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Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression.

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗

Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.

Amino Acid Sequence↗

Enterotoxigenic strains of Escherichia coli O128 are not restricted to subgroup ac but also belong to subgroups ab and abc.

Ninety strains of Escherichia coli O128 isolated in many different countries were examined. The majority (77 strains) belonged to the antigenic subgroup O128ab, and 41 of these strains produced heat-stable enterotoxin (ST), heat-labile enterotoxin (LT), or both. Eight strains were of the antigenic subgroup O128ac; six produced ST only, and two were nontoxigenic. Five strains were of the antigenic subgroup O128abc; two produced ST and LT, and three were nontoxigenic. Ten of the strains studied produced Vero cytotoxin, and all belonged to the antigenic subgroup O128ab. It was concluded that, contrary to the report of Guth et al. (B. E. C. Guth, M. L. M. Silva, I. C. A. Scaletsky, M. R. F. Toledo, and L. R. Trabulsi, Infect. Immun. 47:338-340, 1985), enterotoxigenic strains of E. coli O128 are not restricted to subgroup ac but also belong to the most common subgroup, ab.

Antigens, Bacterial↗

Chimeric subgroup A respiratory syncytial virus with the glycoproteins substituted by those of subgroup B and RSV without the M2-2 gene are attenuated in African green monkeys.

Using the existing reverse genetics system developed for the subgroup A respiratory syncytial virus (RSV), a chimeric virus (designated rA-G(B)F(B)) that expresses subgroup B-specific antigens was constructed by replacing the G and F genes of the A2 strain with those of the 9320 strain of subgroup B RSV. rA-G(B)F(B) grew well in tissue culture, but it was attenuated in the respiratory tracts of cotton rats and African green monkeys. To further attenuate this chimeric RSV, the M2-2 open reading frame was removed from rA-G(B)F(B). rA-G(B)F(B)DeltaM2-2 was highly attenuated in replication in the respiratory tracts of the infected monkeys, but it provided complete protection against wild-type subgroup B RSV challenge following two doses of infection. In this study, rA2DeltaM2-2 (a recombinant A2 RSV that lacks the M2-2 gene) was also evaluated in African green monkeys. The replication of rA2DeltaM2-2 was highly restricted in both the upper and lower respiratory tracts of the infected monkeys and it induced titers of serum anti-RSV neutralizing antibody that were slightly lower than those induced by wild-type rA2. When rA2DeltaM2-2-infected monkeys were challenged with wild-type A2 virus, the replication of the challenge virus was reduced by approximately 100-fold in the upper respiratory tract and 45,000-fold in the lower respiratory tracts. rA2DeltaM2-2 and rA-G(B)F(B)DeltaM2-2 could represent a bivalent RSV vaccine composition for protection against multiple strains from the two RSV subgroups.

Animals↗

Comparison of the oligosaccharide moieties of the major envelope glycoproteins of the subgroup A and subgroup B avian myeloblastosis-associated viruses.

The nature of the oligosaccharide chains of the major envelope glycoprotein, gp85, from avian myeloblastosis-associated viruses has been examined for the subgroup A and subgroup B viruses replicated in fibroblasts from the same chicken embryos. Pronase-digested glycopeptides from [3H]mannose- or [3H]glucosamine-labeled viruses were analyzed by the combined techniques of gel filtration, endo-beta-N-acetylglucosaminidase digestion, and concanavalin A affinity chromatography. The gp85 protein from these two viruses, and also from another subgroup A avian leukosis virus replicated in the same cells, contained a diverse array of asparagine-linked oligosaccharides of the acidic type [(sialic acid +/- galactose-N-acetylglucosamine)2-4-(mannose)3-N-acetylglucosamine2(+/- fucose)-asparagine], hybrid type (sialic acid +/- galactose-N-acetylglucosamine-(mannose)5,4-N-acetylglucosamine2-asparagine), and neutral type [(mannose)5-9-N-acetylglucosamine2-asparagine], with the more highly branched (tri or tetraantennary or both) acidic-type structures representing the predominant class of oligosaccharide. Minor differences were observed between the gp85 of the subgroup B versus subgroup A viruses.

Avian Leukosis Virus↗

Characterization of rotavirus subgroup-specific monoclonal antibodies and use in single-sandwich ELISA systems for rapid subgrouping of human strains.

Two subgroup-specific monoclonal antibodies (MAb) raised in mice against group A human rotavirus were shown to react by immunoblotting with the trimeric form of VP6 of the homologous subgroup and successfully applied to development of new single-sandwich ELISA systems for rapid subgrouping of human strains. All of the 344 strains tested could be subgrouped, but for two of them prior propagation in cell cultures was required.

Antibodies, Monoclonal↗

The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses.

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.

Adenoviridae↗

Development and validation of a PCR-RFLP assay to evaluate TVB haplotypes coding receptors for subgroup B and subgroup E avian leukosis viruses in White Leghorns.

The cellular receptor of subgroup B avian leukosis virus (ALVB) is encoded by a gene at the tumour virus B (TVB) locus. TVB alleles encode specific receptors permitting infection by exogenous ALVB or avian leukosis virus subgroup D (ALVD) as well as endogenous avian leukosis virus subgroup E (ALVE), and thus susceptibility is dominant to resistance. Two single nucleotide polymorphisms at the TVB locus have been reported distinguishing three TVB alleles (TVB*S1, TVB*S3 and TVB*R). We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using the two single nucleotide polymorphisms to define three observed allelic haplotypes and to identify the six possible TVB genotypes consisting of the three haplotypes in defined laboratory strains of chickens. One additional potential allelic haplotype and four genotypes were also briefly discussed. Chickens from parents heterozygous for different TVB alleles were challenged with Rous sarcoma viruses of subgroup ALVB and ALVE to induce wing-web tumours. Tumour incidences were evaluated between chickens of the genotypes determined with this newly developed PCR-RFLP assay. Importantly, chickens typed with this assay as TVB*S3/*S3 were resistant to infection by ALVE only, and those TVB*R/*R were resistant to both ALVE and ALVB. Furthermore, a vast majority of chickens with the susceptible TVB*S1/- genotypes developed a tumour. This PCR-RFLP assay enables a relatively rapid assessment of all six anticipated TVB genotypes in experimental strains of chickens undergoing segregation for TVB*S1, TVB*S3, and TVB*R alleles. This non-infectious assay should be further evaluated for the capacity to select and breed commercial chickens for genetic resistance to infections by ALVB, ALVD and ALVE.

Alleles↗

Early region 3 of adenovirus type 19 (subgroup D) encodes an HLA-binding protein distinct from that of subgroups B and C.

Early region 3 (E3) of human adenoviruses (Ads) codes for proteins that appear to control viral interactions with the host. For example, the most abundant E3 protein, E3/19K, inhibits the transport of newly synthesized class I major histocompatibility molecules to the cell surface, thereby interfering with antigen presentation. So far, the E3 regions of Ad subgroups A, B, C, and F have been characterized. We have cloned the E3A region of Ad type 19a (Ad19a), which belongs to the largest subgroup, D, and causes epidemic keratoconjunctivitis in humans. The sequence reveals five open reading frames (ORFs) with the potential to encode the Ad19 equivalent of pVIII, as well as proteins 12.2K, 16.2K, and 18.6K. The last ORF predicts a novel 49K protein which has no counterpart in other subgroups. Both the sequence and the overall organization of the E3 region from Ad19a shows a closer relationship to group B than to group C Ads. The 18.6K ORF represents the Ad19 homolog of the Ad2 E3/19K protein. By using 293 cells stably transfected with the Adl9a E3A region, we showed by immunoprecipitation, pulse-chase experiments, and fluorescence-activated cell sorter analysis that the Ad19 E3/19K protein binds to and prevents the transport of major histocompatibility complex molecules to the cell surface. The similar but distinct functional activity of the Ad19 E3/19K protein, combined with the new sequence which differs from those of subgroup B and C proteins, allows a more precise definition of amino acids essential for HLA binding.

Adenovirus E3 Proteins↗

Detection of a human rotavirus strain different from types 1 and 2--a new subgroup? Epidemiology of subgroups in a Swedish and an Ethiopian community.

A possible new subgroup of human rotavirus was found by crossed immunoelectrophoresis and enzyme-linked immunosorbent assay. The epidemiology of the established subgroups 1 and 2 and this new variant was studied in two different communities. Of 398 rotavirus isolates from Malmö, Sweden, 26.8% were of type 1, 71.7% of type 2, and 1.5% of the new variant. Corresponding figures for 384 samples from Addis Ababa, Ethiopia, were 33.1% type 1 and 66.9% type 2. A total of 87% of the Swedish and 79% of the Ethiopian rotavirus-positive samples could be classified. The yearly distribution of the subgroups was stable and similar in Sweden and Ethiopia. The new variant could only be found in one outbreak during 1979. Among children with sequential infections eight of ten primary infections were type 1, and no one shed the same type of rotavirus twice.

Child, Preschool↗

Production of subgroup-specific monoclonal antibodies against human rotaviruses and their application to an enzyme-linked immunosorbent assay for subgroup determination.

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid. Using the three monoclonal antibodies (S2-37, YO-5, and YO-156), an enzyme-linked immunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.

Antibodies, Monoclonal↗

Inhibition of feline leukemia virus subgroup A infection by coinoculation with subgroup B.

Feline leukemia virus (FeLV) subgroup B arises de novo through recombination between the env genes of exogenous FeLV subgroup A and endogenous FeLV-like sequences. FeLV-B, which by itself is poorly infectious, will increase to high titer in the presence of FeLV-A, and is associated with FeLV-related neoplastic disease. Although the participation of FeLV-B in disease progression has not been definitively proven, circumstantial evidence supports the hypothesis that the generation of FeLV-B is linked to disease progression. The present study was designed to evaluate whether increasing the levels of FeLV-B early in FeLV-A infection could result in reduction of the incubation period for development of neoplastic disease. For this study, an isolate of FeLV-B, designated FeLV-1B3, was biologically cloned, partially sequenced, and subgroup typed. In in vivo studies, none of the neonatal cats inoculated with FeLV-1B3 alone converted to viremia positive, and all remained healthy throughout the observation period. All of the kittens inoculated with FeLV-A alone became chronically viremic, and those held for long-term observation all developed either neoplastic disease or anemia. However, kittens inoculated with the combination of FeLV-1B3 and FeLV-A showed attenuated infections whereby the majority of cats failed to develop chronic viremia. The apparent interference of FeLV-A infection by FeLV-B was time and titer dependent. This unexpected result suggests that FeLV-B may act as an attenuated virus, causing inhibition of FeLV-A possibly through an immune-mediated mechanism. Partial support for this view was provided by postmortem examination of cats inoculated with FeLV-1B3 alone. Even though none of these cats became viremic, FeLV antigen was detected as focal infections in select tissues, especially salivary gland epithelium, where enough antigen may be expressed to provide an immunizing dose against gag and pol cross-reacting antigens. This work may also provide another approach to vaccine development based on endogenous retrovirus vector systems.

Amino Acid Sequence↗

Classification of a new member of the TBE flavivirus subgroup by its immunological, pathogenetic and molecular characteristics: identification of subgroup-specific pentapeptides.

The antigenic, pathogenic and molecular characteristics of Turkish sheep encephalitis (TSE) virus, strain TTE80, were compared with other members of the tick-borne encephalitis (TBE) virus complex. Monoclonal antibodies with defined specificity for the flavivirus envelope glycoprotein distinguished TSE virus from louping ill (LI), western or far eastern TBE, Langat and Powassan virus in indirect immunofluorescence, haemagglutination-inhibition and neutralization tests. On the other hand, TSE virus, which produces an LI-like disease in sheep, resembled LI virus in mouse neurovirulence tests. Molecular homology data of all the structural genes of TSE virus compared with other tick-borne flaviviruses demonstrated that TSE virus is a distinct member in the TBE virus subgroup. The data are consistent with the conclusion that TSE virus has evolved by a separate evolutionary pathway as compared with the close antigenic relatives, western European, far eastern TBE viruses and LI virus. By aligning the encoded amino acids in the viral envelope glycoprotein of mosquito- and tick-borne flaviviruses, we have also identified subgroup-specific pentapeptide motifs for the tick-borne encephalitis, Japanese encephalitis and dengue subgroup viruses of the genus Flavivirus. These pentapeptides have important implications for the evolution, classification and diagnosis of flaviviruses.

Amino Acid Sequence↗

Pear blister canker viroid is a member of the apple scar skin subgroup (apscaviroids) and also has sequence homology with viroids from other subgroups.

The sequence of pear blister canker viroid (PBCVd), the putative causal agent of pear blister canker (PBC) disease, has been determined. PBCVd consists of a single-stranded circular RNA of 315 nucleotide residues which assumes a branched conformation when it is folded in the model of lowest free energy. PBCVd has highest sequence similarity with grapevine 1B viroid (52.4%), but also contains sequences related to regions present in viroids that belong to different subgroups, suggesting that PBCVd could have developed from RNA recombination between viroids replicating in a common host plant. PBCVd contains almost the entire central sequence which is conserved in the members of the apple scar skin subgroup (apscaviroids) as well as a conserved sequence located in the left-terminal region of apscaviroids and pospiviroids (whose type member is potato spindle tuber viroid). A consensus phylogenetic tree has been obtained in which PBCVd and other viroids previously classified as apscaviroids appear closely related, allowing consideration of PBCVd as a new member of this subgroup.

Base Sequence↗

Microfoam ultrasound-guided sclerotherapy treatment for varicose veins in a subgroup with diameters at the junction of 10 mm or greater compared with a subgroup of less than 10 mm.

OBJECTIVE: The objective was to analyze the effectiveness of foam ultrasound-guided sclerotherapy treatment in saphenous veins and tributary veins with a diameter at the saphenofemoral or saphenopopliteal junction of > or =10 mm and compare these results with a subgroup of veins with diameters of < 10 mm. METHODS: A subgroup of 17 saphenous veins with a diameter at the saphenofemoral or saphenopopliteal junction of > or =10 mm were compared with a subgroup of 98 saphenous veins with a diameter of < 10 mm at the saphenofemoral or saphenopopliteal junction for clinical efficacy and patient satisfaction at a mean 2-year follow-up. RESULTS: A mean number of 2.15 treatments utilizing an average of 8.37 mL of foam sclerosing solution (3% sodium tetradecyl sulfate) were required to close all incompetent varicose veins in the < 10-mm group versus a mean of 2.8 treatments and 13.9 mL foam (3% sodium tetradecyl sulfate) for the > or =10-mm group. A total of 27.5% of saphenous veins of < 10 mm and 37.5% of saphenous veins > or = 10 mm required a second treatment at 3 months. One-hundred percent of patients believed that their legs were successfully treated at 2-year follow-up in both groups with 94% of the < 10-mm group noting improvements in quality of life and 100% in the > or =10-mm group. CONCLUSIONS: Ultrasound-guided foam sclerotherapy is effective in treating all sizes of varicose veins with high patient satisfaction and improvement in quality of life. Duplex ultrasound findings demonstrated a small increase in failure to close the saphenofemoral or saphenopopliteal junction with increasing size of junction diameter, but this does not significantly alter the results with respect to clearance of visible varicosities and patient satisfaction with results.

Adult↗

Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup II human rotaviruses.

We have previously found (O. Nakagomi, T. Nakagomi, H. Oyamada, and T. Suto, J. Med. Virol. 17:29-34, 1985), during an epidemiological study in Japan, a novel human rotavirus that belongs to subgroup I but has a long RNA pattern typical of subgroup II human rotaviruses. From the stool specimen containing this virus, we successfully isolated in MA104 cells a rotavirus, designated AU-1, which possesses these novel characteristics. The possibility that strain AU-1 was a laboratory contaminant of an animal rotavirus previously adapted to tissue culture cells was ruled out, and the identity of the AU-1 strain was established. Genetic analysis by RNA-RNA hybridization revealed that the AU-1 strain is not a simple reassortant between subgroup I and II human rotaviruses but that it shares a high level of sequence homology only with the gene encoding VP7 (the major neutralization protein) of serotype 3 human rotaviruses. Weak homology of the genomic RNA segments was also observed between the AU-1 strain and animal rotavirus strains, including rhesus rotavirus strain RRV and bovine rotavirus strain NCDV. These results suggest that the AU-1 strain may be an animal rotavirus that infected a human.

Animals↗

Molecular epidemiology of subgroup C avian pneumoviruses isolated in the United States and comparison with subgroup a and B viruses.

The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.

Amino Acid Sequence↗

Role of basic residues in the subgroup-determining region of the subgroup A avian sarcoma and leukosis virus envelope in receptor binding and infection.

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.

Alpharetrovirus↗