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Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1–induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1–infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome–related tumor development. (Cancer Res 2006; 66(2): 627-31)

Supplementary Method from HIV-1 Vpr Induces DNA Double-Strand Breaks

Aims We investigated the prevalence of Legionella spp. isolated from shower water in public bath facilities in Toyama Prefecture, Japan. In addition, we analyzed the genetic diversity among Legionella pneumophila isolates from shower water as well as the genetic relationship between isolates from shower water and from stock strains previously analyzed from sputum specimens. Methods The isolates were characterized using serogrouping, 16S rRNA gene sequencing, and sequence-based typing. Results Legionella spp. were isolated from 31/91 (34.1%) samples derived from 17/37 (45.9%) bath facilities. Isolates from shower water and bath water in each public bath facility were serologically or genetically different, indicating that we need to isolate several L. pneumophila colonies from both bath and shower water to identify public bath facilities as sources of legionellosis. The 61 L. pneumophila isolates from shower water were classified into 39 sequence types (STs) (index of discrimination = 0.974), including 19 new STs. Among the 39 STs, 12 STs match clinical isolates in the European Working Group for Legionella Infections database. Notably, ST505 L. pneumophila SG 1, a strain frequently isolated from patients with legionellosis and from bath water in this area, was isolated from shower water. Conclusions Pathogenic L. pneumophila strains including ST505 strain were widely distributed in shower water in public bath facilities, with genetic diversity showing several different origins. This study highlights the need to isolate several L. pneumophila colonies from both bath water and shower water to identify public bath facilities as infection sources in legionellosis cases.

We present the complete genome sequence of an enterohemorrhagic Escherichia coli O111:H8 strain. This strain was isolated from a hemolytic-uremic syndrome patient and was responsible for a large outbreak associated with the consumption of raw beef in 2011.

Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia., A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Mϕs) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally “stitching” together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Mϕs, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/Mϕ, and a Neu. IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia.

Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients’ stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

Key Clinical Message Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

SUMMARYA large outbreak of enterohaemorrhagic Escherichia coli (EHEC) O111 and O157 occurred in Japan in April 2011. We conducted an unmatched case-control study and trace-back investigation to determine the source of EHEC O111 infection and risk factors for severe complications. Pulsed-field gel electrophoresis was performed to help define cases. A total of 86 individuals met the case definition. Of these, 40% experienced haemolytic uraemic syndrome (HUS), 24% acute encephalopathy, and 6% died. Illness was significantly associated with eating the raw beef dish yukhoe (odds ratio 19·64, 95% confidence interval 7·03–54·83), the likely food vehicle. EHEC O111 and its closely related stx-negative variants were found in the beef. HUS occurred most frequently in individuals aged 5–9 years, and this age group was significantly associated with acute encephalopathy. The prevalence of HUS and acute encephalopathy was higher than in previous non-O157-related outbreaks, indicating a high risk of severe complications.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPScin macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt–Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method forin vitroamplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPScby protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPCsubstrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSccontained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPScin the CSF by serial PMCA, and the CSF levels of PrPSctended to increase with disease progression. In addition, PrPScwas detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPScin non-human primate models of CJD.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx 2 and stx -negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx -negative E. coli O111 isolates were closely related to EHEC O111 stx 2 isolates. Although the EHEC O157 strains had diverse stx gene profiles ( stx 1 , stx 2 , and stx 1 stx 2 ), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx 2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx 2 to an stx -negative strain may have occurred during infection.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants.IMPORTANCEInactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.

A water-borne outbreak of Yersinia enterocolitica O:8 associated with a small-scale water system occurred during July-August 2011 in Toyama Prefecture, Japan. Escherichia coli was not detected in tap water from the small-scale water system. However, the maximum concentration of viable bacteria in the tap water was 700CFU/mL, which exceeds the legal standard for purity of tap water (100CFU/mL). Furthermore, Y. enterocolitica O8 was isolated from the tap water with the use of immunomagnetic beads prepared with anti-Y. enterocolitica O8 antibodies. Pulsed-field gel electrophoresis analysis identified 3 isolates from tap water and 5 isolates from 4 patient stool specimens as belonging to the outbreak strain. An epidemiological investigation revealed improper management of the residual chlorine concentration in the tap water. This is the first report of an outbreak of Y. enterocolitica due to tap water from a small-scale water system in Japan.

The rapid detection of infectivity of several agents that cause Creutzfeldt-Jakob disease has previously been achieved by assaying for deposits of abnormal prion protein (PrPSc) in follicular dendritic cells in the spleens of transgenic mice carrying the human prion protein gene. In this study, transgenic mice expressing the bovine prion protein were inoculated intraperitoneally with classical (C-type) or atypical L-type bovine spongiform encephalopathies (BSE). Proteinase-resistant PrPSc were detected in the spleens of all transgenic mice at 75 days after inoculation with both types of BSE. Infectivity in PrPSc-positive spleens of the transgenic mice revealed that prions of C- and L-type BSE replicated. These results suggest that bioassay system by the transgenic mice could be useful for the rapid detection of BSE infectivity with discriminating between C- and L-type BSEs.

We performed comparative analyses of Legionella pneumophila serogroup (SG) 1 isolates obtained during 2005-2012 in Toyama Prefecture, Japan, by sequence-based typing (SBT) and pulsed-field gel electrophoresis (PFGE). Seventy-three isolates of L. pneumophila SG 1, including 17 isolates from patients, 51 from public baths, 4 from cooling towers, and 1 from a shower, were analyzed. The isolates were classified into 43 sequence types (STs) by SBT and 52 types by PFGE. Fourteen STs were unique to Toyama Prefecture, as determined from the SBT database of European Working Group for Legionella Infections (EWGLI), as of October 31, 2012. ST505 strain was identified in 4 isolates from patients and 5 isolates from public baths, and these isolates belonged to 2 PFGE types. These, however, were similar because of the difference with only two restriction fragments, indicating that ST505 strain was prevalent among L. pneumophila SG 1 isolates in this area. ST505 strains isolated from patients and public baths were distributed along the river in a western part of Toyama Prefecture. SBT and PFGE profiles of 3 clinical isolates were identical with those of 3 environmental isolates from the suspected origins of the infection in each case, respectively. This finding suggested that SBT and PFGE were useful for epidemiological study. Furthermore, by SBT analysis, we identified a clonal group formed only by 7 clinical isolates that are not associated with bathwater, suggesting that they were derived from unrecognized sources.

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A + ( n = 6), E + ( n = 6), B + ( n = 4), and J + ( n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A + animals, including two controllers, showed slower disease progression, whereas J + animals exhibited rapid progression. E + and B + animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8 + T-cell responses were efficiently induced in A + animals, while Nef-specific CD8 + T-cell responses were in A + , E + , and B + animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4 + T-cell decline, and SIV-specific CD4 + T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.

For tularemia, a zoonosis caused by the gram-negative coccobacillus Francisella tularensis, research of the relation between skin lesions and lymph node lesions has not been reported in the literature. This report describes skin lesions and lymph node lesions and their mutual relation over time for tularemia in Japan. Around the second day after infection (DAI), a subcutaneous abscess was observed (abscess form). Hand and finger skin ulcers formed during the second to the fourth week. Subcutaneous and dermal granulomas were observed with adjacent monocytoid B lymphocytes (MBLs) (abscess–granulomatous form). From the sixth week, large granulomas with central homogeneous lesions emerged diffusely (granulomatous form). On 2–14 DAI, F. tularensis antigen in skin lesions was detected in abscesses. During 7–12 DAI, abscesses with adjacent MBLs appeared without epithelioid granuloma (abscess form) in regional lymph nodes. During the second to fifth week, granulomas appeared with necrosis (abscess–granulomatous form). After the sixth week, large granulomas with a central homogeneous lesion (granulomatous form) appeared. F. tularensis antigen in lymph node lesions was observed in the abscess on 7–92 DAI. Apparently, F. tularensis penetrates the finger skin immediately after contact with infected hares. Subsequently, the primary lesion gradually transfers from skin to regional lymph nodes. The regional lymph node lesions induced by skin lesion are designated as dermatopathic lymphadenopathy. This study revealed temporal differences of onset among the skin and lymph node lesions.

Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.

A novel detection system was established previously for cells infected with the human cytomegalovirus (HCMV) in vitro that utilizes the unique IE1-dependent nuclear dispersion of promyelocytic leukemia (PML) bodies early in the HCMV replication cycle. This assay system, designated "the PML assay," makes use of the GFP-PML-expressing cell line SE/15, and allows real-time monitoring of infected cells by fluorescence microscopy without any staining procedures. A rapid and quantitative drug susceptibility testing was developed for low-titer clinical isolates propagated in fibroblasts in vitro. The present study sought to exploit the PML assay for evaluating in vivo status of HCMV without virus isolation. Progeny viruses were detected directly from peripheral blood mononuclear cells (PBMCs) infected in vivo obtained from hematopoietic stem cell transplantation recipients. The overall positivity of the PML assay tended to correlate with the levels of genomic DNA. Direct phenotypic susceptibility testing detected one ganciclovir (GCV)-resistant case among 19 samples, which was confirmed further by genomic and plaque reduction assays. However, in another patient with the sequence-proven mutant confirmed by sequencing, the progeny viruses exhibiting GCV-resistance were not detected. Studies on the isolated virus from the latter patient suggested the possibility that replication efficiency may differ between PBMCs and lesions infected in vivo, which may hamper the detection of GCV-resistant viruses by the PML assay, at least in this case. Taken together, the PML assay is sufficiently sensitive to monitor replication-competent HCMV directly from PBMCs infected in vivo, and provides a novel tool for comparing the characteristics of HCMV strains infected in vivo.

Twenty autopsy cases with 2009 pandemic influenza A (2009 H1N1) virus infection, performed between August 2009 and February 2010, were histopathologically analyzed. Hematoxylin-eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalin-fixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five cases, the pathogenesis of 2009 H1N1 was confirmed to be viral infection in pneumocytes, which caused severe alveolar damage and fatal viral pneumonia. Further studies on both host and viral factors in autopsy or biopsy materials will be essential to elucidate the other pathogenic factors involved in influenza virus infection.

Since a combination of flt3 ligand plasmid (pFL) and CpG-oligodeoxynucleotides (ODN)(3) as a dendritic cell (DC)-targeting double mucosal adjuvant elicited ovalbumin-specific secretory IgA (S-IgA) antibody (Ab) responses, we examined whether this double adjuvant could induce influenza-specific protective immunity in aged mice. A double adjuvant plus A/Puerto Rico/8/34 (PR8) hemagglutinin (HA) induced increased numbers of CD11b(+) CD11c(+) DCs and both CD4(+) Th1- and Th2-type responses in the nasopharyngeal-associated lymphoreticular tissue, nasal passages and cervical lymph nodes. Further, increased levels of PR8 HA-specific S-IgA Ab responses were detected in the upper respiratory tact (URT) of aged and young adult mice given nasal PR8 HA with this double adjuvant. Thus, when mice were challenged with PR8 virus via the nasal route, both aged and young adult mice given nasal vaccine exhibited complete protection. Further, IgA-deficient mice nasally immunized with a double adjuvant influenza vaccine failed to provide protection against PR8 challenge. These results indicate that a nasal double adjuvant successfully induces PR8 HA-specific IgA Ab responses in both young adult and aged mice, which are essential for the prevention of influenza infection in the murine URT.

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.

The HIV-1 protein Vpr disrupts higher-order chromatin structure by altering histone modification and displacing important heterochromatin proteins, resulting in chromatid cohesion defects., Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1–encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.

This study reports the experimental transmission of bovine spongiform encephalopathy (BSE) to guinea pigs and describes the cerebellar lesions in these animals. Guinea pigs were inoculated intracerebrally with 10% brain homogenates from BSE-affected cattle. These animals were designated as the first passage. Second and third passages were subsequently performed. All guinea pigs developed infection at each passage. The mean incubation period of the first passage was 370 days post-infection (dpi) and this decreased to 307 dpi and 309 dpi for the second and third passages, respectively. Mild to severe spongiform degeneration and gliosis were observed in the cerebral cortex, thalamus and brainstem. In addition, the affected animals had marked pathological changes in the cerebellum characterized by severe cortical atrophy associated with Bergmann radial gliosis of the molecular layer and reduction in the width of the granular cell layer. Immunohistochemically, intense PrP(Sc) deposition and scattered plaque-like deposits were observed in the molecular and granular cell layers. Cerebellar lesions associated with severe atrophy of the cortex have not been reported in animal prion diseases, including in the experimental transmission of PrP(Sc) to small rodents. These lesions were similar to the lesions of human kuru or the VV2 variant of sporadic Creutzfeldt-Jakob disease, although typical kuru plaques or florid plaques were not observed in the affected animals.

The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C–U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.

A low molecular weight type of atypical bovine spongiform encephalopathy (L-BSE) was transmitted to two cynomolgus macaques by intracerebral inoculation of a brain homogenate of cattle with atypical BSE detected in Japan. They developed neurological signs and symptoms at 19 or 20 months post-inoculation and were euthanized 6 months after the onset of total paralysis. Both the incubation period and duration of the disease were shorter than those for experimental transmission of classical BSE (C-BSE) into macaques. Although the clinical manifestations, such as tremor, myoclonic jerking, and paralysis, were similar to those induced upon C-BSE transmission, no premonitory symptoms, such as hyperekplexia and depression, were evident. Most of the abnormal prion protein (PrP(Sc)) was confined to the tissues of the central nervous system, as determined by immunohistochemistry and Western blotting. The PrP(Sc) glycoform that accumulated in the monkey brain showed a similar profile to that of L-BSE and consistent with that in the cattle brain used as the inoculant. PrP(Sc) staining in the cerebral cortex showed a diffuse synaptic pattern by immunohistochemistry, whereas it accumulated as fine and coarse granules and/or small plaques in the cerebellar cortex and brain stem. Severe spongiosis spread widely in the cerebral cortex, whereas florid plaques, a hallmark of variant Creutzfeldt-Jakob disease in humans, were observed in macaques inoculated with C-BSE but not in those inoculated with L-BSE.

A novel simultaneous detection system for human viruses was developed using a real‐time polymerase chain reaction (PCR) system to identify causes of infection in clinical samples from patients with uncertain diagnoses. This system, designated as the “multivirus real‐time PCR,” has the potential to detect 163 human viruses (47 DNA viruses and 116 RNA viruses) in a 96‐well plate simultaneously. The specificity and sensitivity of each probe–primer set were confirmed with cells or tissues infected with specific viruses. The multivirus real‐time PCR system showed profiles of virus infection in 20 autopsies of acquired immunodeficiency syndrome patients, and detected frequently TT virus, cytomegalovirus, human herpesvirus 6, and Epstein–Barr virus in various organs; however, RNA viruses were detected rarely except for human immunodeficiency virus‐1. Pathology samples from 40 patients with uncertain diagnoses were examined, including cases of encephalitis, hepatitis, and myocarditis. Herpes simplex virus 1, human herpesvirus 6, and parechovirus 3 were identified as causes of diseases in four cases of encephalitis, while no viruses were identified in other cases as causing disease. This multivirus real‐time PCR system can be useful for detecting virus in specimens from patients with uncertain diagnoses. J. Med. Virol. 83:322–330, 2011. © 2010 Wiley‐Liss, Inc.

Amyloid plaques, a well-known hallmark of Alzheimer's disease (AD), are formed by aggregated β-amyloid (Aβ). The cellular prion protein (PrPc) accumulates concomitantly with Aβ in amyloid plaques. One type of amyloid plaque, classified as a neuritic plaque, is composed of an amyloid core and surrounding dystrophic neurites. PrPc immunoreactivity reminiscent of dystrophic neurites is observed in neuritic plaques. Proteinase K treatment prior to immunohistochemistry removes PrPc immunoreactivity from amyloid plaques, whereas Aβ immunoreactivity is enhanced by this treatment. In the present study, we used a chemical pretreatment by a sarkosyl solution (0.1% sarkosyl, 75 mM NaOH, 2% NaCl), instead of proteinase K treatment, to evaluate PrPc accumulation within amyloid plaques. Since PrPc within amyloid plaques is removed by this chemical pretreatment, we can recognize that the PrP species deposits within amyloid plaques were PrPc. We could observe that PrPc accumulation in dystrophic neurites occurred differently compared with Aβ or hyperphosphorylated tau aggregation in the AD brain. These results could support the hypothesis that PrPc accumulation in dystrophic neurites reflects a response to impairments in cellular degradation, endocytosis, or transport mechanisms associated with AD rather than a non-specific cross-reactivity between PrPc and aggregated Aβ or tau.

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Fluoroquinolone (FQ) resistance of Bacillus anthracis is a serious concern in the fields of biodefense and bioterrorism since FQs are very effective antibiotics and are recommended as first-line treatment against this lethal bacterium. In this study, we obtained 2 strains of B. anthracis showing resistance or intermediate resistance to ciprofloxacin (CIP) by a stepwise selection procedure with increasing CIP concentrations. Fifteen genetic variations were identified between the parental and CIP-resistant strains by next-generation sequencing. Nonsynonymous mutations in the quinolone resistance-determining region (QRDR) of type II DNA topoisomerase were identified in the resistant strain but not in the intermediate-resistant strain. The GBAA0834 (TetR-type transcriptional regulator) locus was also revealed to be a novel “mutation hot spot” that leads to the increased expression of multidrug efflux systems for CIP resistance. As an initial step of CIP resistance in B. anthracis , such disruptive mutations of GBAA0834 appear to be more easily acquired than those in an essential gene, such as that encoding type II DNA topoisomerase. Such an intermediate-resistant phenotype could increase a cell population under CIP-selective pressure and might promote the emergence of highly resistant isolates. Our findings reveal, in addition to QRDR, crucial genetic targets for the investigation of intermediate resistance of B. anthracis to FQs.

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues.

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-alpha converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-alpha production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

The synthetic double-stranded RNA polyriboinocinic polyribocytidylic acid [poly(I:C)] is a potent mucosal adjuvant in mice immunized intranasally with an inactivated influenza vaccine. In an attempt, to increase the effectiveness of a nasal poly(I:C)-combined vaccine, the effect of zymosan, a cell wall extract from Saccharomyces cervisiae was investigated, on the adjuvant activity of poly(I:C) in BALB/c mice. The addition of zymosan (10 microg) as an adjuvant in mice which were immunized intranasally with a poly(I:C) (1-5 microg)-combined vaccine (1 microg) enhanced the ability of the mice to mount an effective immune response to a lethal dose of influenza virus, and resulted in a synergistic increase in secretory IgA and serum IgG antibody levels. To define the mechanism by which zymosan enhanced the adjuvant activity of poly(I:C), bone marrow-derived dendritic cells (BM-DCs) were cultured in the presence of poly(I:C) and/or zymosan. There was a synergistic increase in cytokine production (TNF-alpha, IL-6, IL-10, and IFN-beta) in BM-DCs, together with an increase in the expression of co-stimulatory molecules (CD86 and CD40) in response to co-treatment with poly(I:C) and zymosan. This synergistic effect on cytokine production was mimicked by co-treatment with poly(I:C) and a Toll-like receptor 2 (TLR2) ligand, which represented one of the components of zymosan. The results of the current study suggest that one of the mechanisms by which zymosan enhances the adjuvant activity of poly(I:C) is through increased cytokine production by DCs involving the synergistic activation of poly(I:C)-induced TLR3- and zymosan-induced TLR2-mediated signaling pathways. J. Med. Virol. 82:476-484, 2010. (c) 2010 Wiley-Liss, Inc.

We report the pathological and virological findings of the first autopsy case of the 2009 pandemic influenza (A/H1N1pdm) virus infection in Japan. A man aged 33 years with chronic heart failure due to dilated cardiomyopathy, mild diabetes mellitus, atopic dermatitis, bronchial asthma, and obesity died of respiratory failure and multiple organ dysfunction syndrome. Macroscopic examination showed severe pulmonary edema and microscopically the lung sections showed very early exudative-stage diffuse alveolar damage (DAD). Immunohistochemistry revealed proliferation of the influenza (A/H1N1pdm) virus in alveolar epithelial cells, some of which expressed SAalpha2-3Gal on the cell surface. Influenza (A/H1N1pdm) virus genomic RNA and mRNA were also detected in alveolar epithelial cells. Real-time PCR revealed 723 copies/cell in the left lower lung section from which the influenza (A/H1N1pdm) virus was isolated. Electron microscopic analysis revealed filamentous viral particles in the lung tissue. The concentrations of various cytokines/chemokines in the serum and the autopsied lung tissue were measured. IL-2R, IL-6, IL-8, IL-10, IFN-alpha, MCP-1, and MIG levels were elevated in both. These findings indicated a case of viral pneumonia caused by influenza (A/H1N1pdm) virus infection, showing characteristic pathological findings of the early stage of DAD.

Histoplasmosis, caused by Histoplasma capsulatum, is an endemic mycosis in many countries of the world except for Japan. Outbreaks of histoplasmosis among Japanese people are very rare and are mainly imported by travelers. We report an outbreak of histoplasmosis among healthy Japanese people who traveled to a resort area in Southeast Asia. Three young Japanese women traveled to Langkawi island, Malaysia and stayed on the island for five days without visiting caves, a known reservoir of H. capsulatum. All three individuals developed flu-like symptoms with multiple nodule shadows on chest X rays or chest CT scans at around ten days after their return to Japan. Serum samples obtained from the three subjects were positive for anti-Histoplasma antibody and specific PCR for H. capsulatum on lung biopsy specimens and the serum from one patient was positive. The clinical course of all three patients improved without the use of anti-fungal agents and no recurrence has been confirmed. Clinical attendants should consider histoplasmosis when they see patients with flu-like symptoms with abnormal chest X-rays after visiting H. capsulatum endemic areas, especially Southeast Asia.

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection.

Adult T-cell leukemia (ATL) is a T-cell malignancy caused by human T lymphotropic virus type I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have used mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice reproduces a leukemia and lymphoma that is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to stromal cell-derived factor-1alpha (SDF-1alpha). Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1alpha. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1alpha-induced migration and phosphorylation of extracellular signal-related kinase 1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Using the SCID mouse model, it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1alpha/CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL.

Tick-borne encephalitis virus (TBEV) induces acute central nervous system (CNS) disease in humans. In this study, we investigate the pathogenetic mechanisms that correlate with fatal infection with TBEV in a mouse model. Following subcutaneous infection with high challenge doses (>10(7) PFU), mice started to die early (8 days) and mortality rates reached >80%. These doses induced acute and widespread infection of the CNS. On the other hand, following subcutaneous infection with low challenge doses (10(2)-10(6) PFU), mice started to die late (11 days) and approximately one half of the mice survived but exhibited degrees of encephalitis similar to dying mice. However, low dose dying mice exhibited severe systemic stress response, and increased levels of TNF-alpha compared with recovering mice. We therefore conclude that in addition to the development of CNS disease, systemic inflammatory and stress responses contribute to induce a fatal infection following subcutaneous infection of mice with TBEV.

BACKGROUND: Hemagglutination-inhibition (HI) antibody titers correlate with protective immunity to seasonal influenza viruses. However, inactivated H5N1 influenza vaccines from Vietnam 2004 strains afford protection without producing high or even detectable HI antibodies. METHODS: BALB/c mice were immunized twice (at a 3-week interval) with inactivated whole-virus influenza vaccine produced using reverse genetics, with the internal genes of A/PR/8/34 (a high-yield strain) and the hemagglutinin (HA) and neuraminidase (NA) genes of A/Vietnam/1194/04 (H5N1) virus (NIBRG-14) adjuvanted with alum (5 microg of HA). Either HA- or NA-binding antibodies were absorbed from the immune serum. The protective efficacy of these antibodies was determined by injecting the absorbed serum into severe combined immunodeficiency mice, which were then challenged with highly pathogenic H5N1 virus (A/Vietnam/Jp1203/2004; Japanese isolate of A/Vietnam/1203/2004). RESULTS: The NIBRG-14 vaccine elicited levels of anti-HA antibodies similar to levels elicited by the H1N1 vaccines, as well as levels of anti-NA antibodies higher than those elicited by the H1N1 vaccines. The absorption of either anti-HA or anti-NA antibody from immune serum samples obtained from NIBRG-14-vaccinated mice significantly reduced the protective efficacy of the serum. CONCLUSIONS: For NIBRG-14 vaccines to confer protection to vaccinated hosts, both anti-HA and anti-NA antibodies are required. This finding implies that the measurement of both antibody levels may be required for accurate evaluation of vaccine efficacy.

Monkeypox virus is divided into Congo Basin and West African strains. The virulence and pathophysiology of two strains, Zr-599 (a Congo Basin monkeypox virus) and Liberia (a West African monkeypox virus), were evaluated in non-human primates. Four monkeys were infected by the subcutaneous (SC) and two by the intranasal (IN) inoculation routes for Zr-599 and Liberia at a dose of 10(6) p.f.u. One monkey in the Liberia/SC group was demonstrated to be co-infected with Gram-positive cocci and was excluded from analyses. Infections in three of the four Zr-599/SC monkeys and in one of the three Liberia/SC monkeys were fatal. Virus genome levels in blood in the Zr-599/SC monkeys were approximately 10 times higher than those in the Liberia/SC monkeys. Zr-599 affected respiratory, genito-urinary and gastrointestinal tract organs more severely than Liberia. Zr-599 was more virulent than Liberia and one of the factors might be the difference in organ tropism.

Epstein-Barr Virus (EBV)-associated immunoblastic lymphoma occurs in immunocompromised patients such as those with AIDS or transplant recipients after primary EBV infection or reactivation of a preexisting latent EBV infection. In the present study, we evaluated the effect of ritonavir, an HIV protease inhibitor, on EBV-positive lymphoblastoid B cells in vitro and in mice model. We found that it induced cell-cycle arrest at G1-phase and apoptosis through down-regulation of cell-cycle gene cyclin D2 and antiapoptotic gene survivin. Furthermore, ritonavir suppressed transcriptional activation of NF-κB in these cells. Ritonavir efficiently prevented growth and infiltration of lymphoma cells in various organs of NOD/SCID/γcnull mice at the same dose used for treatment of patients with AIDS. Our results indicate that ritonavir targets NF-κB activated in tumor cells and shows anti-tumor effects. These data also suggest that this compound may have promise for treatment or prevention of EBV-associated lymphoproliferative diseases that occur in immunocompromised patients. © 2008 Wiley-Liss, Inc.