Mutant and recombinant avian retroviruses with extended host range.

Avian retroviruses of subgroups B and D efficiently infect chicken (C/E) but not turkey (T/BD) cells. We describe here three variants of subgroup B and D viruses that infect both cell types equally well. One of these viruses, NTRE-4, was a recombinant between transformation-defective Prague (Pr) strain Rous sarcoma virus (RSV) subgroup B and the endogenous virus RAV-0; the second, SR-DE-1, was a recombinant between Schmidt-Ruppin RSV subgroup D and defective endogenous virus information. T1 oligonucleotide fingerprint analysis of the genomes of these two viruses showed only a small alteration in the portion of the env gene responsible for subgroup specificity, as indicated by the presence of a single subgroup E oligonucleotide in an otherwise purely subgroup B or D gene. The third virus, hrBO1Pr-B, was a variant of Pr-RSV-B that did not appear to be a recombinant and whose altered host range we attribute to mutation. Analysis of the host range of all three viruses by infection of selectively resistant cells and by interference testing indicates that all use the subgroup B receptor on chicken cells and the subgroup E receptor on turkey cells. These viruses may be analogous to the polytropic recombinant viruses recently found to be associated with leukemia in some strains of mice.     

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.     

Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus.

The host and tissue specificity of retrovirus infection is largely determined by specific cellular receptors that mediate virus entry. Genes encoding these receptors are widely distributed in the genome, and the receptors identified to date show no sequence similarity. We have identified the cellular receptor for amphotropic murine retroviruses, Ram-1, by screening a rat cDNA expression library introduced into amphotropic virus-resistant hamster cells. The 656-amino acid receptor is homologous to the gibbon ape leukemia virus receptor at both hydrophobic termini but is highly divergent in the central hydrophilic region. Both receptors appear to be integral membrane proteins having multiple membrane-spanning regions. Identification of this family of receptors will help define the evolutionary relationship between retroviruses and their cellular receptors.     

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.     

Highly efficient induction of type C retroviruses by a human tumor in athymic mice.

We have found that 1 of 20 human tumors transplanted and passaged in nude mice was associated with a massive induction of endogenous murine leukemia virus (MuLV). Separation and growth of these viruses on various substrates indicated that both ecotropic and xenotropic MuLV were present in the induced mixture. Tryptic peptide fingerprints of the p30 and gp70 structural elements of the viruses indicated that all of the known endogenous muLVs of BALB/c mice were present in the mixture. In addition, a new xenotropic MuLV was identified. The human tumor that induced the viruses was an oat cell carcinoma. The oat cell carcinoma possibly produced a specific hormone or factor that acts as a potent inducer of endogenous type C retroviruses.     

Safe and efficient mobile robot teleoperation via a network with communication delay

This paper presents the remote control of a mobile robot via the Internet. The delay time is one of the biggest obstacles for many real-time applications, especially for Internet-based control systems. This article presents three architectures to control a robot in an Internet network without guaranteed quality of service. These architectures allow us to control the robot movements accurately and safely. This work presents the use of virtual reality in the context of the remote control. Virtual reality can be used in a conventional manner to simulate the behavior of a system, but also in parallel with the real system to improve the quality of control. To validate our work, we conducted teleoperation experiments in various places, and the experimental results show the advantages and drawbacks of each proposed architecture.     

Gene transfer to primary normal and malignant human hemopoietic progenitors using recombinant retroviruses

To study the feasibility of using retroviruses for gene transfer into human hemopoietic cells, various cell types were exposed to virus carrying the gene for neomycin resistance (neor). In preliminary studies using K562 cells as targets, we found that high viral titer and co-cultivation with viral producer cells rather than incubation in medium exposed to viral producer cells were important variables for achieving high frequencies of G418 resistant (G418r) colonies. The maximum frequency of G418r K562 colonies after co-cultivation with cells producing a neor virus titer of 4 X 10(6) cfu/mL was 60%. When primary human progenitors from normal marrow, fetal liver, or chronic myelogenous leukemia blood were exposed to high titer viral stocks, both with and without helper virus, under conditions optimized for K562 cells, maximum frequencies of G418r colonies were 3% to 16% for granulocyte macrophage progenitors and 2% to 6% for primitive erythroid progenitors. The presence of the neor gene in both G418r K562 and primary hemopoietic colonies was verified by Southern blot. Expression of the neor gene was shown by RNA spot blot. These data demonstrate efficient transfer and expression of the neor gene in both K562 cells and primary human hemopoietic cells from normal and leukemic individuals.     

Identification of human endogenous retroviruses with complex mRNA expression and particle formation.

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti- or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.     

Safe and easy monitoring of anti-rabies antibody in dogs using his-tagged recombinant N-protein

The virus neutralization (VN) test is a reliable indicator of adequate vaccination in animals. However, the VN test is tedious and complicated to perform. Enzyme-linked immunosorbent assay (ELISA), though rapid and simple compared to the VN test, is complicated and hazardous during preparation of the viral antigen. In an effort to overcome the disadvantage of ELISA, the recombinant His-tagged nucleoprotein (His-rNP) expressed in Escherichia coli was used as a safe antigen for ELISA (i.e., live virus was not used). Anti-rabies antibody levels were determined by fluorescent ELISA (FELISA) using His-rNP as an antigen. The presence of anti-rabies VN antibody was determined by the rapid fluorescent focus inhibition test (RFFIT). The VN titers by RFFIT were found to correlate well with the FITC-signal determined by the FELISA (r = 0.616). The sensitivity and specificity of the FELISA were 91.7 and 100%, respectively. This study showed that the His-rNP could be useful as an antigen of ELISA to test for anti-rabies antibody in vaccinated dogs. Several studies in Japan have investigated the antibody level in the sera of vaccinated dogs. A safe and convenient test using His-rNP would contribute to our understanding of the status of herd immunity among not only domestic dogs but also stray dogs in Japan.     

Highly attenuated vaccinia virus mutants for the generation of safe recombinant viruses.

An attenuated vaccinia virus mutant with specific genetic lesions has been used to develop a vehicle for safer live recombinant virus vaccines. The mutant virus 48-7 has an 8-MDa deletion starting 2.2 MDa from the left end of the viral genome and point mutations in the gene encoding the 14-kDa fusion protein that determines the plaque-size phenotype of the virus. Using the highly sensitive reporter gene luciferase, we have shown that this mutant can generate recombinant viruses that infect cultured cells and animals with normal vaccinia virus tropism. Insertion of the envelope and gag genes of human immunodeficiency virus type 1 into the attenuated vaccinia mutant resulted in their efficient expression and precursor processing in infected cultured cells. Infection of mice with human immunodeficiency virus-vaccinia recombinant viruses elicited human immunodeficiency virus-specific antibodies. Using mice pretreated with cyclophosphamide as a model for immunosuppression, the reduced virulence of the mutant recombinant virus was clearly evident. These findings demonstrate that the highly attenuated vaccinia virus mutant 48-7 can be used to generate effective and safer vaccines.     

Friend strain of spleen focus-forming virus is a recombinant between ecotropic murine type C virus and the env gene region of xenotropic type C virus.

The spleen focus-forming virus (SFFV), a replication-defective murine leukemia virus that causes the rapid transformation of certain hematopoietic target cells, has acquired specific xenotropic viral genetic information not contained in Friend helper virus. In the current studies, it is shown that a cDNA that represents a xenotropic virus portion of SFFV detects genetic sequences derived from the env gene region of murine xenotropic virus. The significance of the acquisition of these xenotropic viral sequences by SFFV is discussed with regard to their possible role in the rapid leukemogenicity of SFFV, and an analogy is drawn between the formation of SFFV and the formation of the Kirsten and Harvey sarcoma viruses.     

Identification of DNA fragments carrying ecotropic proviruses of AKR mice.

The proviruses of the N-tropic, ecotropic virus (AKV) of AKR mice (Akv-1, Akv-2) have been studied by the Southern gel--filter transfer technique. These proviruses can be detected by cleavage of cell DNA by BamHI endonuclease, which yields characteristic subgenomic DNA fragments upon cleavage of this type of provirus. Proviruses integrated into different sites in the mouse genome can be resolved with EcoRI endonuclease, which does not cleave the AKV proviruses. Use of congenic and backcrossed mice and a radioactive DNA probe enriched for AKV sequences has allowed identification of the EcoRI fragments carrying the proviruses of the genetically defined Akv-1 and Akv-2 loci. Novel proviruses introduced by superinfection of cultured AKR cells with AKV and present in leukemic cells from AKR mice have also been identified. Comparison of substrains of AKR mice indicates some heterogeneity in their spectra of proviruses.     

Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins

The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.     

双嗜性逆转录病毒感染日本血吸虫细胞的生物学理论与可行性探讨

目的探讨用双嗜性逆转录病毒载体将外源基因导入日本血吸虫（Sj）细胞的生物学理论与实验依据。方法用生物信息学方法对双嗜性逆转录病毒rRam-1受体同源性分布、结构与功能作系统的分析与比较;利用携带外源E77.43基因的双嗜性逆转录病毒感染Sj童虫培养细胞,经PCR和RT-PCR检测感染细胞目的基因（E77.43）的整合与表达。结果根据生物信息学分析结果推断,Sj细胞膜上存在的SjCHGC09605和SjCHGC05362两种蛋白为非分泌性跨膜蛋白,可能具有细胞膜离子转运通道或受体蛋白的功能及双嗜性逆转录病毒感染的膜受体样作用,可能参与病毒对细胞的吸附和穿入过程;利用携带外源E77.43基因的双嗜性逆转录病毒感染Sj童虫培养细胞后,用PCR及RT-PCR检测到目的基因整合与表达,扩增的目的片段大小为330 bp,与理论值相符。结论用载有E77.43基因的双嗜性逆转录病毒感染Sj童虫细胞获得成功,推测SjCHGC09605和SjCHGC05362两种与rRam-1受体同源的蛋白可能是Sj感染过程中起作用的分子。研究结果为下一步用双嗜性逆转录病毒载体转导永生化基因到Sj细胞提供了生物学理论与实验依据。     

Structural basis of P[II] rotavirus evolution and host ranges under selection of histo-blood group antigens

Group A rotaviruses cause severe gastroenteritis in infants and young children worldwide, with P[II] genogroup rotaviruses (RVs) responsible for >90% of global cases. RVs have diverse host ranges in different human and animal populations determined by host histo-blood group antigen (HBGA) receptor polymorphism, but details governing diversity, host ranges, and species barriers remain elusive. In this study, crystal structures of complexes of the major P[II] genogroup P[4] and P[8] genotype RV VP8* receptor–binding domains together with Lewis epitope–containing LNDFH I glycans in combination with VP8* receptor-glycan ligand affinity measurements based on NMR titration experiments revealed the structural basis for RV genotype-specific switching between ββ and βα HBGA receptor–binding sites that determine RV host ranges. The data support the hypothesis that P[II] RV evolution progressed from animals to humans under the selection of type 1 HBGAs guided by stepwise host synthesis of type 1 ABH and Lewis HBGAs. The results help explain disease burden, species barriers, epidemiology, and limited efficacy of current RV vaccines in developing countries. The structural data has the potential to impact the design of future vaccine strategies against RV gastroenteritis.

The major human rotaviruses (RVs), the P[8], P[4], and P[6] genotypes in the P[II] genogroup, are responsible for over 90% of human infections worldwide (1–3). Despite successes of the RotaTeq and Rotarix RV vaccines in many developed countries, their efficacy remains disappointingly poor in developing countries (4–6). Low efficacies of both vaccines in developing countries can be attributed to a lack of cross protection between P[8], which is more common in developed countries, and other P-type RVs, such as P[6] and P[11], that are less common in developed countries but more common in developing countries (7–13).Significant advances have been made in understanding RV evolution under the selection of stepwise synthesis of histo-blood group antigens (HBGAs) in humans. For example, P[II] RVs that mainly infect humans are thought to have originated from P[I] RVs with an animal host origin and evolved the ability to infect humans under selective pressure to bind polymorphic human HBGAs. This deduction is in agreement with a complete VP4 sequence phylogeny analysis that revealed that P[10]/P[12] in P[I] were genetically closer to P[19], P[6], and P[4]/P[8] in P[II] than other genotypes from other genogroups (14, 15). These observations led to the hypothesis that host ranges of P[II] genotypes for certain animal species and different human populations are dictated by the evolutionary stages of their HBGA receptors.P[19] appears to represent an early evolutionary branch of the P[II] genogroup since it recognizes type 1 precursor HBGAs and therefore commonly infects animals (porcine) but rarely humans. On the other hand, P[4] and P[8] appear to be more evolutionarily advanced since they have developed the ability to recognize more mature HBGA products that dominate in humans. P[6] appears to represent an intermediate stage of evolution close to P[19] that commonly infects both animals (porcine) and humans, likely because of its evolutionary status that allows it to recognize less mature type 1 HBGA precursor glycans shared between humans and animals (porcine). The deduced evolutionary path that enabled the transition from animal to human host, which is correlated with the emergence of the P[II] branch from the P[I] branch, may apply to other genotypes and genogroups and may be important for RV classification and epidemiology (16, 17).Evidence for HBGA-controlled RV host ranges and evolution is also available from structural analyses of genotype-specific interactions of RV VP8* domains with their glycan receptor ligands. For example, early structures showed that VP8* domains from animal and human RVs adopted similar galectin-like folds, and they recognize distinct HBGAs either through a ββ or βα site. However, our recent NMR spectroscopy–based docking and crystallographic studies showed that P[4], P[6], P[8], and P[19] VP8*s of P[II] interacted with H type 1 HBGA precursor using a common βα site (17–21), while P[8] VP8* bound Le^b tetra-saccharide and Lewis epitope–containing hexa-saccharide (LNDFH I) in the ββ site (22).To elucidate the molecular basis for receptor-binding bias between βα- and ββ-binding sites, we characterized relative binding affinities of major P[II] RV VP8* domains for glycans representing different HBGA synthetic stages, including the Lewis epitope–containing LNDFH I, using NMR heteronuclear single quantum coherence spectroscopy (HSQC)-monitored titrations. The structural basis for the bias of ββ sites for Lewis epitope HBGAs and βα sites for HBGAs lacking the Lewis epitope was elucidated from crystal structures of P[4] and P[8] bound to LNDFH I and P[6] bound to Lacto-N-tetraose (LNT). Sequence- and structure-based analyses of differences in P[II] VP8* receptor–binding interfaces revealed molecular details responsible for receptor switching between genotype-specific ββ and βα HBGA–binding sites. Overall, the results provide strong evidence for HBGA-controlled P[II] RV evolution from an animal host origin that resulted in diverse genotypes infecting children in different populations and which may impact future strategies for RV disease control and prevention.