Hironori Miyata

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.

Primary prevention of HTLV-I in Japan

The ATL prevention program (AAP) in the Nagasaki Prefecture since 1987 consists of screening of pregnant women and asking the seropositives to refrain from breast-feeding. We screened approximately 90% of gravidas in the Prefecture and > 90% of the seropositive women agreed not to breast-feed. The maternal transmission rate dropped from approximately 20% to approximately 3%. PCR of cord bloods showed that 2.5% were PCR-positive. However, among formula-fed children, none of the cord-positives seroconverted, and none of the seropositives tested had been cord-positive. Breast-feeding for less than six months decreased the transmission rate significantly, but may have a higher transmission rate than the formula feeding.

Prions disturb post-Golgi trafficking of membrane proteins

Conformational conversion of normal cellular prion protein PrP(C) into pathogenic PrP(Sc) is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP(C), insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP(Sc) is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP(C) and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP(Sc) accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.

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.

Dominant-negative Effects of the N-terminal Half of Prion Protein on Neurotoxicity of Prion Protein-like Protein/Doppel in Mice

Prion protein-like protein/doppel is neurotoxic, causing ataxia and Purkinje cell degeneration in mice, whereas prion protein antagonizes doppel-induced neurodegeneration. Doppel is homologous to the C-terminal half of prion protein but lacks the amino acid sequences corresponding to the N-terminal half of prion protein. We show here that transgenic mice expressing a fusion protein consisting of the N-terminal half, corresponding to residues 1-124, of prion protein and doppel in neurons failed to develop any neurological signs for up to 730 days in a background devoid of prion protein. In addition, the fusion protein prolonged the onset of ataxia in mice expressing exogenous doppel. These results suggested that the N-terminal part of prion protein has a neuroprotective potential acting both cis and trans on doppel. We also show that prion protein lacking the pre-octapeptide repeat (Δ25-50) or octapeptide repeat (Δ51-90) region alone could not impair the antagonistic function against doppel.

Evolution in the hypervariable region of hepatitis C virus in infants after vertical transmission.

To elucidate the clonal evolution of hepatitis C virus (HCV) during mother-to-infant transmission, we prospectively analyzed HCV clones of the hypervariable region in four HCV RNA-positive infants and compared them with those of the mother. Cord blood samples from three of the four infants were positive for the HCV RNA (≤103 copies/mL), and all of the four infants had the HCV RNA titer of >106 copies/mL within 2 mo after birth. The hypervariable region clones detected in the infants were closely related to those in the respective mothers. The results suggest the perinatal transmission of HCV. The hypervariable region clones transmitted to infants were not a single selected clone or minor clones in the mother. None of the clones specific to the low-density fraction in the mother was transmitted to the infants. Moreover, the proportion of HCV in the low-density fraction was minimal in the first few months of life, but increased several months after birth in association with the elevation of alanine aminotransferase. These results suggest that the increase of HCV in the low-density fraction reflect the evolution of immune response in infants. We also demonstrated that the emergence of quasispecies in infants precedes the infantile antibody response.

Effects of a Brain-Engraftable Microglial Cell Line Expressing Anti-Prion scFv Antibodies on Survival Times of Mice Infected with Scrapie Prions

We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model.

Measles virus (MeV) vaccine strain, AIK-C, is temperature sensitive (ts), which is thought to be associated with attenuation of virus pathogenicity. In this study, replication and antibody response were examined in cotton rats using viruses carrying different forms of the P gene, which is responsible for the ts phenotype of strain AIK-C and its parental Edmonston strain. When cotton rats were inoculated intranasally, ts viruses neither replicated in lungs, nor reproducibly generated an antibody response. When inoculated intramusculary (i.m.), however, ts strains raised an antibody titer in all animals. This response was not observed when ultraviolet-inactivated virus was used. ts virus, inoculated i.m., was recovered from cotton rat drainage lymph nodes. These results suggest that ts virus, inoculated i.m., could replicate in the cotton rat, presumably at the superficial lymph node, and induce an antibody response. Therefore, cotton rats can serve as a small-animal model for investigating immune responses to safer ts vaccine, as well as recombinant vaccine using AIK-C as a vector for protection against other infectious agents.

Adaptation of wild-type measles virus to cotton rat lung cells: E89K mutation in matrix protein contributes to its fitness.

Wild-type measles virus (wtMeV) adapted well to cotton rat lung (CRL) cells after serial passages. In order to evaluate the contributions of the individual genes of wtMeV for adaptation, whole genome sequences of the adapted and original viruses were determined and analyzed. The results showed that there were two mutations in the whole genome of the adapted virus. One mutation was located at the 265th nucleotide in the open reading frame (ORF) of the M gene, resulting in the substitution of the 89th amino acid from E (glutamate) to K (lysine). The other was a silent mutation located at the 4182nd nucleotide in the ORF of the L gene. It was demonstrated that the E89K mutation in the M protein is responsible for the adaptation of wtMeV MV99Y in CRL cells. Cotton rats were infected with adapted virus and the original strain via intranasal inoculation. Virus titer results showed that adapted strain replicated better than the original strain in cotton rat lungs. It is suggested that the E89K mutation also contributes to the enhancement of wtMeV replication in a cotton rat model infected intranasally. The results revealed that the E89K mutation in the M protein plays a key role in wtMeV adaptation in cotton rat and CRL cells.

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.