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PubMed1988

Immune responses are required to terminate viremia in equine infectious anemia lentivirus infection.

Six normal and four immunodeficient horses were injected with a cloned variant of equine infectious anemia virus (EIAV). The six normal horses had detectable EIAV in their plasma by 7 days postinjection. During their primary viremic episode, which was accompanied by fever and anemia, maximum titers of EIAV in plasma ranged from 10(3.8) to 10(4.8) 50% tissue culture infective doses per ml. All six normal horses cleared detectable virus from their plasma by 21 to 35 days after injection. Horses with combined immunodeficiency became viremic by 9 days postinjection and also developed anemia. In contrast to normal horses, foals with combined immunodeficiency did not eliminate the virus from their plasma.

Animals↗

PubMed1988

Infectivity and complete nucleotide sequence of the genome of a South African isolate of maize streak virus.

The complete infectious genome of a South African isolate of the geminivirus maize streak (MSV-S) has been cloned, characterized, and sequenced. Using an A. tumefaciens Ti plasmid delivery system, the cloned -2.7 kb single circular MSV component was shown to be necessary and sufficient for infection of maize. Based on sequence analysis of the infectious clone, MSV-S is highly homologous to the previously characterized Kenyan and Nigerian isolates. While the genomic organization of MSV-S has elements in common with each of these previously characterized isolates, it is identical to neither and its analysis addresses the discrepancies between them. The result is a somewhat simplified and unified picture of the viral genome, the structural organization of which is essentially identical to that of wheat dwarf virus.

Base Sequence↗

PubMed2006

Monitoring the T-cell receptor repertoire at single-clone resolution.

The adaptive immune system recognizes billions of unique antigens using highly variable T-cell receptors. The alphabeta T-cell receptor repertoire includes an estimated 10(6) different rearranged beta chains per individual. This paper describes a novel micro-array based method that monitors the beta chain repertoire with a resolution of a single T-cell clone. These T-arrays are quantitative and detect T-cell clones at a frequency of less than one T cell in a million, which is 2 logs more sensitive than spectratyping (immunoscope), the current standard in repertoire analysis. Using T-arrays we detected CMV-specific CD4+ and CD8+ T-cell clones that expanded early after viral antigen stimulation in vitro and in vivo. This approach will be useful in monitoring individual T-cell clones in diverse experimental settings, and in identification of T-cell clones associated with infectious disease, autoimmune disease and cancer.

Amino Acid Sequence↗

PubMed2004

Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation.

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Delta132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Delta132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Delta132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.

Animals↗

PubMed1979

Clones of cells from a human embryo lung: their growth and susceptibility to respiratory viruses.

Colonies of cells were obtained from human fetal lung tissue and exposed to recently isolated respiratory viruses. There was a considerable variation in the number of rounded cells found in different colonies exposed to rhinovirus types 2 and 9 (RV2 and 9), human coronavirus 229E (HCV), adenovirus type 3 (Ad3) and respiratory syncytial virus (RSV). Smaller colonies had more rounded cells than larger colonies. Clones were established from 9 out of 11 colonies. They varied in their rate of growth and the pattern formed on a plastic surface. They varied also in their virus susceptibility particularly to "difficult" rhinoviruses such as RV9 and SF1340. One cell clone (HL1/77 Clone 8), was highly susceptible to all these viruses. All cultures were more sensitive to RSV when maintained in F12K medium than in MEM, whereas there was no difference for rhinoviruses. Influenza A and B and parainfluenza 3 viruses sometimes produced cytopathic effect, and always produced haemadsorption, but unlike the previous strains could not be passed serially and presumably produced little infectious virus. All clones were rather insusceptible to Ad3; but the virus could be passed, whereas coxsackie virus B3 produced no CPE. Substantial yields of coronavirus and rhinovirus were obtained in gelatin sponge cultures. Two "very difficult" respiratory viruses which had just been adapted to tissue culture; namely, HS rhinovirus and JK coronavirus grew in 7 of 9 and in 6 of 9 clones respectively.

Adenoviruses, Human↗

PubMed2001

Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses.

A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5' cap. Both RNAs were largely resistant to 3' polyadenylation and ligation, suggesting the presence of an interfering 3' structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5' and 3' non-coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT-PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3' terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 0.4 kb) that was newly synthesized and corresponded to the 3'-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed.

3' Untranslated Regions↗

PubMed1994

Leeway and constraints in the forced evolution of a regulatory RNA helix.

The start of the coat protein gene of RNA phage MS2 adopts a well-defined hairpin structure of 12 bp (including one mismatch) in which the start codon occupies the loop position. An earlier expression study using partial MS2 cDNA clones had indicated that the stability of this hairpin is important for gene expression. For every -1.4 kcal/mol increase in stability a 10-fold reduction in coat protein was obtained. Destabilizations beyond the wild-type value did not affect expression. These results suggested that the hairpin was tuned in the sense that it has the highest stability still compatible with maximal ribosome loading. Employing an infectious MS2 cDNA clone, we have now tested the prediction that the delta G 0 of the coat protein initiator helix is set at a precise value. We have introduced stabilizing and destabilizing mutations into this hairpin in the intact phage and monitored their evolution to viable species. By compensatory mutations, both types of mutants quickly revert along various pathways to wild-type stability, but not to wild-type sequence. As a rule the second-site mutations do not change the encoded amino acids or the Shine-Dalgarno sequence. The return of too strong hairpins to wild-type stability can be understood from the need to produce adequate supplies of coat protein. The return of unstable hairpins to wild-type stability is not self-evident and is presently not understood. The revertants provide an evolutionary landscape of slightly suboptimal phages, that were stable at least for the duration of the experiment (approximately 20 infection cycles).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

PubMed1996

Analysis of the long terminal repeat from a cytopathic strain of equine infectious anemia virus.

Sequential passage of the tissue culture-adapted prototype strain of EIAV in fetal donkey dermal (FDD) cell cultures generated a virus stock which exhibits cytopathic effects in FDD cell cultures. In this study, the effects of the long terminal repeat (LTR) region on virus replication and cytopathogenicity were examined. The FDD-adapted virus LTR was found to contain a number of base pair mutations and a large insertion within the U3 region in comparison with the previously characterized LTR, lambda12. Transient gene expression studies showed that basal promoter activity, in FDD cell cultures, of the FDD-adapted virus LTR was more than seven times greater than that of lambda12. Analysis of an infectious EIAV molecular clone in which the LTR region was replaced with the corresponding region of FDD-adapted virus showed that the LTR increased replication capacity but did not influence cytopathogenicity.

Animals↗

PubMed1988

EIAV genomic organization: further characterization by sequencing of purified glycoproteins and cDNA.

Nucleotide sequence analyses of two different proviral clones of equine infectious anemia virus (EIAV), designated lambda 12 (K. Rushlow et al., 1986, Virology 155, 309-321) and 1369 (T. Kawakami et al., 1987, Virology 158, 300-312), indicate significant differences in the organization of two critical regions of the viral genome, i.e., in the short open reading frames in the pol-env intergenic region and in the 5'-end of the env gene. To determine the correct structure of the EIAV genome, we have performed nucleotide sequence analyses of cDNA clones produced from viral RNA and direct sequencing of purified EIAV envelope glycoproteins (gp90 and gp45). The results of the cDNA sequencing confirm the presence of two short open reading frames in the pol-env intergenic region, as reported previously for the lambda 12 clone. The protein sequencing data correlated exactly with the amino-terminal sequences of gp90 and gp45 deduced from lambda 12 nucleotide sequences. However, the protein sequencing also revealed that the putative signal sequence of EIAV gp90 is not removed during processing. Thus, EIAV apparently contains short open reading frames analogous to human immunodeficiency virus, but differs in its mode of env polyprotein processing.

Amino Acid Sequence↗

PubMed2006

Rescue of the prototypic Arenavirus LCMV entirely from plasmid.

We document a helper-independent reverse genetics system for rescuing infectious arenaviruses from cloned cDNAs. We constructed plasmids containing full-length cDNAs of the antigenomic (ag) L and S segments of the Armstrong (ARM) strain of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) flanked at their 5'- and 3'-termini by the T7 RNA polymerase (T7RP) promoter and ribozyme sequences, respectively. These plasmids directed intracellular synthesis of viral L and S ag RNA species in cells expressing plasmid-supplied T7RP. Co-expression of plasmid-supplied LCMV trans-acting factors, nucleoprotein (NP) and polymerase (L), resulted in replication and expression of L and S ag and genome RNA species, and generation of LCMV infectious progeny termed rT7/LCMV. The recombinant rT7/LCMV was unequivocally identified based on a genetic tag introduced in the recombinant S segment. In addition, rT7/LCMV exhibited growth and biological properties predicted for an ARM-like LCMV. To our knowledge, this is the first documented Arenavirus rescue, as well as of an ambisense negative strand (NS) RNA virus, entirely from cloned cDNAs. Our results extend the use of reverse genetic approaches for DNA-mediated virus rescue to all known virus families with NS RNA genome.

Animals↗

PubMed1990

Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein.

A cDNA clone of the large genomic segment of infectious pancreatic necrosis virus (IPNV) was inserted into transcription vectors and used for production of RNA transcripts of various lengths. These RNA transcripts were used to prime the synthesis of virus-specific polypeptides in a rabbit reticulocyte translation system. Full-length transcripts resulted in the synthesis of processed viral proteins pVP2, NS, and VP3. Transcripts which were deleted in the VP2 coding region did not affect the processing of NS or VP3 and similarly, deletions in VP3 did not affect the formation of NS or VP2. However, deletions which extended into the NS coding region resulted in a loss of protease activity and the production of truncated precursor polypeptides. The virus-specific proteolytic activity could not be inhibited by specific antisera and a trans activity for the viral proteases could not be demonstrated.

Animals↗

PubMed1983

Molecular cloning and characterization of endogenous feline leukemia virus sequences from a cat genomic library.

Recombinant bacteriophage lambda clones from a cat genomic library derived from placental DNA of a specific pathogen-free cat were screened to identify endogenous feline leukemia virus (FeLV) sequences. Restriction endonuclease mapping of four different clones indicates that there are a number of similarities among them, notably the presence of a 6.0- to 6.4-kilobase pair (kbp) EcoRI hybridizing fragment containing portions of sequences homologous to the gag, pol, env, and long terminal repeat-like elements of the infectious FeLV. The endogenous FeLV sequences isolated are approximately 4 kbp in length and are significantly shorter than the cloned infectious FeLV isolates, which are 8.5 to 8.7 kbp in length. The endogenous elements have 3.3- to 3.6-kbp deletions in the gag-pol region and approximately 0.7- to 1.0-kbp deletions in the env region. These deletions would render them incapable of encoding an infectious virus and may therefore be related to the non-inducibility of FeLV from uninfected cat cells and the subgenomic expression of these endogenous sequences in placental tissue. It appears that there is conservation in the ordering of restriction sites previously reported in the proviruses of the infectious FeLVs in sequences corresponding to the pol and env boundary as well as the region spanning the env gene of the endogenous clones, whereas a greater divergence occurs among restriction sites mapped to the gag and part of the pol regions of the infectious FeLV. Such deleted, FeLV-related subsets of DNA sequences could have originated either by germ-line integration of a complete ecotropic virus followed by deletion, or by integration of a preexisting, defective, deleted variant of the infectious virus.

Animals↗

PubMed2001

Analysis of transition from long-term nonprogressive to progressive infection identifies sequences that may attenuate HIV type 1.

Long-term nonprogressive human immunodeficiency virus type 1 (HIV-1) infection and its transition to progressive infection presents an opportunity to identify the molecular determinants of HIV-1 attenuation and pathogenesis. We studied an individual who underwent a transition from long-term nonprogressive to rapidly progressive infection. Because HIV-1 RNA genomes in plasma represent replicating virus, we developed a technique to clone full-length HIV-1 RNA genomes from plasma and used this technique to obtain clones from this individual before and during the transition. Most clones assayed were infectious, demonstrating that the RNA genomes encoded viable virus. Analysis of 20 complete HIV-1 RNA genomic sequences revealed one major difference between sequences found during the two phases of infection. During the nonprogressive phase, the predominant sequences had a large deletion in an Sp1-binding site and adjacent promoter in the U3 part of the long terminal repeat (LTR); when the infection became progressive, all viruses had intact Sp1 and promoter sequences and were derived from a minor species present earlier. Analysis of 184 clones of the LTR region obtained at five time points spanning a 7-year period confirmed this switch. In an in vitro assay, the deletion downregulated LTR-driven transcription of a reporter gene. In addition, analysis of cytotoxic T lymphocyte (CTL) epitopes predicted from the complete viral RNA genomes revealed multiple potential escape mutants that accumulated by the time of progression. These studies suggest that during the nonprogressive phase, the Sp1 enhancer-promoter deletion is likely to have played a role in decreasing replication, thereby attenuating HIV-1. The accumulation of CTL escape mutants suggests that a breakdown in immunologic surveillance may have allowed proliferation of intact virus, thus leading to rapid disease progression. These data reveal the viral and immune interactions characterizing a transition from long-term nonprogressive to rapidly progressive infection.

Base Sequence↗

PubMed1987

Immune response to intermediate filament-associated, Epstein-Barr virus-induced early antigen.

A murine monoclonal antibody (MAb) (15TD3) was found to recognize a unique filamentous structure in Epstein-Barr virus (EBV)-producing lymphoblastoid cell lines. By immunofluorescent morphology, in comparison with a control MAb to vimentin, the 15TD3 filamentous structure was judged to be associated with intermediate filaments of the cytoskeleton. Expression of the 15TD3 antigen and vimentin was induced simultaneously in some EBV genome-positive cell lines either by EBV superinfection or by 12-0-tetradecanoyl-1-phorbol-13-acetate (TPA) and n-butyrate treatment. The 15TD3 antigen was considered to be a restricted component of the EBV-induced early antigen (EA) complex. The 15TD3 antigen was expressed only in EBV genome-activated cells after either spontaneous EBV genome activation, EBV superinfection, or TPA and n-butyrate treatment. The expression of 15TD3 antigen paralleled the induction of EA in several models of induction of EBV antigens, and was detected only in EA+ cells which were stained with anti-EA+ human sera. The reactivity of 15TD3 MAb was blocked with anti-EA+ human serum, but not with anti-EA- serum. The synthesis of 15TD3 antigen was not inhibited with phosphonoacetic acid, was resistant to acetone fixation, and was sensitive to ethanol (or methanol) fixation. Human lymphoblastoid cells from patients with acute infectious mononucleosis were cloned for the production of antibodies which detected EBV-specific or -nonspecific epitopes on filamentous structures. Two human MAb were defined by two-color immunofluorescence to react to the 15TD3 determinants on intermediate filaments of EBV+ cells. This study supports the following views: that EBV genome activation induces a structure associated with intermediate filaments, and that antibodies against both the EBV-specific, intermediate filament-associated epitope and native intermediate filament epitopes are produced by some EBV-transformed lymphoblastoid cell lines from patients with infectious mononucleosis.

Antibodies, Monoclonal↗

PubMed2005

Essential and nonessential elements in the 3' nontranslated region of Bovine viral diarrhea virus.

The 3' nontranslated region (NTR) of the pestivirus Bovine viral diarrhea virus (BVDV), a close relative of human Hepatitis C virus, consists of three stem-loops which are separated by two single-stranded regions. As in other positive-stranded RNA viruses, the 3' NTR of pestiviruses is involved in crucial processes of the viral life cycle. While several studies characterized cis-acting elements within the 3' NTR of a BVDV replicon, there are no studies addressing the significance of these elements in the context of a replicating virus. To examine the functional importance of 3' NTR elements, a set of 4-base deletions and deletions of each of the three stem-loops were introduced into an infectious BVDV cDNA clone. Emerging mutant viruses were characterized with regard to plaque phenotype, growth kinetics, and synthesis of viral RNA. The results indicated that presence of stem-loop (SL) I and the 3'-terminal part of the single-stranded region between stem-loops I and II are indispensable for pestiviral replication. In contrast, deletions within SL II and SL III as well as absence of either SL II or SL III still allowed efficient viral replication; however, a mutant RNA lacking both SL II and SL III was not infectious. The results of this study provide a detailed map of the essential and nonessential elements within the 3' NTR of BVDV and contribute to our understanding of sequence and structural elements important for efficient viral replication of pestiviruses in natural host cells.

3' Untranslated Regions↗

PubMed1970

Functional heterogeneity of virions in human adenovirus types 2 and 12.

Purified preparations of adenovirus types 2 and 12 were used to infect KB cells at different input multiplicities. The resulting infected cultures were scored for inclusion body formation, production of infectious centers, and cloning efficiency. Both preparations were found to contain some defective particles capable of preventing a cell from cloning but unable to induce inclusion bodies or form plaques. The proportion of such defective particles in adenovirus 12 was about 10 times that in adenovirus 2. At high input multiplicities, the percentage of cells displaying an inclusion body was less than that predicted by the Poisson distribution and reached a maximum of 40 to 60% for adenovirus 2 and 12 to 15% for adenovirus 12. This reduction may be due to interference by large numbers of non-plaque-producing particles infecting each cell. The per cent of cells forming infectious centers was substantially greater for adenovirus 2 than for adenovirus 12 when compared at the same input plaque-forming units, reaching a maximum of 35 to 73% for adenovirus 2 and 5 to 10% for adenovirus 12. The low value for adenovirus 12 may be a result of the same interference phenomenon.

Adenoviridae↗

PubMed2003

Intratracheal inoculation as an efficient route of experimental infection with maedi-visna virus.

Maedi-visna virus (MVV) spreads horizontally via the respiratory route. In order to establish an experimental mucosal infection route, we compared intranasal and intratracheal inoculation using the infectious MVV molecular clone KV1772-kv72/67. For intranasal infection 0.5 x 10(3)-0.5 x 10(7) TCID50 of virus was sprayed into the nostrils of the sheep. For the intratracheal infection 10(0)-10(6) TCID50 of virus was injected into the trachea. Successful infection was indicated by development of MVV specific antibodies and virus isolation over a period of 6 months. In the intranasal infection, only the sheep receiving the highest dose i.e., 0.5 x 10(7) TCID50, became infected, suggesting that intranasal application was not an efficient mode of infection. In the intratracheal infection, the sheep infectious dose 50% was 10(1) TCID50 and virus could be isolated from the central nervous system 4 months post infection with 10(4) TCID50. Therefore it is concluded that intratracheal infection is a very efficient route for experimental inoculation with MVV.

Administration, Intranasal↗

PubMed2001

Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection.

Previous studies utilizing Gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (EIAV) Gag p9 protein provides a late assembly function mediated by a critical Y(23)P(24)D(25)L(26) motif (L-domain) to release viral particles from the plasma membrane. To elucidate further the role of EIAV p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of a reference infectious molecular proviral clone, EIAV(uk). Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K(30)K(31) motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAV(uk) in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication.

Amino Acid Sequence↗