Fragment Of Viral Genome Research Articles

An improved reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of two cherry flexiviruses in Prunus spp.

A one-step reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed to detect Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV) in woody indicators and naturally infected Prunus spp. Viral RNA suitable for RT-PCR was obtained by a simple trapping method that did not require either extraction of double-stranded RNA (dsRNA) or total RNA, availability of virus antibodies, or purification of viral particles. Consensus primers, degenerate primers and virus-specific primers, whose designs were based on alignments of available cherry flexivirus sequences, were tested to amplify viral genomic fragments of six CGRMV isolates and one CNRMV isolate. RT-PCR allowed CGRMV detection in total RNA and viral RNA preparations equivalent to 400 μg and 4 μg of infected leaf tissue, respectively. CGRMV was detected in tender shoots, leaves, bark and root tips, and the strongest bands were obtained using young leaves. Detection was less consistent in summer when the temperature was elevated and plant tissues were old. A direct comparison of the RT-PCR and grafting assays indicated that the RT-PCR assay is sensitive, rapid and reliable. The method will improve the routine diagnosis of cherry flexiviruses in Prunus spp.

ViriChip enhances reverse transcriptase polymerase chain reaction in biological fluids and environmental samples

Virus detection is of central importance in clinical diagnosis and treatment, epidemiology, and environmental monitoring. PCR-based detection assays, which detect viral genomic fragments with very high sensitivity, are commonly used diagnostic procedures. However, PCR assays are prone to false positives from introduced contaminates and because of the inability of PCR to distinguish between free viral nucleic acids and nucleic acids obtained from viral particles. False negatives occur due to naturally occurring inhibitors found in the sample or introduced inhibitors from procedures used to isolate or extract the viral nucleic acids [1]. Several reports have addressed these concerns and have demonstrated that aYnity capture of virus prior to ampliWcation reduces PCR inhibition and number of false positives [2,3]. Commonly, the nucleic acid from the aYnity-captured virus capsid is released by heat extraction [4] or by chemical methods [5,6]. We have developed a solid-phase immunocapture substrate, the ViriChip, for the detection and identiWcation of virus particles by atomic force microscopy [7]. In the current report, we demonstrate the use of the ViriChip for capture of virus particles from complex mixtures and ampliWcation of genomic material by reverse transcriptase (RT)-PCR without prior extraction. This methodology reduces handling time, the risk of cross-contamination, and the probability of false positives. In addition, the ViriChip enhanced the sensitivity by RT-PCR to detect viral nucleic acids. Group B coxsackievirus type 4 (CB4) was used to demonstrate the

High prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates.

RNA of the newly identified human metapneumovirus (HMPV) was detected in nasopharyngeal aspirates of 11 of 63 (17.5%) young children with respiratory tract disease. Markers of infection caused by another member of the Pneumovirinae subfamily of the family Paramyxoviridae, respiratory syncytial virus (RSV), were identified in 15 of these patients (23.8%). Three patients were simultaneously infected with HMPV and RSV. Studies of the clinical characteristics of HMPV-infected children did not reveal any difference between HMPV-infected patients and a control population of RSV-infected patients with regard to disease severity, but the duration of symptoms was significantly shorter for HMPV-infected patients. Phylogenetic analysis of the amplified viral genome fragments confirmed the existence and simultaneous circulation within one epidemic season of HMPV isolates belonging to two genetic lineages.

Long PCR amplification of large fragments of viral genomes: a technical overview.

Long PCR amplification of large fragments of viral genomes: a technical overview.

The diagnosis of the tomato variant of pepino mosaic virus: An IC-RT-PCR approach

An immunocapture-retrotranscription-PCR (IC-RT-PCR) procedure for the detection of the tomato-infecting variant of pepino mosaic potexvirus (PepMV) was developed, following an approach that did not require knowledge of viral sequence, availability of commercial antibodies to the virus, or purification of viral particles. Degenerate PCR primers, whose design was based on alignments of published potexviruses sequences to prime theoretically the amplification of a viral genomic fragment of any potexvirus, were used to synthesize cDNA of the potato virus X (PVX) and PepMV RNA polymerases. Tubes coated with antibodies against double-stranded RNA were used in the initial amplifications. Two different non-overlapping fragments of the PepMV polymerase gene were cloned and sequenced, and their putative positions in the viral RNA were determined relative to the PVX sequence. For the diagnosis procedure, new specific PepMV primers were designed based on the virus sequence obtained and their utility to amplify a unique diagnostic band of 835 bp after IC-RT-PCR with specific anti-PepMV antibodies was shown. The method developed should allow the rapid diagnosis of a virus that seriously threatens tomato cultivation in several European countries.

An outbreak of enterically transmitted non-A, non-E viral hepatitis.

Patients with isolated serum transaminase elevations of unknown cause are common in China. An outbreak of such disease took place in a technicians' school during 1996. To define the epidemic and determine the etiology, a study was carried out, which included investigation of epidemiological, clinical and histological features. The symptoms of this disease were mild. The major clinical feature was transaminase elevation, and all serum markers of known hepatitis viruses were negative. Although the course of disease in most patients was self-limiting, in a few it was prolonged and relapsed. Histological findings were mild portal hepatitis or non-specific reactive hepatitis. The disease first appeared in 1994, and this outbreak occurred after October 1996. A total of 381 people were affected and the prevalence was as high as 60.7%. Casual contact and small-scale food transmission were considered to be risk factors for infection and the epidemic was under control 2 months later following the introduction of preventive measures for gastroenteric infection. Viral genomic fragments from the so-called transfusion-transmitted virus (TTV) were detected in acute-phase sera and stool samples collected 2 weeks before onset. Therefore, this disease outbreak might be another form of enterically transmitted viral hepatitis, not related to hepatitis A and E.

Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of specific gene probes.

The ssRNA genome extracted from purified Taura Syndrome Virus (TSV) was transcribed into double-stranded, blunt-ended cDNA and was used to construct cDNA libraries either in pUC 18 or in pBluescript II KS-vectors. Twelve recombinant plasmids chosen after screening of the libraries were subjected to restriction enzyme digestions for determination of size inserts and restriction maps. Two of them, pP15 and pQ1, were selected for probe construction. The inserts, 1500 and 1300 base pairs (bp) respectively, were DIG-11dUTP-labelled and the corresponding probes were named P15 and Q1. On northern blots and dot blots, using different denaturation methods, the 2 probes hybridized specifically with extracted RNA-TSV genome, TSV and infected TS shrimp homogenates. No positive hybridization was obtained with other shrimp viruses tested [Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Hepatopancreatic Parvovirus (HPV)]. The specificity of the 2 probes was confirmed by in situ hybridization on histological sections of TS diseased shrimps.

Molecular epidemiology of animal virus diseases.

In this review the application of methods of molecular epidemiology, particularly the combined approach of amplifying defined fragments of viral genomes using the polymerase chain reaction and subsequent nucleotide sequencing analysis, is described. Emphasis is put on examples of a few important diseases (e.g. those caused by morbilliviruses, rhabdoviruses, pestiviruses and aphthoviruses) to demonstrate the impact of this methodology. Molecular epidemiology is already an important and very sensitive tool to study the evolution of viruses at a level superior to previous methodologies and providing a better understanding of epidemiological relationships.

Identification and characterization of a cDNA derived from multiple splicing that encodes envelope glycoprotein gp105 of human herpesvirus 6.

The glycoprotein complex gp82-gp105 is a major virion envelope glycoprotein complex of human herpesvirus 6 variant A (HHV-6A) and consists of a number of related polypeptides. Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against this glycoprotein complex neutralized HHV-6A infectivity. We have previously reported the isolation, mapping, and characterization of a portion of the viral genomic DNA fragment encoding the gp82-gp105 complex and the identification of the neutralizing epitope (B. Pfeiffer, Z. N. Berneman, F. Neipel, C. K. Chang, S. Tirwatnapong, and B. Chandran, J. Virol. 67:4611-4620, 1993). This gene was further characterized by the identification of a 2.3-kb genomic fragment and by the identification of a 2.5-kb cDNA clone. The genomic sequence contains a short open reading frame (ORF) encoding the epitope recognized by the MAbs. The identified cDNA showed specificity for HHV-6 in Southern blot analysis with viral DNA. In Northern (RNA) blot analysis with total RNA from HHV-6A(GS)-infected cells, the cDNA insert specifically hybridized with several RNA species. Restriction mapping analysis localized this cDNA to the HHV-6A(U1102) genomic BamHI G fragment, at the right end of the unique long segment of the genome and to the SalI L and SalI O fragments within the left and right terminal direct repeat regions, respectively. In vitro transcription and translation of the cDNA revealed a polypeptide of about 88.5 kDa which was glycosylated in the presence of microsomal membranes to a polypeptide of approximately 104.2 kDa. Both polypeptides were immunoprecipiated by MAb 2D6, verifying the identity of the cDNA as encoding the gp105 in the gp82-gp105 complex. Sequence analysis of the cDNA revealed a large ORF potentially encoding a 650-amino-acid protein with 11 potential N-linked glycosylation sites and 18 cysteine residues. A potential membrane-spanning domain is located only near the amino terminus of the putative protein, indicating that gp105 may be a class 2 glycoprotein. Comparison of the cDNA nucleotide sequence with sequences from HHV-6A(U1102) genomic BamHI G and SalI L fragments revealed that the gene encoding gp105 contains 12 exons, spanning over 20 kb of the viral genome, with intron 1 spanning about 8 kb of genomic DNA. The first exon of the cDNA mapped to the right and left terminal direct repeats, while the other exons mapped within the unique long segment of the genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a very early promoter of insect Hz-1 virus using a novel dual-expression shuttle vector.

Very early promoters of viruses control the proper cascade expression of viral genes and are essential for completion of virus life cycles. These promoters are usually rare and weak and do not encode structural proteins. As a result, they are difficult to identify. In order to identify and clone the very early promoters of a large eukaryotic DNA virus, the Hz-1 virus, a novel cloning strategy was applied. This strategy is based on a dual-expression shuttle vector containing a promoter-less lacZ gene. Insertion of eukaryotic promoters upstream permits the efficient expression of LacZ in bacteria cells. The function of the putative promoters was then confirmed by their proper expression in insect cells. The first two productive infection-specific promoters of Hz-1 virus, contained within the shuttle vectors pTSV-2-129 and pTSV-2-49, were cloned from the HindIII-K and HindIII-A fragments of the Hz-1 viral genome, respectively. By primer extension analysis, an immediate and constitutive expression of the promoter in clone pTSV-2-129 was detected after viral infection. Identification of the productive infection-specific promoters has laid down important groundwork for future studies on the molecular mechanism of the transcriptional switch between productive and persistent infections of Hz-1 virus.

A Murine Leukemia Virus Expressed in Aged DBA/2 Mice Is Derived by Recombination of the Emv-3 Locus and Another Endogenous Gag Sequence

Although most inbred strains of mice contain endogenous retroviral sequences, these sequences are usually not capable of producing infectious retroviruses. In some cases, the retroviral sequences are small fragments of viral genomes. In a few cases the sequences for complete retroviruses exist, but contain small defects which prevent the production of infectious virus. While the ability of reversions of these defects to produce retroviruses has been studied by site-directed mutagenesis and chemical-mediated mutagenesis, the presence of spontaneously occurring reversions has not been completely evaluated. We characterized an infectious ecotropic retrovirus spontaneously expressed in aged DBA/2 mice, which carries the complete but defective Emv-3. Although this endogenously produced retrovirus was related to the endogenous Emv-3 sequence, it had undergone recombination with another retroviral sequence to correct the defect which resided in the gag of Emv-3. Thus, recombination of endogenous ecotropic retroviral sequences may be a mechanism to produce infectious retroviruses in adult animals that contributes to pathologic disease.

A New Virus Isolated from an Ash Tree with Die‐Back

AbstractA virus isolated from an ash tree with die‐back has icosahedral virions c. 28 nm in diameter which contain a polyadenlyated and infectious bipartite single stranded RNA genome. The RNAs have M. Wts of c. 2.4 and 2.6 × 106. The virus was transmitted in 5/48 seed from infected Chenopodium quinoa. Serological tests failed to detect any relationships with 10 viruses. cDNA to the viral RNA was cloned. The sequences of the 5′ and 3′ ends of two clones were determined. One of these clones represents the 3′‐terminus of one of the viral genomic fragments. A search of nucleic acid sequence databases showed some similarity of the 3′‐terminal sequence of the virus to the 3′‐terminal sequence of cowpea mosaic virus. The virus is possibly a previously undescribed nepovirus or comovirus.

Analysis of Epstein-Barr virus gene transcription in lymphoma induced by the virus in the cottontop tamarin by construction of a cDNA library with RNA extracted from a tumour biopsy.

Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.

Constitutive synthesis of polyoma antisense RNA renders cells immune to virus infection

Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting polyoma.

Skeletal Muscle Rhabdomyosarcomas in Inbred Laboratory Mice

A total of 14 well differentiated rhabdomyosarcomas were diagnosed at necropsy in 10,000 mice. Of the 14 affected mice, ten were BALB/cJ, and there was one case each of A/HeJ, BALB/cByJ, C58/J, and C.B-17-scid/scid strains. Most often (10/14) tumors originated in the quadriceps muscles and metastases occurred in six cases. When submitted, affected mice were 2 to 8 months of age, with a mean age of 4 months. Tumor frequency for BALB/cJ mice was calculated to be 2.4/100,000 mice retained as breeders. No sexual dimorphisms were determined when data were correlated to actual numbers of each sex in the colony. All 14 primary tumors and metastases were positive by immunohistochemistry for the proteins pan myosin, sarcomeric actin, desmin, actin, and myosin, but were negative for smooth muscle actin, thus confirming the diagnosis. Using cell free homogenates of primary tumors, inoculated by intraperitoneal or intramuscular injection, tumors were not induced in either BALB/cJ or C58/J mice observed over a 22-week period. Southern blot analysis of DNA prepared from tumors and hybridized with a murine leukemia virus probe that recognizes both ecotropic and dualtropic viruses did not demonstrate viral genomic fragments in addition to those known to occur in each strain.

DNA sequence of the nucleoside triphosphate phosphohydrolase I (NPH I) of the Choristoneura biennis entomopoxvirus

The DNA sequence of an open reading frame (ORF) corresponding to a Choristoneura biennis entomopoxvirus putative late gene was determined. Residing within an 8-kp EcoRI viral genomic fragment, this ORF is 1944 nucleotides long, encoding a basic protein (p/9.83) with a predicted molecular weight of 76,000 Da. Computer analysis indicates a 36.4% homology between this ORF and the vaccinia nucleoside triphosphate phosphohydrolase I (NHP I) gene, subh substantially greater homology (60%) in two domains believed to be involved in ATP binding. The entomopoxvirus ORF contains 78% AT residues; and like other poxvirus late genes, it possesses the conserved TAAAT motif at the 5′ teminus of the gene.

Evidence for viral and naked double-stranded RNAs in the basidiomycete Agrocybe aegerita

Double-stranded (ds) RNAs were purified from the vegetative mycelium of the commercial basidiomycete Agrocybe aegerita, by gel-filtration chromatography, and their dsRNA nature was demonstrated. They were shown to be distributed in two different RNase-resistant complexes which were separated by sucrose density gradient ultracentrifugation: the first complex is the encapsidated genome (6200 bp) of an isometric mycovirus; the second consists of three naked dsRNA molecules (1900-1800-1700 bp respectively) associated with large vesicles or mitochondria. This is the first report of the presence of naked dsRNA, unencapsidated, viral genome fragments which have co-evolved with their host, or else represent new replicating genetic elements (dsRNA “plasmids”) of unknown function, in a non pathogenic higher fungus.

Alternative splicing dictates translational start in Epstein-Barr virus transcripts.

Of the 10 viral genes known to be expressed during Epstein-Barr virus (EBV) latency, six encode nuclear antigens (EBNAs), all of which are expressed from a long primary transcript by means of alternative splicing and alternative polyadenylation sites. The primary transcript is generated by either of two promoters which operate in a mutually exclusive fashion in different clonal cell lines. All mRNAs from either promoter have exons in common from the BamHI W viral genomic fragment (the major internal repeat, IR1) which encode the N-terminal portion of one of the nuclear antigens (EBNA 4). In addition to the coding regions for EBNA 4, EBNA mRNAs encode another EBNA (i.e. EBNA 1, 2, 3A, 3B or 3C) downstream. We show that alternative splicing determines whether the translation initiation codon for EBNA 4 is present or absent, thus permitting the generation of mRNAs in which the first translation initiation codon is either that for the EBNA 4 gene or for the other EBNA gene encoded downstream. This mechanism presumably ensures efficient translation of all the EBNA genes.

A mouse linkage testing stock possessing multiple copies of the endogenous ecotropic murine leukemia virus genome

A new linkage testing stock of the laboratory mouse has been constructed. The stock, designated MEV ( multiple ecotropic pro vvirus), was developed by inbreeding and selection beginning with the cross of strains C58 J and AKXD-14. Eleven different murine leukemia virus (MuLV) proviruses have been fixed in the MEV 1Ty strain. Nine of these can be uniquely identified by Southern blotting of PvuII-digested DNA and probing with a cloned fragment of the ecotropic viral genome. Two proviruses had been mapped previously to chromosome 7, while single proviruses had been mapped to chromosomes 2, 9, and 11. The mapping of six additional proviruses, derived from C58 J , to chromosomes 1, 3, 5, 10, 18, and 19 is described. Another C58 J provirus was mapped to chromosome 8 and proved to be identical to the previously mapped C58v-1 virus inducibility gene of strain C58 Lw . Three dominant visible markers, hammer-toe ( Hm), steel ( Sl), and caracul-J ( Ca J ), located on chromosomes 5, 10, and 15, respectively, have been introduced onto the MEV genetic background by repeated backcrosses to provide additional linkage markers. It is estimated that approximately 50% of the genome can be screened by scoring 50 fully informative gametes from a linkage cross of the MEV- Hm, -Sl, -Ca J stock for the combination of viral and visible markers. A strategy for efficiently mapping new recessive visible mutations by pooling tissues for DNA extraction from mutant homozygotes among F 2 progeny is described. Ways of further improving the MEV stock are discussed. The location of the Myb proto-oncogene is defined relative to Sl and one of the C58 J proviruses on chromosome 10.

Characterization of a latent protein encoded by the large internal repeats and the BamHI Y Fragment of the epstein-barr virus (ebv) genome

Analysis of EBV nuclear antigen 1 (EBNA 1) encoding transcripts by cDNA characterization revealed a potentially polycistronic message generated by long-range splicing of several exons (Speck, S., and Strominger, J., Proc. Natl. Acad. Sci. USA 82, 8305–8309, 1985). Besides the open reading frame encoding EBNA 1, two other open reading frames are found in the EBNA 1-specific cDNA. The first reading frame consists of several exons from BamHl W and Y viral genome fragments (Wi, W2, Y1, and Y2). In our experiments, the W1 axon was expressed in the tryptophan-regulated expression vector pATH11. Rabbit sera, raised against the bacterial fusion protein, recognized one or two proteins of molecular weights between 30,000 and 100,000 in several EBV genome harboring Burkitt lymphoma and EBV immortalized peripheral blood cell lines. Although, in a few cell lines from both groups no specific protein could be detected. Immunofluorescence analysis and characterization of subcellular distribution demonstrated that this W/Y fragment encoded latent protein is located, in part, in the cytoskeleton fraction, and in the chromatin. In addition, 2-D immunoblot analysis revealed post-translational modifications of this latent protein, probably due to phosphorylation. In DNA-binding studies on DNA cellulose columns, this W/Y encoded latent protein exhibited specific DNA binding activities.