Seroepidemiology of Feline Chlamydiosis by Microimmunofluorescence Assay with Multiple Strains as Antigens

The prevalence of anti-chlamydia antibodies was examined in 232 cat sera collected in 1985 and from 1993 to 1995 from laboratories and veterinary hospitals located in 11 prefectures of Japan. The antibodies were determined by an indirect microimmunofluorescence test using six strains of feline Chlamydia: one strain each of avian- and guinea pig-derived C. psittaci and one strain each of C. pecorum, C. pneumoniae and C. trachomatis. Positive rates of IgG antibodies to chlamydiae were 34.4% in 1985 and 16.5–21.4% from 1993 to 1995. Positive rates of IgM antibodies to chlamydiae were 8.2% in 1985 and 6.6–14.3% from 1993 to 1995. Variations in antibody reactivity to the different feline strains were observed. The results suggest the wide prevalence of chlamydial infection in cats in Japan, and antigenic diversity in the feline strains of C. psittaci.     

RNA-RNA hybridization identifies a human rotavirus that is genetically related to feline rotavirus.

A human rotavirus AU228 strain which resembled the AU-1 strain (O. Nakagomi, T. Nakagomi, Y. Hoshino, J. Flores, and A. Z. Kapikian, J. Clin. Microbiol. 25:1159-1164, 1987) in its novel characteristics (that it belonged to subgroup I yet possessed a long RNA pattern) was compared with various human and animal strains by RNA-RNA hybridization in solution. This strain showed a high degree of homology with the AU-1 strain but not with either the Wa (subgroup II, long pattern) or the KUN (subgroup I, short pattern) strain, indicating the presence of an additional group of human rotaviruses that do not belong to either of the two human rotavirus families previously identified by RNA-RNA hybridization. It is of particular interest that the AU228 strain showed an unexpectedly high degree of homology with a feline rotavirus isolated recently in Japan. These results indicate transmission of a feline rotavirus to humans and suggest a role of animal rotaviruses in the evolution of human rotaviruses.     

Remarkable similarity in genome nucleotide sequences between the Schwarz FF-8 and AIK-C measles virus vaccine strains and apparent nucleotide differences in the phosphoprotein gene

The Schwarz FF-8 (FF-8) and AIK-C measles virus vaccine strains are currently used for vaccination in Japan. Here, the complete genome nucleotide sequence of the FF-8 strain has been determined and its genome sequence found to be remarkably similar to that of the AIK-C strain. These two strains are differentiated only by two nucleotide differences in the phosphoprotein gene. Since the FF-8 strain does not possess the amino acid substitutions in the phospho- and fusion proteins which are responsible for the temperature-sensitivity and small syncytium formation phenotypes of the AIK-C strain, respectively, other unidentified common mechanisms likely attenuate both the FF-8 and AIK-C strains.     

Establishment of cell strains from human urothelial carcinoma and their morphological characterization

Summary We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain. This work was supported in part by Grants 5319 and 5322 in aid for cancer research from the Ministry of Health and Welfare, Japan.     

Upregulation of surface feline CXCR4 expression following ectopic expression of CCR5: implications for studies of the cell tropism of feline immunodeficiency virus

Feline CXCR4 and CCR5 were expressed in feline cells as fusion proteins with enhanced green fluorescent protein (EGFP). Expression of the EGFP fusion proteins was localized to the cell membrane, and surface expression of CXCR4 was confirmed by using a cross-species-reactive anti-CXCR4 monoclonal antibody. Ectopic expression of feline CCR5 enhanced expression of either endogenous feline CXCR4 or exogenous feline or human CXCR4 expressed from a retrovirus vector, indicating that experiments investigating the effect of CCR5 expression on feline immunodeficiency virus (FIV) infection must be interpreted with caution. Susceptibility to infection with cell culture-adapted strains of FIV or to syncytium formation following transfection with a eukaryotic vector expressing an env gene from a cell culture-adapted strain of virus correlated with expression of either human or feline CXCR4, whereas feline CCR5 had no effect. In contrast, neither CXCR4 nor CCR5 rendered cells permissive to either productive infection with primary strains of FIV or syncytium formation following transfection with primary env gene expression vectors. Screening a panel of Ghost cell lines expressing diverse human chemokine receptors confirmed that CXCR4 alone supported fusion mediated by the FIV Env from cell culture-adapted viruses. CXCR4 expression was upregulated in Ghost cells coexpressing CXCR4 and CCR5 or CXCR4, CCR5, and CCR3, and susceptibility to FIV infection could be correlated with the level of CXCR4 expression. The data suggest that beta-chemokine receptors may influence FIV infection by modulating the expression of CXCR4.     

Two new species of Kazachstania that form ascospores connected by a belt-like intersporal body: Kazachstania zonata and Kazachstania gamospora

Four strains of ascomycetous yeasts were isolated from samples collected at two locations in southern Japan. The strains formed two warty ascospores that were joined together by an intersporal body appearing as a belt. Phylogenetic analysis of rRNA gene nucleotide sequences indicated that the strains represented two new and closely related species of the genus Kazachstania. Isolates of one of the species were from Miyazaki Prefecture and those of the other species were from the Iriomote Islands. Genetic separation of the two species was further confirmed by DNA-DNA reassociation, which gave values of 63.3-78.1%, and from interspecific crosses, which gave nonviable ascospores. On the basis of these data, the isolates from Miyazaki Prefecture are described as Kazachstania zonata sp. nov. [type strain NBRC 100504=CBS 10326, mating types NBRC 101821 (+), NBRC 101822 (-)], and the isolates from the Iriomote Islands are described as Kazachstania gamospora sp. nov. [type strain NBRC 11056=CBS 10328, mating types NBRC 101825 (+), NBRC 101826 (-)].     

Complete nucleotide sequence of the gene encoding VP4 of a human rotavirus (strain K8) which has unique VP4 neutralization epitopes.

In our previous study (K. Taniguchi, Y. Morita, T. Urasawa, and S. Urasawa, J. Virol. 62:2421-2426, 1987) in which the cross-reactive neutralization epitopes on VP4 of human rotaviruses were analyzed, one strain, K8, was found to bear unique VP4 neutralization epitopes. This strain, which belongs to subgroup II and serotype 1, was not neutralized by any of six anti-VP4 neutralizing monoclonal antibodies which reacted with human rotavirus strains of serotypes 1, 3, and 4 or serotypes 1 through 4. We determined the complete nucleotide sequence of the gene encoding VP4 of strain K8 by primer extension. The VP4 gene is 2,359 base pairs in length, with 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. The gene contains a long open reading frame of 2,325 bases capable of coding for a protein of 775 amino acids. When compared with those of other human rotaviruses, VP4 of strain K8 had an insertion of one amino acid after residue 135, as found in simian rotavirus strains, and in addition, it had a deletion of one amino acid (residue 575). The amino acid homology of VP4 of strain K8 and those of other virulent human rotaviruses was only 60 to 70%. This was unusual, since over 90% VP4 homology has been found among the other virulent human rotavirus strains. In contrast, the VP7 amino acid sequence of the K8 strain was quite similar (over 98% homology) to those of other serotype 1 human rotaviruses. Thus, the K8 strain appears to have a unique VP4 gene previously not described.     

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT‐PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9–92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1–65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7–92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5–86.5%) were higher than those with other mammal SaVs (20.7–58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV‐ and SaV‐infected dogs and cats in Japan. The findings suggest there are species‐specific circulations of NoV and SaV among dogs and cats, in Japan.     

Nocardia beijingensis,is a Pathogenic Bacterium to Humans: The First Infectious Cases in Thailand and Japan

Nocardia beijingensis, a recently established new species, is an isolate from soil in China. During our taxonomic studies on 450 nocardial clinical isolates in Thailand and Japan, 17 strains from Thailand and 1 strain from Japan were found to have a similar physiological characteristic to those of N. beijingensis, such as a drug susceptibility pattern to three antimicrobial agents. Our phylogenetic studies on these 18 strains by 16S rRNA gene sequence analysis confirmed that these strains belong to N. beijingensis species. Phylogenetically, these newly isolated N. beijingensis strains were found to be classified into two distinct clades: one is a Japanese clade and other is a Chinese clade, including a reference strain and 17 Thai strains. This is the first report of human infection due to N. beijingensis strains, and we propose that the bacterium be categorized as an opportunistic infectious group regardless of its original isolation from soil.     

丙型肝炎病毒(河北定州株)C33c基因的克隆、序列分析及在大肠杆菌中的表达

本研究通过RT-PCR从河北定州地区献血员血清中克隆了丙型肝炎病毒的C33c抗原基因,序列分析结果表明,该株的C33c基因和中国泰安株DNA同源性为91.0％,氨基酸序列同源性为92.2％;和HCV河北株的同源性分别为91.3％和96.2％;和日本JK1株的同源性分别为91.6％和94.8％。克隆的基因在大肠杆菌中得到表达和纯化,并可用作抗-HCV ELISA试剂的抗原组分。     

Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

Gene flow between sexual and asexual strains of parasitic wasps: a possible case of sympatric speciation caused by a parthenogenesis-inducing bacterium

Sympatric speciation is strictly defined as the emergence of two species from a population in which mating has been random with respect to the place of birth of the mating partners. Mathematical models have shown that sympatric speciation is possible, but very few examples have been documented in nature. In this article, we demonstrate that arrhenotokous and thelytokous strains of a parasitic wasp, Neochrysocharis formosa, speciated sympatrically through infection by a symbiotic bacterium Rickettsia for the following reasons: First, Rickettsia infection was detected in all of the thelytokous strains collected throughout Japan. Second, the arrhenotokous and thelytokous strains have been collected sympatrically. Third, crossing experiments between the two strains did not result in fertilized offspring. In addition, the two strains were genetically isolated at the nuclear and mitochondrial genes. Fourth, the two strains showed a sister relationship in nuclear 28S rRNA gene. Finally, thelytokous females treated with antibiotics produced Rickettsia-free male offspring of the same reproductive form as arrhenotokous females indicating that the thelytokous strain could have speciated sympatrically from an individual of the arrhenotokous strain.     

UL146 variability among clinical isolates of human cytomegalovirus from Japan

Human cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30%), while the success rate for UL145/UL147 gene was 18/56 strains (32%). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.     

Characterization of mumps virus isolated in saitama prefecture, Japan, by sequence analysis of the SH gene

Mumps virus (MuV) strains isolated from cerebrospinal fluid and throat swabs from patients in Saitama Prefecture and Tokyo, Japan, from 1997 to 2000 were examined by analyzing the SH gene nucleotide sequence (316-nt). Eighteen of the 20 strains studied were divided into three genotypes, recognized as B, G, and H in previous reports. Two genotypes (G and H) are believed to be new in Japan. Two of the 20 strains belonged to none of the previously reported genotypes (A-I), but were closely related to two known strains, MP94-H and Loug1/UK97. We propose that the two strains identified in this study together with the previously reported strains, MP94-H and Loug1/UK97, form a new genotype, designated J, based on the divergence of the SH gene nucleotide sequences between these four strains and other strains reported (genotypes A-I). Our results also suggest that more than two genotypes circulated in Saitama Prefecture from 1997 to 1999, but only one, genotype G, was in evidence in 2000. Genotype B was earlier reported as the predominant strain in Japan, but it became undetectable by the year 2000. These results provide important epidemiological data on mumps in Japan.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Mapping the domains of CD134 as a functional receptor for feline immunodeficiency virus

The feline homologue of CD134 is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4+ helper T cells. However, strains of FIV differ in utilization of CD134; the prototypic strain PPR requires a minimal determinant in the first cysteine-rich domain (CRD1) of feline CD134 to confer near-optimal receptor function, while strains such as GL8 require additional determinants in the CD134 CRD2. We map this determinant to a loop in CRD2 governing the interaction between the receptor and its ligand; the amino acid substitutions S78N-S79Y-K80E restored full viral receptor activity to the CDR2 of human CD134 in the context of feline CD134, with tyrosine-79 appearing to be the critical residue for restoration of receptor function.     

Evaluation of tri-combinant vaccine for feline herpesvirus, calicivirus and panleukopenia virus infections in Japanese native cats

Tri-combinant vaccine consisting of attenuated feline herpesvirus (FHV) and feline calicivirus (FCV) and inactivated feline panleukopenia virus (FPLV), were evaluated for safety and efficacy, using Japanese native cats and the viral strains isolated in Japan. Thirty-eight 9- to 12-week-old kittens were inoculated intramuscularly and subcutaneously with the vaccine. Consequently, no adverse reaction was found, and protective efficacy was confirmed by challenge tests with the virulent strains of each virus. Serum-neutralizing antibodies against FCV and FPLV were maintained for at least one year after vaccination, whereas antibody against FHV disappeared in two cases at 24 weeks after vaccination. Application of this vaccine seemed effective for control of feline viral disease in cats for experimental use.     

Construction of an infectious clone of simian foamy virus of Japanese macaque (SFVjm) and phylogenetic analyses of SFVjm isolates

Foamy viruses belong to the genus Spumavirus of the family Retroviridae and have been isolated from many mammalian species. It was reported that simian foamy viruses (SFVs) have co-evolved with host species. In this study, we isolated four strains (WK1, WK2, AR1 and AR2) of SFV (named SFVjm) from Japanese macaques (Macaca fuscata) in main island Honshu of Japan. We constructed an infectious molecular clone of SFVjm strain WK1, termed pJM356. The virus derived from the clone replicated and induced syncytia in human (human embryonic kidney 293T cells), African green monkey (Vero cells) and mouse cell lines (Mus dunni tail fibroblast cells). Phylogenetic analysis also revealed that these four SFVjm strains formed two distinct SFVjm clusters. SFVjm strains WK1 and WK2 and SFV isolated from Taiwanese macaques (Macaca cyclopis) formed one cluster, whereas strains AR1 and AR2 formed the other cluster with SFV isolated from a rhesus macaque (Macaca mulatta).