Takeshi Haga

Relationship between pathogenicity for humans and stx genotype in Shiga toxin-producing Escherichia coli serotype O157

To examine the reason why people infected with Shiga toxin (Stx) producing Escherichia coli (STEC) O157 strains develop varying clinical manifestations, 65 STEC O157 isolates originating from 64 different occurrences of infection in Miyazaki Prefecture in 2001–2003 and their 79 infected individuals were analyzed by stx genotyping, quantitative analysis of reversed passive latex agglutination (RPLA), genomic DNA analysis using pulsed-field gel electrophoresis (PFGE), and clinical manifestations. The isolates were found to carry the following stx genes: stx2vha alone (60.0%), stx1/stx2 (27.7%), stx1/stx2vha (6.1%), stx2 alone (3.1%), and stx2/stx2vha (3.1%). No strain carried the stx1 gene alone. STEC strains carrying stx2 were more frequently associated with clinical manifestations of hemolytic-uremic syndrome (HUS) or bloody diarrhea than those carrying stx2vha. Clusters of PFGE banding patterns were correlated well with the stx genotypes. We conclude that stx genotype is one of the important factors of clinical outcome of STEC O157 infection and that pathogenicity for humans was higher in the stx2 genotype strains than in the stx2vha genotype strains, as reported previously by other researchers. Further, we newly found that four clusters identified by PFGE using restriction enzyme XbaI, stx genotypes and clinical manifestations were well correlated with each other.

Characterization of novel bovine papillomavirus type 12 (BPV-12) causing epithelial papilloma

Bovine papillomavirus type 12 (BPV-12, putative type BAA1) was detected in epithelial papilloma located on the tongue of an infected cow. Then, the whole genome was sequenced, and phylogenetic analysis illustrated that it should be classified as a member of the genus Xipapillomavirus. The viral genome is 7197 base pairs in length and contains five early ORFs (E1, E2, E4, E7 and E8), three late ORFs (L1, L2 and L3), and a long control region that possesses replication regulatory elements. Meanwhile, mRNA of each gene was detected in the papilloma sample. The papilloma was identified as epithelial papilloma by histological and immunohistochemical examination. Based on the genome information and pathological properties, BAA1 was designated as BPV-12 in this study.

Detection of genetic changes in anal intraepithelial neoplasia (AIN) of HIV-positive and HIV-negative men.

Summary: Compared with HIV‐negative individuals, HIV‐positive individuals have a higher prevalence of anogenital human papillomavirus (HPV) infection, as well as a higher incidence of HPV‐associated anal cancer. Little is currently known of chromosomal changes occurring in anal intraepithelial neoplasia (AIN), the probable precursor to anal cancer. Genetic changes in AIN were characterized by comparative genomic hybridization (CGH) in a study of samples obtained from 19 HIV‐positive and 11 HIV‐negative men. The proportion with genetic changes significantly increased with the severity of the histopathologic grade with none diagnosed as (0%) AIN 1; 5 of 17 (29%) as AIN 2; and 5 of 9 (56%) AIN 3 showing genetic changes (p = .02). This correlation was also found in study subjects who had multiple biopsies with different grades of pathology concurrently or serially over time. The most common regional DNA copy number change was gain mapped to chromosome arm 3q (12% of AIN 2 and 33% of AIN 3). This alteration was previously reported to be commonest alteration in cervical cancer, which suggests a common molecular pathway for these two HPV‐associated anogenital neoplasias.

Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus

This paper describes the evaluation of four novel real-time multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for rapid and sensitive diagnosis of foot-and-mouth disease (FMD). In order to overcome the genetic diversity of FMD viruses (FMDV), these multiplex RT-LAMP assay pairs were established by combining four newly designed primer sets with two primer sets that had been previously published. Using a real-time turbidimeter to detect amplification products and a panel of 300 samples collected throughout the world over a 78-year period, the performance of the multiplex RT-LAMP assays was compared with a FMDV-specific real-time RT-PCR assay. The most successful of the four multiplex RT-LAMP assays achieved a diagnostic sensitivity and specificity of 98.0% and 98.1%, and did not falsely detect FMDV in known negatives or samples containing swine vesicular disease virus, vesicular stomatitis virus or vesicular exanthema of swine virus. Furthermore, the analytical sensitivity of this multiplex RT-LAMP assay was at least as good as the individual component RT-LAMP tests. This is the first report of the development of a multiplex RT-LAMP to accommodate the high sequence variability encountered in RNA virus genomes and these results support the use of RT-LAMP as a cost-effective tool for simple diagnosis of FMD.

Specific detection and differentiation of two subspecies of Fusobacterium necrophorum by PCR

The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed.

A new approach to AIDS research and prevention : The use of gene-mutated HIV-1/SIV chimeric viruses for anti-HIV-1 live-attenuated vaccines

The lack of a suitable animal model is a major obstacle to developing anti‐HIV‐1 vaccines. We successfully generated an SIVmac/HIV‐1 chimeric virus (SHIV) (designated as NM‐3rN) that contains the HIV‐1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM‐3rN, we developed an evaluation system for anti‐HIV‐1 Env‐targeted vaccines. For the purpose of making the vaccine, a series of gene‐mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long‐term anti‐virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM‐3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV‐1 but also against an antigenically different HIV‐1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV‐MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene‐deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide‐range immunity against HIV‐1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti‐HIV‐1 live vaccines for humans.

stx genotype and molecular epidemiological analyses of Shiga toxin-producing Escherichia coli O157:H7/H− in human and cattle isolates

The relationship between human diseases caused by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 strains and O157 strains isolated from cattle was investigated in an area where stockbreeding is prolific. For this purpose, the stx genotypes, the molecular epidemiological characteristics of 268 STEC O157 strains including 211 human-origin strains and 57 cattle-origin strains, and clinical manifestations of 210 STEC-infected people were analyzed. Of 211 human-origin strains, 92 strains (44%) were of the stx1/stx2 genotype, and 74 strains (35%) were of the stx2c genotype. Most of the people infected with stx2c genotype strains presented no symptoms or mild symptoms such as slight diarrhea, except for 3 patients with bloody diarrhea. Of the 57 cattle-origin strains, 27 strains (47%) were of the stx2c genotype and 17 strains (30%) were of the stx1/stx2 genotype. Pulsed-field gel electrophoresis (PFGE) and insertion sequence (IS) analysis demonstrated that 11 isolates (41%) of the 27 cattle isolates of the stx2c genotype had high homology (>95% identity) with human isolates. These results suggest that some genetic patterns of the stx2c genotype strains might be preserved in cattle or their surrounding environment for several years, and during these periods, they might have opportunities to infect people through various routes. Because of the mild virulence of the stx2c genotype strains, they seemed to be transmitted asymptomatically from cattle to humans and then spread from person to person. It may be a public health concern. Further, they occasionally cause severe symptoms in humans; therefore, caution is warranted for infections by stx2c genotype O157 strains, in addition to stx2-possessing genotype O157 strains.

Identification of a novel equine infectious anemia virus field strain isolated from feral horses in southern Japan.

Although equine infectious anemia (EIA) was described more than 150 years ago, complete genomic sequences have only been obtained from two field strains of EIA virus (EIAV), EIAV(Wyoming) and EIAV(Liaoning). In 2011, EIA was detected within the distinctive feral Misaki horse population that inhabits the Toi-Cape area of southern Japan. Complete proviral sequences comprising a novel field strain were amplified directly from peripheral blood of one of these EIAV-infected horses and characterized by nucleotide sequencing. The complete provirus of Miyazaki2011-A strain is 8208 bp in length with an overall genomic organization typical of EIAV. However, this field isolate possesses just 77.2 and 78.7 % nucleotide sequence identity with the EIAV(Wyoming) and EIAV(Liaoning) strains, respectively, while similarity plot analysis suggested all three strains arose independently. Furthermore, phylogenetic studies using sequences obtained from all EIAV-infected Misaki horses against known viral strains strongly suggests these Japanese isolates comprise a separate monophyletic group.

Construction of chimeric simian and human immunodeficiency viruses that produce interleukin 12

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for evaluating vaccine candidates against HIV-1 and for investigating the pathogenesis of HIV-1 in vivo. In addition, SHIVs are candidates for a vaccine against HIV-1 because attenuated SHIVs can induce long-lasting anti-HIV-1 Env humoral and cell-mediated immunity in monkeys without AIDS-like diseases. In this study, we inserted IL-12 genes in a nef-deleted SHIV to increase the ability of the SHIV to induce cell-mediated immunity against HIV-1. The SHIV vector was constructed by deleting the nef gene and replacing it with restriction enzyme sites. Since IL-12 consists of two subunit genes, p35 and p40, SHIVs with one or both of these genes were constructed. SHIVs with either one of the subunit genes could replicate without a deletion of the inserted gene, but SHIVs with two subunit genes replicated poorly and the inserted genes were rapidly deleted. Production of IL-12 was detected when both of the single-subunit SHIVs were coinfected. The production of IL-12 by the coinfection reached 800 pg/ml, and IL-12 was detected after serial passage in cell cultures, although this amount of IL-12 heterodimer was 150-1500 times less than that of the p40 subunits. These IL-12-producing SHIVs are candidates for a live-attenuated vaccine to induce effective cellular immunity against HIV-1.

Gene-mutated HIV-1/SIV chimeric viruses as AIDS live attenuated vaccines for potential human use

To develop an AIDS vaccine for human use as well as a suitable animal model for AIDS research, we constructed a series of HIV-1/SIVmac chimeric viruses (SHIVs). We successfully generated a SHIV (designated as NM-3rN) having the HIV-1 env gene, which enabled the evaluation of the efficacy of HIV-1 Env-targeted vaccines in macaque monkeys instead of chimpanzees. Two NM-3rN derivatives (NM-3 and NM-3n) induced long-term anti-virus immunities without manifesting the disease. The monkeys vaccinated with NM-3 or NM-3n became resistant to a challenge inoculation with NM-3rN. Serum from a monkey vaccinated with NM-3 neutralized not only the parental HIV-1 (NL432), but also an antigenically different HIV-1 (MN). In vivo experiments confirmed the heterologous protection against an SHIV having the HIV-1 (MN) env. In addition to specific immunity including neutralizing antibodies and cytotoxic T lymphocyte activity, nonspecific immunity such as natural killer activity is associated with this protection. These data suggest that the live vaccine has the ability to protect individuals against various types of HIVs. These SHIVs should contribute to the development of future anti-HIV-1 live vaccines in humans.