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Self-disinfecting materials that are both safe and scalable for production are increasingly in demand, particularly in healthcare settings where they can be used to combat hospital-acquired infections (HAIs). Here, we employed the natural food colorant chlorophyllin (E140ii) as a photosensitizer to prepare photodynamic antimicrobial materials through both chemical conjugation and electrospinning, resulting in chlorophyllin-grafted cotton fabric (Chl-fabric) and chlorophyllin-embedded polyacrylonitrile nanofibers (Chl-NF), respectively. The materials were characterized by a number of physical methods, as was their ability to generate singlet oxygen upon visible light illumination. The best results with Chl-fabric yielded 99.998% inactivation of vancomycin-resistant E. faecium and 99.994% of methicillin-resistant S. aureus after 60 min visible light illumination (400–700 nm, 80 ± 5 mW/cm2), whereas Chl-NF inactivated both bacteria by 99.9999%. Feline calicivirus was also photodynamically susceptible, with 99.8% inactivation by both materials. Gram-negative Klebsiella pneumoniae was not initially susceptible to photodynamic inactivation by Chl-NF, however addition of the photothermal agent MoS2 fully inactivated (99.9999%) this pathogen under NIR illumination, indicative of synergistic photothermal and photodynamic activities. These findings suggest that chlorophyllin can be used in photodynamic antimicrobial materials against drug-resistant Gram-positive bacteria, and that its efficacy can be synergistically amplified in the presence of a photothermal agent against Gram-negative pathogens.

A substantial increase in the risk of hospital-acquired infections (HAIs) has greatly impacted the global healthcare industry. Harmful pathogens adhere to a variety of surfaces and infect personnel on contact, thereby promoting transmission to new hosts. This is particularly worrisome in the case of antibiotic-resistant pathogens, which constitute a growing threat to human health worldwide and require new preventative routes of disinfection. In this study, we have incorporated different loading levels of a porphyrin photosensitizer capable of generating reactive singlet oxygen in the presence of O2 and visible light in a water-soluble, photo-cross-linkable polymer coating, which was subsequently deposited on polymer microfibers. Two different application methods are considered, and the morphological and chemical characteristics of these coated fibers are analyzed to detect the presence of the coating and photosensitizer. To discern the efficacy of the fibers against pathogenic bacteria, photodynamic inactivation has been performed on two different bacterial strains, Staphylococcus aureus and antibiotic-resistant Escherichia coli, with population reductions of >99.9999 and 99.6%, respectively, after exposure to visible light for 1 h. In response to the current COVID-19 pandemic, we also confirm that these coated fibers can inactivate a human common cold coronavirus serving as a surrogate for the SARS-CoV-2 virus.

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib, paxlovid, and molnupiravir), or in advanced clinical trials. Vandetanib is a kinase inhibitor which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), as well as the RET-tyrosine kinase. In the current study, it was tested in different cell lines and showed promising results on inhibition versus the toxic effect on A549-hACE2 cells (IC500.79 μM) while also showing a reduction of >3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, and TNF-α and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib also decreased CCL2, CCL3, and CCL4 compared to the infected animals. Vandetanib additionally rescued the decreased IFN-1β caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved anticancer drug vandetanib is worthy of further assessment as a potential therapeutic candidate to block the COVID-19 cytokine storm.

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib) or in advanced clinical trials. We have tested 45 FDA-approved kinase inhibitorsin vitroagainst murine hepatitis virus (MHV) as a model of SARS-CoV-2 replication and identified 12 showing inhibition in the delayed brain tumor (DBT) cell line. Vandetanib, which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), and the RET-tyrosine kinase showed the most promising results on inhibition versus toxic effect on SARS-CoV-2-infected Caco-2 and A549-hACE2 cells (IC500.79 μM) while also showing a reduction of > 3 log TCID50/mL for HCoV-229E. Thein vivoefficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, TNF-α, and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load.Vandetanib rescued the decreased IFN-1β caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved vandetanib is a potential therapeutic candidate for COVID-19 positioned for follow up in clinical trials either alone or in combination with other drugs to address the cytokine storm associated with this viral infection.

Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus, however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1’, S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from −8.8 to −7.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from −7.1 to −7.8. In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 µM. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC(50)s from 18 to 50 μM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC(50) of 6.9 μM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC(50) values of 6.6 and 22.0 μM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.

Drug-resistant pathogens, particularly those that result in hospital acquired infections (HAIs), have emerged as a critical priority for the World Health Organization. To address the need for self-disinfecting materials to counter the threat posed by the transmission of these pathogens from surfaces to new hosts, here we investigated if a cationic BODIPY photosensitizer, embedded via electrospinning into nylon and polyacrylonitrile (PAN) nanofibers, was capable of inactivating both bacteria and viruses via antimicrobial photodynamic inactivation (aPDI). Materials characterization, including fiber morphology and the degree of photosensitizer loading, was assessed by scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and UV-visible diffuse reflectance spectroscopy (UV-Vis DRS), and demonstrated that the materials were comprised of nanofibers (125–215 nm avg. diameter) that were thermostable to >300 °C. The antimicrobial potencies of the resultant Nylon-BODIPY(+) and PAN-BODIPY(+) nanofiber materials were evaluated against four strains of bacteria recognized by the World Health Organization as either critical or high priority pathogens: Gram-positive strains methicillin-resistant S. aureus (MRSA; ATCC BAA-44) and vancomycin-resistant E. faecium (VRE; ATCC BAA-2320), and Gram-negative strains multidrug-resistant A. baumannii (MDRAB; ATCC BAA-1605) and NDM-1 positive K. pneumoniae (KP; ATCC BAA-2146). Our results demonstrated the detection limit (99.9999%; 6 log units reduction in CFU mL−1) photodynamic inactivation of three strains upon illumination (30–60 min; 40–65 ± 5 mW cm−2; 400–700 nm): MRSA, VRE, and MDRAB, but only minimal inactivation (47–75%) of KP. Antiviral studies employing PAN-BODIPY(+) against vesicular stomatitis virus (VSV), a model enveloped virus, revealed complete inactivation. Taken together, the results demonstrate the potential for electrospun BODIPY(+)-embedded nanofiber materials as the basis for pathogen-specific anti-infective materials, even at low photosensitizer loadings.

Dengue virus (DV) has become the most prevalent arthropod borne virus due to globalization and climate change. It targets dendritic cells during infection and leads to production of pro-inflammatory cytokines and chemokines. Several DV non-structural proteins (NS) modulate activation of human dendritic cells. We investigated the effect of DV NS1 on human monocyte- derived dendritic cells (mo-DCs) during dengue infection. NS1 is secreted into the serum of infected individuals where it interacts with various immune mediators and cell types. We purified secreted DV1 NS1 from supernatants of 293T cells that over-express the protein. Upon incubation with mo-DCs, we observed NS1 uptake and enhancement of early DV1 replication. As a consequence, mo-DCs that were pre-exposed to NS1 produced more pro-inflammatory cytokines in response to subsequent DV infection compared to DCs exposed to heat-inactivated NS1 (HNS1). Therefore the presence of exogenous NS1 is able to modulate dengue infection in mo-DCs.

Type I interferons (IFNs) are critical for controlling pathogenic virus infections and can enhance immune responses. Hence their impact on the effectiveness of live-attenuated vaccines involves a balance between limiting viral antigen expression and enhancing the development of adaptive immune responses. We examined the influence of type I IFNs on these parameters following immunization with RepliVAX WN, a single-cycle flavivirus vaccine (SCFV) against West Nile virus (WNV) disease. RepliVAX WN-immunized mice produced IFN-α and displayed increased IFN-stimulated gene transcription in draining lymph nodes (LN). SCFV gene expression was over 100 fold-higher on days 1-3 post-infection in type I IFN receptor knockout mice (IFNAR(-/-)) compared to wild-type (wt) mice indicating a profound IFN-mediated suppression of SCFV gene expression in the wt animals. IFNAR(-/-) mice produced nearly equivalent levels of WNV-specific serum IgG and WNV-specific CD4(+) T cell responses compared to wt mice. However, significantly higher numbers of WNV-specific CD8(+) T cells were produced by IFNAR(-/-) mice and a significantly greater percentage of these T cells from IFNAR(-/-) mice produced only IFN-γ following antigen-specific re-stimulation. This altered cytokine expression was not associated with increased antigen load suggesting the loss of type I IFN receptor signaling was responsible for the altered quality of the CD8(+) effector T cell response. Together, these results indicate that although type I IFN is not essential for the intrinsic adjuvanting of RepliVAX WN, it plays a role in shaping the cytokine secretion profiles of CD8(+) effector T cells elicited by this SCFV.

A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.

West Nile virus (WNV), the leading cause of viral encephalitis in the United States, is an arthropod-transmitted member of the family Flaviviridae. We have explored the interaction of this positive-strand RNA virus with signaling pathways involved in induction of the host's innate immune response. Phosphorylation of STAT-1 in response to interferon (IFN) treatment and the ability of IFN to establish an antiviral state were reduced in WNV replicon-bearing cell lines. Similarly, the activation of IRF3 and stimulation of IFN-β transcription in response to the double-stranded RNA (dsRNA) mimetic poly(I:C) were inhibited in replicon-bearing and WNV-infected HeLa cells. In contrast, WNV replicons did not affect IRF3 activation by Sendai virus infection, suggesting that not all IRF3 activating pathways are inhibited by WNV. Taken together, these findings demonstrate that WNV replication in cultured cells interferes with both the response to IFN and synthesis of IFN-β in response to dsRNA.

Considerable controversy surrounds the impact of hepatitis C virus (HCV) protein expression on viability of host cells and regulation of the cell cycle. Both promotion of cellular proliferation and apoptosis have been observed in different experimental systems. To determine whether expression of the entire complement of HCV proteins in the context of ongoing viral RNA replication significantly alters the host cell transcriptome and cell cycle regulatory processes, we carried out high-density oligonucleotide microarray studies and analyzed cell cycle distributions and S-phase entry in Huh7 cell clones harboring selectable, full-length, replicating HCV RNAs that express the entire genotype 1b, HCV-N polyprotein, and clonally related cells in which all viral RNA was eliminated by prior treatment with alpha interferon. Oligonucleotide microarray analyses revealed only subtle, coordinated differences in the mRNA profiles of cells containing replicating viral RNA and their interferon-cured progeny, with variation between different cell clones having a greater influence on the cellular transcriptome than the presence or absence of replicating HCV RNA. Flow cytometric analysis demonstrated no significant differences in cell cycle distribution among populations of asynchronously growing cells of both types. Cell lines containing replicating viral RNA and their interferon-cured progeny were able to reenter the cell cycle similarly after transient G 1 arrest. In contrast, although viral protein expression and genome replication did not alter cell cycle control in these cells, HCV genome replication was highly dependent on cellular proliferation, with viral RNA synthesis strongly decreased in poorly proliferating, confluent, or serum-starved cells and substantially enhanced in the S phase of the cell cycle.

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.

West Nile virus (WNV) is a mosquito-transmitted pathogen, which causes significant disease in humans. The innate immune system is a first-line defense against invading microorganism and many flaviviruses, including WNV, have evolved multifunctional proteins, which actively suppress its activation and antiviral actions. The WNV non-structural protein 1 (NS1) inhibits signal transduction originating from Toll-like receptor 3 (TLR3) and also critically contributes to virus genome replication. In this study we developed a novel FACS-based screen to attempt to separate these two functions. The individual amino acid changes P320S and M333V in NS1 restored TLR3 signaling in virus-infected HeLa cells. However, virus replication was also attenuated, suggesting that the two functions are not easily separated and may be contained within overlapping domains. The residues we identified are completely conserved among several mosquito- and tick-borne flaviviruses, indicating that they are of biological importance to the virus.

Human rhinovirus (HRV) replicons have the potential to serve as respiratory vaccine vectors for mucosal immunization in humans. However, since many vaccine immunogens of interest are glycosylated, an important concern is whether HRV replicons are capable of expressing glycosylated proteins. The human respiratory syncytial virus (RSV) fusion (F) protein was chosen as a model glycoprotein and the HRV replicon DeltaP1FVP3 was generated by inserting the F protein-coding sequence in frame and in lieu of the 5' proximal 1489 nucleotides of the capsid-coding segment in the HRV-14 genome. When transfected into H1-HeLa cells, DeltaP1FVP3 replicated and led to the expression of the F protein. Inhibition with guanidine demonstrated that F-protein expression was dependent on DeltaP1FVP3 replication and did not result from translation of input RNA. Although most of the F protein remained as an immature, glycosylated precursor (F0), a readily detectable fraction of the protein was processed into the mature glycosylated subunit F1, an event known to occur within the Golgi apparatus. Packaged DeltaP1FVP3 replicons were generated in transfected HeLa cells by coexpression of homologous HRV capsid proteins using the vaccinia virus/T7 RNA polymerase hybrid system. Packaged replicon RNAs were capable of infecting fresh cells, leading to accumulation of the F protein as in RNA-transfected cells. Mice immunized with HeLa cell lysates containing F protein expressed from DeltaP1FVP3 produced neutralizing antibodies against RSV. These results indicate that an HRV-14 replicon can express a foreign glycosylated protein, providing further support for the potential of HRV replicons as a vaccine delivery system.

BackgroundExtracts of the creosote bush,Larrea tridentata, have been used for centuries by natives of western American and Mexican deserts to treat a variety of infectious diseases and inflammatory disorders. The beneficial activity of this plant has been linked to the compound nordihydroguaiaretic acid (NDGA) and its various substituted derivatives. Recently, tetra-O-methyl NDGA or terameprocol (TMP) has been shown to inhibit the growth of certain tumor-derived cell lines and is now in clinical trials for the treatment of human cancer. In this report, we ask whether TMP also displays anti-inflammatory activity. TMP was tested for its ability to inhibit the LPS-induced production of inflammatory lipids and cytokinesin vitro. We also examined the effects of TMP on production of TNF-α in C57BL6/J mice following a sublethal challenge with LPS. Finally, we examined the molecular mechanisms underlying the effects we observed.MethodsRAW 264.7 cells and resident peritoneal macrophages from C57BL6/J mice, stimulated with 1 μg/ml LPS, were used in experiments designed to measure the effects of TMP on the production of prostaglandins, cytokines and chemokines. Prostaglandin production was determined by ELISA. Cytokine and chemokine production were determined by antibody array and ELISA.Western blots, q-RT-PCR, and enzyme assays were used to assess the effects of TMP on expression and activity of COX-2.q-RT-PCR was used to assess the effects of TMP on levels of cytokine and chemokine mRNA.C57BL6/J mice injectedi.p.with LPS were used in experiments designed to measure the effects of TMPin vivo. Serum levels of TNF-α were determined by ELISA.ResultsTMP strongly inhibited the production of prostaglandins from RAW 264.7 cells and normal peritoneal macrophages. This effect correlated with a TMP-dependent reduction in levels of COX-2 mRNA and protein, and inhibition of the enzymatic activity of COX-2.TMP inhibited, to varying degrees, the production of several cytokines, and chemokines from RAW 264.7 macrophages and normal peritoneal macrophages. Affected molecules included TNF-α and MCP-1. Levels of cytokine mRNA were affected similarly, suggesting that TMP is acting to prevent gene expression.TMP partially blocked the production of TNF-α and MCP-1in vivoin the serum of C57BL6/J mice that were challengedi.p. with LPS.ConclusionTMP inhibited the LPS-induced production of lipid mediators and several key inflammatory cytokines and chemokines, bothin vitroandin vivo, raising the possibility that TMP might be useful as a treatment for a variety of inflammatory disorders.

The innate immune response is the first line of defense against foreign pathogens. The recognition of virus-associated molecular patterns, including double- and single-stranded RNA, by pattern recognition receptors initiates a cascade of signaling reactions. These result in the transcriptional upregulation and secretion of proinflammatory cytokines that induce an antiviral state. Many viruses have evolved mechanisms to antagonize these responses in order to help them establish a productive infection. We have previously shown that West Nile virus (WNV) is able to inhibit Toll-like receptor 3 (TLR3)-mediated activation of interferon (IFN) regulatory factor 3 (IRF3) (F. Scholle and P. W. Mason, Virology 342:77-87, 2005). In the present study, the WNV nonstructural (NS) proteins were analyzed individually for their ability to antagonize signal transduction mediated by TLR3. We report that expression of WNV NS1 inhibits TLR3-induced transcriptional activation of the IFN-β promoter and of an NF-κB-responsive promoter. This inhibition was due to a failure of the TLR3 ligand poly(I:C) to induce nuclear translocation of IRF3 and NF-κB. Furthermore, NS1 expression also inhibited TLR3-dependent production of interleukin-6 and the establishment of an antiviral state. The function of NS1 in flavivirus infection is not well understood. NS1 is required for viral RNA replication and is also secreted from mammalian cells but not from insect cells. Here, we identify a previously unrecognized role for NS1 in the modulation of signaling pathways of the innate immune response to WNV infection.

West Nile virus (WNV) is a member of the Flavivirus genus of the family Flaviviridae. This genus contains a number of arthropod-borne human pathogens, including dengue virus (DV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus (26). WNV was associated with fever and infrequent encephalitis cases in humans in Africa, the Middle East, and Europe from its discovery in 1938 through the 1990s. In 1999, WNV was first detected in New York City, and from there it rapidly spread across the United States, some regions of Canada, Mexico, and Central America. The majority of WNV infections are asymptomatic; however, a portion of infections result in West Nile fever, and a subset of infections lead to viral invasion of the central nervous system that results in encephalitis, paralysis, and meningitis, outcomes which are especially prevalent in immunocompromised and aged individuals (4). The mechanisms by which WNV causes disease are not completely understood, but studies with a hamster model of the disease suggest that following a brief peripheral replication cycle, the virus crosses the blood-brain barrier, where it infects neurons, causing cell death (47). The direct effect of WNV on neurons or an immune response to their infection may also be responsible for encephalitis and meningoencephalitis in humans (13). Pretreatment of animals with type I interferons (IFN-α/IFN-β) has been shown to block flavivirus disease (3, 23), and animals defective in the IFN response have been shown to be more susceptible to flavivirus infection (19, 28, 39). Although the precise IFN-stimulated genes (ISGs) that are responsible for preventing or controlling flavivirus infections are unknown, a number of studies have demonstrated that flavivirus-infected cells prevent IFN-induced phosphorylation of STAT molecules, which carry the signal from the ligated IFN receptor to the nucleus to activate the transcription of ISGs (1, 11, 20, 24, 25, 32, 37, 44). Interestingly, the precise mechanism by which flaviviruses alter STAT phosphorylation appears to differ among members of the genus (1, 24, 32). IFN is produced by most eukaryotic cells in response to viral infection and/or recognition of virus-associated macromolecules (known as pathogen-associated molecular patterns [PAMPs]), such as single- or double-stranded RNA (ssRNA and dsRNA, respectively). In many mammalian cell types, ligation of Toll-like receptor 3 (TLR3) to extracellular dsRNA or recognition of intracellular dsRNA by the intracellular helicase mda5 or RIG-I induces the phosphorylation and activation of the constitutively expressed IFN regulatory factor 3 (IRF3), leading to nuclear translocation and activation of transcription of the genes for IFN-β and IFN-α subtype 4 (14). Recent studies have indicated that in cell cultures, a subset of viruses appear to activate the mda5 pathway, whereas others, including the flavivirus Japanese encephalitis virus, activate the RIG-I pathway (21). In some cases, IFN-α expression has been linked to protein kinase R activation via binding of intracellular dsRNA (9). Furthermore, multiple IFN-α subtypes can also be induced by PAMP-stimulated signal transduction pathways that lead to the phosphorylation of IRF7, which can be triggered by binding of ssRNA to TLR7/8. Interestingly, IRF7 is constitutively expressed in only a subset of cells, but the IRF7 gene is an ISG, so following IFN binding, many cells can produce IRF7, permitting them to amplify the IFN signal if stimulated by ssRNA (12). For many infections, a subset of cells known as plasmacytoid dendritic cells (pDCs), which express IRF7 constitutively, have been implicated as key IFN producers (16). These cells can produce extremely high levels of IFN in response to stimulation with infectious disease agents or components thereof. Activated pDCs also produce other cytokines, notably interleukin-12 (IL-12), tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and IL-3, and chemokines, such as CCL3, CCL4, CCL5, CCL22, CCL19, and CXCL13 (2, 5, 33). However, pDCs are not the only cell type that express IRF7 constitutively. Other lymphocytes, including monocytes, B cells, and dendritic cell precursors (pDC2) (18), also express IRF7 in the resting state and are hence able to induce synthesis of IFN-α through the IRF7 pathway (reviewed in reference 22). Virus-like particles (VLPs) have been used as tools to study RNA virus infection in vitro and in vivo. VLPs consist of subgenomic replicating genomes lacking structural protein genes (replicons) that have been encapsidated by the missing structural proteins provided in trans by packaging cells. VLPs are thus able to infect cells and initiate genome replication in a manner that mimics that of normal virus, but unlike infections with normal virus, VLP infections cannot spread in the absence of trans-expressed structural proteins. In the case of both alphaviruses (35) and flaviviruses (43), VLPs have been used to identify the first cells that are infected in insect vectors of these viruses. Furthermore, for the alphavirus Venezuelan equine encephalitis virus, VLPs have been used to identify the first cells that are infected in animal models (29), and recent studies with Venezuelan equine encephalitis virus VLPs have shown that these VLPs (referred to as VRPs in the previous study) can strongly stimulate antiviral responses (46). In the studies described in this paper, we used WNV VLPs to study the early events in WNV infection in mice, demonstrating that WNV VLPs induce a rapid IFN response, resulting in very high levels of IFN-α in the serum between 8 and 24 h after either intraperitoneal (i.p.) inoculation or subcutaneous inoculation in the footpad (f.p. inoculation). Since VLPs can undergo only a single round of infection, these studies have demonstrated that the very first cells infected in an animal are capable of stimulating the production of high levels of IFN. IFN production was dependent on the replicative capacity of VLPs, since mice inoculated with UV-inactivated VLPs did not produce IFN. Immunohistochemical (IHC) detection of WNV antigen-positive cells in the popliteal lymph nodes (pLN) after f.p. inoculation with VLPs and the demonstration of high levels of IFN mRNA in pLN collected from VLP-inoculated animals implicate this lymphoid organ as a prominent source of IFN production. Finally, the finding that IRF3−/− animals produced levels of IFN similar to those of wild-type animals suggests that neither the RIG-I/mda5 nor the TLR3 pathway, known to be important for IFN-β induction, is important for this early response to WNV infection.

Different methods of manufacturing permitted the production of air-filled PBCA microparticles (af-pbca-mp) with different physical properties such as size and wall thickness. These differences led to distinctions with respect to mechanical stability and, at the same time, to different levels of biochemical stability when incubated in biofluids. Microparticles, designed as they are to be mechanically more stable (composed of larger nanoparticles resulting in thicker shell wall, no surface hydrolysis), persist longer under in vitro conditions in biofluids such as serum, plasma and whole blood than do the more fragile ones. It was possible when using the measurement of ultrasound attenuation to characterize af-pbca-mp degradation with respect to the disappearance of the ultrasound properties of the particles and therefore to find out how long different formulations can be expected to be active as contrast agents under simulated in vivo conditions. The present examination showed that using either serum, plasma or whole blood leads to results with the same tendencies in terms of the stability and durability of af-pbca-mp in the media, mimicking in vivo conditions. It was thus possible to validate successfully the use of either serum or plasma as substitutes for whole blood. Further studies dealing with the in vitro in vivo correlation will be needed to find out if the situation in this in vitro assay corresponds to the situation in the body.

A trans -packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 10 9 infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.

LMP2A deletion and point mutants, with mutations in phosphotyrosine-containing protein–protein interaction motifs, were transiently expressed in 293 cells and their phosphorylation was examined in immune complex kinase assays as well as in vivo. In vitro LMP2A phosphorylation depended on tyrosine 112. In vivo, mutations of single tyrosines did not eliminate LMP2 phosphorylation, although mutation of the LMP2A ITAM decreased LMP2A phosphorylation. The relationship between LMP2A in vitro phosphorylation and that induced by cell-extracellular matrix (ECM) interactions was also investigated. While LMP2A was phosphorylated to higher levels in whole-cell extracts of stimulated cells, a difference in in vitro kinase assays with extracts from stimulated and unstimulated cells was not detected, indicating that the ECM-mediated regulation of LMP2A phosphorylation is lost in vitro. In the presence of LMP2A, several cellular proteins with molecular weights between 70 and 80 kDa were phosphorylated on tyrosine. This increase in cellular protein phosphorylation depended on the LMP2A ITAM motif and suggests that the ITAM may participate in signal-transduction events in epithelial cells.

Background and Aims: The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood. Indirect evidence suggests that oxidative stress and mitochondrial injury play a role. The aim of this study was to determine if the HCV core protein itself alters mitochondrial function and contributes to oxidative stress. Methods: HCV core protein was expressed in 3 different cell lines, and reactive oxygen species (ROS) and lipid peroxidation products were measured. Results: Core expression uniformly increased ROS. In 2 inducible expression systems, core protein also increased lipid peroxidation products and induced antioxidant gene expression as well. A mitochondrial electron transport inhibitor prevented the core-induced increase in ROS. A fraction of the expressed core protein localized to the mitochondria and was associated with redistribution of cytochrome c from mitochondrial to cytosolic fractions. Sensitivity to oxidative stress was also seen in HCV transgenic mice in which increased intrahepatic lipid peroxidation products occurred in response to carbon tetrachloride. Conclusions: Oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo and may involve a direct effect of core protein on mitochondria. These results provide new insight into the pathogenesis of hepatitis C and provide an experimental rationale for investigation of antioxidant therapy.

The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase–Akt pathway.

Background MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. Results We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. Conclusions Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

West Nile virus (WNV) employs several different strategies to escape the innate immune response. We have previously demonstrated that the WNV NS1 protein interferes with signal transduction from Toll-like receptor 3 (TLR3). NS1 is a glycoprotein that can be found intracellularly or associated with the plasma membrane. In addition, NS1 is secreted to high levels during flavivirus infections. We investigated whether the secreted form of NS1 inhibits innate immune signaling pathways in uninfected cells. Secreted NS1 (sNS1) was purified from supernatants of cells engineered to express the protein. Purified sNS1 associated with and repressed TLR3-induced cytokine production by HeLa cells, and inhibited signaling from TLR3 and other TLRs in bone marrow-derived macrophages and dendritic cells. Footpad administration of sNS1 showed the protein associated predominantly with macrophages and dendritic cells in the draining lymph node. Additionally, sNS1 significantly reduced TLR3 signaling and WNV replicon particle-mediated cytokine transcription in popliteal lymph nodes.

Background Tetra-O-methyl nordihydroguaiaretic acid, also known as terameprocol (TMP), is a naturally occurring phenolic compound found in the resin of the creosote bush. We have shown previously that TMP will suppress production of certain inflammatory cytokines, chemokines and lipids from macrophages following stimulation with LPS or infection with H1N1 influenza virus. In this study our goal was to elucidate the mechanism underlying TMP-mediated suppression of cytokine and chemokine production. We focused our investigations on the response to LPS and the NF-κB protein RelA, a transcription factor whose activity is critical to LPS-responsiveness. Methods Reporter assays were performed with HEK293 cells overexpressing either TLR-3, -4, or -8 and a plasmid containing the luciferase gene under control of an NF-κB response element. Cells were then treated with LPS, poly(I:C), or resiquimod, and/or TMP, and lysates measured for luciferase activity. RAW 264.7 cells treated with LPS and/or TMP were used in ChIP and EMSA assays. For ChIP assays, chromatin was prepared and complexes precipitated with anti-NF-κB RelA Ab. Cross-links were reversed, DNA purified, and sequence abundance determined by Q-PCR. For EMSA assays, nuclear extracts were incubated with radiolabeled probes, analyzed by non-denaturing PAGE and visualized by autoradiography. RAW 264.7 cells treated with LPS and/or TMP were also used in fluorescence microscopy and western blot experiments. Translocation experiments were performed using a primary Ab to NF-κB RelA and a fluorescein-conjugated secondary Ab. Western blots were performed using Abs to IκB-α and phospho-IκB-α. Bands were visualized by chemiluminescence. Results In reporter assays with TLR-3, -4, and -8 over-expressing cells, TMP caused strong inhibition of NF-κB-dependent transcription. ChIP assays showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes for TNF-α, MCP-1/CCL2, and RANTES/CCL5 although the LPS-dependent synthesis of IκB-α was not inhibited. EMSA assays did not reveal an effect of TMP on the binding of RelA to naked DNA templates in vitro. TMP did not inhibit the nuclear translocation of NF-κB RelA nor the phosphorylation of IκB-α. Conclusion TMP acts indirectly as an inhibitor of NF-κB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes.