved domains and a non-essential hypervariable 3′ domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation. © 2006 SGM.

The effect of intranasal (IN) administration of Semliki Forest virus (SFV) recombinant particles expressing interferon-β [IFN-β, a partially effective treatment for multiple sclerosis (MS)] on the progression of experimental autoimmune encephalomyelitis (EAE, a murine model for MS) was investigated. The murine IFN-β gene was cloned from SFV-infected mouse brain by RT-PCR into an SFV-enhanced expression vector, pSFV10-E, from which IFN-β-expressing recombinant particles (rSFV10-E-IFN-β) were prepared. Expression studies using immunohistochemistry and viral inhibition assay in BHK and murine L929 cells confirmed increased expression of IFN-β. High level expression in the central nervous system (CNS) following IN inoculation was confirmed by the excision of olfactory bulbs, brain and spinal cord, and the detection of IFN-β levels in homogenised tissue by ELISA. rSFV10-E-IFN-β particles were administered IN to C57/Bl6 mice that had been induced for EAE using the encephalogenic peptide myelin oligodendrocyte glycoprotein (MOG) 35-55. The progression of EAE was measured by clinical score, weight loss and pathology. As previously shown, treatment with empty rSFV10-E particles moderately exacerbated EAE, as did continuous treatment with rSFV10-E-IFN-β particles. Inhibition of disease with rSFV10-EIFN-β particles was dependent on the number and timing of treatments. Fewer treatments, administered before the effector stage, led to an improvement in clinical and pathology score. In conclusion, the timing and frequency of IN administration of rSFV10-E-IFN-β particles are critical to disease outcome, with treatment prior to the effector stage being most effective.

The immune responses of cattle inoculated with either a virulent or an attenuated vaccine strain of rinderpest virus (RPV) were examined by measuring the proliferation of peripheral blood mononuclear cells (PBMC) to whole RPV antigen preparations and to individual RPV major structural proteins expressed using recombinant adenoviruses. Responses to the T cell mitogen concanavalin A (ConA) were also measured as a control to monitor non-specific effects of infection with RPV on T cell responses. Infection with the vaccine strain of RPV was found to induce a strong CD4+T cell response. A specific response was detected to all RPV proteins tested, namely the haemagglutinin (H), fusion (F), nucleocapsid (N) and matrix (M) proteins, in animals vaccinated with the attenuated strain of the virus. No one protein was found to be dominant with respect to the induction of T cell proliferative responses. As expected, vaccination of cattle with an unrelated virus vaccine, a capripox vaccine, failed to produce a response to RPV antigens. While profound suppression of T cell responses was observed following infection with the virulent strain of RPV, no evidence of impairment of T cell responsiveness was observed following RPV vaccination, or on subsequent challenge of vaccinated animals with virulent virus.

Multiple sclerosis (MS) is a chronic, demyelinating disease of the CNS in which autoimmunity to myelin plays a role in pathogenesis. The epidemiology of MS indicates that it may be triggered by a virus infection before the age of adolescence, but attempts to associate a specific virus with MS have produced equivocal results. Many studies of the aetiology of MS have postulated that a persistent virus infection is involved, but transient virus infection may provide a plausible alternative mechanism that could explain many of the inconsistencies in MS research. The most studied animal model of MS is chronic relapsing experimental autoimmune encephalomyelitis (CREAE), which is induced in susceptible animals following injection of myelin components. While CREAE cannot provide information on the initiating factor for MS, it may mimic disease processes occurring after an initial trigger that may involve transient virus infection. The disease process may comprise separate triggering and relapse phases. The triggering phase may involve sensitisation to myelin antigens as a result of damage to oligodendrocytes or molecular mimicry. The relapse phase could be similar to CREAE, or alternatively relapses may be induced by further transient virus infections which may not involve infection of the CNS, but which may involve the recrudescence of anti-myelin autoimmunity. Although current vaccines have a high degree of biosafety, it is suggested that the measles-mumps-rubella vaccine in particular could be modified to obviate any possibility of triggering anti-myelin autoimmunity. Copyright (C) 2000 John Wiley and Sons, Ltd.

Members of the morbillivirus genus, canine distemper (CDV), phocine distemper virus (PDV), and the cetacean viruses of dolphins and porpoises exhibit high levels of CNS infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, it is possible that all morbilliviruses infect the CNS but in some hosts are rapidly cleared by the immune response. In this study, we assessed whether RPV and PPRV have the potential to be neurovirulent. We describe the outcome of infection, of selected mouse strains, with isolates of RPV, PPRV, PDV, porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), and a wild-type strain of MV. In the case of RPV virus, strains with different passage histories have been examined. The results of experiments with these viruses were compared with those using neuroadapted and vaccine strains of MV, which acted as positive and negative controls respectively. Intracerebral inoculation with RPV (Saudi/81) and PPRV (Nigeria75/1) strains produced infection in Balb/C and Cd1, but not C57 suckling mice, whereas the CAM/RB rodent-adapted strain of MV infected all three strains of mice. Weanling mice were only infected by CAM/RB. Intranasal and intraperitoneal inoculation failed to produce infection with any virus strains. We have shown that, both RPV and PPRV, in common with other morbilliviruses are neurovirulent in a permissive system. Transient infection of the CNS of cattle and goats with RPV and PPRV, respectively, remains a possibility, which could provide relevant models for the initial stages of MV infection in humans.

Yellow fever virus (YFV) is a mosquito-borne flavivirus that is the causative agent of yellow fever (YF), which is a disease that ranges in severity from an influenza-like syndrome to severe liver and renal dysfunction, circulatory shock, and hemorrhage. The disease causes substantial morbidity and mortality; case fatality rates often exceed 20% and, despite the availability of a vaccine, it is still a major public health problem for sub-Saharan Africa and tropical South America, and travelers to endemic areas. The virus is maintained in endemic areas of Africa and South America by enzootic transmission between mosquitoes and monkeys, and the epidemiology of the disease reflects the geographical distribution of the mosquito vectors. There are seven genotypes of this virus with five circulating in different geographic areas of Africa and two in South America. Two live-attenuated YF vaccines were simultaneously developed: the French neurotropic vaccine (FNV), or Dakar vaccine, and the 17D vaccine. In the 1980s the manufacture of FNV was completely discontinued because the World Health Organization (WHO) prohibited vaccination of children under 14 years old due to the high incidence of encephalitis. The development of the highly effective, live-attenuated 17D vaccine in the late 1930s is one of the milestones in vaccine research. The vaccine is safe and highly efficacious with one dose of vaccine giving immunity for at least 10 years, and may be lifelong immunity. Neutralizing antibodies are the correlate of protection. There are three substrains of the YF 17D vaccine in production today (17D-204, 17DD, and 17D-213). The WHO has standardized the seed lot and manufacturing processes, and published guidelines for approval of vaccine production and vaccination certificates. There are a number of contraindications for YF vaccination which include age, thymus disease, pregnancy, breast-feeding, immune suppression, and hypersensitivity to eggs. In recent years, a rare but significant number of severe adverse events have been reported involving vaccine-associated viscerotropic and neurotropic disease. Current vaccine development involves utilizing the 17D vaccine virus backbone, via infectious clone technology, to develop a variety of chimeric virus vaccines, some of which have reached clinical trials for human use.

© 2014 The Royal Entomological Society. The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21days post-infection, 13% and 25% of O.detritus were able to transmit JEV when held at 23°C and 28°C, respectively. Similar results were obtained for C.quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O.detritus, is a potential vector of an exotic flavivirus.

Worldwide central nervous system (CNS) infections in human and animal populations differ with both the geographical location and the immune status of the host. Encephalitis is caused by viruses from many different taxonomic groups but most frequently occurs in rabies and insect-borne virus (arboviruses) infections. However, human herpes viruses cause a range of acute, subacute and long-term neurological diseases in both immunocompetent and immunocompromised individuals and are the most common cause of CNS infection in Western countries. Infection with the retroviruses human immunodeficiency virus type-1 (HIV1) and human T cell leukaemia virus type-1 (HTLV1) can lead to an immunodeficiency marked by depletion of CD4+ T cells and eventually, if untreated, to neurodegenerative diseases, either as a direct result of infection with these agents or due to secondary opportunistic infections. HIV dementia is increasing in prevalence with more people living due to antiviral therapy. Members of the negative-strand RNA viruses (Mononegavirales), including measles, henipaviruses and members of the Bunyaviridae family, cause encephalitis associated with particular regions worldwide. RNA positive-strand toga and alpha virus infections are generally associated with tropical climates, but due to climate changes affecting their insect hosts they are increasingly moving northwards. While not cause by viruses, prion diseases, also known as transmissible spongiform encephalopathies, are invariably fatal and present a major challenge as they are caused by a rogue host protein. While we are close to eradiation of infections such as poliovirus and measles virus, in the next 10 to 20 years new pathogens will emerge. Increasing knowledge of virus-host interactions and the resulting neuroimmunology will allow identification of novel targets to develop future therapies for CNS infection. This edition first published 2014

BACKGROUND: Japanese encephalitis (JE) virus (JEV) is a mosquito-borne flavivirus found across Asia that is closely related to West Nile virus. There is no known antiviral treatment for any flavivirus. Results from in vitro studies and animal models suggest intravenous immunoglobulin (IVIG) containing virus-specific neutralizing antibody may be effective in improving outcome in viral encephalitis. IVIG's anti-inflammatory properties may also be beneficial. METHODOLOGY/PRINCIPAL FINDINGS: We performed a pilot feasibility randomized double-blind placebo-controlled trial of IVIG containing anti-JEV neutralizing antibody (ImmunoRel, 400mg/kg/day for 5 days) in children with suspected JE at two sites in Nepal; we also examined the effect on serum neutralizing antibody titre and cytokine profiles. 22 children were recruited, 13 of whom had confirmed JE; 11 received IVIG and 11 placebo, with no protocol violations. One child (IVIG group) died during treatment and two (placebo) subsequently following hospital discharge. Overall, there was no difference in outcome between treatment groups at discharge or follow up. Passive transfer of anti-JEV antibody was seen in JEV negative children. JEV positive children treated with IVIG had JEV-specific neutralizing antibody titres approximately 16 times higher than those treated with placebo (p=0.2), which was more than could be explained by passive transfer alone. IL-4 and IL-6 were higher in the IVIG group. CONCLUSIONS/SIGNIFICANCE: A trial of IVIG for JE in Nepal is feasible. IVIG may augment the development of neutralizing antibodies in JEV positive patients. IVIG appears an appealing option for JE treatment that warrants further study. TRIAL REGISTRATION: ClinicalTrials.gov NCT01856205.

Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35×10

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Mortality from adult bacterial meningitis exceeds 50% in sub-Saharan Africa. We postulated that-particularly in individuals infected with human immunodeficiency virus (HIV)-herpes simplex virus, varicella zoster virus, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in the cerebrospinal fluid (CSF) contribute to poor outcome. CSF from 149 Malawian adults with bacterial meningitis and 39 controls were analyzed using polymerase chain reaction. EBV was detected in 79 of 149 bacterial meningitis patients. Mortality (54%) was associated with higher CSF EBV load when adjusted for HIV (P =. 01). CMV was detected in 11 of 115 HIV-infected patients, 8 of whom died. The mechanisms by which EBV and CMV contribute to poor outcome require further investigation. © 2011 The Author.

Purpose: We looked for herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA in Malawian adults with clinically suspected meningitis. Methods: We collected cerebrospinal fluid (CSF) from consecutive adults admitted with clinically suspected meningitis to Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi, for a period of 3 months. Those with proven bacterial or fungal meningitis were excluded. Real-time polymerase chain reaction (PCR) was performed on the CSF for HSV-1 and HSV-2, VZV, EBV and CMV DNA. Results: A total of 183 patients presented with clinically suspected meningitis. Of these, 59 (32 %) had proven meningitis (bacterial, tuberculous or cryptococcal), 39 (21 %) had normal CSF and 14 (8 %) had aseptic meningitis. For the latter group, a herpes virus was detected in 9 (64 %): 7 (50 %) had EBV and 2 (14 %) had CMV, all were human immunodeficiency virus (HIV)-positive. HSV-2 and VZV were not detected. Amongst those with a normal CSF, 8 (21 %) had a detectable herpes virus, of which 7 (88 %) were HIV-positive. Conclusions: The spectrum of causes of herpes viral meningitis in this African population is different to that in Western industrialised settings, with EBV being frequently detected in the CSF. The significance of this needs further investigation. © 2012 The Author(s).

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Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the familyFlaviviridae.

West Nile virus (WNV) was first detected in Texas in 2002. During 2003, several isolates exhibiting significant attenuation of mouse neuroinvasiveness, and in some cases a small plaque and temperature sensitive phenotype when compared to other North American WNV isolates, were obtained from birds and mosquitoes in South-East Texas. To determine the attenuation markers of WNV, we have sequenced the genomes of three attenuated isolates and four temporally related virulent isolates and compared the amino acid substitutions in a total of 101 isolates, including three previously published genomes of attenuated strains, to identify mutations that are potentially involved in attenuation. Surprisingly, the attenuated strains fall into three separate "groups", suggesting that the attenuated phenotype evolved on three separate occasions in 2003. None of the groups share the same nucleotide changes or amino acid substitutions, and there are few mutations that can be clearly defined alone as being associated with attenuation. © 2010 Elsevier Inc.

Phylogenetic analysis of West Nile virus in North America has identified replacement of the originally introduced clade, Eastern United States (NY99), by the North American clade. In addition, the transient emergence of other clades and genetic variants has also been observed. In this study, we investigated the potential role of the mosquito in the selection of these clades and genetic variants. We determined the relative susceptibility of Culex quinquefasciatus to infection with isolates from the Eastern U.S. clade, the North American clade, and the Southeast coastal Texas clade. Although significant differences were observed in 50% oral infectious dose values between the Eastern U.S. and two attenuated North American genetic variants compared with the North American and Southeast coastal Texas clade viruses, these differences did not correlate with persistence of the genotype in nature. These results indicate that selection of these viral genotypes was independent of viral oral infectivity in the mosquito.

The alphaviruses Semliki Forest virus (SFV) and Sindbis virus have recently been developed as prototype anti-cancer agents. These are RNA-containing enveloped viruses that code for only 9 proteins of unique sequence. The standard recombinant SFV vector system consists of suicide particles containing recombinant RNA. In addition, alphavirus vectors capable of limited multiplication in the host are also being developed. Several strategies are being adopted to construct prototype SFV vectors for cancer treatment. These include: 1) construction of both prophylactic and therapeutic vaccines to stimulate immunity to tumor-associated antigens, 2) use of apoptosis induction to destroy tumor cells, which includes both the use of the inherent apoptosis-inducing ability of the vector and the action of pro-apoptotic genes cloned into the vector, and 3) expression of cytokines and other immunoregulatory proteins by the vector that enhance anti-tumor immune responses and/or inhibit tumor cell growth. This includes the use of cytokines such as IL-12 that target angiogenesis. Sindbis virus appears to have a natural tropism for tumor cells that may allow targeting both of the wild-type virus and the vector. This approach may also be useful for targeting metastases. For SFV, neurovirulence and/or neurotropism, as well as other tissue damage, may preclude the use of unmodified replication competent wild-type virus in tumor treatment. However, it may be possible to use such a virus in animals that have been vaccinated, using a vector-derived vaccine.

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.

Major virulence determinants ofSemliki Forest virus(SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5′ conserved domains and a non-essential hypervariable 3′ domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.

The immune responses of cattle inoculated with either a virulent or an attenuated vaccine strain of rinderpest virus (RPV) were examined by measuring the proliferation of peripheral blood mononuclear cells (PBMC) to whole RPV antigen preparations and to individual RPV major structural proteins expressed using recombinant adenoviruses. Responses to the T cell mitogen concanavalin A (ConA) were also measured as a control to monitor non-specific effects of infection with RPV on T cell responses. Infection with the vaccine strain of RPV was found to induce a strong CD4

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ABSTRACT

There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.

ABSTRACT

We previously reported mutations in North American West Nile viruses (WNVs) with a small-plaque ( sp ), temperature-sensitive ( ts ), and/or mouse-attenuated ( att ) phenotype. Using an infectious clone, site-directed mutations and 3′ untranslated region (3′UTR) exchanges were introduced into the WNV NY99 genome. Characterization of mutants demonstrated that a combination of mutations involving the NS4B protein (E249G) together with either a mutation in the NS5 protein (A804V) or three mutations in the 3′UTR (A10596G, C10774U, A10799G) produced sp , ts , and/or att variants. These results suggested that the discovery of North American WNV-phenotypic variants is rare because of the apparent requirement of concurrent polygenic mutations.

Japanese encephalitis virus (JEV) causes 35 000–50 000 cases and 10 000 deaths every year and is the most important cause of encephalitis worldwide. There is no known antiviral treatment for any flavivirus. We performed a randomised double-blind placebo-controlled trial of IVIG in 22 Nepalese children with suspected Japanese encephalitis, 13 of whom had serologically confirmed infection. IVIG obtained from areas endemic for JEV had 50% plaque reduction neutralisation test (PRNT50) against wild type JEV, and the IVIG with the highest titre was selected for treatment. Blood was collected pre, mid and post-treatment. Outcome was assessed at discharge and 3–6 month follow-up. IVIG prepared from a JEV endemic population has detectable anti-JEV antibodies. There was evidence of passive transfer of anti-JEV antibody in JEV negative children. Intriguingly JEV positive children treated with IVIG had 3–4 fold higher PRNT50 titres than placebo (p=0.08), more than can be explained by passive transfer alone, suggesting an immune augmenting effect. IL-4 and IL-6 were also higher in the IVIG group. There were no significant differences in outcome. IVIG with neutralising antibody may be an effective treatment for Japanese encephalitis and other flaviviral encephalitis due to anti-inflammatory effects, immune augmentation and JEV neutralisation.

Varicella zoster virus (VZV) is a common cause of viral encephalitis with wide spectrum of morbidity and mortality. The pathophysiology remains poorly understood and few prognostic markers are available. A few studies have attempted to use the viral load of the cerebrospinal fluid (CSF), to investigate the pathophysiology and prognosis, with contradictory results. However, these studies have not assessed the viral load in the context of time. To determine the CSF viral load in VZV encephalitis in relation to time and outcome. The Liverpool Specialist Virology laboratory database was screened to identify patients with CSF positive for VZV over 5 years. Viral load was determined by qPCR. A poor outcome was defined as moderate disability to death (Glasgow outcome score 2–5). 608 patients were screened, 36 had VZV PCR performed, 12 were positive and had clinical features consistent with encephalitis. There was a strong negative correlation between the time from symptom onset to LP and the viral load (τ b=−0.59, p=0.036). There was no significant difference in viral load between those with good and poor outcome. In this study of VZV encephalitis, CSF viral load appears to more closely relate to time of LP from symptom onset than to outcome. Timing of LP should be taken into account in future studies of viral encephalitis which aim to relate viral load to outcome in viral encephalitis.

Yellow fever virus (YFV) is a mosquito-borne flavivirus that is the causative agent of yellow fever (YF), which is a disease that ranges in severity from an influenza-like syndrome to severe liver and renal dysfunction, circulatory shock, and hemorrhage. The disease causes substantial morbidity and mortality; case fatality rates often exceed 20% and, despite the availability of a vaccine, it is still a major public health problem for sub-Saharan Africa and tropical South America, and travelers to endemic areas. The virus is maintained in endemic areas of Africa and South America by enzootic transmission between mosquitoes and monkeys, and the epidemiology of the disease reflects the geographical distribution of the mosquito vectors. There are seven genotypes of this virus with five circulating in different geographic areas of Africa and two in South America. Two live-attenuated YF vaccines were simultaneously developed: the French neurotropic vaccine (FNV), or Dakar vaccine, and the 17D vaccine. In the 1980s the manufacture of FNV was completely discontinued because the World Health Organization (WHO) prohibited vaccination of children under 14 years old due to the high incidence of encephalitis. The development of the highly effective, live-attenuated 17D vaccine in the late 1930s is one of the milestones in vaccine research. The vaccine is safe and highly efficacious with one dose of vaccine giving immunity for at least 10 years, and may be lifelong immunity. Neutralizing antibodies are the correlate of protection. There are three substrains of the YF 17D vaccine in production today (17D-204, 17DD, and 17D-213). The WHO has standardized the seed lot and manufacturing processes, and published guidelines for approval of vaccine production and vaccination certificates. There are a number of contraindications for YF vaccination which include age, thymus disease, pregnancy, breast-feeding, immune suppression, and hypersensitivity to eggs. In recent years, a rare but significant number of severe adverse events have been reported involving vaccine-associated viscerotropic and neurotropic disease. Current vaccine development involves utilizing the 17D vaccine virus backbone, via infectious clone technology, to develop a variety of chimeric virus vaccines, some of which have reached clinical trials for human use.