Explore the vast range of titles available.

Despite the advances in cancer therapies in the past century, malignant melanoma continues to present a significant clinical challenge due to lack of chemotherapeutic response. Systemic therapy with immunostimulatory agents such as interferon and interleukin-2 (IL-2) has shown some promise, though each is associated with significant side effects. Over the past 50 years, oncolytic Newcastle disease virus (NDV) has emerged as an alternative candidate for cancer therapy. The establishment of reverse-genetics systems for the virus has allowed us to further manipulate the virus to enhance its oncolytic activity. Introduction of immunomodulatory molecules, especially IL-2, into the NDV genome was shown to enhance the oncolytic potential of the virus in a murine syngeneic colon carcinoma model. We hypothesize that a recombinant NDV expressing IL-2 would be an effective agent for therapy of malignant melanoma. We show that recombinant NDV possesses a strong cytolytic activity against multiple melanoma cell lines, and is effective in clearing established syngeneic melanoma tumors in mice. Moreover, introduction of murine IL-2 into NDV significantly enhanced its activity against syngeneic melanomas, resulting in increased overall animal survival and generation of antitumor immunity. These findings warrant further investigations of IL-2-expressing NDV as an antimelanoma agent in humans.

From a stock of A/turkey/Oregon/71-delNS1 (H7N3) virus, which has a 10 nucleotide deletion in the coding region of the NS1 gene, we found that several variants with different sizes of NS genes could be produced by passaging the virus in 10- and 14-day-old embryonating chicken eggs (ECE), but not in 7-day-old ECE or Vero cells. We were able to rescue the reassortant virus that has different sizes of the NS genes and confirmed that those NS genes are genetically stable. By conducting in vivo studies in 2-week-old chickens, we found two plaque purified variants (D-del pc3 and pc4) which can be used as a potential live-attenuated vaccine. The variants were highly attenuated in chickens and did not transmit the virus from infected chickens to uninoculated cage mates. At the same time, the variants induced relatively high antibody titers which conferred good protection against a high dose heterologous virus challenge. Our study indicates that naturally selected NS1 deletion variants might be useful in the development of live-attenuated influenza vaccines in poultry. Furthermore, deletion in the NS1 protein can be potentially useful as a negative marker for a differentiating infected from vaccinated animals (DIVA) approach.

The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques ( Macaca fascicularis ) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1α (HIF-1α), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses.

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative-stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics-based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid-only rescue system that allows the rapid generation of high-yield recombinant vaccine strains. Second, we propose developing second-generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.

An assay has been developed that allows the identification of molecules that function as type I IFN antagonists. Using this assay, we have identified an Ebola virus-encoded inhibitor of the type I IFN response, the Ebola virus VP35 protein. The assay relies on the properties of an influenza virus mutant, influenza delNS1 virus, which lacks the NS1 ORF and, therefore, does not produce the NS1 protein. When cells are infected with influenza delNS1 virus, large amounts of type I IFN are produced. As a consequence, influenza delNS1 virus replicates poorly. However, high-efficiency transient transfection of a plasmid encoding a protein that interferes with type I IFN-induced antiviral functions, such as the influenza A virus NS1 protein or the herpes simplex virus protein ICP34.5, rescues growth of influenza delNS1 virus. When plasmids expressing individual Ebola virus proteins were transfected into Madin Darby canine kidney cells, the Ebola virus VP35 protein enhanced influenza delNS1 virus growth more than 100-fold. VP35 subsequently was shown to block double-stranded RNA- and virus-mediated induction of an IFN-stimulated response element reporter gene and to block double-stranded RNA- and virus-mediated induction of the IFN-β promoter. The Ebola virus VP35 therefore is likely to inhibit induction of type I IFN in Ebola virus-infected cells and may be an important determinant of Ebola virus virulence in vivo.

The influenza A virus pandemic of 1918-1919 resulted in an estimated 20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. How the packaging signals regulate the specific incorporation of each RNA segment into virions and whether other viral or host factors are involved in this process is unknown. Here, we show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments. This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). Replacement of conserved SC35M NP residues by those of HL17NL10 NP resulted in RNA packaging defective IAV. Surprisingly, substitution of these conserved SC35M amino acids with HL17NL10 NP residues led to IAV with altered packaging efficiencies for specific subsets of RNA segments. This suggests that NP harbours an amino acid code that dictates genome packaging into infectious virions. The nucleotide sequence of the eight genomic RNA segments of influenza A virus provides essential packaging signals, but how these sequences are recognized is unknown. Here, Moreira et al . identify conserved amino acids in the viral nucleoprotein that regulate packaging of RNA segments.

Dengue virus is a worldwide-distributed mosquito-borne flavivirus with a positive strand RNA genome. Its transcribed polyprotein is cleaved by host- and virus-encoded peptidases into 10 proteins, some of which are of unknown function. Although dengue virus-infected cells seem to be resistant to the antiviral action of IFN, the viral products that mediate this resistance are unknown. Therefore, we have analyzed the ability of the 10 dengue virus-encoded proteins to antagonize the IFN response. We found that expression in human A549 cells of the dengue virus nonstructural proteins NS2A, NS4A, or NS4B enhances replication of an IFN-sensitive virus. Moreover, expression of NS4B and, to a lesser extent, of NS2A and NS4A proteins results in down-regulation of IFN-β-stimulated gene expression. Cells expressing NS4B or infected with dengue virus do not exhibit nuclear signal transducer and activator of transcription (STAT) 1 on treatment with IFN-β or IFN-γ, indicating that NS4B might be involved in blocking IFN signaling during dengue virus infections. This protein, encoded by a positive strand RNA virus, is implicated as an IFN-signaling inhibitor.

Extensive studies on protective immunity to rodent malaria provided the basis for the current experiments in which mice were immunized with recombinant (re) influenza and vaccinia viruses expressing selected sequences of the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum. Mice of different H-2 haplotypes immunized with re influenza viruses expressing the immunodominant B cell epitope of this CS protein produced high titers of antibodies to the parasite. A cytotoxic T lymphocyte epitope of the CS protein of P. falciparum, PF3, recognized by CD8+T cells of H-2kmice, was expressed in a re vaccinia virus (VacPf) and a re influenza virus (FluPf). Immunization of mice with either FluPf or VacPf elicited a modest CS-specific CD8+T cell response detected by interferon γ secretion of individual immune cells. Priming of mice with FluPf, followed by a booster with VacPf, resulted in a striking enhancement of this T cell response. The reverse protocol, i.e., priming with VacPf followed by a booster with FluPf, failed to enhance the primary response. VacPf also greatly enhanced the primary response of mice injected with P. falciparum sporozoites or with a lipopeptide containing PF3. A booster with FluPf also amplified the response of lipopeptide- or sporozoite-primed mice but less than a VacPf booster did. Although mice are not susceptible to infection by P. falciparum sporozoites, we demonstrated that administration of two distinct immunogens expressing PF3 elicited activated, extravasating CS-specific T cells that protected against an intracerebral VacPf challenge.

Signaling through toll-like receptors (TLRs) is essential for dendritic cell (DC) maturation induced by bacteria and other pathogens. The mechanism for virus-induced DC maturation is not known. By use of pairs of live viruses with different abilities to induce the interferon (IFN) pathway, a strong correlation between DC maturation and the ability of the virus to induce type I IFN synthesis was demonstrated. The secreted IFN was not necessary, nor was it sufficient to induce full DC maturation. Intracellular viral replication is necessary for this process, and the transcription factor nuclear factor–κB was crucial for cytokine induction. The double-stranded RNA–dependent protein kinase was not essential for DC maturation. Similar to TLR-induced DC maturation, after virus infection, separate pathways for the induction of cytokine secretion and the up-regulation of major histocompatibility complex and costimulatory molecules were activated. It was demonstrated that these pathways have different sensitivities to the presence of viral stimulus