Biomedical subjects

R J Coelen

Publications and source records attributed to R J Coelen.

14 recordsLinked to original sources

PubMed1992

A one-tube, one manipulation RT-PCR reaction for detection of Ross River virus.

A sensitive, single tube reverse transcription-polymerase chain reaction (RT-PCR) protocol for the detection of Ross River virus (RRV) is described. All components necessary for both reverse transcription and PCR were combined in a single tube, and reverse transcription and PCR carried out sequentially in a single, non-interrupted thermal cycling program. The antisense oligonucleotide from the two primers selected for use in the PCR also served to prime specifically for the reverse transcription. The 549 bp product was detected by electrophoresis and ethidium bromide staining. The detection limit using this system was 18 fg of purified viral RNA or 1.3 pfu of whole virus. Greater sensitivity cannot reasonably be expected unless a more sensitive method than electrophoresis and ethidium bromide staining is used for PCR product detection, such as nested PCR or hybridisation with labelled probe. This PCR detection system will be adapted for detection of RRV in mosquito populations for virus surveillance programs.

Animals↗

PubMed1992

Reverse transcriptase inhibits Taq polymerase activity.

Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.

Avian Myeloblastosis Virus↗

PubMed1991

Detection of immobilised Murray Valley encephalitis virus RNA using oligonucleotide probes with varying degrees of mismatch.

The design of oligonucleotides used for hybridisation studies often utilises available sequence information of the type strain of a particular virus. If hybridisation studies, using such oligonucleotides, are carried out with field isolates of the same virus, the problem of base pair mismatches and consequent difficulties in detection may arise. This study examined the effect of base pair mismatches on the hybridisation between membrane-bound Murray Valley encephalitis virus (MVE) RNA derived from various strains and deliberately mismatched oligonucleotide probes. Under conditions of very low stringency, probes containing up to 5 mismatches were able to detect MVE RNA, but not yeast RNA. Under washing conditions of increased stringency, hybridisation could be detected between MVE virus RNA and probes with only 3 to 4 mismatches. However, the extent of this interaction was dependent on the number and type of mismatches and their relative sequence position.

Animals↗

PubMed1991

Immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus: selection of the appropriate ligand and optimal conditions for elution.

A novel approach was used to select the most suitable antiviral monoclonal antibody (mAb) and elution conditions for immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus (MVE). Crude NS1 protein was subjected to a variety of chemical conditions produced by common elution buffers, and tested with a panel of NS1-specific mAbs by ELISA to determine which buffers denatured antigenic epitopes. Buffers that caused least structural damage to NS1 epitopes were tested by ELISA for dissociation of NS1-mAb complexes adsorbed to the solid phase. For each mAb analysed, the conditions required to break the mAb-NS1 complex on the solid phase were similar to those required to release antigen bound to mAb-sepharose beads. From these results we selected an appropriate antibody for affinity purification of the non-structural viral protein NS1 on CNBr-activated sepharose columns. Elution at pH 11.5 yielded good recoveries of highly pure and antigenically intact NS1 dimer. The results demonstrate that the appropriate ligand and optimal elution conditions can be rapidly determined generally by immunoassay in microtitre plates.

Antibodies, Monoclonal↗

PubMed1991

Persistent infection of Vero cells by the flavivirus Murray Valley encephalitis virus.

Murray Valley encephalitis (MVE) virus strain OR2 was serially passaged on Vero cells to establish a persistent infection which was maintained for over 300 days. Supernatants from infected cells protected Vero cells from c.p.e. and caused up to a 95% reduction of wild-type virus yield. These protective and interfering effects suggest that defective interfering (DI) particles are responsible for the establishment and maintenance of the MVE virus persistent infection. The persistently infected cell supernatant preparations shared several features with DI particle preparations from other viral systems, such as their amplification to detectable levels after two to four passages of virus. However, results from this study suggest that DI particles of MVE virus differ from other studied systems in that they are able to affect only moderately the yield of infectious wild-type virus. The genetic drift of the parental virus during the course of a long term persistent infection in vitro appears to be minimal.

Animals↗

PubMed1991

Comparison of IgE expression at the mRNA and protein levels in vitro.

The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay.

Cell Line↗

PubMed1990

The 5'-terminal non-coding region of Murray Valley encephalitis virus RNA is highly conserved.

The 5' non-coding region of the genomes of 11 isolates of Murray Valley encephalitis virus from Australia and Papua New Guinea were examined by primer extension sequencing. Although the 5' non-coding region of all isolates was found to be highly conserved, three isolates were significantly different in that they contained extra uridine residues. Two of these isolates from Papua New Guinea contained an extra uridine residue, nominally positioned after nucleotide 54, which was absent from all but one of the Australian isolates tested. This isolate (OR 156) contained a further uridine residue at the same site. These results provide further support for earlier observations on the genetic relationships between these isolates, in particular that OR 156 is more closely related to the Papua New Guinea strains than to the Australian strains.

Animals↗

PubMed1989

Two-dimensional gel electrophoresis of RNase T1 resistant oligonucleotides of flavivirus RNA using ultrathin gels.

A convenient method employing a commercially available apparatus for two-dimensional gel electrophoresis of RNase T1 resistant oligonucleotides using ultrathin gels has been developed. The methodology overcomes problems commonly associated with the establishment of good, bubble-free, fusion of the first and second dimension gels. The use of ultrathin gels results in autoradiograms with well resolved oligonucleotide spots.

Electrophoresis, Polyacrylamide Gel↗

PubMed1989

Kunjin virus isolates of Australia are genetically homogeneous.

The genomes of 22 isolates of Kunjin virus (KUN) from Australia were characterized and compared using RNase T1 oligonucleotide fingerprinting. The results show that all isolates belonged to one topotype, the distribution of which covered the entire Australian continent. This finding is similar to that of Murray Valley encephalitis virus, but in contrast to the results reported for some other flaviviruses such as Saint Louis encephalitis virus.

Australia↗

PubMed1988

Genetic variation of Murray Valley encephalitis virus.

The genomes of 21 isolates of Murray Valley encephalitis virus (MVE) from Australia and Papua New Guinea were characterized and compared using RNase T1 oligonucleotide fingerprinting. Most Australian isolates grouped in clusters that were linked with a similarity coefficient of greater than 75%, indicating substantial homogeneity. Two isolates grouped as a cluster that linked with other isolates at a level of 67%. These two isolates, one from the north and one from the south-east of Australia were very similar and could demonstrate the movement of MVE between these areas. This notion is substantiated by genetic homogeneity of isolates from the Kimberley region and from south-eastern Australia. One Australian isolate (OR 156) and the Papua New Guinea isolate (MK 6684) were substantially different from each other as well as from the other isolates. No evidence was found for a poly(A) tract in the genome of MVE.

Animals↗

PubMed1985

Microbiology of chronic otitis media with effusion among Australian Aboriginal children: role of Chlamydia trachomatis.

Serum, eye secretions, post-nasal swabs, external ear swabs and middle ear effusions (MEE) were collected from 131 Australian Aboriginal children with chronic otitis media with effusion (COME). The children were all resident in a trachoma endemic region. Chlamydia trachomatis was recovered from the MEE of 2 children. Probable bacterial pathogens were isolated from 34 (12.7%) ears. The remainder were sterile (52.4%) or contained normal skin flora (34.9%). Serum and secretions were examined by the microimmunofluorescent technique for the presence, titre and serotype of anti-chlamydial antibody. Antibody, predominantly of the C serotype, was found in a high percentage of sera (80%) and secretions (approximately 50%). This serotype is associated with ocular trachoma. It is concluded that C. trachomatis is associated with COME among some Aboriginal children in this trachoma endemic area.

Adolescent↗

PubMed1983

The effect of hexadimethrine bromide (polybrene) on the infection of the primate retroviruses SSV 1/SSAV 1 and BaEV.

Polybrene was shown to enhance the adsorption of simian sarcoma virus-simian sarcoma associated virus complex (SSV-1/SSAV-1) and baboon endogenous virus (BaEV) onto cells in culture. A 16- to 18-fold increased adsorption of both viruses occurred at 8 micrograms/ml polybrene within one hour after infection. The polybrene mediated adsorption was found to be inhibited by the addition of tri-sodium citrate to the culture medium, suggesting the involvement of electrostatic forces. This contention was further supported by the demonstration of temperature independence of the polybrene mediated interaction.

Adsorption↗

PubMed1982

Children's brain tumour cells produce RNA particles with incomplete retrovirus characteristics.

Short-term cultures of cells from human rain tumours have been reported to synthesise RNA particles of density in the range characteristic of C type RNA retroviruses, with associated DNA polymerase activity. Fresh tumour cells obtained from 6 children with astrocytoma and 7 children with medulloblastoma, together with one sample of normal brain tissue and normal leukocytes from brain tumour patients were assayed by several characteristics for the primate retrovirus. 1 or 6 (17%) astrocytomas and 4 of 7 (57%) medulloblastomas released RNA particles which banded in sucrose gradients at a density of 1.16-1.18 g/cm3 together with a short segment of DNA, which was eliminated by prior ribonuclease treatment and two proteins of 28k and 16k daltons. These findings were compatible with the presence of a primate retrovirus. Immune coprecipitation of 125I-labelled proteins from the 1.16-1.18 g/cm3 gradient region failed to show any reactivity with antisera to p28 core antigens or the p70 reverse transcriptase antigens of simian sarcoma virus, baboon endogenous virus or Mason Pfizer virus. Assays for DNA polymerase activity in culture supernatant fluid showed only a low amount of activity with template preferences not characteristic of the retroviral reverse transcriptase enzyme.

Animals↗

PubMed1979

Effect of antiviral lipids, heat, and freezing on the activity of viruses in human milk.

Semliki Forest virus (SFV), herpes simplex virus type 1 (HSV-1), coxsackievirus B4, and cytomegalovirus (CMV) were added to human milk, which was then subjected to treatments that approximated those required for the decontamination or storage of milk. Boiling was the only treatment that eliminated these viruses from the milk. Pasteurization (at 62.5 C for 30 min) did destroy CMV, but the other viruses could still be detected. All of the viruses except HSV-1 were detectable after the contaminated milk samples had been stored at -15 C for 10 days. SFV, HSV-1, and CMV were also decreased by lipid antiviral activity, which was present in 78% of samples that were obtained on day 5 postpartum. Naturally excreted CMV was detected at the highest levels in milk that lacked this antiviral factor; this CMV also survived freezing for 10 days. Thus, whereas the lipid antiviral activity decreased the level of enveloped viruses, whether they were added to or naturally excreted into human milk, further reduction in the levels of these viruses required heat treatment.

Antiviral Agents↗