[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of General Virology 06/2009; 90(Pt 9):2266-71. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
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).
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.
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.
The Journal of Infectious Diseases 05/2009; 199(11):1629-37. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human herpes virus 6 (HHV6) has attracted attention in recent years as an important causative agent for limbic encephalitis after bone marrow transplantation (BMT). We report an autopsy case of HHV6-induced encephalomyelitis that developed after BMT. The patient was a 61-year-old man with acute myeloid leukemia, who developed disorientation and short-term memory disturbance 35 days after allogenic BMT. MRI demonstrated T1-weighted high-signal intensity lesions in the medial temporal lobe and thalamus, and PCR of the CSF disclosed an increase in the copy numbers of the HHV6 genome. The patient died after a clinical course of 6 months, and at autopsy the brain showed remarkable atrophy of the hippocampus. Histopathologically, neuronal loss with astrocytosis and patchy necrosis with infiltration of macrophages were found predominantly in the hippocampus, amygdala, mamillary body, claustrum, and thalamus. Perivascular and intraparenchymal lymphocytic infiltration was slight. Similar lesions were also scattered in the cerebral neocortex, midbrain, pontine base, cerebellar white matter, and lumbar cord. In some of these lesions, axons were relatively preserved in comparison with myelin sheaths. Significant increase in the copy numbers of the HHV6 genome was demonstrated in the postmortem brain tissue by PCR. Neuropathological features of the present case were similar to those described in previously reported cases, but the distribution of lesions was more widespread. Demyelination was supposed to be involved in the pathogenesis of some of the lesions.
[show abstract][hide abstract] ABSTRACT: We investigated the level of telomerase activity (TA) in 17 specimens of non-genital Bowen's disease (BD) and in 14 specimens of skin without sun exposure (non-exposed skin) using a non-isotopic PCR-based telomeric repeat amplification protocol (TRAP) assay. Expression of human telomerase reverse transcriptase (hTERT; the catalytic subunit of telomerase) was also evaluated by immunochemistry in the non-genital BD tissues. Moderate to high levels of TA were detected in 41.2% of 17 non-genital BD specimens (P = 0.001). In contrast, TA was not evident in non-exposed skin. Recently, nucleolin was reported to be associated with hTERT, so we used this antibody instead of hTERT antibody. Immunohistochemistry showed that nucleolin expression was associated with high TA levels in non-genital BD. Our results also revealed differences of TA levels among non-genital BD specimens. High levels of TA in those specimens were not age related. Five out of 7 specimens (71.4%) with moderate to high TA levels were from sun-exposed sites, while the remaining 10 specimens with low levels of TA were from non-exposed sites. These results suggested that cellular DNA damage caused by ultraviolet irradiation might be associated with an increase of TA in non-genital BD. Among non-genital BD specimens, 4 out of 17 (23.5%) showed high levels of TA (median relative TA value: 79.8%; P = 0.003), which might be associated with immortalization or transformation to invasive squamous cell carcinoma.
Journal of the European Academy of Dermatology and Venereology 03/2009; 23(6):668-72. · 2.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Telomerase, a ribonucleoprotein enzyme, is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric repeats at the eukaryotic chromosomal ends. We previously reported that topoisomerase I dissociates HIV-1 reverse transcriptase from genomic RNAs, and binding of topoisomerase I to RNA template regulates cDNA synthesis. We also found that a monoclonal antibody (MAb) against topoisomerase I, designated as MAb 1, suppresses the reverse transcription efficiency using a detergent-disrupted HIV-1 virion. In this study, we describe how MAb 1 suppresses telomerase activity in cellular lysates. In addition, siRNAs of topoisomerase I has attenuated telomerase activity in culture cells. These results suggest that topoisomerase I is involved in telomerase activity, as well as HIV-1 reverse transcription.
[show abstract][hide abstract] ABSTRACT: Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine.
[show abstract][hide abstract] ABSTRACT: Since high levels of prions, the causative agent of bovine spongiform encephalopathy (BSE), accumulate in the brain and spinal cord, contamination of beef carcasses with central nervous system tissue (CNST) may occur at post-slaughter process. In this study, we investigated CNST contamination on the surface of beef carcasses using glial fibrillary acidic protein as a marker after splitting and evaluated the effects of washing procedures on contamination removal. High levels of CNST contamination was detected immediately after splitting, especially in the area close to the spinal column. This suggests that spinal cord fragments are attached to carcasses at the time of splitting even though the spinal cords have been removed by vacuum before splitting. Steam cleaning or manually washing with normal pressure water around the spinal column, performed prior to washing with high-pressure water, was found to be effective for reducing the level of CNST contamination. Furthermore, manually washing with high-pressure water could reduce CNST contamination to almost negligible levels. These results are useful for preparation of appropriate sanitation standard operating procedures to reduce the risk of CNST contamination of carcasses for prevention of exposure to BSE prion via the food chain.
Journal of Veterinary Medical Science 12/2008; 70(11):1225-30. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The herpes simplex virus 1 (HSV-1) UL7 gene is highly conserved among herpesviridae. Since the construction of recombinant HSV-1 with a mutation in the UL7 gene has not been reported, the involvement of HSV-1 UL7 in viral replication has been unclear. In this study, we succeeded in generating a UL7 null HSV-1 mutant virus, MT102, and characterized it. Our results were as follows. (i) In Vero cells, MT102 was replication-competent, but formed smaller plaques and yielded 10- to 100-fold fewer progeny than the wild-type virus, depending on the multiplicity of infection. (ii) Using mass spectrometry-based proteomics technology, we identified a cellular mitochondrial protein, adenine nucleotide translocator 2 (ANT2), as a UL7-interacting partner. (iii) When ANT2 was transiently expressed in COS-7 cells infected with HSV-1, ANT2 was specifically co-precipitated with UL7. (iv) Cell fractionation experiments with HSV-1-infected cells detected the UL7 protein in both the mitochondrial and cytosolic fractions, whereas ANT2 was detected only in the mitochondrial fraction. These results indicate the importance of HSV-1 UL7's involvement in viral replication and demonstrate that it interacts with ANT2 in infected cells. The potential biological significance of the interaction between UL7 and ANT2 is discussed.
[show abstract][hide abstract] ABSTRACT: 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-kappaB in these cells. Ritonavir efficiently prevented growth and infiltration of lymphoma cells in various organs of NOD/SCID/gammacnull mice at the same dose used for treatment of patients with AIDS. Our results indicate that ritonavir targets NF-kappaB 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.
International Journal of Cancer 10/2008; 124(3):622-9. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 +/- 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 +/- 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrP(Sc) were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice. The molecular weight and glycoform ratio of PrP(Sc) in BSE/JP24 were different from those in C-BSE, and PrP(Sc) in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 +/- 3.1 days). This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.
[show abstract][hide abstract] ABSTRACT: Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.
Journal of Virology 08/2008; 82(13):6172-89. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Advanced age is a risk factor of severe acute respiratory syndrome (SARS) in humans. To understand its pathogenesis, we developed an animal model using BALB/c mice and the mouse-passaged Frankfurt 1 isolate of SARS coronavirus (SARS-CoV). We examined the immune responses to SARS-CoV in both young and adult mice. SARS-CoV induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. Moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. Adult murine lungs, which had significantly higher interleukin (IL)-4 and lower IL-10 and IL-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-alpha). On day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also IL-2, interferon-gamma, IL-10, and IL-13. Adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after SARS-CoV infection, which led to severe pulmonary edema and diffuse alveolar damage. Intravenous injection with anti-tumor necrosis factor-alpha antibody 3 hours after infection had no effect on SARS-CoV infection. However, intraperitoneal interferon-gamma injection protected adult mice from the lethal respiratory illness. The experimental model described here may be useful for elucidating the pathophysiology of SARS and for evaluating therapies to treat SARS-CoV infection.
American Journal Of Pathology 07/2008; 172(6):1625-37. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epstein-Barr virus (EBV) causes EBV-associated lymphoproliferative diseases in patients with profound immune suppression. Most of these diseases are life-threatening and the prognosis of AIDS-associated lymphomas is extremely unfavorable. Polyclonal expansion of virus infected B-cell predisposes them to transformation. We investigated the possibility of nuclear factor kappa B (NF-kappaB) inhibition by dehydroxymethylepoxyquinomicin (DHMEQ) for the treatment and prevention of EBV-associated lymphoproliferative diseases. We examined the effect of DHMEQ on apoptosis induction in four EBV-transformed lymphoblastoid cell lines as well as peripheral blood mononuclear cells infected with EBV under immunosuppressed condition. DHMEQ inhibits NF-kappaB activation in EBV-transformed lymphoblastoid cell lines and induces apoptosis by activation of mitochondrial and membranous pathways. Using an in vivo NOD/SCIDgammac mouse model, we showed that DHMEQ has a potent inhibitory effect on the growth of lymphoblastoid cells. In addition, DHMEQ selectively purges EBV-infected cells expressing latent membrane protein (LMP) 1 from peripheral blood mononuclear cells and inhibits the outgrowth of lymphoblastoid cells. These results suggest that NF-kappaB is a molecular target for the treatment and prevention of EBV-associated lymphoproliferative diseases. As a potent NF-kappaB inhibitor, DHMEQ is a potential compound for applying this strategy in clinical medicine.
Microbes and Infection 07/2008; 10(7):748-56. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, respectively. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.
Journal of Virology 07/2008; 82(11):5198-211. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Severe acute respiratory syndrome coronavirus (SARS-CoV) is a high-risk infectious pathogen. In the proposed model of respiratory
failure, SARS-CoV down-regulates its receptor, angiotensin-converting enzyme 2 (ACE2), but the mechanism involved is unknown.
We found that the spike protein of SARS-CoV (SARS-S) induced TNF-α-converting enzyme (TACE)-dependent shedding of the ACE2
ectodomain. The modulation of TACE activity by SARS-S depended on the cytoplasmic domain of ACE2, because deletion mutants
of ACE2 lacking the carboxyl-terminal region did not induce ACE2 shedding or TNF-α production. In contrast, the spike protein
of HNL63-CoV (NL63-S), a CoV that uses ACE2 as a receptor and mainly induces the common cold, caused neither of these cellular
responses. Intriguingly, viral infection, judged by real-time RT-PCR analysis of SARS-CoV mRNA expression, was significantly
attenuated by deletion of the cytoplasmic tail of ACE2 or knock-down of TACE expression by siRNA. These data suggest that
cellular signals triggered by the interaction of SARS-CoV with ACE2 are positively involved in viral entry but lead to tissue
damage. These findings may lead to the development of anti-SARS-CoV agents.
Proceedings of the National Academy of Sciences 06/2008; 105(22):7809-7814. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations that result in escape from CTL recognition with viral fitness costs. The replication in vivo of such viruses carrying not single but multiple escape mutations in the absence of the CTL pressure has remained undetermined. Here, we have examined the replication of simian immunodeficiency virus (SIV) with five gag mutations selected in a macaque possessing the major histocompatibility complex haplotype 90-120-Ia after its transmission into 90-120-Ia-negative macaques. Our results showed that even such a "crippled" SIV infection can result in persistent viral replication, multiple reversions, and AIDS progression.
Journal of Virology 06/2008; 82(10):5093-8. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: In a nasal vaccine against influenza, the activation of natural killer T (NKT) cells by intranasal coadministration of alpha-galactosylceramide (alpha-GalCer) can potently enhance protective immune responses. The results of this study show that the NKT cell-activated nasal vaccine can induce an effective cross-protection against different strains of influenza virus, including H5 type. To analyze the mechanism of NKT cell activation by this nasal vaccine, we prepared fluorescence-labeled alpha-GalCer by which we detect a direct interaction between NKT cells and alpha-GalCer-stored dendritic cells in nasal mucosa-associated tissues. Accordingly, although very few NKT cells exist at mucosa, the nasal vaccination induced a localized increase in NKT cell population, which is partly dependent on CXCL16/CXCR6. Furthermore, we found that NKT cell activation stimulates mucosal IgA production by a mechanism that is dependent on interleukin (IL)-4 production. These results strengthen the basis of nasal vaccination via NKT cell activation, which can induce immune cross-protection.