Isolation and characterization of virus-specific double-stranded DNA from tissues infected by bean golden mosaic virus

A double-stranded (ds) DNA which may be a replication intermediate was isolated from bean (Phaseolus vulgaris L. “Top Crop”) leaves systemically infected with bean golden mosaic virus, a whitefly-transmitted plant virus with a genome of circular single-stranded (ss) DNA. The isolation method used phenol/chloroform extraction, hydroxyapatite column chromatography, and rate-zonal centrifugation. The dsDNA had sequences complementary to those of viral DNA. The guanine-plus-cytosine content was 35%, and the sedimentation coefficient in alkaline sucrose density gradients was similar to that of viral ssDNA. Digestion of the dsDNA by Hha I endonuclease produced fragments that corresponded exactly in number and size with those produced by complete digestion of circular viral ssDNA by Hha I, when the fragments were denatured and analyzed on polyacrylamide gels. The dsDNA molecule was a circular structure with one discontinuity in one strand; hybridization results suggest that some of a the dsDNA has a discontinuity in the viral strand and some has a discontinuity in the nonviral strand. On the basis of these structures for the dsDNA, a preliminary model for replication of viral DNA is discussed.     

JC virus DNA is present in the mucosa of the human colon and in colorectal cancers

JC virus (JCV) is a polyoma virus that commonly infects humans. We have found T antigen DNA sequences of JCV in the mucosa of normal human colons, colorectal cancers, colorectal cancer xenografts raised in nude mice, and in the human colon cancer cell line SW480. A larger number of viral copies is present in cancer cells than in non-neoplastic colon cells, and sequence microheterogeneity occurs within individual colonic mucosal specimens. The improved yield of detection after treatment with topoisomerase I suggests that the viral DNA is negatively supercoiled in the human tissues. These results indicate that JCV DNA can be found in colonic tissues, which raises the possibility that this virus may play a role in the chromosomal instability observed in colorectal carcinogenesis.     

In vivo recombination of cauliflower mosaic virus DNA

Ligation and recombination of the DNA of cauliflower mosaic virus (CaMV) is demonstrated by the following experiments: (i) Ligation: Different noninfectious fragments of the CaMV genome (obtained after insertion into plasmid pBR322 followed by enzymatic excision) regained infectivity when mixtures of them were used to inoculate their host. The symptom appearance was delayed by comparison with a typical CaMV infection, and only the newly formed leaves were affected. (ii) Recombination: Pairs of noninfectious recombinant full-length CaMV genomes (integrated into pBR322 at different restriction endonuclease sites) regained infectivity upon simultaneous inoculation of a sensitive host. The symptomatology of the resulting infection was indistinguishable from that of a typical CaMV infection. We show that progeny DNA had the same characteristics (size, structure, restriction endonuclease digestion pattern) as bona fide CaMV DNA, and that the vector pBR322 had been completely eliminated. A cloned tandem dimer of CaMV DNA with a partial deletion similarly was infectious in the plant assays. This system should be useful to study the expression of mutant genomes, thus allowing characterization of the CaMV genes.     

Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus.

The extraction of a template-dependent and template-specific RNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from a eukaryotic source is described. The enzyme, extracted from barley leaves infected with brome mosaic virus (BMV), is capable of incorporating high levels of radioactivity into trichloroacetic acid-insoluble products. The purification procedure included solubilization with nonionic detergent and precipitation with polyethylene glycol. The enzyme was more than 50 times more active than was a comparable preparation from mock-inoculated leaves and was stimulated more than 15-fold by the addition of BMV RNA to the reaction. Other viral RNA templates were less than 25% as efficient as was BMV RNA in stimulating UMP incorporation; poly(A), tRNA, and mRNA gave little stimulation and rRNA was inactive. Autoradiographic analysis after electrophoretic separation of the radioactive products from reaction mixtures containing BMV RNA template revealed prominent bands that coelectrophoresed with replicative forms of BMV RNAs. When BMV RNA template was enriched in RNA3 or RNA4, larger proportions of the products were replicative forms of RNA3 or RNA4, respectively.     

Infectious DNA of spleen necrosis virus is integrated at a single site in the DNA of chronically infected chicken fibroblasts.

The infectious DNAs of a number of avian leukosis-sarcoma and reticuloendotheliosis viruses were digested with six nucleotide-specific restriction endonucleases, and the digests were tested for infectivity. All of the enzymes inactivated the viral infectivities except for EcoRI, which did not inactivate the infectivity of the DNA of two of the reticuloendotheliosis viruses, spleen necrosis and chick syncytial viruses. The infectious DNA of spleen necrosis virus after digestion with EcoRI had a buoyant density in CsCl solution greater than the density of the high-molecular-weight infectious viral DNA. The infectious EcoRI-digested spleen necrosis virus DNA from chronically infected chicken cells was uniform in size, 10 megadaltons, which indicated a single site of integration. The infectious EcoRI-digested spleen necrosis virus DNA from acutely infected cells was heterogeneous in size, ranging from 8-14 megadaltons, which indicated multiple sites of integration. These results are consistent with the hypothesis that cells that integrate infectious spleen necrosis virus DNA at a single site survive and multiply, whereas cells that integrate infectious viral DNA at additional sites either die or selectively lose or inactivate the DNA in the additional sites.     

经输血传播病毒感染恒河猴的嗜肝性

目的 了解经输血传播病毒（ＴＴＶ）的嗜肝性。方法 从５只实验感染病毒的恒河猴各组织中抽提ＤＮＡ,分别用病毒双链探针或单链负义探针,与吸附在纤维膜上的组织ＤＮＡ进行斑点杂交。结果 双链探针与肝、脾、胃、小肠和大肠杂交阳性,表明各该组织中都有无包膜ＤＮＡ病毒的存在。单链负义探针与各该组织杂交,仅肝和小肠为阳性,表示存在可能作为病毒复制中间体的正义单链ＤＮＡ。结论 肝和小肠可能是病毒的复制场所,故无包膜     

HIV Type 1 Nef is released from infected cells in CD45(+) microvesicles and is present in the plasma of HIV-infected individuals

HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10?ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV(MN). Interestingly, plasma mvNef levels in HIV(+) patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.     

Recovery of infectious proviral DNA from mammalian cells infected with respiratory syncytial virus.

The DNA fraction from a line of bovine embryonic kidney cells originally exposed as primary cultures several months earlier to a temperature-sensitive (ts) mutant of respiratory syncytial (RS) virus could be used to transfect human HEp-2 cells with the production of infectious RS virus. The DNA donor cells, designated BEK/RS ts, retained their healthy fibroblastic appearance during continuous cultivation at a temperature (39 degrees) restrictive for growth of the original infecting mutant and showed no evidence for RS virus replication or viral antigen synthesis when directly examined for these activities by conventional methods. The infectious property of the DNA from BEK/RS ts cells was abolished by exposure of the nucleic acid preparation to DNase (but not RNase) or by pretreatment of recipient HEp-2 cells with actinomycin D or mitomycin C. The latter drug treatments substantially enhanced the replication of infecting wild-type RS virus in HEp-2 cells. Viral isolates derived from the progeny of a DNA transfection included clones possessing several genetic markers of the RS ts mutant originally used to infect BEK/RS ts cells and other virus clones that appeared to be either hybrid or wild-type for phenotypic properties such as their temperature sensitivity. An infectious proviral DNA was also detected in a line of virogenic HEp-2 cells (HEp-2/RS) persistently infected with respiratory syncytial virus after exposure to the wild-type strain 2 years earlier.     

Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells.

The lack of an endonuclease EcoRI site in the AKR murine leukemia virus (MuLV) DNA genome was utilized to molecularly clone, in Charon 4A lambda DNA, integrated infectious AKR MuLV DNA isolated from productively infected mouse cells. Three lambda-mouse recombinants (clones 614, 621, and 623) were selected by virtue of their reactivity with AKR MuLV [32P]cDNA. Clones 614 and 623 contained the complete AKR MuLV DNA flanked by nonviral cell sequences of which no more than 100 base pairs beyond the viral DNA appear to be shared. DNAs from both clones 614 and 623 were highly infectious for mouse cells and yielded N-tropic ecotropic MuLV; the specific infectivity of the DNA and the titer of the derived virus was more than 10-fold higher with 623. Clone 621 contained only some viral DNA and was not infectious under similar conditions.     

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SRI), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS: Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.     

Human immunodeficiency virus DNA is present in a high percentage of CD4+ lymphocytes of seropositive individuals.

Human immunodeficiency virus type 1 (HIV-1) infects predominantly CD4+ cells in human peripheral blood and infection is associated with CD4+ lymphocyte dysfunction in patients with AIDS. To determine the frequency of HIV-1 infection in CD4+ lymphocytes in vivo, peripheral blood CD4+ lymphocytes were isolated by fluorescence-activated cell sorting from HIV-1-infected persons with clinical disease ranging from asymptomatic to AIDS. Using standard and booster polymerase chain reaction analyses, study patients with AIDS and AIDS-related complex (ARC) were found to harbor the HIV-1 genome in at least 10% of CD4+ lymphocytes, and approximately 10-fold less infected cells were found in those with asymptomatic infection. In addition, the peripheral blood mononuclear cells from patients with ARC frequently contained a higher absolute number of HIV-1-infected CD4+ lymphocytes than those with AIDS or asymptomatic infection. It is likely that this high level of infection of CD4+ lymphocytes is the primary cause for the progressive immunologic deficiency observed in patients infected with HIV-1.     

Relatively well preserved DNA is present in the crystal aggregates of fossil bones

DNA from fossil human bones could provide invaluable information about population migrations, genetic relations between different groups and the spread of diseases. The use of ancient DNA from bones to study the genetics of past populations is, however, very often compromised by the altered and degraded state of preservation of the extracted material. The universally observed postmortem degradation, together with the real possibility of contamination with modern human DNA, makes the acquisition of reliable data, from humans in particular, very difficult. We demonstrate that relatively well preserved DNA is occluded within clusters of intergrown bone crystals that are resistant to disaggregation by the strong oxidant NaOCl. We obtained reproducible authentic sequences from both modern and ancient animal bones, including humans, from DNA extracts of crystal aggregates. The treatment with NaOCl also minimizes the possibility of modern DNA contamination. We thus demonstrate the presence of a privileged niche within fossil bone, which contains DNA in a better state of preservation than the DNA present in the total bone. This counterintuitive approach to extracting relatively well preserved DNA from bones significantly improves the chances of obtaining authentic ancient DNA sequences, especially from human bones.     

Longer-than-unit-length viroid minus strands are present in RNA from infected plants

Nucleic acids isolated from uninfected and potato spindle tuber viroid-infected Rutgers tomato plants were fractionated on agarose gels under two different sets of denaturing conditions and hybridized to ¹²⁵I-labeled viroid in a series of blot hybridization experiments. Complementary strand nucleic acids detected in extracts of infected plants were heterogeneous in size, with four discrete bands containing molecules approximately 700, 1050, 1500, and 1800 nucleotides long. Enzymatic studies indicated that these viroid minus strands are composed exclusively of RNA and, as extracted, are present in complexes containing extensive double-stranded regions. After treatment with several RNases under conditions favoring digestion of single-stranded regions, the high molecular weight minus strands can no longer be detected and roughly unit-length minus strands appear. A model for the structure of the viroid replication intermediate is proposed.     

A translational enhancer derived from tobacco mosaic virus is functionally equivalent to a Shine-Dalgarno sequence.

When present at the 5' end of mRNAs, the untranslated leader sequence (omega) of tobacco mosaic virus RNA significantly enhances translation in eukaryotes and prokaryotes. We have tested a deletion derivative of the omega sequence, omega delta 3, for its enhancing ability on gene constructs in which the ribosomal binding site was either present or deleted, in several Gram-negative bacterial species including Escherichia coli, Agrobacterium tumefaciens, Xanthomonas campestris pv. vitians, Erwinia amylovora, and Salmonella typhimurium. In vivo production of chloramphenicol acetyltransferase from a gene construct lacking its native ribosomal binding site was enhanced 40- to 120-fold by the presence of omega delta 3. Similar levels of enhancement (30- to 240-fold) were observed when the gene encoding beta-glucuronidase was tested. With a chloramphenicol acetyltransferase construct containing a ribosomal binding site, enhancement was markedly less, between 1- and 3.8-fold. Omega delta 3 appeared to enhance translation independent of its position upstream of the AUG codon used for initiation.     

Varicella zoster virus deoxythymidine kinase is present in serum before the onset of varicella

A sensitive enzyme assay with 125I-iododeoxyuridine as substrate and cytidine triphosphate as phosphate donor was used for the direct detection of varicella zoster virus (VZV) deoxythymidine kinase (TK) in human serum. Sera sampled during the incubation period of varicella from 2 patients, a 42-year-old man and his 11-year-old son, have been analysed for TK activity. A simultaneous increase in cellular and VZV TK activity, starting 5 to 3 days before the onset of clinical varicella, was observed.     

Chromosomal DNA from a variety of bacterial species is present in synovial tissue from patients with various forms of arthritis

OBJECTIVE: We and others have reported the presence of Chlamydia and other bacterial species in joint specimens from patients with reactive arthritis (ReA). The present study was conducted to investigate whether bacteria other than those specified by diagnostic criteria for ReA could be identified in synovial fluid (SF) or tissue from patients with various arthritides, and whether the presence of such organisms corresponds to particular clinical characteristics in any patient set or subset. METHODS: DNA in synovial biopsy samples and SF obtained from 237 patients with various arthritides, including ReA, rheumatoid arthritis, and undifferentiated oligoarthritis, was assayed by polymerase chain reaction (PCR) using "panbacterial" primers; we chose only samples known to be PCR negative for Chlamydia, Borrelia, and Mycoplasma species. PCR products were cloned, and cloned amplicons from each sample were sequenced; DNA sequences were compared against all others in GenBank for identification of bacterial species involved. RESULTS: Ten percent of patient samples were PCR positive in panbacterial screening assays. Bacterial species identified belonged to the genera Neisseria, Acinetobacter, Moraxella, Salmonella, Pseudomonas, and others. Thirty-five percent of PCR-positive patients showed the presence of DNA from more than a single bacterial species in synovium; overall, however, we could identify no clear relationship between specific single or multiple bacterial species in the synovium and any general clinical characteristics of any individual or group of patients. CONCLUSION: This analysis provides the first systematic attempt to relate bacterial nucleic acids in the synovium to clinical characteristics, joint findings, and outcomes. Many patients with arthritis have bacterial DNA in the joint, and, in some cases, DNA from more than a single species is present. However, except for 1 case of a control patient with staphylococcal septic arthritis, it is not clear from the present study whether the synovial presence of such organisms is related to disease pathogenesis or evolution in any or all cases.