Toshio Kamahora

Three Spliced mRNAs of TT Virus Transcribed from a Plasmid Containing the Entire Genome in COS1 Cells

ABSTRACT A permuted whole-genome construct of a TT virus (TTV), named VT416, had 3,852 nucleotides (nt) 98.2% similar to the prototype TA278 genome. To allow the transcription of TTV from the internal promoter, pBK*VT416(1.3G), carrying 1.3 units of VT416, was constructed. The poly(A)+ RNAs expressed in COS1 cells 48 h posttransfection contained three TTV mRNA species 3.0, 1.2, and 1.0 kb in length, which were recovered in the 13 DNA clones from a λ phage cDNA library. These mRNAs in the antigenomic orientation possessed in common the 3′ terminus downstream of a poly(A) signal (A3073ATAAA) and the 5′ terminus downstream of a cap site (C98ACTTC). A common splicing to join nt 185 with nt 277 was detected in all mRNAs. The coding region of the largest open reading frame (ORF) was maintained in 3.0-kb mRNA, because this splicing was located upstream of its initiation codon (A589TG). The second splicing was detected in 1.2-kb mRNA to join nt 711 with nt 2374 and in 1.0-kb mRNA to bind nt 711 to nt 2567. They linked a proposed ORF2 to another ORF for creating new ORFs over nt 2374 to 2872 in frame 2 and nt 2567 to 3074 in frame 3. The donor and acceptor sites of all three splicings matched the consensus sequence and were conserved in most of the 16 TTVs of distinct genotypes retrieved from the database. The observed transcription profile is unique to TTV among known members in the familyCircoviridae.

A new method for extracting dna or rna for polymerase chain reaction

The use of glass powder suspension for the extraction of RNA or DNA was studied to simplify the procedures of polymerase chain reaction (PCR). Using this procedure, proviral DNA of human T-lymphotropic virus type-1 (HTLV-1) in the blood of an asymptomatic virus carrier and viral RNA of human immunodeficiency virus (HIV) in the blood of an AIDS patient were easily detected by PCR employing glass powder. The use of glass powder is a simple and highly efficient procedure for the extraction of DNA or RNA, and can be applied for routine PCR.

High frequency of postnatal transmission of TT virus in infancy

Abstract. DNA of TT virus (TTV), a novel human circovirus, was tested for in 116 mother-infant pairs who had participated in the adult T-cell leukemia prevention program (APP) in Nagasaki, Japan, and refrained from breast-feeding. By polymerase chain reaction with Okamoto’s seminested primers, 36 of the 115 (31%) mothers were positive. At the age of 6–8 months, 7 of 29 (24%) and 6 of 72 (8%) infants born to infected and uninfected mothers were positive, respectively (P = 0.047; RR, 2.90). Maternal TTV DNA load did not correlate with infantile infections. Since 99 of 100 (99%) cord blood samples were negative and all the mothers refrained from breast-feeding, the infantile TTV transmission would not be intrauterine or milk-borne. Between 6–8 and 12–21 months of age, 4 of 12 (33%) and 5 of 22 (23%) children born to infected and uninfected mothers turned positive, respectively (NS). At 12–21 months of age, 8 of 21 (38%) and 12 of 32 (38%) children born to infected and uninfected mothers were positive, respectively (NS). These results indicate that the TTV infection prevails in children at a frequency comparative to that in their mothers within the first 2 years of life, regardless of the maternal TTV status.

Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque teno virus (TTV)

To test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(-), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(-). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(-) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT(108)I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T(108)I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10-36, 31-59, 59-88 and 80-112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(-). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV.

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.

Deleted HTLV-1 provirus in cord-blood samples of babies born to HTLV-1-carrier mothers

We screened 596 cord‐blood samples by nested short PCRs for the gag and pX regions of HTLV‐1 which are capable of detecting a single copy. The samples were derived from 449 and 147 babies born to seropositive and seronegative mothers respectively. Of these, 20 samples were positive in at least one of the PCRs: 9 (45%) were positive in both PCRs, but 10 and 1 samples in either the pX or the gag PCR respectively. These samples were tested further in nested long PCRs directed for gag‐pX, gag‐pol and pol‐pX regions capable of detecting 8, 1 and 2 copies respectively. Of 9 dually positive samples, 7 (77%) showed the predicted 6.2‐kbp band in the gag‐pX PCR; only 2 of them had the predicted band alone; 7 samples had discrete bands shorter than the predicted size. In the gag‐pol PCR, all 9 samples showed the predicted 2.2‐kbp band alone. In the pol‐pX PCR, 8/9 samples showed the predicted 4.2‐kbp band, including one with an additional 2.1‐kbp band, and the last a 1.0‐kbp band alone. Thus, all of the dually positive samples had proviruses harboring gag, pol and pX priming sites. In contrast, none of the 11 singly positive samples showed the predicted band in the gag‐pX PCR: 5 had no visible band, and the other 6 had shorter bands only. None of these 11 samples showed any positive signal in either gag‐pol or pol‐pX PCR. Our results suggest that HTLV‐1 proviruses in the cord blood are frequently defective. Int. J. Cancer 77:701–704, 1998.

Hepatitis C virus infection in a Japanese leprosy sanatorium for the past 67 years.

Oku‐Komyo‐En is one of the national leprosy sanatoria, located on a small island in Setouchi city, Okayama prefecture of Japan since 1938. Since autopsies were carried out routinely on almost all patients who had died in the sanatorium up to 1980, approximately 1,000 formalin‐fixed autopsy tissue samples were available for analysis. When these samples were reviewed, the pathological data indicated a sharp rise in the death rate caused by cirrhosis of the liver and hepatocellular carcinoma (HCC) since 1960 and 1970, respectively. Hepatitis C virus (HCV) infection is a common cause of HCC in Japan. The presence of HCV RNA was demonstrated in paraffin sections prepared from the autopsied liver tissue fixed in formalin for a prolonged period of time, by employing nested RT‐PCR using type‐specific primers. The data showed that HCV RNA was detectable in samples of the liver archived as early as 1940, representing the liver tissues kept in formalin for up to 67 years. HCV genotypes 1b and 2a were found by RT‐PCR at 85.7% and 14.3%, respectively, in patients with leprosy. J. Med. Virol. 82:556–561, 2010.

Prevalence of Anti-Borna Disease Virus Antibody in Horses and Their Caretakers in Bangladesh

To elucidate the spread of Borna disease virus (BDV) in Asian countries, we surveyed 48 normal horses in Bangladesh and their 26 caretakers for the BDV antibody by electrochemiluminescence immunoassay. Eleven horses (23%) were found positive. None of the 5 horses at the age of < 1 year was positive. Seven of 23 horses (30%) at the age of 1 year were positive, as well as 4 of 16 horses (25%) at the age of ≥ 3 years. The geometric average of the ECLIA titer of the antibody positive horses at the age of 1 year, 3041, was significantly lower than that found at the age of ≥ 3 years, 6887, by the MannWhitney test (P = 0.012). Sexual preference in the prevalence of anti-BDV was not evident. None of the 26 male horse caretakers between the ages of 12 to 54 years was positive, including those who were taking care of the antibody positive horses. Total RNA extracted from the peripheral blood nucleated cells was tested by polymerase chain reaction coupled with reverse transcription capable of detecting 200 molecules of BDV p40 RNA per reaction. None of the 11 seropositive horses and the 5 randomly selected seronegative horses was positive. The results showed that BDV is penetrating the Bangladeshi labor horse population with similar levels reported in Germany, Iran and Japan, although the viral genome in the blood was not detected.

Isolation and characterization of a cold-sensitive strain of coxsackievirus A10

A coxsackievirus A10 strain, isolated from a clinical specimen from a patient with pharyngitis, was characterized with respect to its growth properties in different cultured cells and at different incubation temperatures. This virus multiplied within cultured cells and produced cytopathogenic effects, whereas a prototype strain of coxsackievirus A10 did not. The isolate multiplied efficiently in cultured cells at 37 degrees C but its replication was markedly restricted at 32 degrees C. Temperature shift experiments indicated that the cold-sensitive event affected the late function(s) of the virus.

Changes in RNA Fingerprints of Measles Virus after Passages in Chicken Cells

Little is known about measles virus concerning its mechanisms of attenuation or adaptation to hosts other than natural hosts. Moreover, the relationship between attenuation and adaptation is not well understood. The Tanabe strain (4) of measles virus was derived from a field isolate and this strain was passaged repeatedly in chick embryos to obtain further attenuated live measles vaccine strains (5, 6, 10). Using these vaccine strains, field trials of vaccination have been extensively performed (7-9), and we now have sufficient information about their grades of attenuation against humans. We tried to compare the original Tanabe strain and vaccine strains derived from it by oligonucleotide fingerprints. Vero cells grown in Eagles minimal essential medium (MEM) (Nissui, Tokyo) supplemented with 5% fetal calf serum were used. Confluent monolayers were infected with measles virus at a m.o.i. of 0.1-1 and were further incubated for 2 to 3 days until 10 to 20% of the monolayer formed syncytia. After rinsing the monolayer with phosphate-free MEM three times, the same medium supplemented with 1 /lg/ml of actinomycin D (AMD) and 5% dialyzed fetal calf serum was added and incubated at 37 C for I hr. The medium was replaced by 3 ml of fresh phosphate-free MEM containing AMD, 5% dialyzed fetal calf serum and 300 /lCi/ml of (32P]orthophosphate and incubated for a further 6 hr. The medium was sucked off and the cells were harvested by scraping the monolayers from the Petri dishes with a rubber policeman. Viral nucleocapsids were collected from the infected cells according to Compans and Choppin (1). The scraped cells were suspended in 10 volumes of ice-cold 0.01 MTris-HCl, pH 7.4, and placed on ice for 20 min to allow swelling. The cell suspension was centrifuged at 2,000 rpm for 10 min, and the precipitate was again allowed to swell in the same buffer. These procedures were repeated for three cycles. After the final centrifugation, the pooled supernatants from each extraction were centrifuged for 13.5 hr at 8,000 rpm onto a 30 (w/v)% sucrose cushion. The precipitate was suspended in STE (0.1 M NaCl, 0.01 M Tris-HCl, pH 7.4, and 1 mM EDTA) buffer. After centrifugation of the suspension through 30 (w/v)% sucrose onto a 60 (w/v)% sucrose cushion for 45