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Biomedical subjects

J S Mackenzie

Publications and source records attributed to J S Mackenzie.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1986

Two types of Newcastle disease viruses isolated from feral birds in western Australia detected by monoclonal antibodies.

Thirteen viruses isolated from feral birds and one isolated from a domestic duck, obtained in 1979-1980 during a survey of birds in Western Australia, were shown to be Newcastle disease viruses of low virulence for chickens. The binding of mouse monoclonal antibodies, raised against NDV-Ulster 2C to MDBK cells infected with the isolates was assessed using an indirect immunoperoxidase test. Five viruses caused binding of all 9 monoclonal antibodies tested, whereas the other 9 isolates induced binding of only 4 monoclonal antibodies.

Animals↗
PubMed1984

Isolation of ortho- and paramyxoviruses from wild birds in Western Australia, and the characterization of novel influenza A viruses.

As part of the World Health Organization's international programme on the ecology of influenza, cloacal swabs were collected from 3,736 birds belonging to 67 species over a 3-year period in Western Australia for the isolation of ortho- and paramyxoviruses. A total of 24 influenza A viruses were isolated from various species of ducks, shearwaters , noddies , terns and a coot , and were subtyped as H1N9 , H3N8 , H4N4 , H4N6 , H6N2 , H6N4 , H?N2, H?N6 and H? N9 . The H? haemagglutinins did not react in tests with reference antisera. Whether they represent a novel haemagglutinin subtype or atypical members of an established subtype remains to be determined, although preliminary results indicate that they may be atypical members of the H7 subtype. The H1N9 isolate is the first reported isolate of this particular antigenic combination. A total of 17 Newcastle disease viruses was isolated from ducks, noddies , terns and a black- fronted plover : preliminary results suggest that they are avirulent for domestic chickens. This study indicates that ortho- and paramyxoviruses are present in a variety of wild birds in Australia.

Animals↗
PubMed1983

Isolation and characterization of avipoxviruses from wild birds in Western Australia.

Avipoxviruses were isolated from wart-like lesions in an Australian magpie (Gymnorhina tibicen) and a silvereye (Zosterops lateralis), and the poxvirus aetiology of wart-like lesions in a magpie-lark (Grallina cyanoleuca) was confirmed. The three viruses produced typical pock lesions on the chorioallantoic membrane of embryonated eggs and were able to replicate in trypsin-dispersed chick embryo fibroblast cultures but not confluent monolayer cultures. Pock neutralization and immunodiffusion studies showed that the three wild bird isolates were distinct from fowlpox, although antigenically closer to fowlpox than pigeonpox. The magpie and silvereye isolates were more closely related to each other than to the magpie-lark isolate.

Animal Population Groups↗
PubMed1982

Autocytotoxic cells against normal leucocytes identified in mouse mesenteric lymphnodes.

Plaques were formed in monolayers of leucocytes prepared from mesenteric lymphnodes (LN) of normal mice. The plaques were first detected after 2 h incubation, followed by a sudden increase in number at 7 h, and reached a peak between 11 and 21 h. Large cells were observed in almost all plaques under the microscope. The large cells attached surrounding leucocytes via cytoplasmic extensions, and held aggregated leucocytes on their cell surface. Aggregated leucocytes were lyzed and/or phagocytized by the large cells. Results suggest that plaque formation was due to the direct, indiscriminate killing of normal leucocytes by the large cells. The large cells increased with age. The large cells were contained in the adherent cell fraction but not the non-adherent fraction, and were removed from leucocytes by the carbonyl iron method. Thus, the large effector cells may be related to macrophages.

Animals↗