New Protocol for Predicting the Ligand-Binding Site and Mode Based on the 3D-RISM/KH Theory.

An efficient algorithm to find the binding position and mode of small ligands bound at an active site of protein is proposed based on the spatial distribution function (SDF) obtained from the three-dimensional reference interaction site model (3D-RISM) theory with the Kovalenko-Hirata (KH) closure relation. The ligand examined includes hydrophobic, acidic, and basic molecules and zwitterions. Eighteen different types of proteins, which serve as targets for those ligands, are selected to examine the robustness of the algorithm. An imaginary atom, referred to as an "anchor site", is defined at the center of geometry of a ligand molecule that serves as a center for searching the binding position and mode of the ligand molecule in the translational and rotational spaces. The probable binding sites (PBSs) are identified based on the SDFs of the ligand molecules around the protein, and the PBS is ranked according to the peak height of SDF. The deviations from the mean height of the peak values of SDFs for 50 PBSs are analyzed based on the z-score, which is a measure of prominence of the site. The PBS found at the closest distance from the anchor site of the crystal structure is referred to as the "nearest site". The orientation of the ligand molecule at each PBS is explored by changing the Euler angles, and the most probable binding mode is determined based on the superposition approximation. The binding position of ligand molecules is successfully predicted as one of the distinct peaks in SDF of the anchor site, with a few exceptions. The binding mode of the ligand molecule predicted based on the superposition approximation is consistent with the X-ray crystal structure in nine systems, a half of the systems investigated. The significance of the results is discussed in detail. An application of the new protocol to fragment-based drug discovery is suggested.

Emergence of infectious malignant thrombocytopenia in Japanese macaques (Macaca fuscata) by SRV-4 after transmission to a novel host.

We discovered a lethal hemorrhagic syndrome arising from severe thrombocytopenia in Japanese macaques kept at the Primate Research Institute, Kyoto University. Extensive investigation identified that simian retrovirus type 4 (SRV-4) was the causative agent of the disease. SRV-4 had previously been isolated only from cynomolgus macaques in which it is usually asymptomatic. We consider that the SRV-4 crossed the so-called species barrier between cynomolgus and Japanese macaques, leading to extremely severe acute symptoms in the latter. Infectious agents that cross the species barrier occasionally amplify in virulence, which is not observed in the original hosts. In such cases, the new hosts are usually distantly related to the original hosts. However, Japanese macaques are closely related to cynomolgus macaques, and can even hybridize when given the opportunity. This lethal outbreak of a novel pathogen in Japanese macaques highlights the need to modify our expectations about virulence with regards crossing species barriers.

Dynamics of cellular immune responses in the acute phase of dengue virus infection.

In this study, we examined the dynamics of cellular immune responses in the acute phase of dengue virus (DENV) infection in a marmoset model. Here, we found that DENV infection in marmosets greatly induced responses of CD4/CD8 central memory T and NKT cells. Interestingly, the strength of the immune response was greater in animals infected with a dengue fever strain than in those infected with a dengue hemorrhagic fever strain of DENV. In contrast, when animals were re-challenged with the same DENV strain used for primary infection, the neutralizing antibody induced appeared to play a critical role in sterilizing inhibition against viral replication, resulting in strong but delayed responses of CD4/CD8 central memory T and NKT cells. The results in this study may help to better understand the dynamics of cellular and humoral immune responses in the control of DENV infection.

CD16(+) natural killer cells play a limited role against primary dengue virus infection in tamarins.

CD16 is a major molecule expressed on NK cells. To directly assess the role of natural killer (NK) cells in dengue virus (DENV) infection in vivo, CD16 antibody-treated tamarins were inoculated with a DENV-2 strain. This resulted in the transient depletion of CD16(+) NK cells, whereas no significant effects on the overall levels or kinetics of plasma viral loads and antiviral antibodies were observed in the treated monkeys when compared to control monkeys. It remains elusive whether the CD16(-) NK subpopulation could play an important role in the control of primary DENV infection.

Needle-free jet injection of small doses of Japanese encephalitis DNA and inactivated vaccine mixture induces neutralizing antibodies in miniature pigs and protects against fetal death and mummification in pregnant sows.

Japanese encephalitis (JE) virus causes abortion and stillbirth in swine, and encephalitis in humans and horses. We have previously reported that immunogenicity of a DNA vaccine against JE was synergistically enhanced in mice by co-immunization with a commercial inactivated JE vaccine (JEVAX) under a needle-free injection system. Here, we found that this immunization strategy was also effective in miniature pigs. Because of the synergism, miniature pigs immunized twice with a mixture of 10 µg of DNA and a 1/100 dose of JEVAX developed a high neutralizing antibody titer (1:190 at 90% plaque reduction assay). Even using 1 µg of DNA, 3 of 4 pigs developed neutralizing antibodies. Following challenge, all miniature pigs with detectable neutralizing antibodies were protected against viremia. Pregnant sows inoculated with 10 or 1 µg of DNA mixed with JEVAX (1/100 dose) developed antibody titers of 1:40-1:320. Following challenge, fetal death and mummification were protected against in DNA/JEVAX-immunized sows.

A novel genetic marker to differentiate feline herpesvirus type 1 field isolates.

Five recent field isolates of feline herpesvirus type 1 (FHV-1) were compared by digestion with a restriction endonuclease, SalI or MluI. The SalI digestion showed a potentially useful difference in one isolate 00-035 that had an approximately 3.0 kbp fragment instead of a 2.6 kbp fragment in the other strains. After cloning the 3.0 and 2.6 kbp fragments, the nucleotide sequences were analyzed. The result showed that the 3.0 kbp fragment of 00-035 included a complete open reading frame of the herpes simplex virus 1 (HSV-1) homologue of the UL17 gene and a 5'-part of UL16 gene and that only one nucleotide substitution was found in the 5'-region of UL17 gene where the SalI site of the 2.6 kbp fragment locates. Based on these nucleotide sequences, two PCR primers were designed to amplify the region around the SalI site in the UL17 gene and the PCR was carried out using 78 field isolates from various parts of Japan. The SalI digestion of the PCR products revealed an interesting profile in that the genotype without the SalI site in UL17 gene was dominant in Tottori and Yamagata prefectures (69% and 75%, respectively) but minor in the other regions of Japan (0-10%). These results suggest that the SalI digestion method described in the present study can be used as a genetic marker to differentiate some FHV-1 field isolates and this is the first report that showed different distributions of FHV-1 genotypes using the novel genetic marker.

Identification of another B-cell epitope in the type-specific region of equine herpesvirus 4 glycoprotein G.

Recently, a novel 12-mer B-cell epitope, MKNNPIYSEGSL, in the type-specific region of equine herpesvirus 1 (EHV-1) glycoprotein G (gG) was identified and used as an antigen for enzyme-linked immunosorbent assay (Maeda et al., J. Clin. Microbiol. 42:1095-1098, 2004). Although our prototype strain, TH20p, possesses two repeat sequences containing the B-cell epitope, the EHV-4 NS80567 strain has two repeat sequences that are not identical. One repeat sequence stretch contained the B-cell epitope, while the other contained the 11-mer, MKNNPVYSESL (underlining indicates a different amino acid). In this study, heterogeneity of the type-specific region was compared among Japanese EHV-4 isolates. The 11-mer peptide, MKNNPVYSESL, specifically reacted with sera from horses naturally infected with EHV-4 but not with sera from horses experimentally infected with EHV-4 TH20p. The 11-mer peptide may be another B-cell epitope in the type-specific region.

Development of an equine herpesvirus type 4-specific enzyme-linked immunosorbent assay using a B-cell epitope as an antigen.

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol. 67:6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci. 60:1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci. 62:687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione S-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.

Genetic rearrangements in the gC gene of the feline herpesvirus type 1.

In the field isolate, 91-58, of feline herpesvirus type 1 (FHV-1), one of the major immunogenic proteins was found to have different molecular masses of 75 and 130 kDa from those in the other field isolates (Maeda et al., J Vet Med Sci 57, 147-150, 1995). Immunoblot analysis using monoclonal antibodies (MAbs) indicated that the protein is glycoprotein C (gC). The gC gene of 91-58 was amplified by polymerase chain reaction (PCR) and shown to have an inserted fragment of approximately 160 base pairs (bp). Restriction endonuclease analysis of the PCR product with various restriction enzymes was carried out, indicating that the insertion located within 262 bp between Eco RV and DraI sites. Nucleotide sequence analysis indicated that the inserted fragment was 156 bp encoding 52 amino acids and composed repeat sequences. Next, five recent isolates were also examined by immunoblot analysis using anti-FHV-1 cat serum or MAbs. The result showed that one isolate, 98-064, also had the gC with different molecular weights. PCR and nucleotide sequence analyses indicated that 98-064 had an inserted sequence of 78 bp at the corresponding region identified in the gC gene of 91-58, although the inserted sequence was different from that of 91-58. These results indicated that some of FHV-1 isolates had the genetic rearrangements in the gC gene and detection of such mutations would be useful for differentiation among FHV-1 field isolates.

Experimental infection of recent field isolates of feline herpesvirus type 1.

Two field isolates of feline herpesvirus type 1 (FHV-1) designated as 00-015 and 00-035, were obtained from cats diagnosed as feline viral rhinotracheitis (FVR) in Japan. To analyze the character of recent FHV-1, these two isolates and our laboratory strain C7301 were inoculated experimentally to specific-pathogen-free cats. Although all cats showed typical FVR symptoms, more severe clinical symptoms were observed on cats infected with the isolates 00-015 and 00-035 compared with those of C7301-infected cats. Severe ocular lesions including conjunctivitis were found in the cats infected with the isolates, indicating that the recent FHV-1 has a potential to induce severe FVR symptoms including ocular lesions.

IgG antibody subclass response against equine herpesvirus type 4 in horses.

In this study, IgG subclass responses against equine herpesvirus type 4 (EHV-4) were examined by enzyme-linked immunosorbent assay (ELISA) using a type-specific region of EHV-4 glycoprotein G (gG). ELISA using sera collected from horses experimentally infected with EHV-4 revealed that IgGa and IgGb antibodies were detected at high level, but IgGc and IgG(T) antibody responses were detected at low level or were undetectable. The IgGa antibody response reached its peak on day 10 post-infection, and then dropped. The IgGb antibody response reached its maximum level on day 12 post-infection, and then the level was sustained during at least 28 days after infection. Forty healthy racehorses that had already been infected with EHV-4 possessed antibody against EHV-4. Although IgGa antibodies specific for EHV-4 were not detected in any horses, IgGb antibodies were detected and the levels correlated with total IgG antibodies against EHV-4 gG. The results suggest that EHV-4-specific IgGa and IgGb antibodies are induced in EHV-4-infected horses, and that IgGb antibody, but not IgGa, is long lasting.