Defensins are antimicrobial peptides that participate in the innate immunity of hosts. Humans constitutively and/or inducibly express α- and β-defensins, which are known for their antiviral and antibacterial activities. This review describes the application of human defensins. We discuss the extant experimental results, limited though they are, to consider the potential applicability of human defensins as antiviral agents. Given their antiviral effects, we propose that basic research be conducted on human defensins that focuses on RNA viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), respiratory syncytial virus (RSV), and dengue virus (DENV), which are considered serious human pathogens but have posed huge challenges for vaccine development for different reasons. Concerning the prophylactic and therapeutic applications of defensins, we then discuss the applicability of human defensins as antivirals that has been demonstrated in reports using animal models. Finally, we discuss the potential adjuvant-like activity of human defensins and propose an exploration of the ‘defensin vaccine’ concept to prime the body with a controlled supply of human defensins. In sum, we suggest a conceptual framework to achieve the practical application of human defensins to combat viral infections.

It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

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.

The human immune system has evolved to fight against foreign pathogens. It plays a central role in the body's defense mechanism. However, the immune memory geared to fight off a previously recognized pathogen, tends to remember an original form of the pathogen when a variant form subsequently invades. This has been termed 'original antigenic sin'. This adverse immunological effect can alter vaccine effectiveness and sometimes cause enhanced pathogenicity or additional inflammatory responses, according to the type of pathogen and the circumstances of infection. Here we aim to give a simplified conceptual understanding of virus infection and original antigenic sin by comparing and contrasting the two examples of recurring infections such as influenza and dengue viruses in humans.

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In addition to influenza A subtypes, two distinct lineages of influenza B virus also cause seasonal epidemics to humans. Recently, Dudas et al. have done evolutionary analyses of reassortment patterns of the virus and suggested genetic lineage relationship between PB1, PB2, and HA genes. Using genetic plasmids and reassortant viruses, we here demonstrate that a homologous lineage PB1-PB2 pair exhibits better compatibility than a heterologous one and that the lineage relationship between PB1 and HA is more important for viral replication than that between PB2 and HA. However, co-adaptation of PB1-PB2-HA genes appears to be affected by complete gene constellation.

Clamp loaders assemble sliding clamps onto 3 primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3 primed site, and unable to bind a 5 site. We demonstrate here that the Escherichia coli ␥ complex clamp loader can load the ␤ clamp onto a 5 primed site, although with at least 20-fold reduced efficiency relative to loading at a 3 primed site. Preferential clamp loading at a 3 site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with ␤ at a 5 site. Preferential loading at a 3 versus a 5 site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3 site. Although the single-stranded template strand extends in opposite directions from 3 and 5 primed sites, thus making it a favorite candidate for distinguishing between 3 and 5 sites, the single-strand polarity at a primed template junction does not determine 3 site selection for clamp loading. Instead, we find that clamp loader recognition of a 3 site lies in the duplex portion of the primed site, not the singlestrand portion. We present evidence that the ␤ clamp facilitates its own loading specificity for a 3 primed site. Implications to eukaryotic clamp loader complexes are proposed.Chromosomal replicases are tethered to DNA by a circular ring-shaped clamp (1). The clamp encircles DNA and slides along the duplex behind the polymerase, holding it to the primed site for highly processive DNA synthesis. Sliding clamps are assembled onto primed sites by a clamp loader machine that harnesses the energy of ATP hydrolysis to open and close the sliding clamp around DNA (2). Bacterial sliding clamps are homodimers called ␤, while eukaryotic and archaeal sliding clamps are homotrimers referred to as PCNA.Structural studies of clamp loader complexes show that they are circular AAAϩ heteropentamers (2-4). The N-terminal domains contain the AAAϩ region of homology and are arranged in a spiral to form a central chamber that binds double-stranded (ds) DNA. The C-terminal domains form a closed circular cap above the DNA binding chamber. This results in a screw cap architecture that underlies the structure-specific

Herbal medicine has been used in the orient for thousands of years to treat large and small ailments, including microbial infections. Although there are treatments for influenza virus infection, there is no treatment for drug-resistant viruses. It is time that we explored and exploited the multi-component nature of herbal extracts as multi-drug combination therapies. Here, we present data on the anti-influenza virus effect of a medicinal mushroom, Phellinus igniarius. The P. igniarius water extract was effective against influenza A and B viruses, including 2009 pandemic H1N1, human H3N2, avian H9N2, and oseltamivir-resistant H1N1 viruses. Virological assays revealed that the extract may interfere with one or more early events in the influenza virus replication cycle, including viral attachment to the target cell. Therefore, our results provide new insights into the use of P. igniarius as an anti-influenza medicine.