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At least 865 records · Page 48Linked to original sources

PubMed1992

Detection of anti-gag antibodies of feline immunodeficiency virus in cat sera by enzyme-linked immunosorbent assay.

Using gag protein of feline immunodeficiency virus (FIV) expressed in Escherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIV gag protein in cat sera. With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIV gag protein were observed in all cases by the ELISA at early stage of infection. When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9%) out of 70 IFA positive sera were judged as negative by the ELISA. However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gp130 but not to p26 by a radioimmunoprecipitation assay. The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.

Animals↗

PubMed1995

Functional analysis of human spuma retrovirus genome.

Human spuma retrovirus (HSRV) belongs to retroviruses that possess a complex genome organization. HSRV carries at least three extra genes in the region between env and the 3' long terminal repeat, which are not found in simple retroviruses. Via alternative splicing, these HSRV genes can encode several proteins. To genetically study the requirements of these viral proteins for viral replication in tissue cultures, a number of mutant viruses were constructed from an infectious molecular clone of HSRV. All mutants grew normally in the cell lines tested, except for those lacking transcriptional transactivator activity. By reporter-based transient assay systems, no Rev/Rex equivalent was detected in the HSRV proteins.

Alternative Splicing↗

PubMed2003

The role of the defective interfering particle DI9c in mucosal disease in cattle.

Mucosal disease occurs in cattle persistently infected with a noncytopathogenic strain of bovine viral diarrhoea virus (BVDVnc) following in utero infection. The disease can be initiated by superinfection with a cytopathogenic biotype (BVDVc) of the virus with antigenic "homology" to the persisting virus. A BVDVc isolated from a clinical case of mucosal disease has been discovered to consist of a defective interfering particle, DI9, and an associated BVDVnc helper virus. A defective virus corresponding to DI9 was recently recovered from an infectious cDNA clone and was named DI9c. To evaluate the role of DI9 in the pathogenesis of mucosal disease a two-part experimental study was carried out which included clinical, haematological, pathological and virological investigations. Eight of nine calves persistently infected with BVDVnc were experimentally inoculated with DI9c. The defective virus was propagated in cells preinfected with the same strain of virus used to persistently infect the calves in utero. The calves were euthanased on days 4, 7, 14, 21, 28, 40, 40 or 87 post inoculation. None of the inoculated animals developed classical mucosal disease, neither clinically nor pathologically. DI9c was not found in serum, nasal swab or tissue samples from the calves by observing cytopathogenic effect and/or using a polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. DI9c did not replicate to a detectable extent in these assays, and its participation in the pathogenesis of mucosal disease could not be proven.

Animals↗

PubMed2003

The quantity and diversity of infectious viruses in various tissues of SHIV-infected monkeys at the early and AIDS stages.

To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV(KU-2)-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had the largest number of infectious viruses at the AIDS stage more than at the early stage of infection. These results suggested that the gastrointestinal tract was a major site of viral replication. In the brain, proviral DNA was detected at the early and AIDS stage of infection, but infectious viruses were detected at only the AIDS stage. Moreover, we analyzed the nucleotide sequences of the env V3 region in infectious virus clones isolated from each plaque. The viruses in the lymphoid tissues of the monkey that developed AIDS diverged from the inoculated virus and had the same three amino acid substitutions. However, the viruses in the brain were almost identical to the inoculated virus, suggesting that the virus entered the brain early after infection and persisted without replication and genetic diversion until the AIDS stage.

Amino Acid Sequence↗

PubMed2004

Stable expression of foreign proteins in herbaceous and apple plants using Apple latent spherical virus RNA2 vectors.

Infectious cDNA clones of Apple latent spherical virus (ALSV)-RNA1 (pEALSR1) and -RNA2 (pEALSR2) were constructed using an enhanced 35S promoter. A viral vector was constructed from pEALSR2 by creating artificial protease processing sites by duplicating the Q/G protease cleavage site between 42KP and Vp25. Eight RNA2-derived vectors expressing GFP with varied sizes of duplications around the 42KP/Vp25 junction were constructed and tested for infectivity in Chenopodium quinoa. The results indicated that greater than five aa from the C-terminus of 42KP and N-terminus of Vp25 in duplication are necessary for systemic infection. In infected C. quinoa plants, GFP fluorescence was observed in both inoculated and upper leaves. Serial passages of the viruses derived from the above vectors in C. quinoa showed that the size of duplications affected the stability of the GFP gene. The version of the RNA2-vector (pER2L5R5GFP) with the shortest duplications and its silent mutant version could stably express GFP in leaves even after at least nine serial passages. ALSV-RNA2 vector has a capacity to maintain a DNA insert as long as 1300 bp because Apple chlorotic leaf spot virus movement protein (50KP) gene could be expressed in C. quinoa. Inoculation of a virus derived from pER2L5R5GFP to apple seedlings resulted in the expression of GFP fluorescence in uninoculated upper leaves, indicating that the vector is available for the expression of foreign genes in apple trees.

Blotting, Western↗

PubMed1997

Feline leukaemia virus proviral DNA detected by polymerase chain reaction in antigenaemic but non-viraemic ('discordant') cats.

A nested polymerase chain reaction (PCR) was established to detect exogenous feline leukaemia virus (FeLV) proviral DNA in feline peripheral blood leukocytes (PBL). The assay detected a single copy of plasmid DNA of an infectious molecular clone of FeLV subgroup A in the sample by ethidium bromide staining in agarose gels. The utility of the nested PCR in the diagnosis of FeLV infections was compared with the detection of FeLV p27 antigen by enzyme-linked immunosorbent assay (ELISA) and virus isolation (VI). FeLV genomes were detected by PCR in all 4 samples that were positive by ELISA and VI but in none of 7 samples that were negative by the two methods. FeLV genomes were found by PCR in 13 of 39 samples from cats that were antigenaemic but from which no virus was isolated ('discordant' cats). These results demonstrated that a proportion of discordant cats harboured FeLV genome in their PBL.

Animals↗

PubMed1997

Non-random deletions in human foamy virus long terminal repeat during viral infection.

We have characterized a new form of human foamy virus (HFV) non-random deleted long terminal repeat (LTR) sizing 1078-bp, deleted in its U3 region, sensitive to the viral transactivator and functional in an infectious proviral clone. Besides two known HFV LTRs of 1260-bp and 1123-bp, this LTR represents the smallest, designed S. Analysis of the LTR sequence shows the presence of short direct repeats surrounding the deletions, suggesting a mechanism generating deletion by misalignment of the growing strand during replication. Our data suggest that the deleted LTRs, preferentially associated with chronic viral infection, could be related with viral persistence.

Animals↗

PubMed1998

Mapping the genetic determinants of human immunodeficiency virus type 2 for cell tropism and replication efficiency.

Two distinct infectious molecular clones of human immunodeficiency type 2 (HIV-2) were analyzed for their biological properties in six cell lines. Fourteen chimeric and ten mutant viruses were constructed from these two viral genomes to localize the genetic determinants responsible for the phenotypes. Growth property of the viruses in the cell lines, together with the biochemical data, showed that a major determinant for the viral tropism resides in the env gene. In addition, in some cell lines, the accessory genes vif and nef affected the efficiency of virus replication. Thus, like HIV-1, mutations in the auxiliary and env genes of HIV-2 contributed much to the differences in virological characteristics.

Base Sequence↗

PubMed1998

Cassava vein mosaic virus (CsVMV), type species for a new genus of plant double stranded DNA viruses?

The complete sequence of 8159 nucleotides of the double stranded DNA genome of cassava vein mosaic virus (CsVMV) was determined (# U59751) and revealed a significant difference in genome organization when compared with a previous report (# U20341). When transferred to cassava plants by microbombardment, the full length CsVMV clone was infectious, confirming the genome organization here described. Sequence comparisons between CsVMV and members of the genera Caulimovirus and Badnavirus revealed high homologies between consensus sequences of several proteins that are indispensable for virus replication, including a potential transactivator factor not reported previously. The presence of a sequence complementary to a plant Met tRNA confirms that CsVMV is a plant pararetrovirus and is most closely related to members of the genus Caulimovirus as previously assessed. However, differences in genome organization, number and size of the ORFs, in addition to sequence comparisons with other plant pararetroviruses, shows that either the genetic variability of caulimoviruses is much greater than previously thought, or that CsVMV is the unique representative of a new genus within the Caulimoviridae family. On the basis of this study, it is proposed to upgrade the floating genus Caulimovirus to the family level and to divide the Caulimoviridae family into at least three genera with CsVMV being the type member of a new genus.

Amino Acid Sequence↗

PubMed1999

Classical swine fever virus is genetically stable in vitro and in vivo.

Phylogenetic analyses of large numbers of classical swine fever strains have revealed a high degree of sequence conservation in the genomic regions examined, suggesting either a recent common ancestor or a low evolution rate. This low variability is in contrast to findings with other RNA viruses. To investigate the consequence of this apparent genetic stability on phylogenetic examinations, the Belgian field isolate Wingene'93 was passaged in pigs as well as in cell culture by various methods. Sequence analyses of viruses collected after various passages in three target regions proposed for phylogenetic studies (5' NTR, E2, and NS5B) revealed a complete sequence conservation. Only when the amount of passaged virus was lowered, mimicking a genetic bottleneck, a single point mutation was observed in the E2 gene. Additionally, only four nucleotide substitutions were observed when the genome of a virus obtained after 96 cell passages in persistently infected cells was compared with its parental virus, the recombinant virus derived from an infectious cDNA clone of CSFV strain Alfort/187. This low mutation frequency observed both in vitro and in vivo demonstrates that classical swine fever virus is genetically stable. Hence, even minor mutations can be considered significant in molecular epidemiological studies.

Animals↗

PubMed2001

Determination of the nucleotide sequences at the extreme 5' and 3' ends of swine hepatitis E virus genome.

The nucleotide sequences at the extreme 5' and 3' ends of swine hepatitis E virus (swine HEV) genome were determined, and genomic sequence of swine HEV is now complete. Sequence analysis revealed that the 3' and 5' non-coding regions (NCRs) of swine HEV are closely related to that of the US-1 and US-2 strains of human HEV. Like the two U.S. strains of human HEV, an extra G residue immediately proceeding the poly(A) tail was identified in swine HEV. The 5' NCR of swine HEV also differed from many HEV strains: it lacks an A residue at its 5' very end, and the extra 9 nucleotides in the US-2 strain. In the 3' NCR, swine HEV shared 90-91% nucleotide sequence identities with the US-1 and US-2 strains but only about 58-65% identities with other HEV strains. This study further suggests that the US-1 and US-2 strains of human HEV may be of swine origin. The availability of the complete sequence of swine HEV should facilitate the construction of an infectious cDNA clone of swine HEV.

Animals↗

PubMed2005

A 45-nucleotide insertion in the NS2 gene is responsible for the cytopathogenicity of a bovine viral diarrhoea virus strain.

Cytopathogenicity (cp) markers have recently been investigated in the genomes of field isolates of bovine viral diarrhoea virus (BVDV). Most of the isolates originated from mucosal disease (MD) cases observed after vaccination with a live attenuated vaccine, termed here BVDV-X. The NS2-3 genes of these isolates and of the vaccine proved to be identical, including a 45-nucleotide (nt) viral insertion at nt position 4355. The insertion originated from the NS4B/5A junction region of the BVDV genome. Interestingly, in BVDV strain CP7 a 27-nt insertion originating from the NS2 is located exactly at the same position. Complete genome analysis of BVDV-X did not reveal further potential cp markers. Furthermore, expression studies indicated that the insertion promotes NS2-3 cleavage. In order to examine the possible role of the 45-nt insertion in viral cytopathogenicity in details, a full-length infectious cDNA clone of BVDV-X was generated, and bovine turbinate (BT) cells were transfected with RNA transcribed from the clone. The recovered virus, termed BVDV-XR, showed slight retardation in growth in comparison with the original BVDV-X, and induced cytopathogenic effect (CPE). Since the natural non-cytopathogenic (ncp) counterpart of the vaccine virus was not available, an insertion-negative mutant cDNA clone was generated from BVDV-XR by PCR-directed mutagenesis. The recovered virus, termed BVDV-XR-INS-, showed the same growth characteristics as its cp counterpart BVDV-XR, but caused no CPE. These findings provide a direct proof that the 45-nt insertion at position 4355 has a basic role in the cytopathogenic character of this BVDV strain.

Animals↗

PubMed2006

Production of a full-length infectious GFP-tagged cDNA clone of Beet mild yellowing virus for the study of plant-polerovirus interactions.

The full-length cDNA of Beet mild yellowing virus (Broom's Barn isolate) was sequenced and cloned into the vector pLitmus 29 (pBMYV-BBfl). The sequence of BMYV-BBfl (5721 bases) shared 96% and 98% nucleotide identity with the other complete sequences of BMYV (BMYV-2ITB, France and BMYV-IPP, Germany respectively). Full-length capped RNA transcripts of pBMYV-BBfl were synthesised and found to be biologically active in Arabidopsis thaliana protoplasts following electroporation or PEG inoculation when the protoplasts were subsequently analysed using serological and molecular methods. The BMYV sequence was modified by inserting DNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene close to its 3' end. A. thaliana protoplasts electroporated with these RNA transcripts were biologically active and up to 2% of transfected protoplasts showed GFP-specific fluorescence. The exploitation of these cDNA clones for the study of the biology of beet poleroviruses is discussed.

Beta vulgaris↗

PubMed1992

Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1.

Preparations of coxsackievirus B1 (CVB1) derived from an infectious cDNA clone have been crystallized in multiple crystal forms. Using high intensity synchrotron radiation, an orthorhombic form of the crystals was shown to diffract X-rays to at least 2.9 A resolution. The unit cell has a primitive lattice with dimensions a = 323 A, b = 450 A, and c = 522 A. A crystallographic asymmetric unit of these CVB1 crystals probably contains an entire virus particle, implying the presence of 60-fold non-crystallographic redundancy. This CVB1 crystal form appears to be suitable for high-resolution structure determination by X-ray crystallography.

Crystallography↗

PubMed1984

The nucleotide sequence of the Akv murine leukemia virus genome.

The nucleotide sequence of an infectious molecular clone of the Akv murine leukemia virus has been determined by the dideoxy chain termination method after subcloning in bacteriophage M13 vectors. The sequence predicts an RNA genome of 8371 nucleotides containing three large open reading frames corresponding to the gag, pol, and env genes. Signal sequences for transcription, splicing, and translation have been identified. The positions of 95 major RNase T1 resistant oligonucleotides of the Akv RNA genome have been located.

AKR murine leukemia virus↗

PubMed1987

Nucleotide sequence of a radiation leukemia virus genome.

The complete nucleotide sequence of an infectious molecular clone of a radiation murine leukemia proviral DNA RadLV/VL3(T+L+) has been determined. The sequence of the RNA genome is 8318 nucleotides long and contains three large open reading frames encoding the gag, pol, and env gene products. With the exception of a xenotropiclike R peptide and the LTR which bears structural similarities to a xenotropic LTR, displaying typical enhancerlike sequences, the remaining sequences are strikingly similar to the endogenous, ecotropic Akv murine leukemia virus. Therefore, it could be postulated that the leukemogenic properties of RadLV/VL3(T+L+) were generated by a recombination event between a xenotropic virus and an Akv-like ecotropic virus.

Base Sequence↗

PubMed1989

Mutation rate of the hepadnavirus genome.

An essential factor for charting the evolution of hepadnaviruses is an estimation of the mutation rate of the virus genome during replication in the host. In order to determine the mutation rate of the hepadnavirus genome under defined experimental conditions, we transfected 10 neonatal woodchucks with an infectious molecular clone of woodchuck hepatitis virus (WHV). By 4 months post-transfection, all 10 animals showed serological evidence for WHV infection. Subsequently, 1 animal became chronically infected and was used for further study. At 16 months post-transfection WHV DNA from serum virions was cloned and the nucleotide sequence of three independent progeny genomes compared directly with that of the input recombinant DNA. Although the consensus nucleotide sequence remained unchanged, we found three differences in individual progeny genomes when compared to the parental genome sequence. Thus, we estimate the mutation rate of the WHV genome to be less than or equal to 2 X 10(-4) base substitutions/site/year. This figure is one to two orders of magnitude lower than the mutation rates previously calculated for the positive- and negative-strand RNA viruses, but is similar to the mutation rate of the gag gene which is the most slowly evolving gene of retroviruses. Therefore, we find that the hepadnavirus genome is relatively stable during replication in host tissues when compared to other viruses that lack polymerase-associated proofreading functions.

Animals↗

PubMed1989

Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag.

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.

Acquired Immunodeficiency Syndrome↗