Naohiko Hattori

Anti-influenza Virus Activities of 2-Alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides

A series of 2-alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides and related compounds were synthesized and their antiviral activities against human influenza A virus were assessed. Studies of the structure-activity relationships revealed the strongest antiviral activity when position-5 of the isoxazoline ring was substituted with a tert-butyl group. When the alkoxyimino moiety was substituted with a methyl, ethyl, isopropyl or allyl group, good antiviral activity was obtained. Among the geometrical isomers at the oxime moiety, the E-isomers were more active than the Z-isomers. Among the compounds examined, (E)-2-allyloxyimino-2-cyano-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)acetamide (1j) was the most active inhibitor with an EC(50) of 3 microg/mL in vitro.

Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin.

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1–14, 1999). Stachyflin‐resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA‐expressing cells and octadecylrhodamine (R18)‐labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin‐resistant phenotype of HA protein. The molecular mechanism of anti‐HA conformational change activity of Stachyflin is discussed.

Maturation of human immunodeficiency virus, strain LAV, in vitro

The budding process of human immunodeficiency virus (HIV), strain LAV, starts with the formation of a crescent electron-dense layer directly underneath the cell membrane of infected CCRF-CEM cells. After completion of the formation of the circle of inner dense layer, immature virions with an electron-lucent center are released from the cells. Serial thin sections and stereo observation of thick sections showed that most of the immature virions adjacent to the cell surface had already come off the cell and some still had very thin connections to the cell. However, on rare occasions, virions at an intermediate stage between immature stage and mature virions with bar-shaped electron-dense cores were observed. Virions with dense cores were never observed to be connected to the cell surface. These observations support the idea that the last step of the maturation of HIV occurs outside the cell and that the electron-dense core seems to develop by rearrangement and dispersion of the substance of the inner dense layer of immature virions.

Oligonucleotide Fingerprint Analysis of Coxsackievirus A10 Isolated in Japan

Eight coxsackievirus A10 strains isolated in 1978 and in 1981 and 1982 from patients with hand, foot-and-mouth disease and with herpangina at a dispensary in Matsue city were compared by RNA fingerprinting techniques. The oligonucleotide maps of the four 1978 isolates were related to each other by 85 to 93% with respect to their large T1 oligonucleotides. In contrast, the oligonucleotide maps of the four 1981 and 1982 isolates were very different from each other. Co-electrophoresis experiments revealed that the 1981 and 1982 strains shared only 17 to 34% of their large oligonucleotides. In addition, some large oligonucleotides were found in most of the fingerprint maps of isolates from 1978 to 1982, suggesting that there are regions in the genome of coxsackievirus A10 which are not subject to mutational changes.

Budding Process and Fine Structure of Lymphadenopathy-associated Virus (LAV)

The budding process and fine structure of lymphadenopathy‐associated virus (LAV), were studied by indirect immunofluorescence (IF) and electron microscopy (EM). By IF, LAV antigen was seen to be distributed focally within infected CCRF‐CEM cells. Consistent with this finding, electron micrographs showed that LAV particles occurred in a focally aggregated state in a restricted area of the surface of the infected cells. LAV particles possessed bar‐shaped, dense and central or eccentric cores. In addition, two or more cores were occasionally observed in one virus particle, or the cores were sometimes absent when thin sections were examined. The envelope of the virus particles had an irregular structure, although LAV particles were approximately spherical.

Replication of Human Immunodeficiency Virus in Two T-Cell Lines and Their Application as Antigens for Immunofluorescence

Two T‐cell lines, TALL‐1 and CCRF‐CEM, were infected with human immunodeficiency virus (HIV), strain LAV, to explore the time course of the appearance of various virus specific antigens, and to establish an antibody assay system by indirect immunofluorescence (IF). These cells were infected with LAV at two different input multiplicity of infection (MOI). Antigens were tested by Western blot analysis (WB) and IF. Antigens for WB were extracted from the infected cells at various times after infection, but pooled sera of American HIV carriers could not recognize gp41 or gp160. Antigen expression was highest in CCRF‐CEM, but, as the antigen for IF, TALL‐1 infected at the MOI of 8.0 was the most suitable 7 days after infection, because it includes a fairly large number of uninfected cells, which served as the internal control.