Biomedical subjects

M Arella

Publications and source records attributed to M Arella.

At least 19 recordsLinked to original sources

PubMed1995

Cloning, sequencing and expression of the S1 gene of avian reovirus.

The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced. The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature. The three ORFs from 5' to 3' were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively. Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation. The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots. The protein was concluded to be sigma 3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral sigma 3 polypeptide. Sequence comparison of the avian reovirus S1 gene with its mammalian counterpart did not show any significant similarity between the two. However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of alpha-helix structures in the amino terminal portion of sigma 3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus sigma 1 protein.

ATP-Binding Cassette Transporters↗

PubMed1995

Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA. The larger segment contains a small ORF partly overlapping the 5' end of the polyprotein reading frame. Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5). The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing. Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others. Moreover, the deduced amino acid sequences did not appear to be well conserved. Despite the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides. To verify whether the small ORF is translated into protein in fish cells, the 17 kDa polypeptide of the VR-299 strain was expressed as fusion protein in a prokaryotic expression vector and used to produce a specific antiserum. This antiserum reacted with concentrated virus in an immunodot assay indicating that VP5 is synthesized in infected cells, but probably only in small quantities. When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299. Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF.

Amino Acid Sequence↗

PubMed1995

Alterations in cell-cell communication in human papillomavirus type 16 (HPV16) transformed rat myoblasts.

A reduction of gap-junctional intercellular communication (GJIC) often accompanies neoplastic transformation. The present work demonstrates that transformation by the oncogenic human DNA virus, human papilloma virus 16(HPV16), also reduces GJIC between L6 rat myoblasts. HPVs are associated with anogenital cancers, the incidence of which is increasing in HIV positive patients of both sexes. Using videofluorescence imaging of Fura-2 loaded cells a lack of GJIC between transformed HPV16-L6 cells was first indicated by uncoordinated brief [Ca2+]i spikes in clusters of DMSO-treated HPV16-L6 cells instead of the synchronous, sustained [Ca2+]i surges in clusters of DMSO-treated L6 cells. Reduced GJIC between HPV16-L6 cells was demonstrated directly by a much reduced transfer of lucifer yellow dye from HPV16-L6 cells, which had been loaded with the dye through electroporation with an EPIZAP II in situ electroporator, to neighbouring nonelectroporated HPV16-L6 cells. One reason for this reduced GJIC between HPV16-L6 cells could have been their dramatically enhanced activity of membrane-associated PKC which is known to phosphorylate connexins and down-regulate gap junctions. However, the main reason was the viral-induced inhibition of the expression of a major gap junction component, Cx43 (Connexin 43), in the transformed myoblasts.

Animals↗

PubMed1995

HIV-1 gp120/160 expressing cells upregulate HIV-1 LTR directed gene expression in a cell line transfected with HIV-1 LTR-reporter gene constructs.

The results described in this paper demonstrate that HIV-1 gp120 can upregulate gene expression directed by the HIV-1 LTR. Briefly, exposing responder CD4+CEM-T4 ID5 cells to stimulator CEMgp120/160 expressing cells (stably transfected with HIV-1 LTR-CAT and HIV-1 gp160, respectively) resulted in the increased synthesis of the CAT enzyme. Control non-transfected CEM-T4 cells did not induce the synthesis of CAT. In addition, when the responder cell line, U937-1C5 which also contains stably transfected HIV-1 LTR-CAT plasmid was exposed to irradiated CEM gp120/160 cells, there was no synthesis of the CAT enzyme. Neither recombinant gp120 nor gp160 were able to stimulate the synthesis of CAT in the responder cells. These results indicate that the mechanism by which gp120/160 expressed on transfected cells increase CAT synthesis in responder cells may be dependent on the manner which the protein is presented in association with accessory molecules. Moreover, recombinant soluble CD4 and anti-CD4 monoclonal antibodies inhibited CEM gp120/160 induced expression of HIV-1 LTR-directed expression in CEM-1D5 cells. Based on these results we hypothesize that HIV or its envelope protein, gp120, upon interaction with its receptor, the CD4 molecule on T helper cells, transduces a signal which translates into the upregulation of the gene expression directed by the HIV-1 LTR.

Antibodies, Monoclonal↗

PubMed1994

HIV-1 gp120/160 expressing transfected cell clones to evaluate the antibody-dependent cellular cytotoxicity (ADCC) during the course of HIV-1 infection.

Transfection by electroporation of the CEM.NKR cell line with a plasmid containing the HIVNL43 gp160 gene resulted in the establishment of HIV-1 gp120/160 expressing CEM.NKR cell clones. The cell-surface expression of the recombinant viral envelope is stable and does not lead to syncytium formation among a substantial number of cells expressing envelope glycoprotein, suggesting that these cells are suitable as targets to monitor HIV-specific ADCC activities with the stage or the progression of HIV-1 disease. By using these cells, the ability of sera from HIV-1 seropositive individuals to mediate ADCC against HIV-1 gp120/160 transfected cells in an antigen-specific manner was established. Approximately 63% of the HIV-infected individuals representing all stages of infection have virus-specific ADCC antibodies. Moreover, sera from healthy seropositive (CDC class II) subjects mediated substantially higher levels of ADCC activity (25.7 +/- 11.5%) than did sera from individuals in CDC class III (10.3 +/- 8.9%) or IVC1 (6.4 +/- 8.1%). The mean ADCC activity for the sera from the seronegative volunteers was 2 +/- 1.3%. The correlation between the ADCC level and CD4 cell depletion remains uncertain. Therefore, the established transfected cell line may be used to probe the role of gp120/160-specific ADCC activity in the development of AIDS and may also prove useful in screening human and murine monoclonal antibodies with potential ADCC activity. Such monoclonals may be useful for future immunotherapeutic agents in conjunction with antiviral chemotherapy.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1993

Expression in Escherichia coli of the cloned polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus.

Cloned cDNA of genomic segment 10 of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was placed downstream from the lambda PL promoter in expression plasmid pRC23 and expressed in Escherichia coli cells. A polypeptide of the same molecular weight (28 kDa) as natural polyhedrin was synthesized at the level of approximately 10% of total host cell protein. This polypeptide was identified as CPV polyhedrin (r-polyhedrin) after comparative studies. The r-polyhedrin did not form any crystalline structure in E. coli cells but instead accumulated in the form of an insoluble inclusion body, even though natural polyhedrin is known to form a crystalline matrix (polyhedra) in infected insect cells. The purified r-polyhedrin complex, like natural polyhedra, was not soluble in neutral or acidic buffer but soluble in alkaline buffer. Upon solubilization, the r-polyhedrin complex did not undergo proteolytic degradation, while natural polyhedra were digested into small peptides by the associated protease. Incubation of r-polyhedrin with natural polyhedra in alkaline buffer, however, degraded the r-polyhedrin, resulting in an identical profile of peptide products to that of natural polyhedra. These results indicate that even though r-polyhedrin molecules produced in E. coli cells are not in the natural conformation, the molecules can present the identical cleavage sites to the polyhedra-associated alkaline protease. Experiments showed that the alkaline protease was associated with the matrix of polyhedra and not with virus particles.

Alkalies↗

PubMed1993

Comparison of amino acid sequences deduced from a cDNA fragment obtained from infectious pancreatic necrosis virus (IPNV) strains of different serotypes.

Infectious pancreatic necrosis is an important viral disease of salmonid fish reared in hatchery. Its etiological agent, IPNV, showed a high degree of antigenic heterogeneity. Up to 10 serotypes and 2 serogroups were proposed. Yet, very little is known about genomic variations among viruses of different origin. In order to investigate these variations, a 310-bp cDNA fragment was prepared from 17 IPNV strains by reverse transcription of the viral genome and amplification by the polymerase chain reaction. These fragments were then cloned and sequenced. Comparison of the 17 sequences obtained with 3 previously published ones, at the amino acid level, showed that serologically related viruses are highly homologous (over 96% homology) but some strains which were reported to belong to different serotypes also appeared closely related. Thus, only three major groups, clearly distinct from each other, could be formed. Apart from this, a search for the exact cleavage site of the unprocessed polyprotein of IPNV was done since the amplified fragment used for sequencing was localized at the junction between two polypeptides of the virus, pVP2 and NS. No obvious sequence or dipeptide appeared conserved in all birnaviruses.

Amino Acid Sequence↗

PubMed1993

Antigenic characterization of serogroup 'A' of infectious pancreatic necrosis virus with three panels of monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against three serotypes of infectious pancreatic necrosis virus (IPNV): A1 (LWVRT 60-1, U.S.A.), A2 (d'Honnincthun, France) and A9 (Jasper, Canada). Each panel of MAbs (identified as LW, HF and JA) was analysed by ELISA with the 10 proposed serotypes of IPNV and their specificity defined by immunoprecipitation and Western immunoblotting analysis. A first group of MAbs, directed against the outer capsid protein VP2, reacted with linear or conformational epitopes. A second group of MAbs, directed against the internal protein VP3, reacted with linear epitopes. There was no relationship between the neutralizing property of anti-VP2 MAb and the configuration of the epitope that it recognized. The MAbs were used for antigenic characterization of serogroup A. Each panel of MAbs showed a characteristic pattern of reactivity. The European HF series was predominantly cross-reactive and detected conserved epitopes among the 10 serotypes for both VP2 and VP3. The North American LW and JA series identified a group of conserved epitopes on VP3 and new specific epitopes on VP2 and VP3. The higher variability observed for VP2 in comparison with VP3 is one example of how external pressures may promote natural selection of those epitopes required for virus survival. Our results are consistent with an ancestral relationship of the European to the North American strains, the latter having developed new antigenic determinants upon evolution in their new geographical location.

Animals↗

PubMed1992

A simple method for the extraction of baculovirus DNA.

We have developed a method using lysozyme for DNA extraction from Baculoviruses using as a model Lymantria dispar nuclear polyhedrosis virus (LdNPV) obtained from infected larvae. This method proved to be quick, inexpensive and the extracted DNA was successfully used in molecular hybridization experiments.

Animals↗

PubMed1992

Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles.

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of an important disease in hatchery-reared salmonid fish in North America, Europe and Japan. It belongs to the family Birnaviridae and shows a high degree of antigenic heterogeneity. However, genomic variations between the 10 identified serotypes have not yet been studied. In order to correlate genomic heterogeneity with the different serotypes, oligonucleotides were synthesized according to the published sequence of the Jasper strain (serotype A9). They were used as primers for the amplification of a 359 bp cDNA fragment of the viral genome using the polymerase chain reaction. Fragments amplified from 37 strains were digested with five different restriction enzymes. Restriction fragment profiles obtained an agarose gels showed heterogeneity not only between strains of different serotypes, but also among those belonging to serotype A1. A cluster analysis of the restriction patterns showed that IPNV strains can be divided into three major groups, corresponding approximately to serotypes A1, A2 and A3, and 10 subgroups which do not correlate with the serotyping of the strains.

Animals↗

PubMed1992

Antigenic and genomic differences of two Jasper strains of infectious pancreatic necrosis virus.

Strains of infectious pancreatic necrosis virus (IPNV-Jasper) obtained from two different laboratories were compared serologically with polyclonal and monoclonal antibodies. Nucleotide sequence and restriction endonuclease patterns of 359-bp fragment of genome segment A cDNA were also compared. Substantial differences were found in both analyses that will support the fact that the two Jasper strains are not identical.

Animals↗

PubMed1992

Diagnosis of vertical HIV-1 transmission using the polymerase chain reaction and dried blood spot specimens.

We have used the polymerase chain reaction (PCR) to detect HIV proviral sequences in minute amounts of peripheral blood collected onto newborn screening blotters. Forty-three newborns, infants, and children of HIV-infected mothers were serially studied: dried blood spot (DBS) specimens were processed for PCR; serum was assayed for HIV antibodies, p24 antigen, and immunoglobulins; mononuclear cells were cultured and CD4 cells were quantitated by immunofluorescence. There was excellent agreement between the results of blood spot PCR, viral culture, and clinical and immunological indicators of HIV infection. Eighteen of 19 infected children tested positive by both PCR and culture, including six asymptomatic infants who were less than 10 weeks of age. As expected, p24 antigen capture assays were insensitive, detecting only 13 of the 19 infected children. One infected infant tested positive by PCR, but negative by culture and antigen. This infant was seropositive at 27 months and had pronounced hypergammaglobulinemia in association with non-specific symptoms. Twenty-four of the 43 infants were asymptomatic with normal immune profiles, declining antibody levels and no evidence of infection. These children tested repeatedly negative by PCR, culture, and p24 antigen assays. Our results indicate that DBS PCR is a sensitive, specific, and cost-effective alternative to viral culture for the early diagnosis (or exclusion) of perinatal HIV infection. DBS sampling opens the way for large-scale prospective studies to determine the exact rates of vertical HIV transmission in industrialized, as well as, nonindustrialized countries.

Child, Preschool↗

PubMed1991

A highly conserved nucleotide string shared by all genomes of human papillomaviruses.

The nucleotide string TAAAACGAAAGT is the longest perfect homology shared by all sequenced human papillomavirus genomes. This nucleotide string, which was also found to be highly specific for human papillomavirus genomes, shares the same genomic position in all viral types (5' end of the E1 open reading frame) and putatively codes in every case for the same amino acids. One possible evolutionary model was used to estimate the probability of random occurrence of the nucleotide string in 10 human papillomavirus genomes. It assumed that the universal string had been subjected to the same mutation rate as the entire E1 open reading frame. The estimated probability was found to be very low, suggesting that the conservation of the string could not have resulted from random divergence and that its conservation among human papillomaviruses is likely to reflect the occurrence of biological constraints. It is speculated that this nucleotide string may be required to code for amino acids indispensable for the nuclear localization of E1-coded peptides or to bind cellular factors affecting viral replicative functions. Definitive evidence is expected to come from oligonucleotide-protein binding experiments and from site-directed mutagenesis of cloned HPV genomes. This motif, universal among human papillomaviruses, is being successfully used in the design of consensus primers from the early region.

Amino Acid Sequence↗

PubMed1991

Improved detection of HIV-1 envelope sequences using optimized PCR and inosine-substituted primers.

We describe inosine-substituted 'consensus' primers for the detection of HIV-1 envelope sequences by the polymerase chain reaction. The primers, modifications of SK68 and SK69, are highly specific (100%) and sensitive (greater than 94%) and they prevent false-negative results due to variation in the HIV-1 genome. Consensus primers are needed to ensure the detection of most, if not all, variants of HIV-1 including American, African and newly emerging strains.

AIDS-Related Complex↗

PubMed1991

Use of dried blood spot specimens in the detection of human immunodeficiency virus type 1 by the polymerase chain reaction.

Dried blood spots (DBSs) constitute a potentially valuable source of material for human immunodeficiency virus (HIV) serologic and molecular testing. To facilitate molecular testing, we have adapted the polymerase chain reaction (PCR) to the detection of HIV proviral DNA in DBS samples. The method is highly reproducible, with 75 microliters of whole dried blood providing sufficient DNA for duplicate testing with three primer sets. By using DBS PCR, 66 of 69 (95.6%) seropositive at-risk individuals tested positive by at least two primer sets and 85 of 85 (100%) low-risk seronegative blood donors tested negative by all three sets of primers. The frequency of HIV DNA detection in seronegative at-risk individuals was low, with only 1 of 58 (1.7%) individuals testing positive. These results show that in a clinical environment, HIV PCR analysis of DBS specimens is specific and sensitive. The method is cost effective and presents a useful alternative to the isolation of HIV from seropositive babies with an undefined infection status.

Blood↗

PubMed1989

Nucleotide sequence of the polyhedrin gene of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV).

The double-stranded RNA genome of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) was reversely transcribed to the double-stranded DNA and cloned into pIBI30. The complete nucleotide sequence of cloned genome segment 10, which encodes the virus polyhedrin polypeptide, was determined. The EsCPV polyhedrin gene consists of 881 bp and possesses an open reading frame that codes for a polypeptide of 269 amino acids (MW 30.1K), consistent with an apparent MW of 30K determined by SDS-PAGE for purified polyhedrin. The sequence is identical to that reported for the amino terminus of polyhedrin from the CPV of Orgyia pseudotsugata; however, no amino acid or nucleotide sequence homology was found between the EsCPV polyhedrin and that from Bombyx mori CPV (BmCPV) or several nuclear polyhedrosis viruses. The hydrophilic profiles and predicted secondary structures of both EsCPV and BmCPV polyhedrin show some similarities, mainly in the amino half of the polypeptides. These data should be helpful in identifying the domains responsible for the polyhedrin crystallizing properties.

Amino Acid Sequence↗

PubMed1989

Amplification of human papillomavirus DNA sequences by using conserved primers.

The polymerase chain reaction has potential for use in the detection of small amounts of human papillomavirus (HPV) viral nucleic acids present in clinical specimens. However, new HPV types for which no probes exist would remain undetected by using type-specific primers for the polymerase chain reaction before hybridization. Primers corresponding to highly conserved HPV sequences may be useful for detecting low amounts of known HPV DNA as well as new HPV types. Here we analyze a pair of primers derived from conserved sequences within the E1 open reading frame for HPV sequence amplification by using the polymerase chain reaction. The longest perfect homology among HPV sequences is a 12-mer within the first exon of E1M. A region of conserved amino acids coded by the E1 open reading frame allowed the detection of another highly conserved region about 850 base pairs downstream. Two 21-mers derived from these conserved regions were used to amplify sequences from all HPV DNAs used as templates. The amplified DNA was shown to be specific for HPV sequences within the E1 open reading frame. DNA from HPVs whose sequences were not available were amplified by using these two primers. HPV DNA sequences in clinical specimens could also be amplified with the primers.

Animals↗

PubMed1988

Molecular cloning and characterization of cytoplasmic polyhedrosis virus polyhedrin and a viable deletion mutant gene.

The double-stranded RNA genome of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was converted to double-stranded DNA and cloned into plasmid pBR322. The complete nucleotide sequence of cloned genome segment 10, which encodes virus polyhedrin polypeptide, was determined. The CPV polyhedrin gene consists of 942 based pairs and possesses a long open reading frame that codes for a polypeptide of 248 amino acids (molecular weight, 28,500), consistent with an apparent molecular weight of 28,000 previously determined for purified polyhedrin. No sequence homology was found between CPV polyhedrin and polyhedrins from several nuclear polyhedrosis viruses. In addition to the polyhedrin gene, we completed the sequence analysis of a small deletion mutant gene derived from the polyhedrin gene. This mutant gene consists of two subset domains of the polyhedrin gene, i.e., the 5'-terminal 121 base pairs and the 3'-terminal 200 base pairs. An in vitro transcription demonstrated that the small mutant gene is transcribed by virion-associated RNA polymerases. These data confirm the importance of CPV terminal sequences in virus genome replication.

Amino Acid Sequence↗