The development of flavivirus vaccines

Mosquito and tick-borne flaviviruses are the causative agents of some of the world’s most important diseases, including dengue fever, yellow fever, Japanese encephalitis, tick-borne encephalitis and West Nile fever. Cumulatively, these viruses cause many millions of infections each year and impose a significant burden on public health resources, particularly in developing and newly developed countries. Vaccine development to eliminate flaviviral infections has been marked by uneven progress and a large number of setbacks. To date, no single approach has proved successful in leading to vaccine development against a wide range of flaviviruses, but the application of modern techniques to the problem is opening up new avenues of approach. This review summarizes some of the developments in vaccine research aimed at inducing protective immunity against flaviviral infections

Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms

Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice

The effect of tumor necrosis factor alpha (TNF-α) on rabies virus (RV) infection of the mouse central nervous system (CNS) was studied, using recombinant RV engineered to express either soluble TNF-α [SPBN-TNF-α(+)] or insoluble membrane-bound TNF-α [SPBN-TNF-α(MEM)]. Growth curves derived from infections of mouse neuroblastoma NA cells revealed significantly less spread and production of SPBN-TNF-α(+) than of SPBN-TNF-α(MEM) or SPBN-TNF-α(−), which carries an inactivated TNF-α gene. The expression of soluble or membrane-bound TNF-α was not associated with increased cell death or induction of alpha/beta interferons. Brains of mice infected intranasally with SPBN-TNF-α(+) showed significantly less virus spread than did mouse brains after SPBN-TNF-α(−) infection, and none of the SPBN-TNF-α(+)-infected mice succumbed to RV infection, whereas 80% of SPBN-TNF-α(−)-infected mice died. Reduced virus spread in SPBN-TNF-α(+)-infected mouse brains was paralleled by enhanced CNS inflammation, including T-cell infiltration and microglial activation. These data suggest that TNF-α exerts its protective activity in the brain directly through an as yet unknown antiviral mechanism and indirectly through the induction of inflammatory processes in the CNS

Identification of viral genomic elements responsible for rabies virus neuroinvasiveness

Attenuated tissue culture-adapted and natural street rabies virus (RV) strains differ greatly in their neuroinvasiveness. To identify the elements responsible for the ability of an RV to enter the CNS from a peripheral site and to cause lethal neurological disease, we constructed a full-length cDNA clone of silver-haired bat-associated RV (SHBRV) strain 18 and exchanged the genes encoding RV proteins and genomic sequences of this highly neuroinvasive RV strain with those of a highly attenuated nonneuroinvasive RV vaccine strain (SN0). Analysis of the recombinant RV (SB0), which was recovered from SHBRV-18 cDNA, indicated that this RV is phenotypically indistinguishable from WT SHBRV-18. Characterization of the chimeric viruses revealed that in addition to the RV glycoprotein, which plays a predominant role in the ability of an RV to invade the CNS from a peripheral site, viral elements such as the trailer sequence, the RV polymerase, and the pseudogene contribute to RV neuroinvasiveness. Analyses also revealed that neuroinvasiveness of an RV correlates inversely with the time necessary for internalization of RV virions and with the capacity of the virus to grow in neuroblastoma cells

Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses

<p>Abstract</p> <p>Background</p> <p>Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF), dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications.</p> <p>Results</p> <p>Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA) was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA) emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow chromatographic assays and a fluorescent sandwich assay on the surface of magnetic microbeads is also demonstrated.</p> <p>Conclusions</p> <p>This article catalogues numerous DNA aptamer sequences which can bind various important pathogenic arboviruses and have, in some cases, already demonstrated diagnostic potential. These aptamer sequences are proprietary, patent-pending, and partially characterized. Therefore, they are offered to the scientific community for potential research use in diagnostic assays, biosensor applications or for possible passive immunity and prophylaxis against pathogenic viruses.</p