Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes.

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1's role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain's duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5' and 8 bp 3' to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

Glycosylation generates an efficacious and immunogenic vaccine against H7N9 influenza virus.

Zoonotic avian influenza viruses pose severe health threats to humans. Of several viral subtypes reported, the low pathogenic avian influenza H7N9 virus has since February 2013 caused more than 1,500 cases of human infection with an almost 40% case-fatality rate. Vaccination of poultry appears to reduce human infections. However, the emergence of highly pathogenic strains has increased concerns about H7N9 pandemics. To develop an efficacious H7N9 human vaccine, we designed vaccine viruses by changing the patterns of N-linked glycosylation (NLG) on the viral hemagglutinin (HA) protein based on evolutionary patterns of H7 HA NLG changes. Notably, a virus in which 2 NLG modifications were added to HA showed higher growth rates in cell culture and elicited more cross-reactive antibodies than did other vaccine viruses with no change in the viral antigenicity. Developed into an inactivated vaccine formulation, the vaccine virus with 2 HA NLG additions exhibited much better protective efficacy against lethal viral challenge in mice than did a vaccine candidate with wild-type (WT) HA by reducing viral replication in the lungs. In a ferret model, the 2 NLG-added vaccine viruses also induced hemagglutination-inhibiting antibodies and significantly suppressed viral replication in the upper and lower respiratory tracts compared with the WT HA vaccines. In a mode of action study, the HA NLG modification appeared to increase HA protein contents incorporated into viral particles, which would be successfully translated to improve vaccine efficacy. These results suggest the strong potential of HA NLG modifications in designing avian influenza vaccines.

Short-Term Effectiveness of Oral Nirmatrelvir/Ritonavir Against the SARS-CoV-2 Omicron Variant and Culture-Positive Viral Shedding.

BACKGROUND: Information on the effectiveness of nirmatrelvir/ritonavir against the omicron is limited. The clinical response and viral kinetics to therapy in the real world need to be evaluated. METHODS: Mild to moderate coronavirus disease 2019 (COVID-19) patients with risk factors for severe illness were prospectively enrolled as a treatment group with nirmatrelvir/ritonavir therapy versus a control group with supportive care. Serial viral load and culture from the upper respiratory tract were evaluated for seven days, and clinical responses and adverse reactions were evaluated for 28 days. RESULTS: A total of 51 patients were analyzed including 40 in the treatment group and 11 in the control group. Faster symptom resolution during hospitalization (P = 0.048) was observed in the treatment group. Only minor adverse reactions were reported in 27.5% of patients. The viral load on Day 7 was lower in the treatment group (P = 0.002). The viral culture showed a positivity of 67.6% (25/37) vs. 100% (6/6) on Day 1, 0% (0/37) vs. 16.7 (1/6) on Day 5, and 0% (0/16) vs. 50.0% (2/4) on Day 7 in the treatment and control groups, respectively. CONCLUSIONS: Nirmatrelvir/ritonavir against the omicron was safe and resulted in negative viral culture conversion after Day 5 of treatment with better symptomatic resolution.

Indoor Human Detection from a Building's Exterior Using 433 MHz Wireless Transceivers.

This study introduces a novel system for detecting humans inside a building by utilizing RF signals from the building's exterior. Existing RF communication devices encounter signal attenuation issues when passing through walls, limiting their effectiveness. In contrast, our system employs a low-power, long-distance communication signal operating at 433 MHz to enhance signal permeability, enabling the accurate detection of individuals within the building. The system analyzes received signal strength indicator (RSSI) data using variance and mean analysis algorithms to determine the presence or absence of people. The evaluation results indicate promising average accuracies of 88% for the variance analysis algorithm and 97.7% for the mean analysis algorithm. The proposed system holds potential for real-world deployment, particularly in challenging scenarios such as fire incidents, where pre-installation is challenging. Continued research and development efforts aim to enhance the system's performance and address any limitations, making it more effective and robust in various practical applications.

Scanning transmission X-ray microscopy study of subcellular granules in human platelets at the carbon K- and calcium L2,3-edges.

Platelets and their subcellular components (e.g., dense granules) are essential components in hemostasis. Understanding their chemical heterogeneities at the sub-micrometer scale, particularly their activation during hemostasis and production of platelet-derived extracellular vesicles, may provide important insights into their mechanisms; however, this has rarely been investigated, mainly owing to the lack of appropriate chemical characterization tools at nanometer scale. Here, the use of scanning transmission X-ray microscopy (STXM) combined with X-ray absorption near edge structure (XANES) to characterize human platelets and their subcellular components at the carbon K-edge and calcium L2,3-edge, is reported. STXM images can identify not only the spatial distribution of subcellular components in human platelets, such as dense granules (DGs) with sizes of ~200 nm, but also their granule-to-granule chemical heterogeneities on the sub-micrometer scale, based on their XANES spectra. The calcium distribution map as well as the principal component analysis of the STXM image stacks clearly identified the numbers and locations of the calcium-rich DGs within human platelets. Deconvolution of the carbon K-edge XANES spectra, extracted from various locations in the platelets, showed that amide carbonyl and carboxylic acid functional groups were mainly found in the cytoplasm, while ketone-phenol-nitrile-imine, aliphatic, and carbonate functional groups were dominant in the platelet DGs. These observations suggest that platelet DGs are most likely composed of calcium polyphosphate associated with adenosine triphosphate (ATP) and adenosine diphosphate (ADP), with significant granule-to-granule variations in their compositions, while the cytoplasm regions of platelets contain significant amounts of proteins.

Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Novel Small Molecule Targeting the Hemagglutinin Stalk of Influenza Viruses.

Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.

Off-Target Editing by CRISPR-Guided DNA Base Editors.

Base editing is a genome editing strategy that induces specific single-nucleotide changes within genomic DNA. Two major DNA base editors, cytosine base editors and adenine base editors, that consist of a Cas9 protein linked to a deaminase enzyme that catalyzes targeted base conversion directed by a single-guide RNA have been developed. This strategy has been used widely for precise genome editing because, unlike CRISPR-Cas nuclease-based genome editing systems, this strategy does not create double-strand DNA breaks that often result in high levels of undesirable indels. However, recent papers have reported that DNA base editors can cause substantial off-target editing in both genomic DNA and RNA. The off-target editing described in these studies is primarily independent of guide RNA and arises from the promiscuous reactivity of the deaminase enzymes used in DNA base editors. In this Perspective, we discuss the development of DNA base editors, the guide RNA-independent off-target activity reported in recent studies, and strategies that improve the selectivity of DNA base editors.

Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function.

Screening of chemical libraries with 2,000 synthetic compounds identified salinomycin as a hit against influenza A and B viruses, with 50% effective concentrations ranging from 0.4 to 4.3 µM in cells. This compound is a carboxylic polyether ionophore that exchanges monovalent ions for protons across lipid bilayer membranes. Monitoring the time course of viral infection showed that salinomycin blocked nuclear migration of viral nuclear protein (NP), the most abundant component of the viral ribonucleoprotein (vRNP) complex. It caused cytoplasmic accumulation of NP, particularly within perinuclear endosomes, during virus entry. This was primarily associated with failure to acidify the endosomal-lysosomal compartments. Similar to the case with amantadine (AMT), proton channel activity of viral matrix protein 2 (M2) was blocked by salinomycin. Using purified retroviral Gag-based virus-like particles (VLPs) with M2, it was proved that salinomycin directly affects the kinetics of a proton influx into the particles but in a manner different from that of AMT. Notably, oral administration of salinomycin together with the neuraminidase inhibitor oseltamivir phosphate (OSV-P) led to enhanced antiviral effect over that with either compound used alone in influenza A virus-infected mouse models. These results provide a new paradigm for developing antivirals and their combination therapy that control both host and viral factors.IMPORTANCE Influenza virus is a main cause of viral respiratory infection in humans as well as animals, occasionally with high mortality. Circulation of influenza viruses resistant to the matrix protein 2 (M2) inhibitor, amantadine, is highly prevalent. Moreover, the frequency of detection of viruses resistant to the neuraminidase inhibitors, including oseltamivir phosphate (OSV-P) or zanamivir, is also increasing. These issues highlight the need for discovery of new antiviral agents with different mechanisms. Salinomycin as the monovalent cation-proton antiporter exhibited consistent inhibitory effects against influenza A and B viruses. It plays multifunctional roles by blocking endosomal acidification and by inactivating the proton transport function of M2, the key steps for influenza virus uncoating. Notably, salinomycin resulted in marked therapeutic effects in influenza virus-infected mice when combined with OSV-P, suggesting that its chemical derivatives could be developed as an adjuvant antiviral therapy to treat influenza infections resistant or less sensitive to existing drugs.

A Single Amino Acid in the Polymerase Acidic Protein Determines the Pathogenicity of Influenza B Viruses.

Influenza B virus (IBV) is one of the human respiratory viruses and one of the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find a molecular clue(s) to the increased pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in the PB2, PB1, PA, BM2, and/or NS1 protein-coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using viruses rescued through reverse genetics, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication in the respiratory tracts of ferrets. In a subsequent minireplicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.IMPORTANCE To investigate molecular pathogenic determinants of IBVs, which are one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein-coding region. When analyzed using viruses rescued through reverse genetics, the PA mutation alone appeared to contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and to the replication of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate the viral polymerase complex activity of both IBV lineages. These results indicate that the PA mutation could be a newly defined molecular pathogenic determinant of IBVs that substantiates our understanding of the viral pathogenicity and public health risks of IBVs.

Distinct molecular evolution of influenza H3N2 strains in the 2016/17 season and its implications for vaccine effectiveness.

Influenza virus is a respiratory pathogen that causes seasonal epidemics by resulting in a considerable number of influenza-like illness (ILI) patients. During the 2016/17 season, ILI rates increased unusually earlier and higher than previous seasons in Korea, and most viral isolates were subtyped as H3N2 strains. Notably, the hemagglutinin (HA) of most Korean H3N2 strains retained newly introduced lysine signatures in HA antigenic sites A and D, compared with that of clade 3C.2a vaccine virus, which affected antigenic distances to the standard vaccine antisera in a hemagglutination inhibition assay. The neuraminidase (NA) of Korean H3N2 strains also harbored amino acid mutations. However, neither consistent amino acid mutations nor common phylogenetic clustering patterns were observed. These suggest that Korean H3N2 strains of the 2016/17 season might be distantly related with the vaccine virus both in genotypic and phenotypic classifications, which would adversely affect vaccine effectiveness.

Biocatalytic Fabrication of α-Glucan-Coated Porous Starch Granules by Amylolytic and Glucan-Synthesizing Enzymes as a Target-Specific Delivery Carrier.

In this study, we created biocatalytically coated porous starch granules (PSGs) using amylosucrase from Neisseria polysaccharea to apply them as an encapsulant for target-specific delivery. Field-emission scanning electron and confocal laser scanning microscopic images showed that the PSGs were completely concealed by the α-glucan coating layer. This carbohydrate-based encapsulant displayed higher amount of resistant glucan contents due to the elongated chains of the glucan coating, resulting in lower digestibility of these PSGs in simulated digestive fluid systems. Among the various PSGs evaluated, the highest loading efficiency for the bioactive molecule crocin was observed with the ß-amylase-induced PSGs (ß-PSGs) that had the smallest nanosize pores. Furthermore, α-glucan-coated ß-PSGs showed the highest capacity to preserve the loaded crocin when incubated in simulated digestive fluids. This suggests that the α-glucan-coated ß-PSGs can potentially be used for the delayed release of the core material in the upper region of the gastrointestinal tract. Therefore, this system can be potentially utilized as an effective carrier for colon-specific delivery, and the release of the bioactive compound can be triggered by beneficial intestinal microbiota.

Selective Recognition of RNA Substrates by ADAR Deaminase Domains.

Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA and is catalyzed by ADAR1 and ADAR2 in humans. ADAR1 and ADAR2 have different substrate selectivity, which is believed to mainly originate from the proteins' deaminase domains (hADAR1d and hADAR2d, respectively). RNA-seq of the Saccharomyces cerevisiae transcriptome subjected to ADAR-catalyzed RNA editing identified substrates with common secondary structure features preferentially edited by hADAR1d over hADAR2d. The relatively small size and efficient reaction of one of these substrates suggested it could be useful for further study of the hADAR1d reaction. Indeed, a short hairpin stem from the S. cerevisiae HER1 mRNA was efficiently deaminated by hADAR1d and used to generate an hADAR1d-specific fluorescent reporter of editing activity. Using substrates preferred by either hADAR1d or hADAR2d in vitro, we found that a chimeric protein bearing an RNA-binding loop from hADAR2d grafted onto hADAR1d showed ADAR2-like selectivity. Finally, a high-throughput mutagenesis analysis (Sat-FACS-Seq) of conserved residues in an RNA-binding loop of hADAR1d revealed essential amino acids for function, advancing our understanding of RNA recognition by this domain.

Evolutionary relationships of the hexon and penton base genes of novel squirrel adenovirus.

Squirrel adenovirus (SqAdV) was reported previously. However, only partial sequences of its hexon and polymerase genes have been revealed. For the first time, we report the full-length genome of SqAdV including the complete hexon and penton base genes. From internal body organs of 59 red squirrels archived in Korea Bank for Pathogenic Viruses, the hexon, penton base, and full-length genome of SqAdV were determined by a PCR method. Of the internal body organs examined, the spleen showed the highest detection rate (25.42%) for SqAdV whereas the kidney and lung exhibited 18.64% and 3.39% rates, respectively. Based on the phylogenetic relationships of the hexon and penton base genes, SqAdV appears to belong to the genus Mastadenovirus, and, at least in our study, the hexon of SqAdV exhibits the closest relationship to that of an alpaca AdV. Compared with the hexon, the penton base of SqAdV appears to be genetically more divergent from that of other mastadenoviruses. It was also revealed that the full-length SqAdV genome retained AT nucleotide content similar level to AT-rich atadenoviruses, which is unusual for mastadenoviruses. Our results emphasize that SqAdV is classified into the genus Mastadenovirus and demonstrate the AT-biased nucleotide constitution of SqAdV.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs.

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance.

Hymenobacter terrae sp. nov., a bacterium isolated from soil.

A Gram-negative, UV tolerant bacterial strain, DG7A(T), was isolated from soil samples collected in Seoul city, South Korea. The cells were grown on R2A agar at 25 °C and were pink to red in color. The DNA G+C content of the novel strain DG7A was 63.5 mol%. Chemotaxonomic data revealed that the strains contain the major fatty acids iso-C15:0, anteiso-C15:0, and summed feature 3 (16:1 ω7c/16:1 ω6c), with phosphatidylethanolamine as the major polar lipid. Phylogenetic analysis of the 16S rRNA gene sequences showed that strain DG7A(T) formed a distinct phylogenetic line along with Hymenobacter soli PB17(T), and they shared approximately 98.35 % 16S rRNA gene sequence similarity. However, these two strains shared only 5.3 % pairwise similarity (reciprocal analysis, 36.3 %) in their genomic DNA. The next highest degree of 16S rRNA gene sequence similarity after H. soli PB17(T) was found with H. glaciei VUG-A130(T) (96.78 %), H. antarcticus VUG-A42aa(T) (96.66 %), and H. saemangeumensis GSR0100(T) (96.57 %). Based on the phylogenetic analysis and analysis of the physiological and biochemical characteristics, this isolate was considered to represent a novel species, for which we propose the name Hymenobacter terrae sp. nov., with type strain DG7A(T) (= KCTC 32554(T) = KEMB 9004-164(T )= JCM 30007(T)).

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus.

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.

Genetic requirement for hemagglutinin glycosylation and its implications for influenza A H1N1 virus evolution.

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.

Inhibition of Pseudomonas aeruginosa with a recombinant RNA-based viral vector expressing human ß-defensin 4.

BACKGROUND: Harassed with extensive epithelial burn wounds, patients can be affected by complications, such as infection, hypovolemic shock, hypothermia, and respiratory failure. Immediate first aid and followed supportive cares are critical for the prevention of severe complications. However, secondary bacterial infection is hard to be controlled in burn patients, and Pseudomonas aeruginosa (P. aeruginosa) is one of the top listed pathogens perturbing burn wounds beyond the antibiotics spectrum. RESULTS: To find the way for efficacious protection from the pseudomonas-mediated complications in burn patients, we assessed the in vitro and in vivo inhibitory values of human ß-defensin 4 (hBD4), which is known as a member of the cationic, antimicrobial peptides found in human cells of many kinds. The Newcastle disease virus (NDV) was used as a viral vector for the expression of hBD4 in burn wounds. Expressed from the recombinant NDV (rNDV-hBD4), hBD4 effectively inhibited the pseudomonal growths in cell culture media. In a mouse model, severely burn-injured skin was recovered by the direct installation of the rNDV-hBD4 infected cells in the burn wounds whereas that of control mice remained severely damaged. CONCLUSIONS: We suggest that the application of hBD4 may protect burn patients from secondary pseudomonal infection and provide a therapeutic potential for burn wound treatment.

Effects of a hemagglutinin D222G substitution on the pathogenicity of 2009 influenza A (H1N1) virus in mice.

The surface glycoprotein hemagglutinin (HA) of influenza virus initiates the infection process by binding to sialic acid receptors on upper respiratory cells in the host. In contrast to avian influenza viruses, which bind to sialic acids connected by an α2-3 linkage to the penultimate galactose, human influenza viruses prefer sialic acids with an α2-6 linkage. Recently, there have been multiple cases of severe human infections associated with an HA D222G mutant influenza virus. In this study, we have investigated the pathogenic effects of the HA D222G substitution in a 2009 pandemic H1N1 virus in mice. Compared with the A/Korea/01/2009 (K/09) virus, the HA D222G mutant showed reduced growth in cells and reduced binding avidity to human and turkey red blood cells. In a BALB/c mouse infection model, infection with the HA D222G mutant virus resulted in less body weight loss when compared to the parental K/09 virus. Altogether, our data suggest that the HA D222G substitution in the K/09 virus might be deleterious to viral fitness.