Biomedical subjects

J S Mackenzie

Publications and source records attributed to J S Mackenzie.

At least 19 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

Murray Valley encephalitis acquired in Western Australia.

OBJECTIVE: To report a recent fatal case of encephalitis associated with evidence of Murray Valley encephalitis virus infection, only the second fatality from this infection in Western Australia. CLINICAL FEATURES: An 18-month-old Aboriginal boy was admitted to hospital in northwest Western Australia with proven Haemophilus influenzae type b meningitis. INTERVENTION AND OUTCOME: After an initial good response to antibiotics (amoxycillin and cefotaxime) he relapsed and died with evidence of encephalitis. Analysis of serum showed a high titre of antibody to Murray Valley encephalitis (MVE) virus with the presence of specific IgM. No evidence was found for other infective agents. CONCLUSION: It is likely that this child died from MVE which followed his bacterial meningitis. Of the strains of mosquitoes trapped in the area of suspected infection 77.8% were Culex annulirostris, the major vector species for MVE. No MVE virus was isolated from these mosquitoes, but serum from one of the sentinel chickens contained MVE virus antibodies, indicating the presence of the virus in that region.

Encephalitis↗

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

Effect of respiratory tract viral infection on murine airway beta-adrenoceptor function, distribution and density.

1. The effects of a respiratory tract viral infection on beta-adrenoceptor density, distribution and function were investigated in murine airways. 2. Following intranasal inoculation of CBA/CaH mice with influenza A/PR-8/34 virus, the virus proliferated rapidly in trachea (peak titres 2 days post-inoculation) and lung (peak titres 4-6 days post-inoculation). Respiratory tract viral infection was associated with a significant increase in lung weight (88% higher than control mice at day 6 post-inoculation) that was related temporally to the development of peripheral lung inflammation and consolidation. 3. Analysis of specific binding of [125I]-cyanopindolol to beta-adrenoceptors revealed that on days 2, 4 and 8 post-inoculation with virus, mouse isolated tracheal sections contained, on average, 40% more beta-adrenoceptors than tracheal sections from time matched control mice. Subsequent quantitative autoradiographic studies demonstrated that this increase in total tracheal beta-adrenoceptors was due primarily to a 90% increase in the density of beta-adrenoceptors in the tracheal epithelium in virus-infected mice. 4. In contrast, virus-infection had no significant effect on the density of beta-adrenoceptors in tracheal airway smooth muscle, although within 2 days of inoculation with virus, mouse tracheal smooth muscle segments were approximately 2 fold less sensitive to the beta-adrenoceptor agonist, noradrenaline (mean pD2 = 6.57 +/- 0.04, n = 24) and to the adenylyl cyclase-activator forskolin (mean pD2 = 6.78 +/- 0.04, n = 12) compared to segments from control mice (mean pD2 = 6.84 +/- 0.06 for noradrenaline; mean pD2 = 7.03 +/- 0.07 for forskolin). Similar values were obtained 8 days post-inoculation. At day 2, but not day 8 post-inoculation with virus, relaxation responses to theophylline were also marginally attenuated compared with controls.5. Mouse isolated tracheal segments obtained 2 days after virus inoculation and segments from timematched control mice were equisensitive to the spasmogenic actions of the muscarinic cholinoceptor agonist, carbachol. However, tracheal segments from mice inoculated with virus were less responsive to carbachol on day 4 (mean pD2 = 6.45 + 0.04, n = 8) and day 8 (mean pD2 = 6.45 +/- 0.02, n = 12) compared to control preparations (day 4, mean pD2 = 6.73 +/- 0.06, n = 8; day 8, mean pD2= 6.65 +/- 0.04, n = 12, P < 0.05). In contrast, endothelin-l-induced contractions of tracheal smooth muscle were notaffected by virus-infection.6. These data demonstrate that respiratory tract viral infection can produce significant tissue-selective changes in airway /beta-adrenoceptor density as well as small reductions in airway smooth muscle muscarinic cholinoceptor and /beta-adrenoceptor function.

Animals↗

PubMed1990

Serum IgG subclass responses of humans to inactivated and live influenza A vaccines compared to natural infections with influenza A.

Studies with various viral agents have suggested that a preferential production of IgG subclasses may occur during infection, but limited information has been reported on the IgG isotypes produced during vaccination with live or killed virus preparations. The serum IgG subclass responses to influenza A infection or inoculation with live or killed influenza A vaccines were examined by an enzyme-linked immunosorbent assay, and results were expressed using a 4-parameter logistic model. It was observed that IgG1 was induced by both natural infections and the live virus vaccine depending on the dose given. Inactivated vaccines induced significant titres of IgG1, IgG2, and IgG3 isotypes in vaccinees, again depending upon the amount of virus preparation administered.

Enzyme-Linked Immunosorbent Assay↗

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↗

PubMed1989

The IgG subclass responses to influenza virus haemagglutinin in the mouse: effect of route of inoculation.

The influence of the route of infection, regime of inoculation and virus preparation on murine IgG subclass responses to the haemagglutinin of influenza A virus were examined. Virus preparations inoculated by the intraperitoneal, intravenous, intramuscular and intranasal routes were found to induce different IgG subclass profiles after a primary and secondary dose. IgG2a was found to be a major contributor to the responses elicited by all virus preparations irrespective of the route of inoculation. The magnitude of the response varied with the number of doses of virus and the route of inoculation; the intravenous and intramuscular routes produced the largest responses after two doses of virus. Evidence for the local production of antibody is presented and the influence of antigen presentation on the induction of different subclass is discussed.

Administration, Intranasal↗

PubMed1989

The immunoglobulin G subclass responses of mice to influenza A virus: the effect of mouse strain, and the neutralizing abilities of individual protein A-purified subclass antibodies.

The IgG subclass responses to cold-adapted (ca) influenza A/Queensland/6/72 virus and purified haemagglutinin H3 was assessed in C57BL/6 and BALB/c mice. In BALB/c mice IgG2a was present as the major subclass in serum, lung and salivary secretions after two doses of ca virus. In contrast, the serum response in C57BL/6 mice was predominantly IgG1 after primary and secondary inoculations of ca virus. However, in lung and salivary secretions no specific subclass was dominant. When purified H3 was used as the inoculum, serum responses were dominated by IgG1 in BALB/c mice after two inoculations whereas all four subclasses were present at equal levels in C57BL/6 mice. Overall the lung and salivary responses detected in C57BL/6 mice were lower than those observed in BALB/c mice with all four subclasses contributing equally to the response in BALB/c mice. The neutralizing and haemagglutination inhibition abilities of the four Protein A-Sepharose-purified IgG subclasses differed between the BALB/c, C57BL/6 and CBA/CaH mice strains. IgG1 and IgG2a were most effective in BALB/c mice and in C57BL/6 and CBA/CaH mice, IgG2a and IgG2b. These results are discussed in terms of the differing abilities of replicating and non-replicating virus to stimulate differential responses in mice and the TH1 and TH2 helper cell concept.

Animals↗

PubMed1988

The IgG subclass responses induced by wild-type, cold-adapted and purified haemagglutinin from influenza virus A/Queensland/6/72 in CBA/CaH mice.

The IgG subclass and IgA responses were investigated in CBA/CaH mice after inoculation with wild-type (wt) and cold-adapted (ca) derivatives of influenza A/Queensland/6/72 virus, and with purified haemagglutinin (H3) derived from the wt strain of the same virus. Intranasal inoculation of the wt and ca viruses resulted in responses dominated by IgG2a in serum, saliva and lung secretions, whereas an intramuscular injection of purified H3 elicited the production of all four IgG subclasses in serum and IgG2b and IgG3 in saliva and lung secretions. The source of IgG on mucosal surfaces was from local production and was not a transudate from serum, as demonstrated by the lack of albumin in saliva and lung secretions, and by the appearance in saliva and lung samples of IgG subclasses not present in serum at the time of sampling. The level of IgA on mucosal surfaces was influenced by the growth restrictions of intranasally inoculated ca virus, resulting in higher levels of IgA in saliva, whereas wt virus, able to replicate at higher temperatures, induced higher levels of IgA in lung secretions. The purified H3 inoculated by the intramuscular route elicited lower levels of IgA in serum, saliva and lung secretions than either the wt or ca viruses.

Animals↗

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↗

PubMed1988

Functional studies on macrophage populations in the airways and the lung wall of SPF mice in the steady-state and during respiratory virus infection.

Collagenase digestion of slices of lavaged and perfused murine lung from SPF animals yielded, on average, 104 x 10(6) mononuclear cells per gram tissue, including approximately 14% F4/80+ macrophages and 35% lymphocytes. The lung tissue-associated (digest) macrophage (LDM) population was 10-fold higher in number than the alveolar macrophage (AM) population recoverable by lavage. Comparative functional analysis of these populations was performed, employing assays for immune receptors (Fc and C3), complement-induced spreading, endogenous peroxidase, IL-1 secretion and tumour cytolysis; resident and activated peritoneal macrophages and blood monocytes were examined in parallel. The resident LDM population exhibited an activation status intermediate between blood monocytes and the relatively more activated AM from the airways. Murine lung macrophages were also examined during an acute influenza infection and LDM and AM recoveries increased significantly. The AM population exhibited activation during an acute infection. In contrast, LDM remained quiescent, despite the influx of a large number of T lymphocytes into the lung wall. These results suggest that LDM may be intrinsically resistant to the signals generated during T-cell-mediated eradication of influenza, or else local tissue factors modulate T-cell-derived activation signals.

Animals↗

PubMed1987

An enzyme-linked immunosorbent assay for detection of flavivirus antibodies in chicken sera.

An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of flavivirus antibodies to Murray Valley encephalitis (MVE) and Kunjin viruses in sentinel chicken sera. The development of a quick, reliable assay to detect antibodies to MVE was an essential part of a large-scale surveillance programme to monitor arbovirus activity in Western Australia. This assay was developed for use with alkaline phosphatase conjugated goat anti-mouse IgG using mouse anti-chicken globulin in an intermediate step. There was a significant difference in absorbance values between neutralization test positive and pre-bled sera. However, some sera obtained from sentinel chickens and deemed negative by neutralization demonstrated adsorbance levels above the cutoff level in the ELISA, which reflects the increased sensitivity of this technique. The ELISA test detected antibodies to MVE in chicken sera 7-10 days after infection, whereas these antibodies were only consistently detected by the neutralization test 24 days after infection. Antibodies to both MVE and Kunjin reacted positively with the MVE antigen, but there was little cross-reactivity between this antigen and antibodies to other togaviruses. The main advantages of the ELISA over the neutralization test for detecting antibodies to MVE virus in the sera of sentinel chickens are its greater sensitivity and the speed with which tests can be performed. Results are available within 48 h of receiving specimens and emergency mosquito control measures may then be implemented.

Animals↗

PubMed1979

The effect of long-term exposure to cigarette smoke on the height and specificity of the secondary immune response to influenza virus in a murine model system.

The effect of long-term exposure to cigarette smoke on the height and specificity of the secondary humoral immune response to influenza was investigated in a murine model system. It was shown that if mice were pre-immunized with a sub-lethal infection of influenza virus and then exposed to cigarette smoke daily for 36 weeks, they were able to mount a secondary immune response of normal height on subsequent challenge with the homologous virus strain. The response however, was less specific than that elicited in control mice, with high titres of cross-reacting antibody by haemagglutination-inhibition to the following strain in the same antigenic series. Recall of antibody to the previous strain in the antigenic series was not observed in either control or smoke-exposed animals. These results serve to correct an earlier discrepancy between the murine system and human studies in which the response to influenza infection in mice was depressed by prolonged exposure to cigarette smoke, whereas in man the response of smokers did not differ significantly from that of non-smokers. This apparent discrepancy had been caused by a lack of previous experience of influenza in the mice, which had therefore mounted a primary response, compared with the secondary response observed in the human studies.

Animals↗