Broad-Spectrum Antiviral Therapeutics

Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered.National Institute of Allergy and Infectious Diseases (U.S.) (grant AI057159)New England Regional Center of Excellence for Biodefense and Emerging Infectious DiseasesUnited States. Dept. of Defense (Director of Defense Research & Engineering)United States. Defense Threat Reduction AgencyUnited States. Defense Advanced Research Projects Agenc

Variation in the Ovine Abomasal Lymph Node Transcriptome between Breeds Known to Differ in Resistance to the Gastrointestinal Nematode

Texel lambs are known to be more resistant to gastrointestinal nematode (GIN) infection than Suffolk lambs, with a greater ability to limit infection. The objectives of this study were to: 1) profile the whole transcriptome of abomasal lymph node tissue of GIN-free Texel and Suffolk lambs; 2) identify differentially expressed genes and characterize the immune-related biological pathways and networks associated with these genes. Abomasal lymph nodes were collected from Texel (n = 6) and Suffolk (n = 4) lambs aged 19 weeks that had been GIN-free since 6 weeks of age. Whole transcriptome profiling was performed using RNA-seq on the Illumina platform. At the time of conducting this study, a well annotated Ovine genome was not available and hence the sequence reads were aligned with the Bovine (UMD3.1) genome. Identification of differentially expressed genes was followed by pathway and network analysis. The Suffolk breed accounted for significantly more of the differentially expressed genes, (276 more highly expressed in Suffolk v 162 in Texel; P < 0.001). The four most significant differentially expressed pathways were all related to immunity and were classified as: Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses, Activation of IRF by Cytosolic Pattern Recognition Receptors, Role of RIG-I-like Receptors in Antiviral Innate Immunity, and Interferon Signaling. Of significance is the fact that all of these four pathways were more highly expressed in the Suffolk. These data suggest that in a GIN-free environment, Suffolk lambs have a more active immune profile relative to the Texel: this immune profile may contribute to the poorer efficiency of response to a GIN challenge in the Suffolk breed compared to the Texel breed

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape

Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

BACKGROUND: Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown. PRINCIPAL FINDINGS: Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection. SIGNIFICANCE: Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection

Is Nitric Oxide Decrease Observed with Naphthoquinones in LPS Stimulated RAW 264.7 Macrophages a Beneficial Property?

The search of new anti-inflammatory drugs has been a current preoccupation, due to the need of effective drugs, with less adverse reactions than those used nowadays. Several naphthoquinones (plumbagin, naphthazarin, juglone, menadione, diosquinone and 1,4-naphthoquinone), plus p-hydroquinone and p-benzoquinone were evaluated for their ability to cause a reduction of nitric oxide (NO) production, when RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS). Dexamethasone was used as positive control. Among the tested compounds, diosquinone was the only one that caused a NO reduction with statistical importance and without cytotoxicity: an IC25 of 1.09±0.24 µM was found, with 38.25±6.50% (p<0.001) NO reduction at 1.5 µM. In order to elucidate if this NO decrease resulted from the interference of diosquinone with cellular defence mechanisms against LPS or to its conversion into peroxynitrite, by reaction with superoxide radical formed by naphthoquinones redox cycling, 3-nitrotyrosine and superoxide determination was also performed. None of these parameters showed significant changes relative to control. Furthermore, diosquinone caused a decrease in the pro-inflammatory cytokines: tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Therefore, according to the results obtained, diosquinone, studied for its anti-inflammatory potential for the first time herein, has beneficial effects in inflammation control. This study enlightens the mechanisms of action of naphthoquinones in inflammatory models, by checking for the first time the contribution of oxidative stress generated by naphthoquinones to NO reduction

Eurasian Tree Sparrows, Risk for H5N1 Virus Spread and Human Contamination through Buddhist Ritual: An Experimental Approach

Background: The Highly Pathogenic Avian Influenza H5N1 virus has dramatically spread throughout Southeast Asia since its first detection in 1997. Merit Release Birds, such as the Eurasian Tree Sparrow, are believed to increase one's positive karma when kissed and released during Buddhist rituals. Since these birds are often in close contact with both poultry and humans, we investigated their potential role in the spread of H5N1 virus. Methodology/Principal Findings: Seven series of experiments were conducted in order to investigate the possible interactions between inoculated and exposed birds, including sparrow/sparrow, sparrow/chicken, duck/sparrow. Daily and post-mortem samples collected were tested for H5N1 virus by real-time RT-PCR and egg inoculation. When directly inoculated, Eurasian Tree Sparrows were highly susceptible to the H5N1 virus, with a fatality rate approaching 100% within 5 days post-inoculation. Although transmission of fatal infection between sparrows did not occur, seroconversion of the exposed birds was observed. Up to 100% chickens exposed to inoculated sparrows died of H5N1 infection, depending on the caging conditions of the birds, while a fatality rate of 50% was observed on sparrows exposed to infected ducks. Large quantities of H5N1 virus were detected in the sparrows, particularly in their feathers, from which infectious particles were recovered. Conclusions/Significance: Our study indicates that under experimental conditions, Eurasian Tree Sparrows are susceptible to H5N1 infection, either by direct inoculation or by contact with infected poultry. Their ability to transmit H5N1 infection to other birds is also demonstrated, suggesting that the sparrows may play a role in the dissemination of the virus. Finally, the presence of significant quantities of H5N1 virus on sparrows' feathers, including infectious particles, would suggest that Merit Release Birds represent a risk for human contamination in countries where avian influenza virus is circulating and where this religious ritual is practiced. © 2011 Gutiérrez et al.published_or_final_versio

Investigation of Griffithsin's Interactions with Human Cells Confirms Its Outstanding Safety and Efficacy Profile as a Microbicide Candidate

Many natural product-derived lectins such as the red algal lectin griffithsin (GRFT) have potent in vitro activity against viruses that display dense clusters of oligomannose N-linked glycans (NLG) on their surface envelope glycoproteins. However, since oligomannose NLG are also found on some host proteins it is possible that treatment with antiviral lectins may trigger undesirable side effects. For other antiviral lectins such as concanavalin A, banana lectin and cyanovirin-N (CV-N), interactions between the lectin and as yet undescribed cellular moieties have been reported to induce undesirable side effects including secretion of inflammatory cytokines and activation of host T-cells. We show that GRFT, unlike CV-N, binds the surface of human epithelial and peripheral blood mononuclear cells (PBMC) through an exclusively oligosaccharide-dependent interaction. In contrast to several other antiviral lectins however, GRFT treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells or human PBMC, has no measureable effect on cell viability and does not significantly upregulate markers of T-cell activation. In addition, GRFT appears to retain antiviral activity once bound to the surface of PBMC. Finally, RNA microarray studies show that, while CV-N and ConA regulate expression of a multitude of cellular genes, GRFT treatment effects only minimal alterations in the gene expression profile of a human ectocervical cell line. These studies indicate that GRFT has an outstanding safety profile with little evidence of induced toxicity, T-cell activation or deleterious immunological consequence, unique attributes for a natural product-derived lectin

The 3′ Untranslated Regions of Influenza Genomic Sequences Are 5′PPP-Independent Ligands for RIG-I

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5′-triphosphate (PPP) group or by unphosphorylated dsRNA up to ∼300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5′ end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5′ and 3′ untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5′ UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5′PPP group. In contrast, activation of RIG-I by the 3′ UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5′PPP-dependence, as capping the 5′ end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3′ UTR was completely 5′PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5′PPP moiety is needed for interferon induction by RIG-I

Cross-Protection against Lethal H5N1 Challenge in Ferrets with an Adjuvanted Pandemic Influenza Vaccine

Background. Unprecedented spread between birds and mammals of highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype has resulted in hundreds of human infections with a high fatality rate. This has highlighted the urgent need for the development of H5N1 vaccines that can be produced rapidly and in sufficient quantities. Potential

SARS Coronavirus 3b Accessory Protein Modulates Transcriptional Activity of RUNX1b

BACKGROUND: The causative agent of severe acute respiratory syndrome, SARS coronavirus (SARS-CoV) genome encodes several unique group specific accessory proteins with unknown functions. Among them, accessory protein 3b (also known as ORF4) was lately identified as one of the viral interferon antagonist. Recently our lab uncovered a new role for 3b in upregulation of AP-1 transcriptional activity and its downstream genes. Thus, we believe that 3b might play an important role in SARS-CoV pathogenesis and therefore is of considerable interest. The current study aims at identifying novel host cellular interactors of the 3b protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor RUNX1b (Runt related transcription factor, isoform b) as a novel interacting partner for SARS-CoV 3b protein. Chromatin immunoprecipitaion (ChIP) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the RUNX1 binding element that led to an increase in RUNX1b transactivation potential on the IL2 promoter. Kinase assay and pharmacological inhibitor treatment implied that 3b also affect RUNX1b transcriptional activity by regulating its ERK dependent phosphorylation levels. Additionally, mRNA levels of MIP-1α, a RUNX1b target gene upregulated in SARS-CoV infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing U937 monocyte cells. CONCLUSIONS/SIGNIFICANCE: These results unveil a novel interaction of SARS-CoV 3b with the host factor, RUNX1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of SARS virus infection