Genome wide association study of HTLV-1-associated myelopathy/tropical spastic paraparesis in the Japanese population.

HTLV-1-associated myelopathy (HAM/TSP) is a chronic and progressive inflammatory disease of the central nervous system. The aim of our study was to identify genetic determinants related to the onset of HAM/TSP in the Japanese population. We conducted a genome-wide association study comprising 753 HAM/TSP patients and 899 asymptomatic HTLV-1 carriers. We also performed comprehensive genotyping of HLA-A, -B, -C, -DPB1, -DQB1, and -DRB1 genes using next-generation sequencing technology for 651 HAM/TSP patients and 804 carriers. A strong association was observed in HLA class I (P = 1.54 × 10-9) and class II (P = 1.21 × 10-8) loci with HAM/TSP. Association analysis using HLA genotyping results showed that HLA-C*07:02 (P = 2.61 × 10-5), HLA-B*07:02 (P = 4.97 × 10-10), HLA-DRB1*01:01 (P = 1.15 × 10-9) and HLA-DQB1*05:01 (P = 2.30 × 10-9) were associated with disease risk, while HLA-B*40:06 (P = 3.03 × 10-5), HLA-DRB1*15:01 (P = 1.06 × 10-5) and HLA-DQB1*06:02 (P = 1.78 × 10-6) worked protectively. Logistic regression analysis identified amino acid position 7 in the G-BETA domain of HLA-DRB1 as strongly associated with HAM/TSP (P = 9.52 × 10-10); individuals homozygous for leucine had an associated increased risk of HAM/TSP (odds ratio, 9.57), and proline was protective (odds ratio, 0.65). Both associations were independent of the known risk associated with proviral load. DRB1-GB-7-Leu was not significantly associated with proviral load. We have identified DRB1-GB-7-Leu as a genetic risk factor for HAM/TSP development independent of proviral load. This suggests that the amino acid residue may serve as a specific marker to identify the risk of HAM/TSP even without knowledge of proviral load. In light of its allele frequency worldwide, this biomarker will likely prove useful in HTLV-1 endemic areas across the globe.

Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain Using an Engineered High-Fidelity Viral Polymerase.

RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence after their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two-times-lower mutation rates and decreased misincorporation rates, compared to the wild type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus.IMPORTANCE The LAIV currently in use is prescribed for actively immunizing individuals aged 2 to 49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1 that displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.

Development of multiplexing gene silencing system using conditionally induced polycistronic synthetic antisense RNAs in Escherichia coli.

Although efficient methods of gene silencing have been established in eukaryotes, many different techniques are still used in bacteria due to the lack of a standardized tool. Here, we developed a convenient and efficient method to downregulate the expression of a specific gene using ∼140 nucleotide RNA with a 24-nucleotide antisense region from an arabinose-inducible expression plasmid by taking Escherichia coli lacZ and phoA genes encoding ß-galactosidase and alkaline phosphatase, respectively, as target genes to evaluate the model. We examined the antisense RNA (asRNA) design, including targeting position, uORF stability elements at the 5'-end, and Hfq-binding module at the 3'-end, and inducer amount required to obtain effective experimental conditions for gene silencing. Furthermore, we constructed multiplexed dual-acting asRNA genes in the plasmid, which were transcribed as polycistronic RNA and were able to knockdown multiple target genes simultaneously. We observed the highest inhibition level of 98.6% when lacZ was targeted using the pMKN104 asRNA expression plasmid, containing a five times stronger PBAD -10 promoter sequence with no requirement of the Hfq protein for repression. These features allow the system to be utilized as an asRNA expression platform in many bacteria, besides E. coli, for gene regulation.

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium.

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G16, -G18, -G20, -G22, and -G24). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G20 clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.

Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq- conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species.

Tyr82 Amino Acid Mutation in PB1 Polymerase Induces an Influenza Virus Mutator Phenotype.

In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single-amino-acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivoIMPORTANCE Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live attenuated vaccine candidates.

Unique Directional Motility of Influenza C Virus Controlled by Its Filamentous Morphology and Short-Range Motions.

Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ICV strain, Taylor/1233/47 (Taylor), moved randomly, similar to IAV. The AA and Taylor viruses each moved with a combination of gradual (crawling) and rapid (gliding) motions, but the distances of crawling and gliding for the AA virus were shorter than those of the Taylor virus. Our findings indicate that like IAV, ICV also has a motility that is driven by the receptor exchange mechanism. However, compared with IAV movement, filamentous ICV movement is highly regulated in both direction and speed. Control of ICV movement is based on its specific motility employing short crawling and gliding motions as well as its own filamentous morphology.IMPORTANCE Influenza virus enters into a host cell for infection via cellular endocytosis. Human influenza virus infects epithelial cells of the respiratory tract, the surfaces of which are hidden by abundant cilia that are inactive in endocytosis. An open question is the manner by which the virus migrates to endocytosis-active domains. In analyzing individual virus behaviors through single-virus tracking, we identified a novel function of the hemagglutinin and esterase of influenza C virus (ICV) as the motility machinery. Hemagglutinin iteratively exchanges a viral receptor, causing virus movement. Esterase degrades the receptors along the trajectory traveled by the virus and prevents the virus from moving backward, causing directional movement. We propose that ICV has a unique motile machinery directionally controlled via hemagglutinin sensing the receptor density manipulated by esterase.

EOS, an Ikaros family zinc finger transcription factor, interacts with the HTLV-1 oncoprotein Tax and is downregulated in peripheral blood mononuclear cells of HTLV-1-infected individuals, irrespective of clinical statuses.

BACKGROUND: EOS plays an important role in maintaining the suppressive function of regulatory T cells (Tregs), and induces a regulated transformation of Tregs into T helper-like cells, which are capable of secreting proinflammatory cytokines in response to specific inflammatory signals. Meanwhile, significant reduction in Treg activity along with production of proinflammatory cytokines has been reported in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). METHODS: In this study, to examine whether there is an alteration in EOS expression in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals especially HAM/TSP, we investigated the expression of HTLV-1 tax genotype, proviral load (PVL), and the mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals including adult T-cell leukemia/lymphoma (ATL), HAM/TSP, or asymptomatic carriers. The expression levels of EOS mRNA and protein in various HTLV-1-infected or uninfected human T-cell lines were also investigated. RESULTS: EOS was highly expressed at the protein level in most HTLV-1 infected T-cell lines, and was augmented after the HTLV-1 regulatory factor Tax was induced in a Tax-inducible JPX-9 cell line. Immunoprecipitation experiments demonstrated a physical interaction between EOS and the viral regulatory protein Tax, but not HBZ. Meanwhile, there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals irrespective of their clinical statuses. We found an inverse correlation between EOS mRNA levels and HTLV-1 PVL in ATL patients, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP patients, whereas this correlation was not observed in other clinical statuses. CONCLUSIONS: These findings suggest that both Tax and HBZ can alter the expression of EOS through undetermined mechanisms, and dysregulated expression of EOS in PBMCs of HTLV-1 infected individuals may contribute to the pathological progression of HTLV-1-associated diseases, such as ATL and HAM/TSP.

Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM/TSP.

BACKGROUND: Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 tax subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein-protein interaction. RESULTS: (1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2-3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B. CONCLUSIONS: Our results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP.

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain.

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Inhibitory effects of dietary soyasaponin on 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice.

Soyasaponins (SSs) abundant in soybean have anti-inflammatory activities; however, their therapeutic effects on allergic contact dermatitis (ACD) remain unknown. To assess the effects of SS-enriched diets on ACD, we used a mouse model of contact hypersensitivity (CHS). Mice were fed low-dose or high-dose SS-containing diets for 3 weeks prior to CHS induction with 2,4-dinitrofluorobenzene (DNFB). The low-dose SS diet attenuated DNFB-induced ear swelling and tissue oedema, and reduced the number of infiltrating Gr-1-positive myeloid cells. Low-dose, but not high-dose, SSs decreased chemokine (C-X-C motif) ligand 2 (CXCL2) and triggering receptor expressed on myeloid cells (TREM)-1 production in ear tissues, compared to a control. Taxonomic 16S rRNA analysis revealed significant alterations in faecal microbiota caused by CHS, which were reversed by low-dose SSs. The low-dose SS and non-CHS groups clustered together, while the high-dose SS group split between CHS and non-CHS clusters. Our results demonstrated that low-dose SSs alleviated CHS symptoms by attenuating inflammation and improving the intestinal microbiota composition, suggesting that dietary SSs may have beneficial effects on ACD.

The CC chemokine ligand (CCL) 1, upregulated by the viral transactivator Tax, can be downregulated by minocycline: possible implications for long-term treatment of HTLV-1-associated myelopathy/tropical spastic paraparesis.

BACKGROUND: Chemokine (C-C motif) ligand 1 (CCL1) is produced by activated monocytes/ macrophages and T-lymphocytes, and acts as a potent attractant for Th2 cells and a subset of T-regulatory (Treg) cells. Previous reports have indicated that CCL1 is overexpressed in adult T-cell leukemia cells, mediating an autocrine anti-apoptotic loop. Because CCL1 is also known as a potent chemoattractant that plays a major role in inflammatory processes, we investigated the role of CCL1 in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). RESULTS: The results showed that: (1) CCL1 was preferentially expressed in HAM/TSP-derived HTLV-1-infected T-cell lines, (2) CCL1 expression was induced along with Tax expression in the Tax-inducible T-cell line JPX9, (3) transient Tax expression in an HTLV-1-negative T-cell line activated the CCL1 gene promoter, (4) plasma levels of CCL1 were significantly higher in patients with HAM/TSP than in HTLV-1-seronegative patients with multiple sclerosis and HTLV-1-infected asymptomatic healthy carriers, and (5) minocycline inhibited the production of CCL1 in HTLV-1-infected T-cell lines. CONCLUSIONS: The present results suggest that elevated CCL1 levels may be associated with the pathogenesis of HAM/TSP. Although further studies are required to determine the in vivo significance, minocycline may be considered as a potential candidate for the long-term treatment of HAM/TSP via its anti-inflammatory effects, which includes the inhibition of CCL1 expression.

Prevalence of plasma autoantibody against cancer testis antigen NY-ESO-1 in HTLV-1 infected individuals with different clinical status.

BACKGROUND: Detection of specific immune responses against cancer/testis antigen NY-ESO-1 was recently reported in patients with adult T-cell leukemia/lymphoma (ATL) and human T-cell leukemia virus type 1 (HTLV-1)-infected asymptomatic carriers (ACs). However, the relationship of the responses with the HTLV-1 proviral load (PVL) and the levels of viral gene expression remain unclear. FINDINGS: We measured plasma levels of autoantibodies to NY-ESO-1 immunogenic tumor antigen in HTLV-1-infected individuals with different clinical status, and in healthy controls. Data were compared to tax and HBZ mRNA levels, and PVL. Plasma anti-NY-ESO-1 antibody was detectable in 13.7% (7/51) of ACs, 29.2% (38/130) of patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 18.9% (10/53) of patients with ATL. Anti-NY-ESO-1 plasma levels were significantly higher in patients with HAM/TSP than in patients with ATL or ACs. Anti-NY-ESO-1 levels were not associated with PVL or the expression levels of tax and HBZ mRNA among HTLV-1-infected individuals, regardless of clinical status. CONCLUSIONS: The present results indicate the strong humoral immune response against NY-ESO-1 in natural HTLV-1 infection, irrespective of the clinical status. The higher immunoreactivity against NY-ESO-1 is not simply associated with the levels of both HTLV-1 gene expression and the number of infected cells in vivo. Rather, it might reflect chronic and generalized immune activation in infected individuals.

Intracellular morphological changes in Staphylococcus aureus induced by treatment with sodium hypochlorite.

Sodium hypochlorite (NaOCl) is commonly used as a disinfectant; however, its bactericidal mechanism has not yet been clarified. In the present study, the bactericidal mechanism of NaOCl was examined using microscopy and gel electrophoresis techniques with Staphylococcus aureus strain 209P. S. aureus cells treated with 500 and 1000 ppm NaOCl for 5 and 15 min were observed by SEM and TEM. SEM images of the bacterial cells treated with NaOCl showed an irregular surface, with cells being partially invaginated. TEM images of the bacterial cells showed cytoplasmic alterations, accompanied by a partially irregular cellular surface. Under a fluorescence microscope, we clearly observed fluorescence quenching in the 1000 ppm NaOCl-treated cells. Based on these observations, which indicated that NaOCl damaged chromosomal DNA, we next extracted chromosomal DNA from bacterial cells treated with NaOCl and performed agarose gel electrophoresis. Chromosomal DNA was absent in the DNA sample from the bacterial cells treated with 500 ppm NaOCl. From these biochemical results, it was strongly suggested that NaOCl degrades the chromosomal DNA of S. aureus. We consider that the morphological changes in the cytoplasm induced by NaOCl may be related to NaOCl-induced degradation of S. aureus chromosomal DNA.

Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status.

BACKGROUND: Human T cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), which is encoded by a minus strand mRNA, is thought to play important roles in the development of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, a comprehensive analysis of HBZ, including mRNA and protein expression, humoral immunoreactivity against HBZ, and HTLV-1 proviral load (PVL), in HTLV-1-infected individuals with different clinical status has not been reported previously. RESULTS: In this study, using novel monoclonal antibody-based in-house enzyme-linked immunosorbent assay systems, we report the absolute quantification of HBZ protein and its plasma antibody in clinical samples from HTLV-1-infected individuals with different clinical status. The data were compared to both HBZ mRNA levels and PVL. The results showed that plasma anti-HBZ antibody was detectable only in 10.4 % (5/48) of asymptomatic carriers (ACs), 10.8 % (13/120) of HAM/TSP patients, and 16.7 % (7/42) of ATL patients. HBZ protein was detected in three out of five patients with acute ATL, but was not detected in patients with HAM/TSP (0/10) or ACs (0/4). Thus, an antibody response to HBZ was not associated with the PVL or the expression of HBZ (both at the mRNA and protein levels) or the clinical status of the infection. CONCLUSIONS: The present results emphasize the extremely low expression and immunogenicity of HBZ in natural HTLV-1 infection. However, there is a possibility that the low but distinct expression of HBZ protein in PBMCs is associated with the survival of HTLV-1-infected cells and the development of ATL.

HTLV-1 subgroups associated with the risk of HAM/TSP are related to viral and host gene expression in peripheral blood mononuclear cells, independent of the transactivation functions of the viral factors.

Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, the risk of developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) across lifetime differs between ethnic groups. There is an association between HTLV-1 tax gene subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. In this study, we investigated the full-length proviral genome sequences of various HTLV-1-infected cell lines and patient samples. The functional differences in the viral transcriptional regulators Tax and HTLV-1 bZIP factor (HBZ) between each subgroup and the relationships between subgroups and the clinical and laboratory characteristics of HAM/TSP patients were evaluated. The results of these analyses indicated the following: (1) distinct nucleotide substitutions corresponding to each subgroup were associated with nucleotide substitutions in viral structural, regulatory, and accessory genes; (2) the HBZ messenger RNA (mRNA) expression in HTLV-1-infected cells was significantly higher in HAM/TSP patients with subgroup-B than in those with subgroup-A; (3) a positive correlation was observed between the expression of HBZ mRNA and its target Foxp3 mRNA in HAM/TSP patients with subgroup-B, but not in patients with subgroup-A; (4) no clear differences were noted in clinical and laboratory characteristics between HAM/TSP patients with subgroup-A and subgroup-B; and (5) no functional differences were observed in Tax and HBZ between each subgroup based on reporter gene assays. Our results indicate that although different HTLV-1 subgroups are characterized by different patterns of viral and host gene expression in HAM/TSP patients via independent mechanisms of direct transcriptional regulation, these differences do not significantly affect the clinical and laboratory characteristics of HAM/TSP patients.

Re-emergence of H3N2 strains carrying potential neutralizing mutations at the N-linked glycosylation site at the hemagglutinin head, post the 2009 H1N1 pandemic.

BACKGROUND: Seasonally prevalent H1N1 and H3N2 influenza A viruses have evolved by antigenic drift; this evolution has resulted in the acquisition of asparagine (N)-linked glycosylation sites (NGSs) in the globular head of hemagglutinin (HA), thereby affecting the antigenic and receptor-binding properties, as well as virulence. An epidemiological survey indicated that although the traditional seasonal H1N1 strain had disappeared, H3N2 became predominant again in the seasons (2010-11 and 2011-12) immediately following the H1N1 pandemic of 2009. Interestingly, although the 2009 pandemic H1N1 strain (H1N1pdm09) lacks additional NGSs, clinically isolated H3N2 strains obtained during these seasons gained N (Asn) residues at positions 45 and 144 of HA that forms additional NGSs. METHODS: To investigate whether these NGSs are associated with re-emergence of H3N2 within the subtype, we tested the effect of amino acid substitutions on neutralizing activity by using the antisera raised against H3N2 strains with or without additional NGSs. Furthermore, because the N residue at position 144 of HA was identified as the site of mismatch between the vaccine and epidemic strains of 2011-2012, we generated mutant viruses by reverse genetics and tested the functional importance of this particular NGS for antibody-mediated neutralization by intranasal inoculation of mice. RESULTS: The results indicated that amino acid substitution at residue 144 significantly affected neutralization activity, acting as an escape mutation. CONCLUSIONS: Our data suggest that the newly acquired NGSs in the HA globular head may play an important role in the re-emergence of endemic seasonal H3N2 strain by aiding the escape from humoral immunity.

The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/γcnull (NOG) mice.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections. FINDINGS: In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24 h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected. CONCLUSIONS: Our data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection.

Neuroimmunological aspects of human T cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis.

Human T cell leukemia virus type 1 (HTLV-1) is a human retrovirus etiologically associated with adult T cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Only approximately 0.25-4 % of infected individuals develop HAM/TSP; the majority of infected individuals remain lifelong asymptomatic carriers. Recent data suggest that immunological aspects of host-virus interactions might play an important role in the development and pathogenesis of HAM/TSP. This review outlines and discusses the current understanding, ongoing developments, and future perspectives of HAM/TSP research.

Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

BACKGROUND: OX40 is a member of the tumor necrosis factor receptor family that is expressed primarily on activated CD4+ T cells and promotes the development of effector and memory T cells. Although OX40 has been reported to be a target gene of human T-cell leukemia virus type-1 (HTLV-1) viral transactivator Tax and is overexpressed in vivo in adult T-cell leukemia (ATL) cells, an association between OX40 and HTLV-1-associated inflammatory disorders, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), has not yet been established. Moreover, because abrogation of OX40 signals ameliorates chronic inflammation in animal models of autoimmune disease, novel monoclonal antibodies against OX40 may offer a potential treatment for HTLV-1-associated diseases such as ATL and HAM/TSP. RESULTS: In this study, we showed that OX40 was specifically expressed in CD4+ T cells naturally infected with HTLV-1 that have the potential to produce pro-inflammatory cytokines along with Tax expression. We also showed that OX40 was overexpressed in spinal cord infiltrating mononuclear cells in a clinically progressive HAM/TSP patient with a short duration of illness. The levels of the soluble form of OX40 (sOX40) in the cerebrospinal fluid (CSF) from chronic progressive HAM/TSP patients or from patients with other inflammatory neurological diseases (OINDs) were not different. In contrast, sOX40 levels in the CSF of rapidly progressing HAM/TSP patients were higher than those in the CSF from patients with OINDs, and these patients showed higher sOX40 levels in the CSF than in the plasma. When our newly produced monoclonal antibody against OX40 was added to peripheral blood mononuclear cells in culture, HTLV-1-infected T cells were specifically removed by a mechanism that depends on antibody-dependent cellular cytotoxicity. CONCLUSIONS: Our study identified OX40 as a key molecule and biomarker for rapid progression of HAM/TSP. Furthermore, blocking OX40 may have potential in therapeutic intervention for HAM/TSP.