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Masayuki Saijo

Publications and source records attributed to Masayuki Saijo.

At least 19 recordsLinked to original sources
PubMed2006

Participation of both host and virus factors in induction of severe acute respiratory syndrome (SARS) in F344 rats infected with SARS coronavirus.

To understand the pathogenesis and develop an animal model of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), the Frankfurt 1 SARS-CoV isolate was passaged serially in young F344 rats. Young rats were susceptible to SARS-CoV but cleared the virus rapidly within 3 to 5 days of intranasal inoculation. After 10 serial passages, replication and virulence of SARS-CoV were increased in the respiratory tract of young rats without clinical signs. By contrast, adult rats infected with the passaged virus showed respiratory symptoms and severe pathological lesions in the lung. Levels of inflammatory cytokines in sera and lung tissues were significantly higher in adult F344 rats than in young rats. During in vivo passage of SARS-CoV, a single amino acid substitution was introduced within the binding domain of the viral spike protein recognizing angiotensin-converting enzyme 2 (ACE2), which is known as a SARS-CoV receptor. The rat-passaged virus more efficiently infected CHO cells expressing rat ACE2 than did the original isolate. These results strongly indicate that host and virus factors such as advanced age and virus adaptation are critical for the development of SARS in rats.

Age Factors↗
PubMed2006

Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.

Human respiratory syncytial virus (RSV) is a major causative agent of lower respiratory tract infections in children and the elderly. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed assay to amplify the genome of RSV subgroups A and B, in order to improve current diagnostic methods for RSV infection. The primer sets for RT-LAMP were designed using highly conserved nucleotide sequences in the matrix protein region of subgroups A and B, and were specific for each subgroup. The RT-LAMP efficiency was compared to virus isolation and a commercially available enzyme immunoassay (EIA) for RSV detection (BD Directigen EZ RSV test), using nasopharyngeal aspirates from 59 children with respiratory tract infections. The RT-LAMP was specific for RSV and could not detect other respiratory pathogens. 61% (36/59) of children were positive by RT-LAMP, 34% (20/59) by viral isolation, and 56% (26/46) by EZ RSV. Of 16 specimens that were negative by both antigen detection and virus isolation, 12.5% (2/16) were RT-LAMP positive. These results suggest that the RT-LAMP is more sensitive than other methods used to detect RSV. The RT-LAMP assay developed in this study may be useful for diagnostic and epidemiological studies of RSV infection.

Base Sequence↗
PubMed2006

Mechanisms of establishment of persistent SARS-CoV-infected cells.

Previously, we reported the establishment of cells with persistent SARS-CoV infection after apoptotic events and showed that both JNK and PI3K/Akt signaling pathways are important for persistence by treatment with inhibitors at the early stages of SARS-CoV infection. However, the mechanisms of establishment of persistent infection are still unclear. In this study, we investigated which signaling pathways play important roles in escape from apoptosis in cells infected with SARS-CoV. In persistently infected cells at 50h.p.i., PI3K/Akt, JNK, p38 MAPK and Bcl-2 were phosphorylated and the protein levels of Bcl-2 and Bcl-xL were increased. When surviving cells were treated with the JNK-specific inhibitor, SP600125, at 50h.p.i., all cells died, suggesting that the JNK signaling pathway is necessary for maintenance of persistently infected cells. Among the signaling pathways in persistently infected cells, Akt and JNK were phosphorylated in SARS-CoV-nucleocapsid (N) protein-expressing Vero E6 cells using vaccinia viral vector (DIs), strongly suggesting that N protein-induced phosphorylation of Akt and JNK are necessary to establish persistence. These results indicated that at least four proteins, Akt, JNK, Bcl-2 and Bcl-xL, are necessary for survival of persistently SARS-CoV-infected cells.

Animals↗
PubMed2006

Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells.

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.

Animals↗
PubMed2006

Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to SARS-CoV.

Severe acute respiratory syndrome (SARS)-coronavirus (SARS-CoV) is the causative agent of SARS. The S protein of SARS-CoV is a major target for neutralizing antibodies (Nabs) in infected patients. We developed a neutralization assay using a recombinant vesicular stomatitis virus (VSV) bearing SARS-CoV-S protein (VSV-SARS-St19). A total of 56 serum samples collected from 22 healthcare workers in the Hanoi French Hospital during the SARS epidemic in 2003 were evaluated and compared to the conventional neutralizing assay using infectious SARS-CoV. The results of the neutralization assay using VSV-SARS-St19 pseudotype showed good correlations with those using infectious SARS-CoV. The newly developed neutralization assay was more sensitive to low antibody titers in serum samples. Thus, the VSV-SARS-St19 is a useful tool for detecting Nabs against SARS-CoV.

Animals↗
PubMed2006

Inhibition of cell proliferation by SARS-CoV infection in Vero E6 cells.

Severe acute respiratory syndrome (SARS) is caused by SARS-coronavirus (SARS-CoV). Infection of Vero E6 cells with SARS-CoV inhibits cell proliferation. Our previous study indicated that Akt, which is poorly phosphorylated in confluent cultures of Vero E6 cells, is phosphorylated and then dephosphorylated upon infection by SARS-CoV. In the present study, we showed that a serine residue of Akt was phosphorylated in Vero E6 cells in subconfluent culture and that Akt was dephosphorylated rapidly after SARS-CoV infection without up-regulation of its phosphorylation. Phosphorylation of glycogen synthase kinase-3beta, which is one of the downstream targets of Akt, was prevented in SARS-CoV-infected cells. However, treatment with glycogen synthase kinase-3beta small interfering RNA indicated that the glycogen synthase kinase-3beta signaling pathway was not related to inhibition of cell proliferation. Treatment of Vero E6 cells with the phosphatidylinositol 3'-kinase/Akt inhibitor, LY294002, which induces dephosphorylation of Akt, inhibited cell proliferation. As shown in our previous studies, apoptosis occurred in virus-infected cells within 18 h postinfection. Cellular mRNA transcription, which was reported to be up-regulated in SARS-CoV-infected Caco-2 cells, was not up-regulated in virus-infected Vero E6 cells, partially as a result of apoptosis. These results suggested that inhibition of cell proliferation is regulated by both the phosphatidylinositol 3'-kinase/Akt signaling pathway and by apoptosis in SARS-CoV-infected Vero E6 cells. This is the first study to analyze SARS-CoV-induced cell growth inhibition.

Animals↗
PubMed2006

LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox.

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.

Animals↗
PubMed2006

Marburgvirus nucleoprotein-capture enzyme-linked immunosorbent assay using monoclonal antibodies to recombinant nucleoprotein: detection of authentic Marburgvirus.

There have recently been large outbreaks of Marburg hemorrhagic fever (MHF) caused by Marburgvirus (MARV) in the Democratic Republic of Congo and Angola. The development of reliable diagnostic systems for MHF is urgently needed. An antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) using either of the two monoclonal antibodies (2A7 and 2H6) produced by immunizing mice with recombinant nucleoprotein of MARV was described (Journal of Medical Virology, 76, 111-118, 2005). In the present study, it was revealed that the Ag-capture ELISA specifically detected authentic MARV antigen and that the sensitivity of the Ag-capture ELISA was at a level similar to that of reverse-transcription polymerase chain reaction. These results suggest that the Ag-capture ELISA using the monoclonal antibodies, 2A7 and 2H6, is applicable to the diagnosis of MHF.

Animals↗
PubMed2005

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells.

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.

Animals↗
PubMed2005

Inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (SARS)-associated coronavirus.

The activity of inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, mizoribine and ribavirin, against severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) was determined by plaque reduction and yield reduction assays. Mizoribine and ribavirin selectively inhibited replication of SARS-CoV. The 50% inhibitory concentration (IC50) of mizoribine for SARS-CoV Frankfurt-1 and SARS-CoV HKU39849, as determined by plaque reduction was 3.5 microg/ml and 16 microg/ml, respectively, and the IC50 of ribavirin for SARS-CoV Frankfurt-1 and SARS-CoV HKU39849 was 20 microg/ml and 80 microg/ml, while the 50% cytotoxic concentration of mizoribine and ribavirin for Vero E6 cells exceeded 200 microg/ml. In a yield reduction assay, mizoribine (10 microg/ml) and ribavirin (40 microg/ml) inhibited the replication of SARS-CoV and reduced the infectious SARS-CoV titers to one-tenth or less. Mizoribine inhibited replication of SARS-CoV more strongly than ribavirin. However, neither drug could completely inhibit replication of SARS-CoV even at concentrations up to 100 microg/ml.

Animals↗
PubMed2005

Recombinant nucleoprotein-based serological diagnosis of Crimean-Congo hemorrhagic fever virus infections.

An enzyme-linked immunosorbent assay (ELISA) using recombinant nucleoprotein (rNP) was reported for the detection of immunoglobulin G (IgG) antibodies to Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV). The immunoglobulin M (IgM)-capture ELISA was developed for the diagnosis of CCHFV infections, using CCHFV rNP as an antigen. These newly developed assays were applied to a study of a CCHF-outbreak and evaluated with sera collected from patients diagnosed as having CCHF by positive reverse transcription-polymerase chain reaction (RT-PCR) and by detection of IgG response. IgM antibodies to CCHFV were detected in 10 of the 13 patients. IgM antibodies to the rNP of CCHFV were detected by the CCHFV rNP-based IgM-capture ELISA in all 6 patients in whom IgG responses were demonstrated, while it was not detected in the 10 patients in whom IgG responses were not demonstrated. Furthermore, the IgM antibodies were detected in 6 of the 61 residents living a CCHF endemic area during the endemic season, while it was not detected in any of the 48 Japanese residents that had never visited the CCHF endemic area. It is concluded that this newly developed CCHFV rNP-based IgM-capture ELISA is a useful method for the diagnosis of CCHFV infections.

Adolescent↗
PubMed2005

Characterization of monoclonal antibodies to Marburg virus nucleoprotein (NP) that can be used for NP-capture enzyme-linked immunosorbent assay.

After the first documented outbreak of Marburg hemorrhagic fever identified in Europe in 1967, several sporadic cases and an outbreak of Marburg hemorrhagic fever have been reported in Africa. In order to establish a diagnostic system for Marburg hemorrhagic fever by the detection of Marburg virus nucleoprotein, monoclonal antibodies to the recombinant nucleoprotein were produced. Two clones of monoclonal antibodies, MAb2A7 and MAb2H6, were efficacious in the antigen-capture enzyme-linked immunosorbent assay (ELISA). At least 40 ng/ml of the recombinant nucleoprotein of Marburg virus was detected by the antigen-capture ELISA format. The epitope of the monoclonal antibody (MAb2A7) was located in the carboxy-terminus of nucleoprotein from amino acid position 634 to 647, while that of the MAb2H6 was located on the extreme region of the carboxy-terminus of the Marburg virus nucleoprotein (amino acid position 643-695). These monoclonal antibodies strongly interacted with the conformational epitopes on the carboxy-terminus of the nucleoprotein. Furthermore, these two monoclonal antibodies were reacted with the authentic Marburg virus antigens by indirect immunofluorescence assay. These data suggest that the Marburg virus nucleoprotein-capture ELISA system using the monoclonal antibodies is a promising technique for rapid diagnosis of Marburg hemorrhagic fever.

Amino Acid Sequence↗
PubMed2005

Antigen-capture enzyme-linked immunosorbent assay for the diagnosis of crimean-congo hemorrhagic fever using a novel monoclonal antibody.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed for the diagnosis of Crimean-Congo hemorrhagic fever (CCHF) using a novel monoclonal antibody, 1B7, to the recombinant nucleoprotein (rNP) of CCHF virus (CCHFV) Chinese strain 8402. This ELISA detected at least 2 ng/100 microl of CCHFV rNP of 8402 and of the Nigeria strain Ibr 10200, and also detected authentic nucleoproteins (NP) of Chinese strains. Although the sensitivity of the ELISA was lower than that of nested reverse transcription polymerase chain reaction (RT-PCR), it was able to detect nucleoproteins in acute sera of CCHF patients. The presence of anti-CCHFV IgG decreased the sensitivity of the ELISA, possibly due to competition with 1B7, and this would tend to limit application of the ELISA. However, the method may be useful for the diagnosis of CCHF patients in the acute stage of illness, especially in laboratories not equipped with RT-PCR testing capabilities.

Animals↗