Biomedical subjects
Publications and source records attributed to M Arella.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Addition of Tween 80 to a 1.5% solution of beef extract was found to enhance the elution of bacteriophages adsorbed to electronegative filters. When reconcentration of the eluate was attempted by ammonium sulfate precipitation, a floating layer containing most of the viruses was formed. This floating layer can be obtained with several nonionic detergents including Tween 80 and under a salt saturation of 55% with ammonium sulfate, potassium tartrate, and sodium phosphate. Virus recovery ranged from 91 to 103% and was obtained with several bacteriophage strains. With poliovirus type 1, coxsackievirus B-4, and rotavirus SA-11 the recoveries were 100, 20, and 80%, respectively, but toxicity to cell culture was encountered: after removal of the detergent by a second floating layer method the recovery was 32% for poliovirus. Compared with organic flocculation, this method also had both improved recovery for bacteriophages and protective properties for samples frozen at -70 degrees C.
The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order Lepidoptera, Euxoa scandens, was studied in eight different lepidopterean cell lines. Lymantria dispar cells, which were found to support viral replication, more efficiently, were used to follow the kinetics of appearance of viral-specific polypeptides by a 2-h pulse with [(35)S]methionine. Five polypeptides (ca. 120,000 molecular weight [120K], 105K, 66K, 46K, and 28K) were identified as components of the polyhedral inclusion bodies, and two polypeptides (112K and 39K) were assigned as viral-particle polypeptides. All these polypeptides were present after 24 h and were still being produced 96 h after infection. The rate of synthesis of the major polyhedral polypeptide (28K) increased in the time course of infection, whereas the background of cellular polypeptides seemed to be unaffected. An indirect immunoperoxidase technique, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was blotted to a nitrocellulose membrane, showed that traces of the major polyhedral polypeptide were found from 8 h postinfection.
The behaviour of different Invertebrate cell lines iridovirus type 6 (CIV) infection was compared. The results allow us to distinguish at least four types of cellular systems: non-permissive systems (a. albopictus), semi-permissive systems (L. dispar) and two types of permissive systems in which the viral replication cycle is complete A. aegypti (slow replication cycle) and C. fumiferana (rapid replication cycle)
Lymantria dispar cells wee exposed to different doses of gamma radiation one hour after infection with cytoplasmic polyhedrosis virus (CPV). It was found that irradiated cells can produce infectious polyhedra. Modifications in the structure and in the process of maturation of the polyhedra were noted. The number of polyhedra per cell increased significantly after cell irradiation at 10(4) and 10(5) rads but no change was noted after cell treatment at 10(2) rads. On the other hand, morphological changes and a high mortality rate were noted in cell cultures treated at intensities higher than 10(3) rads. Therefore, the total yield of polyhedra produced when using 10(2) or 10(4) rads was similar to that obtained in normal cells but dropped significantly after cell irradiation at 10(5) rads.
As part of a screening program for pseudomonad enzymes having an industrial interest, we selected ribonuclease (RNase) producing strains. Of the 150 pseudomonads screened, 6 were found to produce an extracellular RNase activity when grown on solid medium. In broth culture, the RNase activity from these six species remained bound to the cells unless gelatin was added to the medium. Gelatin was essential for the release of RNase in the broth culture, but the pH of the medium, addition of potential inducers such as nucleic acids, or addition of cations did not affect this release. However, gelatin did not appear to induce the synthesis of the enzyme. Strain B-88, identified as Pseudomonas maltophilia, was selected for further study of the enzyme. The extracellular RNase isolated from B-88 broth cultures could be separated in two fractions on the basis of the molecular weight by the ultrafiltration technique. The low molecular weight fraction reacts optimally at temperatures between 55 and 60 degrees C and optimal pH values varying from 7.4 to 9.5. At neutral or alkaline pH, the enzyme was stable at temperatures below 37 degrees C but was inactivated at 55 degrees C. The RNase was inhibited by mercury and cobalt and stimulated by magnesium.