Search

Ecotropic murine leukemia viruses (MuLVs) are classified into B-N-, or NB-tropic MuLV by their host range determined by the Fv-1 gene product. B-tropic MuLV is restricted in N-type mouse cells (Fv-1 n/n) and N-tropic MuLV is restricted in B-type mouse cells (FV-1 b/b). Although forced passages in a restrictive host grant a wider host range (NB-tropism), we show here a host range conversion from B to N tropism. The conversion was most likely a result of recombination between the exogenously infected B-tropic MuLV and an endogenously expressed N-tropic MuLV in a C57BL/6 mouse cell line, YH-7.

We previously demonstrated that a human T-cell line, HPBMa10-2 derived from HPBALL, was capable of supporting a productive infection of hepatitis C virus (HCV). We subsequently found Daudi cells, a human B-cell line, to be susceptible to HCV infection. Employing these cell lines infected with HCV as well as liver obtained during the acute phase of hepatitis C from a chimpanzee, we observed intracellular HCV particles by electron microscopy (EM). We detected viruslike particles with a diameter of approximately 50 nm in the cytoplasmic vesicles. In Daudi cells harvested 15 days after virus inoculation, the appearance of cytoplasmic tubular structures which we originally described for chimpanzee hepatocytes in association with HCV infection was noted. Immunoperoxidase EM using antibodies against HCV core and envelope demonstrated that the particles contained the viral antigens.

SUMMARY. The mode of replication of the hepatitis C virus (HCV) remains poorly understood. Attempts to produce a tissue culture model containing replicating HCV have been largely unsuccessful. Recent studies on sera from patients chronically infected with HCV have shown that viral particles may be found in high-or low-density fractions. High-density fractions contain non-infectious virions whilst infectious particles can be derived from low-density material. Using appropriate infectious fractions we have successfully infected a number of human cell lines allowing studies of HCV replication to be initiated.

In spite of the recent progress in molecular biology of the hepatitis C virus (HCV) genome, the biological characteristics of this virus remain poorly known. This is primarily because biological assays for HCV have been limitted to the experimental inoculation of chimpanzees. It is imperative to develop either a less expensive animal model or a cell-culture system for propagating HCV. Several studies, including ours, have provided evidence for replication of the HCV genome in cell cultures. Although the reported systems are not yet fully satisfactory for wide application to in vitro studies of HCV, they are useful at least for examining the infectivity of HCV materials.

Hypersonic aerodynamic heating induced by the complex shock-shock interactions, has been investigated for a first stage winged reusable rocket combined with two rocket boosters. Hypersonic wind tunnel tests have been made by using NAL Mach 10 HWT and heat transfer distributions have been measured mainly on the booster nose by NAL infrared systems. Severe local peak heating is generated by strong bow shock wave impingement. Moreover, in the present Mach 10 conditions, separation shock wave is caused on the forward lower fuselage surface influenced by the boosters. These shock waves complicate shock interaction flowfields in the booster nose region and oil flow tests are made to clarify these flow structures. Corresponding numerical computations has been made by using overset grid before the HWT tests. Present numerical simulation can predict the occurrence of the separation shock wave and local peak heating characteristics have been captured well quantitatively. It is known that combined study, using wind tunnel tests and numerical simulations simultaneously, is significantly effective for the aerodynamic analysis of future reusable space transport systems including shock-shock interactions.

The 441-nucleotide (nt) region (nt 5325 to 5766) around the splice acceptor (SA) site (nt 5491) was found to be necessary for high-level expression of gag -containing unspliced RNA of Moloney murine leukemia virus (M. Oshima, T. Odawara, T. Matano, H. Sakahira, K. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286–2295, 1996). Detailed genetic dissection of the 441-nt region revealed that the 5′-end 64 nt (nt 5325 to 5389) were necessary for high-level expression of the unspliced RNA when the spliced RNA was not produced, while the 3′-side 301 nt (nt 5466 to 5766) containing the SA site were necessary for producing spliced RNA. When the spliced RNA was produced, the unspliced RNA could be expressed at a high level even when the 5′-end 64 nt were absent. Probably the virus sequence ensuring the splicing could produce an RNA structure able to compensate for the function of the 5′-end 64-nt region responsible for the expression of the unspliced RNA.

An excisable retroviral vector, TSN-lox, was developed by exploiting Cre-loxP homologous recombination. An integrated TSN-lox provirus could be excised, leaving a solo long terminal repeat at the integration site; inverse PCR, taking advantage of the solo long terminal repeat, was used to characterize cellular flanking sequences. A TSN-lox-transduced Rat2 cell clone, lox-7, was found to harbor the provirus in an intron of the procollagen C-proteinase enhancer protein (PCPE) gene, whose expression was lowered compared with that of the parental Rat2. When the vector provirus in lox-7 cells was excised, PCPE expression was elevated. The level of PCPE expression seemed to affect cell growth properties such as morphology, contact inhibition, and anchorage-independent growth. These results suggested that the excisable retroviral vector may be useful for studying the molecular basis for proviral insertion mutagenesis, and that PCPE may play a significant role in controlling cell growth and differentiation.

Comparison of the whole p30 (CA) sequence between B-tropic WN1802B murine leukemia virus and its NB-tropic derivative revealed a single base change at the 313th nucleotide. We constructed hybrid proviruses differing only at the 313th nucleotide of p30 along the whole genome, and examined the host range of the produced viruses. The single point mutation in p30 was found sufficient for the B to NB tropism conversion.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.

We previously reported that a clone of a human lymphocytic cell line, HPBMa clone 10-2, supports the genome replication of hepatitis C virus (HCV). In the present study, the multicycle transmission of HCV from infected cells to new cells was demonstrated by coculture with drug-resistant cells as recipients; i.e., the system was proved productive for infectious HCV. Inhibition of viral replication by alpha and beta interferons was observed.

Mouse cell lines (L, NIH3T3, and RMA cells) infected with the Edmonston strain of measles virus (MV) did not exhibit cytopathic effects (CPE), consistent with the finding that mice are not susceptible to MV. Northern blot analysis, however, revealed that MV genes were transcribed in infected L and NIH3T3 cells, although expression levels were much lower than those in lytically infected Vero cells. Expression of MV genes was not detected in infected RMA cells. L and NIH3T3 cells were found to synthesize viral proteins and produce infectious virions after infection. These cell lines did not express on the surface the molecule detectable by antibodies directed against human CD46, the recently identified MV receptor. L cell transfectants expressing human CD46 exhibited CPE after MV infection, and produced 50 times more viral transcripts and 20 times more infectious virions than the parental L cells. The lowest titer of MV that induced viral multiplication in L cells as detected by cocultivation with Vero cells was larger than that in CD46+ L cells by two orders of magnitude. Our results indicate that MV can infect some mouse cells in the absence of CD46, yet the presence of CD46 facilitates multiplication and cytopathogenicity of MV in mouse cells.

An L cell clone developing cytopathic plaques upon infection with measles virus (Edmonston strain) was obtained. The sensitivity as measured by the newly devised UV-Vero assay was not significantly different between plaque-forming and non-plaque-forming L cell clones. Cytopathogenicity and sensitivity to the virus infection appear to be under different host cell regulations.

A consensus primer‐mediated polymerase chain reaction devised to amplify a short sequence in LI region (L1‐PCR) efficiently detected genital human papillomaviruses (HPVs) in clinical materials. Nucleotide sequencing of the amplified fragment showed that L1‐PCR is also applicable to hitherto unsequenced HPVs. By comparing the amplified 210 nucleotides HPVs were classified into six groups, which are consistent with clinical pictures and a grouping based on cross‐hybridization under the stringent condition.

Among Moloney murine leukemia viruses (Mo-MuLVs) having stop codons other than UAG at the gag-pol junction, Mo-MuLV with UAA, but not with UGA, had a replication disadvantage. Mo-MuLV with a glutamine codon (CAG) at the junction did not replicate. A revertant of this virus consisted of the original virus and a virus with a deletion of the pol region. Protease and Pr65gag encoded by their respective genomes complemented each other.

MLg cells originating from a newborn ddY mouse began to release C-type virus by treatment with 5-iodo-2′-deoxyuridine (5 μg/ml) for 48 h, or spontaneoulsy after prolonged cultivation. The data indicated the presence of three categories of the viruses spontaneously induced, i.e., virus equipped with the abilities of infecting and transforming the S+L- C182 cells, virus which can infect C182 cells but is unable to transform C182 cells, and virus which cannot infect nor transform the C182 cells, but is positive for the XC test. The type of the virus produced depended upon the cell clone. With the advance in the passage level, the cell population was dominated by the cells producing, uniquely, the last category of the virus.

G mouse cells were resistant to N- and NB-tropic Friend leukemia viruses and to B-tropic WN 1802B. Though the cells were resistant to focus formation by the Moloney isolate of murine sarcoma virus, they were relatively sensitive to helper component murine leukemia virus. To amphotropic murine leukemia virus and to focus formation by amphotropic murine sarcoma virus, G mouse cells were fully permissive. When the cell lines were established starting from the individual embryos, most cell lines were not resistant to the murine leukemia viruses. Only one resistant line was established. Cloning of this cell line indicated that the resistant cells constantly segregated sensitive cells during the culture; i.e., the G mouse cell cultures were probably always mixtures of sensitive and resistant cells. Among the sensitive cell clones, some were devoid of Fv-1 restriction. Such dually permissive cells, and also feral mouse-derived SC-1 cells, retained glucose-6-phosphate dehydrogenase-1 and apparently normal number 4 chromosomes. The loss of Fv-1 restriction in these mouse cells was not brought about by any gross structural changes in the vicinity of Fv-1 on number 4 chromosomes.

The 32S RNA of the Friend strain of spleen focus-forming virus (SFFV) contains two sets of sequences: about half is specific to SFFV, and the other half is in common with the sequence of the helper lymphatic leukemia virus. Fingerprinting analysis of RNase T1 oligonucleotides showed that the SFFV-specific sequences were located in two distinct regions: in the 3' half and near the 5' terminus of the genome. Translation of SFFV RNA in a cell-free system yielded three SFFV-specific polypeptides: two main products with molecular weights of about 47,000 (P47) and 16,000 (P16) and a variable amount of a product with a molecular weight of 40,000 (P40). P47 was translated from polyadenylic acid-containing fragments of 1,500 to 3,000 nucleotides with SFFV-specific sequences from the 3' half of the genome, whereas P16, which contained peptides in common with those of P47, was synthesized by smaller RNA. P47 formed in vitro was found to be structurally related to the protein portion of a glycoprotein, gp55, specifically found in SFFV-infected cells in vitro. It is concluded from the results that a defective env gene containing SFFV-specific sequences in the 3' half of the genome codes for SFFV-specific gp55.

Summary Using an MLg cell line originating from a single newborn ddY mouse, N-tropic C-type virus able to infect the cells of origin was induced by 5-iodo-2′-deoxyuridine (IUdR). All the clones isolated from the MLg cell line released the virus after IUdR-treatment. There was more than a 100-fold difference in the virus inducibility between the highly and lowly inducible clones. The [125I]-IUdR incorporation was, however, the same for the both types of clones. When the induced virus was titrated in the S + L - C182 cells, the XC plaque titre was about tenfold higher than murine sarcoma virus rescue focus titre.

Summary Intracellular replication of murine sarcoma virus was followed by Luria-Latarjet experiments. In experiments with unsynchronized cultures ultraviolet sensitivity of the cells infected with murine sarcoma virus as focus centres increased with the lapse of time after infection, and 16 hr after infection u.v.-resistant cells appeared which were probably producing mature virus. The u.v.-resistant population continued to increase thereafter. Ultraviolet sensitivities as focus centres of synchronized cells infected with murine sarcoma virus at different periods of the cell cycle were compared 23 hr after infection. The cells infected with the virus in the G1 phase remained more sensitive to u.v. light than those infected with the virus in the S phase, i.e. the latent period was longer after G1 phase infection than after S phase infection. An antimitotic agent colchicine, prevented the increase of u.v. resistance of the cells as focus centres. These facts suggest that murine sarcoma virus infection requires the division of the host cells and that virus replication is synchronized at this stage of the cell cycle.

A transient depression of mitotic activity occurred immediately after addition of fresh calf serum to the cultures of C3H2K cells; the extent of the depression depended upon the serum concentration.

It has been reported that the maximum efficiency of the infection of RNA tumour viruses, such as Rous sarcoma virus, Friend leukaemia virus or murine sarcoma virus, coincided with the maximum cellular DNA synthesis (Bader, 1966; Yoshikura, 1968; Yoshikura et al. 1968). I present here a confirmatory experiment. The C3H2K cell line originating from the kidney tissues of a newborn C3H/He mouse is sensitive to contact inhibition of cell growth and stops growing at a low cell density in the G1 phase. After the renewal of the culture medium, all the cells replicate in the synchronous fashion. DNA synthesis begins to increase at 10 hr and reaches its maximum at 20 hr. Mitosis occurs 10 hr later than DNA synthesis (Fig. 1A) (Yoshikura & Hirokawa, 1968). Synchronous cell division was also induced by scraping the confluent monolayer as in the case of 3T3 cells (Todaro, Lazar & Green, 1965), 1S1 cells (Castor, 1968), or chick embryo fibroblasts (Gurney, 1969).

The N-type MLg cells transformed by B-tropic MuSV (WN1802B) produced defective MuSV without detectable among of MuLV; the focus formation by the culture fluid required the co-infection of exogenous MuLV. After one passage of the virus in MLg cells, we obtained a virus preparation which was capable of inducing foci without the exogenous MuLV, but which remained negative in the XC test.

When the MLg Mouse cells were treated with the chemical carcinogens, in the presence of microsomal enzymes and NADPH2 after 5-iododeoxyuridine (IUdR) treatment, the induction frequency of the endogenous C-type virus was increased by nearly 10-fold in comparison with the cultures treated with IUdR only. In the preliminary screening of several polycyclic hydrocarbons including carcinogenic and non-carcinogenic products, apparently there was no correlation between the carcinogenecity in vivo and the stimulation capacity of IUdR induced C-type virus activation in our test conditions.

Summary N-tropic Friend leukaemia virus (FLV) was serially passaged in a B-type C57BL/6 mouse cell line, YH-7. After a single passage, the infectious efficiency of FLV in the restrictive YH-7 cells was significantly increased. This adapted character of FLV could be reversed by a single passage in permissive N-type MLg cells. The true host range conversion from N to NB was accomplished after eleven to twelve passages in YH-7 cells. In contrast, the host range conversion of N-tropic murine sarcoma pseudotype, MuSV(FLV), took place after two passages.

This paper reports the epidemiological trend of emergence of antibiotic resistant isolates of Shigella in Japan over a period of recent 25 years, and a method for in vivo evaluation of chemotherapeutic agents against shigellosis by infection of tissue culture monolayers of virulent Shigella.

Formation of proviral DNAs by B-tropic murine leukaemia viruses (MLVs) was examined in N-type and dually permissive mutant cells derived from two inbred mouse strains, DDD and G, both of which are N-type. In the N-type cells, formation of circular proviral DNA was strongly suppressed relative to that of linear DNA. Mutation resulting in loss of the N-type Fv-1 restriction resulted in efficient formation of circular DNA by the previously restricted B-tropic MLV. This showed that Fv-1 restriction and inhibition of closed circular DNA formation were controlled by the same gene. The efficiency of formation of circular proviral DNA by the defective Kirsten murine sarcoma virus was determined by the tropism of the helper virus.

Treatment of C3H2K cells arrested in the G 1 phase with arabinosyl-cytosine (araC) for 2 h caused an unexpected delay of DNA synthesis when the cells were subsequently induced to divide by medium change. The araC-induced delay was reversed by excess amount of deoxycytidine. The higher the concentration of araC, the longer the delay period. The delay period was invariable whether the cells were treated with araC from 0 to 2 h or from 6 to 8 h, i.e., early or late G 1. Similar delay was produced by ethidium bromide and fluorodeoxyuridine.

ENDOGENOUS C-type viruses can be induced by treating the rodent cells in which they reside with halogenated pyrimiding analogues, notably 5-iodo-2′-deoxyuridine (IUdR) (refs 1, 2). Here I show that caffeine, which inhibits repair of photolesions and interacts with DNA in various ways3, inhibits the induction of the endogenous virus by IUdR.

Human T-cell leukemia virus type I (HTLV I) propagated in human diploid fibroblast IMR90 was transmitted to human promyelocytic leukemia HL60 cells by coculture. Of 14 provirus-positive HL60 clones, five harbored only defective proviruses, five had defective proviruses in addition to full-sized HTLV I, and four had full-sized proviruses integrated in their chromosomes. The frequency of defective proviruses was unexpectedly high (41% of total proviruses). Analysis of the genomic structure of these defective proviruses revealed polarity of deletion, that is, preferred conservation of the 3' end of the proviral genome (pX and the 3' long terminal repeat). The implication of these findings are discussed with reference to the replication and pathogenesis of HTLV I.

A 3D display equipped with a linearly moving mirror reflecting a series of 2D cross sections pictures has been devised. The pictures were displayed by a CRT in perfect synchronization with the back-and-forth movements of the mirror. The 80*80*80 mm/sup 3/ 3D space contained 128*128 pixels; with each pixel having 256 grades of intensity, enough to produce a satisfactory 3D image of an ultrasonic echogram of hepatic vessels. The images can be observed from different angles by changing the observer's position or by prompt rotation of the image through a console. Mechanical problems inherent in using moving parts have been solved. >

The effects of physical restraint on the development of Friend erythroleukemia in ddY mice were examined. Restraint was applied by immobilizing the mice in wire cages. In both sexes there was a reduction in the weight of the whole body, thymus and spleen, though the food intake was not reduced. The incidence of leukemia was reduced in the males, but not in the females, while the latent period was prolonged in both sexes. This sex dependency is characteristic of the stress response.

The authors devised a 3-D display with a linearly moving mirror to reflect a series of 2-D cross sections of the 3-D image on a CRT. The crystallography data in a protein data bank can be instantaneously converted into real 3-D images. Structures of several proteins and t-RNAs are presented as examples. >

Interferon(IFN)-alpha and -beta strongly inhibited syncytia formation of human T-cell leukemia virus (HTLV). They also inhibited transmission of HTLV to normal human fibroblasts. These phenomena suggest a physiological role of IFNs in defense against HTLV infection.