Biomedical subjects

J S Mackenzie

Publications and source records attributed to J S Mackenzie.

At least 73 records · Page 4Linked to original sources

PubMed1995

Low resolution mapping around the flavivirus resistance locus (Flv) on mouse chromosome 5.

Although the phenomenon of innate resistance to flaviviruses in mice was recognized many years ago, it was only recently that the genetic locus (Flv) controlling this resistance was mapped to mouse Chromosome (Chr) 5. Here we report the fine mapping of the Flv locus, using 12 microsatellite markers which have recently been developed for mouse Chr 5. The new markers were genotyped in 325 backcross mice of both (C3H/HeJ x C3H/RV)F1 x C3H/HeJ and (BALB/c x C3H/RV)F1 x BALB/c backgrounds, relative to Flv. The composite genetic map that has been constructed identifies three novel microsatellite loci, D5Mit68, D5Mit159, and D5Mit242, tightly linked to the Flv locus. One of those loci, D5Mit159, showed no recombinations with Flv in any of the backcross mice analyzed, indicating tight linkage (< 0.3 cM). The other two, D5Mit68 and D5Mit242, exhibited two and one recombinations with Flv (0.6 and 0.3 cM) respectively, defining the proximal and distal boundaries of a 0.9-cM segment around this locus. The proximal flanking marker, D5Mit68, maps to a segment on mouse Chr 5 homologous to human Chr 4. This, together with the previous data produced by our group, locates Flv to a region on mouse Chr 5 carrying segments that are conserved on either human Chr 4, 12, or 7, but present knowledge does not allow precise identification of the syntenic element.

Animals↗

PubMed1995

A novel complex formed between the flavivirus E and NS1 proteins: analysis of its structure and function.

We examined the structural features and functional significance of a novel complex which forms between the envelope (E) protein and nonstructural protein NS1 of Murray Valley encephalitis (MVE) virus. Western blot analysis of virus-infected C6/36 cell lysates revealed that the undenatured form of this E-NS1 complex was a heat-sensitive E-(NS1 dimer) complex. Furthermore, the E-NS1 complex was observed in cells infected with Kunjin, Japanese encephalitis, West-Nile and Kokobera viruses which indicates the complex is a common feature of flavivirus infection. E-NS1 complex which had been immunoaffinity purified from MVE-infected cell lysates or eluted from gel slices exhibited partial breakdown into the individual monomers, demonstrating that the complex arose from the association of E and NS1 proteins and was not a single polypeptide created from incomplete gene cleavage. Radioimmunoprecipitation and western blot analysis of MVE-infected cell lysates and culture fluid preparations collected at various times after infection revealed that the E-NS1 complex has a long half life, accumulates in the virus-infected cell with time and is not secreted into the extracellular fluid. We have postulated that the E-NS1 complex, or at least a major portion of the complex, is a non-specific aggregation with no functional significance in the viral life cycle.

Animals↗

PubMed1995

Molecular epidemiology and evolution of mosquito-borne flaviviruses and alphaviruses enzootic in Australia.

Three distinct patterns in the molecular epidemiology and evolution are evident among the alphaviruses and flaviviruses enzootic in Australia. One pattern, exemplified by MVE and KUN viruses, is of a single genetic type evolving slowly and uniformly in geographically widely separated regions of Australia with no evidence of independent divergence. The second pattern, exemplified by RR virus, is of separate genotypes evolving in different geographic regions with significant nucleotide divergence between genotypes. The third pattern, exemplified by SIN virus, is of a succession of temporally related genotypes that extend over most of the Australian continent, with relatively low levels of nucleotide divergence within a genotype, and which are each replaced by the subsequent genotype. These patterns are associated in part due to the nature and dispersal of their vertebrate hosts. Nucleotide divergence rates for Australian alphaviruses are similar to those reported elsewhere. Genomic relationships between Australian flavivirus members of the JE virus serological complex and between Australian alphaviruses are discussed, and evidence is presented for a possible new genomic lineage of SIN virus.

Alphavirus↗

PubMed1995

Immunodominant epitopes on the NS1 protein of MVE and KUN viruses serve as targets for a blocking ELISA to detect virus-specific antibodies in sentinel animal serum.

Two mosquito-borne flaviviruses, Murray Valley encephalitis (MVE) and Kunjin (KUN), are the aetiological agents of Australian encephalitis. MVE causes a severe and potentially fatal form of the disease while KUN is responsible for only a few relatively mild cases. Therefore it is important that serological tests used in flavivirus surveillance differentiate between infections with these two viruses. However, this has been hampered in the past by the close antigenic relationships between flaviviruses in traditional serological assays. An epitope blocking ELISA using MVE-specific and KUN-specific monoclonal antibodies (mAb) reacting to the non-structural protein NS1 of these viruses and a flavivirus group-specific mAb reacting to the envelope (E) protein was assessed for testing sentinel animals for seroconversion to specific flavivirus infections. Using these assays we were able to detect serum antibodies to a variety of flavivirus in laboratory infected rabbits, and naturally infected chickens and in the case of primary infections, differentiate those caused by KUN or MVE. These assays are now used routinely in our laboratory for testing chicken sera from sentinel flocks in the Kimberley and Pilbara regions of north Western Australia.

Animals↗

PubMed1995

Australian X disease, Murray Valley encephalitis and the French connection.

Epidemics of a severe encephalitis occurred in eastern Australia between 1917 and 1925, in which over 280 cases were reported with a fatality rate of 68%. The disease had not been described previously and was called Australian X disease. The next epidemic occurred in south-east Australia in the summer of 1950-51. The disease was given its name of Murray Valley encephalitis as this was the area from which most cases were reported. A virus was isolated by Eric French in Victoria, and about the same time by John Miles and colleagues in South Australia. The virus Murray Valley encephalitis (MVE) virus, was shown to be a Group B arbovirus (flavivirus) which was related to, but distinct from, Japanese encephalitis virus. Early seroepidemiological studies showed that the most likely vertebrate hosts were water birds. MVE virus was first isolated from Culex annulirostris mosquitoes in 1960. The most recent epidemic of Murray Valley encephalitis occurred in 1974, at which time it was renamed Australian encephalitis. Since 1974, however, all cases have been confined to northern Australia, particularly the north of Western Australia. Indeed, the Kimberley region of Western Australia contains the only confirmed enzootic foci of virus activity. A closely related flavivirus, Kunjin virus, has also been shown to be an aetiological agent of Australian encephalitis. Since the first isolation of MVE and Kunjin viruses, considerable information has been accumulated on their ecology and epidemiology, some aspects of which are briefly described.

Animals↗

PubMed1995

Specific enzyme immunoassays for the rapid detection of Ross River virus in cell cultures inoculated with infected mosquito homogenates.

BACKGROUND: Ross River (RR) virus is a mosquito-borne alphavirus and one of the aetiological agents of epidemic polyarthritis in humans. Early detection of increased virus activity in mosquito populations enables public health authorities to implement measures to reduce the number of human infections during epidemics. However, current surveillance techniques require a minimum of four weeks for viruses to be isolated and identified. OBJECTIVES: This study was carried out to assess the use of enzyme immunoassays (EIA) as rapid alternatives to traditional cell culture techniques for detection of RR virus in mosquitoes. STUDY DESIGN: Enzyme immunoassays and immunoperoxidase assays were developed using RR-specific monoclonal antibodies and compared to traditional methods for detection of RR virus in field-caught mosquito samples. RESULTS: By inoculation of C6/36 cell cultures with mosquito homogenates and testing monolayers and culture supernatant by EIA, RR virus was detected and identified in all infected samples within 6 days. CONCLUSIONS: The use of EIA provides a rapid, sensitive and specific alternative to traditional methods for the detection of RR virus in mosquito vectors.

Journal Article↗

PubMed1995

Detection of Ross River virus in clinical samples using a nested reverse transcription-polymerase chain reaction.

BACKGROUND: Ross River virus (RRV) is a mosquito borne alphavirus that has been found in Australia, Papua New Guinea and the Pacific Islands. It is aetiological agent of epidemic polyarthritis, a debilitating illness whose symptoms are arthritis, arthralgia, lethargy, rash and fever which may persist for weeks or months. Diagnosis is made on a serological basis, but in many cases is presumptive rather than definite. OBJECTIVES: To apply the polymerase chain reaction (PCR) to detection of RRV in human sera to assess its suitability for application in disease diagnosis. STUDY DESIGN: Sensitivity of the nested RT-PCR assay was determined by detection of virus of known titre diluted in uninfected serum. Clinical serum samples from patients serologically diagnosed of having RRV infection were tested by nested RT-PCR to assess its diagnostic value. RESULTS: Sensitivity of the nested RT-PCR assay was determined to be detection of 0.01 PFU of virus stock in 100 mul serum. Clinical samples tested showed that 10 of 26 (38%) serum samples with low or negative (non-diagnostic) virus-specific antibody titres were PCR-positive, whereas all 22 specimens with high antibody titres were PCR-negative. PCR positivity was unaffected by repeated freezing and thawing of samples. CONCLUSIONS: While PCR cannot replace serology as a means of RRV diagnosis, it may be useful in conjunction with serological testing, particularly for forming definitive diagnoses in those samples with low (inconclusive) antibody titres. It is faster and more sensitive than virus isolation by tissue culture, and could also prove useful in investigations of disease pathogenesis.

Journal Article↗

PubMed1995

Two possible mechanisms for survival and initiation of Murray Valley encephalitis virus activity in the Kimberley region of Western Australia.

Two possible mechanisms are described for the initiation of Murray Valley encephalitis (MVE) virus activity in arid, epizootic regions of tropical Australia. Virus isolations were made from mosquitoes trapped shortly after the first heavy wet season rains and flooding in the east Kimberley, which followed approximately nine months of drought. A number of isolates of MVE virus were obtained, including isolates from pools of blood-engorged Culex annulirostris mosquitoes and from a single pool of male Aedes tremulus mosquitoes. The results strongly suggested that MVE virus activity was due both to its introduction in viremic vertebrate hosts, from which first-generation mosquitoes became infected following blood meals, and also to reactivation of vertically transmitted virus from desiccation-resistant eggs of Ae. tremulus. Both mechanisms are discussed with respect to environmental conditions.

Aedes↗

PubMed1994

Arboviruses causing human disease in the Australasian zoogeographic region.

Over 65 arboviruses have been reported from countries in the Australasian zoogeographic region, but only a few have been implicated in human disease. These include the flaviviruses Murray Valley encephalitis (MVE), Kunjin (KUN), Kokobera (KOK), and dengue, particularly types 1 and 2; the alphaviruses Ross River (RR), Barmah Forest (BF), and Sindbis (SIN); and the bunyaviruses, Gan Gan and Trubanaman. In this paper recent epidemiological and clinical results pertaining to these viruses are reviewed, with major emphasis on MVE and RR viruses. The extensive early studies of Australian arboviruses have been reviewed by Doherty [49, 50], and their ecology and vectors more recently by Kay and Standfast [87]. In addition, the biology of MVE and KUN [113] and RR [87, 114] viruses have been the subjects of more detailed reviews. The Australasian zoogeographic region is defined as countries east of the Wallace and Weber lines, two hypothetical lines in the Indo-Australian archipelago where the fauna of the Australasian and Oriental regions meet. Seroepidemiological studies of human arboviral infections have suggested that the Japanese encephalitis flavivirus and the chikungunya alphavirus occur only in the Oriental region, whereas the related MVE and RR viruses, respectively, are restricted to the Australasian region [85, 148]. Serological results from Wallacea, the zone between the Wallace and Weber lines, are not so clear-cut [85]. This review is therefore restricted to countries east of Wallacea, specifically New Guinea and Australia.

Animals↗

PubMed1994

Sensitive detection of Ross River virus--a one-tube nested RT-PCR.

A sensitive nested RT-PCR that can be carried out in a single tube is described. The sensitivity of this system was determined, and compared to that of a single round of PCR, and a single round of PCR followed by hybridisation with a radiolabelled oligonucleotide probe. We found that with the one-tube nested RT-PCR we were able to detect 0.1 pfu/ml of Ross River virus. The nested RT-PCR was 100-times more sensitive than a single round of RT-PCR followed by hybridisation, and 10,000-times more sensitive than a single round of RT-PCR alone. This system provides a sensitive detection of Ross River virus, and can be adapted for detection of RNA from any source. The test material is added to a single tube at the outset, and by subsequent addition of two sets of reagents, the entire nested RT-PCR can be carried out in the same tube. This system has maximum sensitivity, minimises risk of contamination, and is amenable to automation.

Alphavirus Infections↗

PubMed1994

Mapping the Flv locus controlling resistance to flaviviruses on mouse chromosome 5.

Genetically determined resistance to flaviviruses in mice is a dominant trait conferred by alleles at a single autosomal locus designated Flv, but no gene products have been associated with this locus and the mechanism of resistance is not well understood. To further characterize this model of genetic resistance, we conducted mapping studies to determine the chromosomal location of Flv. Because of evidence suggesting that the Flv locus is on chromosome 5, three-point backcross linkage analyses were used to define the location of Flv relative to previously assigned chromosome 5 markers. The results confirm the chromosome 5 location of Flv and indicate a map position between the anchor loci rd and Gus-s. The chromosomal localization of Flv is the first step in the production of a detailed linkage map of the Flv region, which may open approaches to positional cloning of the resistance gene.

Animals↗

PubMed1993

Australian encephalitis in Western Australia, 1978-1991.

OBJECTIVE: To review the various clinical manifestations of Murray Valley encephalitis (MVE) or Kunjin virus encephalitis in patients in Western Australia. DESIGN: Review of clinical records, 1978 to 1991. PATIENTS: Of 26 reported cases of Australian encephalitis, four were excluded from study because the patient's symptoms were not definitely associated with MVE virus or Kunjin virus infection. Two further cases of MVE were not reviewed as case records were not available. Of the remaining 20 patients, 18 had MVE and two had Kunjin virus encephalitis. RESULTS: Sixteen cases were in the Kimberley, a tropical region where the viruses are endemic. Four were in the subtropical Pilbara and Gascoyne regions. Thirteen of the 20 cases were in Aborigines, of whom 11 were children. The seven non-Aboriginal patients were adults. Seventeen of 20 cases were in males. The range of neurological disease and outcome was similar to that in previously reported cases, with convulsions, brainstem disease or respiratory failure in severe and fatal cases, and involvement of the spinal cord, cranial nerve or cerebellum in the moderate cases. One mild cases without neurological involvement was caused by Kunjin virus. CONCLUSIONS: The poor outcome in young Aboriginal children indicated that disease resulting from exposure early in life was more likely to be severe. The disease in adults, irrespective of facial background, was similar to that in cases reported previously from south-eastern Australia, but generally milder.

Adolescent↗

PubMed1993

Genetic heterogeneity among isolates of Ross River virus from different geographical regions.

The RNase T1 maps of 80 isolates of Ross River virus from different regions of mainland Australia and the Pacific Islands were compared. Four different clusters of isolates with greater than an estimated 5 to 6% diversity at the nucleotide level were found. There was a pattern of differences between eastern and western Australian strains; however, the pattern was disturbed by overlaps and incursants. Pacific Islands isolates belonged to the eastern Australian topotype. Our findings suggest that certain genetic types of Ross River virus predominate in different geographical regions. In contrast, populations of other important Australian arboviruses (Murray Valley encephalitis, Kunjin, and Sindbis viruses) are distributed across the Australian continent as minor variants of one strain. Our data also show that in one region, strains of Ross River virus with identical RNase T1 maps circulate during both years when epidemics occur and years when they do not. This finding suggests that Ross River virus epidemics are not dependent on the introduction or evolution of new strains of the virus. Two strains, belonging to the eastern Australian topotype, were isolated in Western Australia. It is likely that viremic humans or possibly domestic livestock travelling by aircraft were responsible for this movement.

Australia↗

PubMed1993

Ross River virus isolations from mosquitoes in arid regions of Western Australia: implication of vertical transmission as a means of persistence of the virus.

Outbreaks of mosquito-borne Ross River (RR) virus disease (epidemic polyarthritis) occur suddenly in the arid north and interior of the State of Western Australia, often within a few weeks of heavy rainfall. Between outbreaks, these regions may undergo long periods of drought, with little or no mosquito or arbovirus activity. The means by which RR virus is reintroduced or reactivated in these areas when environmental conditions favor mosquito-borne virus activity are unknown. In this paper, we describe isolations of RR virus from eight mosquito species trapped at two different locations, one coastal and one inland, in the arid Pilbara region of Western Australia, prior to outbreaks of epidemic polyarthritis. The isolation of RR virus has not been previously reported for five of these species and the isolations from the other three species are new records for Western Australia. The timing and number of isolations of RR virus from Aedes (Ochlerotatus) vigilax (Skuse, 1889) implicate that species as a vector of the virus on the Pilbara coast. Significantly, RR virus was isolated from pools of male Ae. vigilax and male Ae. (Macleaya) tremulus (Theobald, 1903) mosquitoes. This is the first report of RR virus (or other Australian arbovirus) isolates from wild-caught male mosquitoes. Both Ae. vigilax and Ae. tremulus have desiccation-resistant eggs that can survive long periods of drought, making them ideal candidates for the overwintering of arboviruses. The findings implicate vertical transmission as a means of persistence of RR virus in arid regions of Australia and therefore offer a likely explanation for the sudden recurrence of virus activity after heavy rains.

Aedes↗

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↗