(Fig

(Fig.?3j). antibodies. Syncytial giant cells (SGCs) expressing viral and CD11b antigens were also detected among these inflammatory cells. These antigen\positive cells appeared in the subarachnoidal space prior to viral antigen spread into the brain parenchyma, indicating that viral encephalitis starts with the infection of infiltrating monocytes which express MHVR. Furthermore, the observation indicates that viral infection has cytopathic effects on the monocyte lineage, which plays Rabbit Polyclonal to 14-3-3 zeta a critical role in innate immunity, leading to the rapid spread of viruses during the early stage of infection. and experiments have shown that the infection of glial progenitors, oligodendrocytes and astrocytes, was blocked by pretreatment with the anti\MHVR antibody MAb CC1 or Ab\655, 12 which suggested that MHVR is essential for Pikamilone the initiation of MHV infection in the brain. In mixed neural cell cultures, cl\2 induced syncytia Pikamilone in most of the cells including neurons. 11 On the other hand, soluble\receptor\resistant (srr)7, which infects and spreads solely in an MHVR\dependent fashion, 13 infected a limited number of microglia marker\positive cells and infection did not spread, indicating that microglial cells are the initial target for MHV infection and that the wt spreads from initially infected microglia to a variety of cells in an MHVR\independent fashion, which suggested that MHVR is essential for the initiation of MHV infection in the brain. srr7 was isolated as an srr mutant, that is, the mutant virus is not neutralized with the soluble form of MHV receptor proteins. In general, the soluble receptor neutralizes virus infectivity. 14 , 15 , 16 , 17 This neutralization may be due to the ability of the soluble receptor to compete with the membrane\anchored receptor for virus binding. 18 Alternatively, the neutralization could be due to receptor\induced conformational changes of the envelope protein, which can no longer bind to the membrane\anchored receptor. 13 Although srr7 surface proteins show binding activity through the S1 region, an N\terminal subunit of the S protein, to the viral receptor, similar to that of wild\type cl\2 protein, srr7 is less virulent than cl\2. The reduced virulence and infectivity of srr7 compared with those of cl\2 could be attributed to the mutation of a single amino acid at position 1114 (Leu to Phe) in the S2 subunit of the viral surface protein, 19 which is not involved in receptor binding activity. This substitution in the S2 subunit could have brought about a structure less vulnerable to conformational changes in the S glycoprotein of srr7 virus compared to that of cl\2, 18 and causes reduced infectivity which occurs only in a receptor\dependent manner, leading to the reduced neurovirulence of srr7 compared to that of the wild\type, cl\2 virus. 13 The conformational changes in the S glycoprotein occur after binding of S1 to the receptor protein to induce fusogenic activities of the membrane\anchored fusion subunit, S2. 20 This paper Pikamilone focused on viral spread during the Pikamilone initial phase of infection, especially at 24?h post\inoculation (p.i.), to determine the events that facilitate the dense exposure of the viruses to the cell surfaces of receptor\negative cells including neurons, providing an opportunity for receptor\independent infection in the micro\environments of the brain. METHODS Animals and viruses Specific pathogen\free inbred BALB/c mice purchased from Charles River (Tokyo, Japan) were maintained according to the guidelines set by the committee of our university. At 5 weeks old, 21 and 46 mice were inoculated with 1??101C103 of JHMV cl\2 or srr7 virus, 21 respectively, as indicated Pikamilone in Figure?1, into the right frontal lobe under deep anesthesia. Infected mice were killed at intervals, and organs and peripheral blood were aseptically isolated from animals and stored at ?80C until titration. These organs in PBS were homogenized with a glass homogenizer and centrifuged at 620?for 15?min. The infectivity in the supernatants was measured by a plaque assay using DBT cells, as described previously. 21 DBT cells were grown in Dullbecco’s modified minimal essential medium (DMEM; Nissui, Tokyo, Japan) supplemented with 5% fetal bovine serum (FBS; Japan Bioserum,.