Hiroko Minagawa

Measles Viruses on Throat Swabs from Measles Patients Use Signaling Lymphocytic Activation Molecule (CDw150) but Not CD46 as a Cellular Receptor

ABSTRACT Both CD46 and signaling lymphocytic activation molecule (SLAM) have been shown to act as cellular receptors for measles virus (MV). The viruses on throat swabs from nine patients with measles in Japan were titrated on Vero cells stably expressing human SLAM. Samples from all but two patients produced numerous plaques on SLAM-expressing Vero cells, whereas none produced any plaques on Vero cells endogenously expressing CD46. The Edmonston strain of MV, which can use either CD46 or SLAM as a receptor, produced comparable titers on these two types of cells. The results strongly suggest that the viruses in the bodies of measles patients use SLAM but probably not CD46 as a cellular receptor.

SLAM (CD150)-Independent Measles Virus Entry as Revealed by Recombinant Virus Expressing Green Fluorescent Protein

ABSTRACT Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.

Virus Entry Is a Major Determinant of Cell Tropism of Edmonston and Wild-Type Strains of Measles Virus as Revealed by Vesicular Stomatitis Virus Pseudotypes Bearing Their Envelope Proteins

ABSTRACT The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVΔG*). MV glycoproteins were efficiently incorporated into VSVΔG*, producing the VSV pseudotypes. VSVΔG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVΔG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVΔG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVΔG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.

V Domain of Human SLAM (CDw150) Is Essential for Its Function as a Measles Virus Receptor

ABSTRACT Human signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been shown to be a cellular receptor for measles virus (MV). Chinese hamster ovary cells transfected with a mouse SLAM cDNA were not susceptible to MV and the vesicular stomatitis virus pseudotype bearing MV envelope proteins alone, indicating that mouse SLAM cannot act as an MV receptor. To determine the functional domain of the receptor, we tested the abilities of several chimeric SLAM proteins to function as MV receptors. The ectodomain of SLAM comprises the two immunoglobulin superfamily domains (V and C2). Various chimeric transmembrane proteins possessing the V domain of human SLAM were able to act as MV receptors, whereas a chimera consisting of human SLAM containing the mouse V domain instead of the human V domain no longer acted as a receptor. To examine the interaction between SLAM and MV envelope proteins, recombinant soluble forms of SLAM were produced. The soluble molecules possessing the V domain of human SLAM were shown to bind to cells expressing the MV hemagglutinin (H) protein but not to cells expressing the MV fusion protein or irrelevant envelope proteins. These results indicate that the V domain of human SLAM is necessary and sufficient to interact with the MV H protein and allow MV entry.

Induction of the measles virus receptor SLAM (CD150) on monocytes.

Wild-type strains of measles virus (MV) isolated in B95a cells use the signalling lymphocyte activation molecule (SLAM; also known as CD150) as a cellular receptor, whereas the Edmonston strain and its derivative vaccine strains can use both SLAM and the ubiquitously expressed CD46 as receptors. Among the major target cells for MV, lymphocytes and dendritic cells are known to express SLAM after activation, but monocytes have been reported to be SLAM-negative. In this study, SLAM expression on monocytes was examined under different conditions. When freshly isolated from the peripheral blood, monocytes did not express SLAM on the cell surface. However, monocytes became SLAM-positive after incubation with phytohaemagglutinin, bacterial lipopolysaccharide or MV. Anti-SLAM monoclonal antibodies efficiently blocked infection of activated monocytes with a wild-type strain of MV. These results indicate that SLAM is readily induced and acts as a monocyte receptor for MV.

Human SCARB2-Dependent Infection by Coxsackievirus A7, A14, and A16 and Enterovirus 71

ABSTRACT Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.

Detection of human parechoviruses from clinical stool samples in Aichi, Japan.

ABSTRACT Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5′ untranslated region (5′UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema, “hand, foot, and mouth disease,” aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.

One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses

Abstract Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R 2 =0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses.

Experimental allergic encephalomyelitis in the Lewis rat ☆: A model of predictable relapse by cyclophosphamide

Relapse of experimental allergic encephalomyelitis (EAE) was achieved in Lewis rats by cyclophosphamide (CY). All rats, immunized with an emulsion of guinea pig spinal cord homogenate in complete Freunds adjuvant and treated with 100 mg/kg of CY 21 days postimmunization (pi), developed moderate to severe paralysis 9-14 days following CY injection. A second relapse was observed in 4 of 11 rats reinjected with CY 49 days pi. Histologically, focal mononuclear cell infiltration with or without demyelination of the white matter of the central nervous system was observed. Cyclophosphamide administration caused transient leukopenia and T-cell defect, the resolution of which coincided with relapse of clinical EAE. Lymphocyte proliferative responses to myelin basic protein (BP) and concanavalin A (Con A) and antibody titers to BP were preserved in CY-treated rats. Adoptive transfer of EAE to naive recipients with Con A-activated spleen cells from donors with CY-induced relapse was unsuccessful.

Amino acid substitutions at position 481 differently affect the ability of the measles virus hemagglutinin to induce cell fusion in monkey and marmoset cells co-expressing the fusion protein

Summary. The hemagglutinin (H) protein of the measles virus (MV) Edmonston strain induced cell fusion in Cos (monkey) and B95a (marmoset) cells, when co-expressed with the fusion (F) protein, whereas the H protein of the wild-type KA strain induced fusion in B95a cells, but not in Cos cells. Asparagine residue at position 481 of the KA H protein was replaced by various amino acids through site-directed mutagenesis. Substitution with tyrosine, which was found at position 481 of the Edmonston H protein, enabled the mutant KA H protein (N481Y) to induce cell fusion in Cos cells co-expressing the F protein, which could be completely blocked by anti-CD46 antibody. This mutant, however, did not cause CD46 downregulation, unlike the Edmonston H protein. The other H protein mutants (N481S, N481T, N481D, N481H, N481F) did not produce syncytia in Cos cells. On the other hand, all of the mutants retained the ability to induce cell fusion in B95a cells. Thus, while tyrosine at position 481 was indispensable for the MV H protein’s interaction with CD46, the residue at this position does not appear to be critically involved in the interaction with the receptor for wild-type strains present on B95a cells.