Co-distribution and co-infection of chikungunya and dengue viruses

BACKGROUND Chikungunya and dengue infections are spatio-temporally related. The current review aims to determine the geographic limits of chikungunya, dengue and the principal mosquito vectors for both viruses and to synthesise current epidemiological understanding of their co-distribution. METHODS Three biomedical databases (PubMed, Scopus and Web of Science) were searched from their inception until May 2015 for studies that reported concurrent detection of chikungunya and dengue viruses in the same patient. Additionally, data from WHO, CDC and Healthmap alerts were extracted to create up-to-date global distribution maps for both dengue and chikungunya. RESULTS Evidence for chikungunya-dengue co-infection has been found in Angola, Gabon, India, Madagascar, Malaysia, Myanmar, Nigeria, Saint Martin, Singapore, Sri Lanka, Tanzania, Thailand and Yemen; these constitute only 13 out of the 98 countries/territories where both chikungunya and dengue epidemic/endemic transmission have been reported. CONCLUSIONS Understanding the true extent of chikungunya-dengue co-infection is hampered by current diagnosis largely based on their similar symptoms. Heightened awareness of chikungunya among the public and public health practitioners in the advent of the ongoing outbreak in the Americas can be expected to improve diagnostic rigour. Maps generated from the newly compiled lists of the geographic distribution of both pathogens and vectors represent the current geographical limits of chikungunya and dengue, as well as the countries/territories at risk of future incursion by both viruses. These describe regions of co-endemicity in which lab-based diagnosis of suspected cases is of higher priority.

Prevalence, codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom

Most studies exploring the role of upper airway viruses and bacteria in paediatric acute respiratory infections (ARI) focus on specific clinicaldiagnoses and/or do not account for virus–bacteria interactions. We aimed to describe the frequency and predictors of virus and bacteria codetection in children with ARI and cough, irrespective of clinical diagnosis. Bilateral nasal swabs, demographic, clinical and risk factor data were collected at enrollment in children aged <15 years presenting to an emergency department with an ARI and where cough was a symptom. Swabs were tested by polymerase chain reaction for 17 respiratory viruses and seven respiratory bacteria. Logistic regression was used to investigate associations between child characteristics and codetection of the organisms of interest. Between December 2011 and August 2014, swabs were collected from 817 (93.3%) of 876 enrolled children, median age 27.7 months (interquartile range13.9–60.3 months). Overall, 740 (90.6%) of 817 specimens were positive for any organism. Both viruses and bacteria were detected in 423 specimens (51.8%). Factors associated with codetection were age (adjusted odds ratio (aOR) for age <12 months = 4.9, 95% confidence interval (CI) 3.0, 7.9; age 12 to <24 months = 6.0, 95% CI 3.7, 9.8; age 24 to <60 months = 2.4, 95% CI 1.5, 3.9), male gender (aOR 1.46; 95% CI 1.1, 2.0), child care attendance (aOR 2.0; 95% CI 1.4, 2.8) and winter enrollment (aOR 2.0; 95% CI 1.3, 3.0). Haemophilus influenzae dominated the virus–bacteria pairs. Virus–H. influenzae interactions in ARI should be investigated further, especially as the contribution of nontypeable H. influenzae to acute and chronic respiratory diseases is being increasingly recognized.

Development of a Rep-inducible, BBTV-based expression system in banana

Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV. The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed. An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed. The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication. Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed. Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids. The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.

Anti-metabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system

The common brown leafhopper, Orosius orientalis (Matsumura) (Homoptera: Cicadellidae), previously described as Orosius argentatus (Evans), is an important vector of several viruses and phytoplasmas worldwide. In Australia, phytoplasmas vectored by O. orientalis cause a range of economically important diseases, including legume little leaf (Hutton & Grylls, 1956), tomato big bud (Osmelak, 1986), lucerne witches broom (Helson, 1951), potato purple top wilt (Harding & Teakle, 1985), and Australian lucerne yellows (Pilkington et al., 2004). Orosius orientalis also transmits Tobacco yellow dwarf virus (TYDV; genus Mastrevirus, family Geminiviridae) to beans, causing bean summer death disease (Ballantyne, 1968), and to tobacco, causing tobacco yellow dwarf disease (Hill, 1937, 1941). TYDV has only been recorded in Australia to date. Both diseases result in significant production and quality losses (Ballantyne, 1968; Thomas, 1979; Moran & Rodoni, 1999). Although direct damage caused by leafhopper feeding has been observed, it is relatively minor compared to the losses resulting from disease (P Tr E bicki, unpubl.).

Sewage disposal and wildlife health in Antarctica

Sewage and its microbiology, treatment and disposal are important to the topic of Antarctic wildlife health because disposal of untreated sewage effluent into the Antarctic marine environment is both allowed and commonplace. Human sewage contains enteric bacteria as normal flora, and has the potential to contain parasites, bacteria and viruses which may prove pathogenic to Antarctic wildlife. Treatment can reduce levels of micro-organisms in sewage effluent, but is not a requirement of the Environmental Protocol to the Antarctic Treaty (the Madrid Protocol). In contrast, the deliberate release of non-native organisms for any other reason is prohibited. Hence, disposal of sewage effluent to the marine environment is the only activity routinely undertaken in Antarctica knowing that it will likely result in the release of large numbers of potentially non-native species. When the Madrid Protocol was negotiated, the decision to allow release of untreated sewage effluent was considered the only pragmatic option, as a prohibition would have been costly, and may not have been achievable by many Antarctic operators. In addition, at that time the potential for transmission of pathogens to wildlife from sewage was not emphasised as a significant potential risk. Since then, the transmission of disease-causing agents between species is more widely recognised and it is now timely to consider the risks of continued discharge of sewage effluent in Antarctica and whether there are practical alternatives.

Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% hostspecificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10_5, 10_4 and 10_3 respectively.HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10_4 whilst EVs were detected up to dilution 10_5. The ability of the HS-HF183 marker to detect freshsewagewas3–4 orders ofmagnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.

Seasonal activity and abundance of Orosius orientalis (Hemiptera : Cicadellidae) at agricultural sites in Southeastern Australia

Orosius orientalis is a leafhopper vector of several viruses and phytoplasmas affecting a broad range of agricultural crops. Sweep net, yellow pan trap and yellow sticky trap collection techniques were evaluated. Seasonal distribution of O. orientalis was surveyed over two successive growing seasons around the borders of commercially grown tobacco crops. Orosius orientalis seasonal activity as assessed using pan and sticky traps was characterised by a trimodal peak and relative abundance as assessed using sweep nets differed between field sites with peak activity occurring in spring and summer months. Yellow pan traps consistently trapped a higher number of O. orientalis than yellow sticky traps.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Phylogenetic analysis of polyomaviruses based on their complete genomes

Perez-Losada et al. [1] analyzed 72 complete genomes corresponding to nine mammalian (67 strains) and 2 avian (5 strains) polyomavirus species using maximum likelihood and Bayesian methods of phylogenetic inference. Because some data of 2 genomes in their work are now not available in GenBank, in this work, we analyze the phylogenetic relationship of the remaining 70 complete genomes corresponding to nine mammalian (65 strains) and two avian (5 strains) polyomavirus species using a dynamical language model approach developed by our group (Yu et al., [26]). This distance method does not require sequence alignment for deriving species phylogeny based on overall similarities of the complete genomes. Our best tree separates the bird polyomaviruses (avian polyomaviruses and goose hemorrhagic polymaviruses) from the mammalian polyomaviruses, which supports the idea of splitting the genus into two subgenera. Such a split is consistent with the different viral life strategies of each group. In the mammalian polyomavirus subgenera, mouse polyomaviruses (MPV), simian viruses 40 (SV40), BK viruses (BKV) and JC viruses (JCV) are grouped as different branches as expected. The topology of our best tree is quite similar to that of the tree constructed by Perez-Losada et al.

Monitoring and analysis of internet traffic targeting unused address spaces

Today’s evolving networks are experiencing a large number of different attacks ranging from system break-ins, infection from automatic attack tools such as worms, viruses, trojan horses and denial of service (DoS). One important aspect of such attacks is that they are often indiscriminate and target Internet addresses without regard to whether they are bona fide allocated or not. Due to the absence of any advertised host services the traffic observed on unused IP addresses is by definition unsolicited and likely to be either opportunistic or malicious. The analysis of large repositories of such traffic can be used to extract useful information about both ongoing and new attack patterns and unearth unusual attack behaviors. However, such an analysis is difficult due to the size and nature of the collected traffic on unused address spaces. In this dissertation, we present a network traffic analysis technique which uses traffic collected from unused address spaces and relies on the statistical properties of the collected traffic, in order to accurately and quickly detect new and ongoing network anomalies. Detection of network anomalies is based on the concept that an anomalous activity usually transforms the network parameters in such a way that their statistical properties no longer remain constant, resulting in abrupt changes. In this dissertation, we use sequential analysis techniques to identify changes in the behavior of network traffic targeting unused address spaces to unveil both ongoing and new attack patterns. Specifically, we have developed a dynamic sliding window based non-parametric cumulative sum change detection techniques for identification of changes in network traffic. Furthermore we have introduced dynamic thresholds to detect changes in network traffic behavior and also detect when a particular change has ended. Experimental results are presented that demonstrate the operational effectiveness and efficiency of the proposed approach, using both synthetically generated datasets and real network traces collected from a dedicated block of unused IP addresses.

Evaluating sewage associated JCV and BKV polyomaviruses for sourcing human fecal pollution in a coastal river in Southeast Queensland Australia

In this study, the host-sensitivity and -specificity of JCV and BKV polyomaviruses were evaluated by testing wastewater/fecal samples from nine host groups in Southeast Queensland, Australia. The JCV and BKV polyomaviruses were detected in 48 human wastewater samples collected from the primary and secondary effluent suggesting high sensitivity of these viruses in human wastewater. Of the 81 animal wastewater/fecal samples tested, 80 were PCR negative for this marker. Only one sample from pig wastewater was positive. Nonetheless, the overall host-specificity of these viruses to differentiate between human and animal wastewater/fecal samples was 0.99. To our knowledge, this is the first study in Australia that reports the high specificity of JCV and BKV polyomaviruses. To evaluate the field application of these viruses to detect human fecal pollution, 20 environmental samples were collected from a coastal river. Of the 20 samples tested, 15% and 70% samples exceeded the regulatory guidelines for E. coli and enterococci levels for marine waters. In all, 5 (25%) samples were PCR positive for JCV and BKV indicated the presence of human fecal pollution in the studied river. The results suggest that JCV and BKV detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

The development of a serological-based diagnostic test for Dasheen mosaic potyvirus (DsMV)

Dasheen mosaic potyvirus (DsMV) is an important virus affecting taro. The virus has been found wherever taro is grown and infects both the edible and ornamental aroids, causing yield losses of up to 60%. The presence of DsMV, and other viruses,prevents the international movement of taro germplasm between countries. This has a significant negative impact on taro production in many countries due to the inability to access improved taro lines produced in breeding programs. To overcome this problem, sensitive and reliable virus diagnostic tests need to be developed to enable the indexing of taro germplasm. The aim of this study was to generate an antiserum against a recombinant DsMV coat protein (CP) and to develop a serological-based diagnostic test that would detect Pacific Island isolates of the virus. The CP-coding region of 16 DsMV isolates from Papua New Guinea, Samoa, Solomon Islands, French Polynesia, New Caledonia and Vietnam were amplified,cloned and sequenced. The size of the CP-coding region ranged from 939 to 1038 nucleotides and encoded putative proteins ranged from 313 to 346 amino acids, with the molecular mass ranging from 34 to 38 kDa. Analysis ofthe amino acid sequences revealed the presence of several amino acid motifs typically found in potyviruses,including DAG, WCIE/DN, RQ and AFDF. When the amino acid sequences were compared with each other and the DsMV sequences on the database, the maximum variability was21.9%. When the core region ofthe CP was analysed, the maximum variability dropped to 6% indicating most variability was present in the N terminus. Within seven PNG isolates ofDsMV, the maximum variability was 16.9% and 3.9% over the entire CP-coding region and core region, respectively. The sequence ofPNG isolate P1 was most similar to all other sequences. Phylogenetic analysis indicated that almost all isolates grouped according to their provenance. Further, the seven PNG isolates were grouped according to the region within PNG from which they were obtained. Due to the extensive variability over the entire CP-coding region, the core region ofthe CP ofPNG isolate Pl was cloned into a protein expression vector and expressed as a recombinant protein. The protein was purified by chromatography and SDS-PAGE and used as an antigen to generate antiserum in a rabbit. In western blots, the antiserum reacted with bands of approximately 45-47 kDa in extracts from purified DsMV and from known DsMV -infected plants from PNG; no bands were observed using healthy plant extracts. The antiserum was subsequently incorporated into an indirect ELISA. This procedure was found to be very sensitive and detected DsMV in sap diluted at least 1:1,000. Using both western blot and ELISA formats,the antiserum was able to detect a wide range ofDsMV isolates including those from Australia, New Zealand, Fiji, French Polynesia, New Caledonia, Papua New Guinea, Samoa, Solomon Islands and Vanuatu. These plants were verified to be infected with DsMV by RT-PCR. In specificity tests, the antiserum was also found to react with sap from plants infected with SCMV, PRSV-P, PRSV-W, but not with PVY or CMV -infected plants.