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PubMed1976

Hepatitis-B: a review.

The recent literature on various aspects of hepatitis-B is reviewed with emphasis on the interrelationships of viral structure, antigenic components, and host immune response in acute, chronic, and asymptomatic carrier states of the infection. The mode of replication and mechanisms of transmission are discussed. Special attention is paid to potential non-parenteral routes of spread. The role of hepatitis-B in associated immune complex diseases and in hepatoma is outlined. A guide to the interpretation of serologic tests for hepatitis-B associated antigen and antibody patterns is presented in relation to the clinical stage and prognosis of the infection. Therapy, except in conceptual terms, is not covered but a summary of the current status of active and passive immunization is given. The unresolved question of the infectivity of carrier medical staff for their patient contacts, and the reverse, is discussed.

Acute Disease↗

PubMed2005

Heterogeneity analysis of the hepatitis B virus genome in intrauterine infection.

There are many factors leading to intrauterine infection with hepatitis B virus (HBV). These factors include viral structure, HBV mutations, HBV DNA level, placenta barrier, immune status of the mother, and susceptibility of the fetus. The purpose of this study was to investigate the possible relationship between intrauterine infection with and HBV mutations of the genome of the virus. In this study, HBsAg-positive mothers were divided into two groups: intrauterine infection group and non-infection group according to whether the newborn infants were infected or not. The intrauterine infection group included four pairs of mother and their newborn infants infected in utero, and non-infection group included five HBsAg-positive mothers. HBV sequences from the two groups were analyzed and compared. The predominant strains in the mothers and infants from the intrauterine infection group were not completely consistent. This suggested that both HBV predominant strains and minority strains in the mothers could infect their infants through intrauterine transmission. Some HBV mutations probably related to intrauterine infection were examined and it was found that the frequencies of mutations were low in isolates of the virus of infants from the intrauterine infection group and high in the non-infection group. These results suggest that some strains of HBV from the mother may be transmitted selectively to the fetus in utero because of viral heterogeneity. The strains without screened mutations such as P21L in the pre-S1 region may infect the fetus more readily.

Adult↗

PubMed1996

Cell-free HIV-1Zr6 vif mutants are defective in binding to peripheral blood mononuclear cells and in internalization.

The vif gene of the human immunodeficiency virus (HIV-1) is required for productive virus infection of primary blood mononuclear cells (PBMCs) and macrophages in vitro. Replication of HIV-1 vif- mutants in T-lymphoid cell lines varies and is dependent on the cell line used for virus production. To further understand the role of Vif in HIV-1 infection, we constructed to vif deletion mutants from a molecular clone derived from an African patient (HIV-1Zr6). Cell-free Zr6 vif- virus pools made from transfected rhabdomyosarcoma (RD) cells do not replicate when added to cultures of stimulated PBMCs. However, vif mutants were able to spread from transfected RD cells to PBMCs if cell-to-cell contact was permitted. By Western blot analysis, viral structural proteins expressed after transfection of RD cells by wild-type or vif mutant proviruses were indistinguishable. However, binding of vif mutants to PBMCs or to purified CD4 and virus internalization were significantly reduced when compared with wild-type virus. The defects in cell-free infection, CD4 binding, and internalization were rescued by transcomplementation using a vif expression plasmid. Our results suggest a novel level at which the HIV-1 vif gene product acts to enhance cell-free infection and indicate that vif plays an important role in promoting HIV-1 binding and internalization. Combined with the previous reports of vif's effect at other steps in infection, this suggests that vif is a pleuripotent gene product that affects multiple stages of the infective process.

CD4 Antigens↗

PubMed1993

Interleukin-1 production and cell-activation response to cytomegalovirus infection of vascular endothelial cells.

Human cytomegalovirus (HCMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in HCMV infection is well documented. Traditionally laboratory strains of HCMV have been used in experimental investigations in vitro; however the continuous propagation of these strains in fibroblasts have attenuated the virus making it unsuitable for infecting other cell systems such as endothelial cells. In this study a recent clinical isolate of HCMV was propagated through several passages in endothelial cells and was used to investigate the effect of HCMV infection of human umbilical vein endothelial cells (HUVEC) on IL-1 production and cell proliferation. Infection of HUVEC with the clinical isolate of HCMV (at multiplicity of infection 5:1) suppressed the production of IL-1 alpha (82%) and IL-1 beta (99%) at 5 h post infection; the levels returned to that of the control within 24h post infection. Ultraviolet inactivated (but not heat killed) virus produced similar suppression confirming that a thermolabile viral structural protein or intact virion were responsible for this suppression. Infection of HUVEC with the clinical isolate increased the number of these cells and the rate of their proliferation. An increase of infected HUVEC number under quiescent growth conditions continued as the infection progressed (6-10 days post infection), exhibiting, at 3 days post infection, 5 times the number of uninfected HUVEC (control) which did not tolerate the quiescent culture conditions for more than 4 days. Live virus is responsible for this increase because UV-inactivated virus did not maintain the proliferation of HUVEC. These studies suggest that while infection of HUVEC with a recent clinical isolate of HCMV suppressed the production of IL-1 at early hours after infection, it increased the proliferation of these cells at later stages of infection.

Antigens, Viral↗

PubMed1991

Construction of a recombinant adenovirus containing the denV gene from bacteriophage T4 which can partially restore the DNA repair deficiency in xeroderma pigmentosum fibroblasts.

The denV gene from bacteriophage T4 encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Cells from excision repair-deficient xeroderma pigmentosum (XP) patients are able to carry out excision repair initiated by the denV gene product and introduction of the denV gene into XP cells results in the partial restoration of colony-forming ability after irradiation with UV light. In this work we have constructed a helper-independent recombinant human adenovirus, Ad5denV, which contains the denV gene. A 1.9 kb cartridge consisting of the denV gene flanked by the long terminal repeat (LTR) promoter from Rous sarcoma virus (RSV) and the simian virus 40 (SV40) polyadenylation (poly A) splice signals, was inserted into the E3 region of an E3 deletion mutant (Ad5d1E3) of adenovirus type 5. Infection of human fibroblasts and other permissive human cells with Ad5denV resulted in lytic infection and expression of the denV gene was confirmed by primer extension of infected cell RNA. The ability of the denV gene to restore the DNA repair deficiency in XP fibroblasts was examined using host cell reactivation of viral structural antigen formation for UV-irradiated adenovirus. The control virus, Ad5VSV, was also a recombinant which contained the gene for vesicular stomatitis virus glycoprotein G inserted into the E3 region of Ad5d1E3. UV survival of Ad5denV was similar to that of Ad5VSV following infection of two normal fibroblast strains and a Cockayne syndrome fibroblast strain, CS7SE, from complementation group B. In contrast, UV survival of Ad5denV was significantly greater than that for Ad5VSV after infection of three unrelated XP fibroblast strains from complementation groups A, C and E. However, UV survival of Ad5denV in the XP fibroblasts did not reach levels obtained in normal fibroblasts, indicating that restoration of the XP defect was partial.

Adenoviridae↗

PubMed1997

Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki Forest virus.

Recombinant Semliki Forest viruses (SFV) that express one or none of the viral structural proteins were used to infect cells and to analyze the fate of incoming superinfecting wild-type viruses. It was found that in addition to the previously described block in replication that superinfecting viruses encounter within 15 min of infection, other mechanisms of superinfection inhibition occurred at later times. Over a 6-hr infection period, inhibition was seen in binding of virus to the cell surface, in acid-activated penetration into the cytoplasm, and in uncoating of nucleocapsids. For each of these processes, the inhibitory mechanism was investigated. In summary, we found that infection evoked several independent mechanisms for blocking the entry and uncoating of superinfecting viruses. The results also offered new insights into the normal processes of penetration and uncoating of SFV.

Animals↗

PubMed2007

Expression of VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes of Epstein-Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma.

EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results in screening for NPC.

Adult↗

PubMed1982

Biochemical characterization of transformation-specific proteins of acute avian leukemia and sarcoma viruses.

The biological and biochemical properties of the transformation-specific proteins of three avian oncornaviruses with different oncogenic potentials were compared, namely the gag-myc protein of the avian myelocytomatosis virus MC29, the gag-erb A protein of the avian erythroblastosis virus AEV, and the gag-fps protein of Fujinami sarcoma virus FSV. These oncogenes were analyzed in transformed fibroblasts that expressed only the transforming proteins but showed no virus replication. Monoclonal antibodies against the viral structural protein p19, which is the N-terminus of the proteins, were used for indirect immunofluorescence, for immunoprecipitation of the proteins from subcellular fractions, and for immunoaffinity column chromatography. With this last method a 3000-fold purification of the proteins was obtained. By indirect immunofluorescence it was shown that the gag-myc protein was located in the nucleus, and bound to DNA after purification. The gag-erb A protein was not nuclear but probably located in the cytoplasm and did not bind to DNA after purification. Neither of the two proteins exhibited protein kinase activity. In contrast, the gag-fps protein did not bind to DNA but showed protein kinase activity after purification. It was not located in the nucleus either.

Animals↗

PubMed1994

Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA.

Self-propagating infectious particles were produced in animal cells transfected with an RNA replicon encoding a single viral structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). The replicon is derived from an alphavirus, Semliki Forest virus (SFV), and encodes the SFV RNA replicase, but none of the SFV structural proteins. After transfection of the replicon into tissue culture cells, expression of G protein spread from small foci throughout the culture. Supernatants from the cells contained infectious, virus-like particles that could be passaged and were neutralized by anti-VSV serum. The majority of the infectious particles were smaller and less dense than either VSV or SFV. Characterization by electron microscopy showed membrane-enveloped vesicles that contained the VSV-G protein. Infectious particles were apparently generated by budding of vesicles containing VSV-G protein and the RNA replicon. These experiments reveal that an enveloped infectious agent can be much simpler than previously thought.

Animals↗

PubMed1983

U.V. enhanced reactivation of U.V.-and gamma-irradiated adenovirus in Cockayne syndrome and Xeroderma pigmentosum fibroblasts.

U.V.-enhanced reactivation (UVER) of both U.V.-irradiated and gamma-irradiated human adenovirus type 2 (Ad 2) was examined following the infection of a variety of Cockayne Syndrome (CS) and Xeroderma pigmentosum (XP) fibroblast strains which had been pre-irradiated with U.V. light. U.V.-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or irradiated Ad2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Normal levels of UVER (i.e. 2-4 fold) of U.V.- and of gamma-irradiated Ad 2 were detected in 2 CS strains (CS IBE and CS 3BE), 2 XP complementation group A strains (XP 12BE and XP 25RO), and 2 XP complementation group D strains (XP 5BE and XP 6BE), although the U.V. doses to these mutant cells which resulted in peak UVER values (0 . 2 Jm-2 for XP 25RO, 0 . 14 Jm-2 for XP 12BE, 0 . 8 Jm-2 for XP 5BE and XP 6BE, and 1 . 6-5 . 0 Jm-2 for CS 1BE and CS 3BE) were considerably lower than those yielding peak UVER in normal strains (10-15 Jm-2). XP variant strains (XP 4BE and XP 115LO), however, showed substantially lower levels of UVER than normal strains.

Adenoviruses, Human↗

PubMed1990

Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction.

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.

Animals↗

PubMed1987

Molecular characterization of the genomic S RNA segment from lymphocytic choriomeningitis virus.

We have used cDNA clones derived from the genomic S RNA segment of lymphocytic choriomeningitis virus (LCMV), Armstrong strain, as hybridization probes to monitor virus gene expression during acute infections. Our results with strand-specific probes confirm the ambisense character of the LCMV S RNA segment and document the presence of both genomic sense and genomic complementary sense RNA species over the time course of infection. We have used nucleotide sequence information to predict primary amino acid sequences for the major viral structural proteins, nucleoprotein (NP) and glycoprotein (GP-C). Antibodies raised against synthetic peptides derived from these predicted protein sequences have indicated that the gene order for the S segment is 3' NP----5' GP-C and provided direct demonstration that the GP-1 portion of the GP-C precursor is encoded nearest the 5' end of the S segment. Comparison of the predicted amino acid sequences for NP and GP-C between the Armstrong CA-1371 strain and the WE strain shows over 90% amino acid identity. This suggests that significant differences described for the pathogenic potential of the Arm and WE strains in C3H mice reside in one or a very few critical amino acid changes.

Amino Acid Sequence↗

PubMed2003

Blocking the dengue virus 2 infections on BHK-21 cells with purified recombinant dengue virus 2 E protein expressed in Escherichia coli.

Dengue viruses (DVs) are mosquito-borne infectious pathogens. They have become an expanding public health problem in the tropics and subtropics. The dengue envelope (E) protein is one of the viral structure proteins responsible mainly for the virus attachment and entry onto host cells. It is also the major immunogen for virus neutralization. In this study, we have constructed a recombinant plasmid expressing a truncated E protein of DV-2 virus PL046 strain. The C-terminal hydrophobic domain of the E protein was removed and replaced with the sequence of S peptide to facilitate expression and purification. When expressed in Escherichia coli, the recombinant E proteins were found to be in the form of aggregated state. Through denaturation and dialysis processes, the receptor-interacting function of the purified recombinant E proteins was maintained, which was demonstrated by its ability to inhibit the DV-2 plaque-forming efficiency on mammalian BHK-21 host cells.

Animals↗

PubMed1991

Transdominant repressors for human T-cell leukemia virus type I rex and human immunodeficiency virus type 1 rev function.

Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I rex genes. Transient expression experiments demonstrated that 3 of 28 mutant proteins are functionally inactive on the homologous HTLV-I rex response element, whereas an additional 2 mutant proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex protein in trans on the homologous rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev protein in a dominant-negative manner.

Amino Acid Sequence↗

PubMed1999

Activation of a cryptic splice donor in human immunodeficiency virus type-1.

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function as cis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5' splice donor of HIV-1 was mutated in the context of other gag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5' splice donor and the tat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.

Animals↗

PubMed2000

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains.

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.

Animals↗

PubMed1992

Nonreplicating vaccinia vector efficiently expresses recombinant genes.

Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

Animals↗

PubMed1994

Rapid regeneration of virus from cells infected with a retroviral vector.

Recombinant retroviral vectors usually encode the genes of interest in place of the viral structural genes, which must be provided in trans. These viruses are therefore defective for replication: infected cells cannot produce progeny virus. However, in some cases it may be desirable to generate virus from an infected cell clone displaying a phenotype of interest. We describe a rapid method for producing virus, which involves fusing the infected cells to fresh packaging cells. Stable producer lines are generated after fusion by co-selecting for the Ecogpt+ marker in the packaging cells and the G418 resistance (neor) marker in the infected cells. We have used this method to develop cell lines that produce retroviruses encoding a Leu 61-activated c-Ha-ras oncogene as well as a neor gene. The viruses confer oncogenic transformation on 95%-100% of infected target cells as assayed by altered morphology, focus formation and soft agar growth.

Animals↗