Long-term follow-up of a randomized trial of biliary drainage in perihilar cholangiocarcinoma.

BACKGROUND AND AIMS: The DRAINAGE trial was a randomized controlled trial comparing preoperative endoscopic (EBD) and percutaneous biliary drainage (PTBD) in patients with potentially resectable, perihilar cholangiocarcinoma (pCCA). The aim of this study was to compare the long-term outcomes. METHODS: Patients were randomized in four tertiary referral centers. Follow-up data were available for all included patients. Primary outcome was overall survival (OS). Secondary outcomes were readmissions, and re-interventions not including in-trial interventions. RESULTS: A total of 54 patients were randomized; 27 in both groups. Median follow-up for both groups was 62 months (95% CI 54-70). The median OS was 13 months (95% CI 7.9-18.1) in the EBD and 7 months (95% CI 0.0-17.2) in the PTBD group (P = 0.28). Twenty (37%, n = 8 EBD vs n = 12 PTBD, P = 0.43) of 54 patients were readmitted at least once, mostly due to drainage-related complications (n = 13, 24%). Of note, 14 out of the 54 patients died within the trial. A total of 76 drainage procedures (32 EBD and 44 PTBD) were performed in 28 patients. The median number of stent or drain placements was 2 (2-4) for the EBD group and 2 (1-3) for the PTBD group (P = 0.77). DISCUSSION: Although this follow-up study represented a small cohort, no long-term differences in survival, readmissions, and drainage procedures for EBD and PTBD were found, even when comparing the resected and unresected group. However, this study demonstrates the complexity of biliary drainage for patients with potentially resectable pCCA, even in tertiary referral centers.

Translating the ABC-02 trial into daily practice: outcome of palliative treatment in patients with unresectable biliary tract cancer treated with gemcitabine and cisplatin.

BACKGROUND: Biliary tract cancer (BTC) is an uncommon cancer with an unfavorable prognosis. Since 2010, the standard of care for patients with unresectable BTC is palliative treatment with gemcitabine plus cisplatin, based on the landmark phase III ABC-02 trial. This current study aims to evaluate the efficacy and safety of gemcitabine and cisplatin in patients with unresectable cholangiocarcinoma and gallbladder cancer in daily practice that meet the criteria for the ABC-02 trial in comparison to patients who did not. METHODS: Patients diagnosed with unresectable BTC between 2010 and 2015 with an indication for gemcitabine and cisplatin were included. We divided these patients into three groups: (I) patients who received chemotherapy and met the criteria of the ABC-02 trial, (II) patients who received chemotherapy and did not meet these criteria and (III) patients who had an indication for chemotherapy, but received best supportive care without chemotherapy. Primary outcome was overall survival (OS) and secondary outcome was progression-free survival (PFS). RESULTS: We collected data of 208 patients, of which 138 (66.3%) patients received first line chemotherapy with gemcitabine and cisplatin. Median OS of 69 patients in group I, 63 patients in group II and 65 patients in group III was 9.6 months (95%CI = 6.7-12.5), 9.5 months (95%CI = 7.7-11.3) and 7.6 months (95%CI = 5.0-10.2), respectively. Median PFS was 6.0 months (95%CI = 4.4-7.6) in group I and 5.1 months (95%CI = 3.7-6.5) in group II. Toxicity and number of dose reductions (p = .974) were comparable between the two chemotherapy groups. CONCLUSION: First-line gemcitabine and cisplatin is an effective and safe treatment for patients with unresectable BTC who do not meet the eligibility criteria for the ABC-02 trial. Median OS, PFS and treatment side effects were comparable between the patients who received chemotherapy (group I vs. group II).

Systematic review of transarterial embolization for hepatocellular adenomas.

BACKGROUND: Hepatocellular adenoma (HCA) larger than 5 cm in diameter is considered an indication for elective surgery, because of the risk of haemorrhage and malignant transformation. Transarterial embolization (TAE) is used to manage bleeding HCA and occasionally to reduce tumour size. TAE might have potential as an elective therapy, but its current role in this context is uncertain. This systematic review provides an overview of clinical outcomes after TAE, in bleeding and non-bleeding HCA. METHODS: Two independent reviewers performed a systematic search of literature in PubMed and Embase. Outcomes were change in tumour size, avoidance of surgery, complications and malignant transformation after TAE in bleeding and non-bleeding HCA. The Critical Appraisal Skills Programme tool for cohort studies was used for quality assessment of included studies. RESULTS: From 320 potential articles, 20 cohort studies and 20 case reports including 851 patients met the inclusion criteria. TAE was performed in 151 of 851 patients (17·7 per cent), involving 196 tumours, of which 95 (48·5 per cent) were non-bleeding. Surgical treatment was avoided in 68 of 151 patients (45·0 per cent). Elective TAE was performed in 49 patients involving 66 HCAs, with 41 of these patients (84 per cent) not requiring surgery. Major complications occurred in eight of 151 patients (5·3 per cent); no death was reported. Among cohort studies, complete tumour disappearance was observed in 10 per cent of patients, and regression in 75 per cent. CONCLUSION: Acute or elective TAE in the management of HCA is safe. In the elective setting, TAE provides a potential alternative to surgery.

In vitro detection of cholangiocarcinoma cells using a fluorescent protein-expressing oncolytic herpes virus.

Pathological confirmation is desired prior to high-risk surgery for suspected perihilar cholangiocarcinoma (PHC), but preoperative tissue diagnosis is limited by poor sensitivity of available techniques. This study aimed to validate whether a tumor-specific enhanced green fluorescent protein (eGFP)-expressing oncolytic virus could be used for cholangiocarcinoma (CC) cell detection. Extrahepatic CC cell lines SK-ChA-1, EGI-1, TFK-1 and control cells (primary human liver cells) were exposed to the oncolytic herpes simplex type 1 virus NV1066 for up to 24 h in adherent culture. The technique was validated for cells in suspension and cultured cells that had been exposed to crude patient bile. Optimal incubation time of the CC cells with NV1066 at a multiplicity of infection of 0.1 was determined at 6-8 h, yielding 15% eGFP-expressing cells, as measured by flow cytometry. Cells were able to survive 2-h crude bile exposure and remained capable of producing eGFP following NV1066 infection. Detection of malignant cells was possible at the highest dilution tested (10 CC cells among 2 × 105 control cells), though hampered by non-target cell autofluorescence. The technique was not applicable to cells in suspension due to insufficient eGFP production. Accordingly, as yet the technique is not suitable for standardized clinical diagnostics in PHC.

Skeletal Muscle Quality is Associated with Worse Survival After Pancreatoduodenectomy for Periampullary, Nonpancreatic Cancer.

BACKGROUND: Body composition measures may predict outcomes of cancer surgery. Whereas low muscle mass shown on preoperative computed tomography (CT) scans has been associated with worse outcomes after surgery for pancreatic cancer, less consideration has been given to low muscle attenuation, reflecting poor muscle quality. Studies relating muscle mass and muscle attenuation with outcomes for patients with periampullary, nonpancreatic cancer are lacking. METHODS: Skeletal muscle mass and attenuation were assessed in 166 consecutive patients undergoing pancreatoduodenectomy (PD) for periampullary, nonpancreatic cancer at a single center between 2000 and 2012. The skeletal muscle index (SMI) was calculated from cross-sectional muscle area on preoperative CT imaging at the third lumbar vertebra level (L3) and normalized for height. The skeletal muscle attenuation index (MAI) was calculated by measuring the average Hounsfield units of the total muscle area at the L3 level. Overall survival (OS) and the rate of major postoperative complications (Clavien-Dindo ≥3) were extracted from prospectively maintained databases. RESULTS: Low SMI was present in 78.3 % and low MAI in 48.8 % of the patients. The multivariate analysis showed lymph node metastasis [hazard ratio (HR) 1.8; 95 % confidence interval (CI) 1.1-2.9], microscopic radicality (HR 2.0; 95 % CI 1.2-3.4), and low MAI (HR 2.0; 95 % CI 1.2-3.3), but not low SMI to be significantly associated with decreased OS. Low MAI (HR 1.9; 95 % CI 1.0-3.8) was the only independent risk factor for major postoperative complications. CONCLUSION: Skeletal muscle quality, but not muscle mass, predicted survival and major complications after PD for periampullary, nonpancreatic cancer. Preoperative CT-derived body composition measures may stratify patients into risk categories and support shared decision making.

The introduction of a laparoscopic liver surgery programme: a cost analysis of initial experience in a university hospital.

BACKGROUND AND AIMS: In the era of expanding costs of healthcare, this study was conducted to perform a cost analysis of introducing a laparoscopic liver surgery programme for left sided liver lesions. MATERIALS AND METHODS: Consecutive patients treated by laparoscopic liver resections of left lateral segments were included. Controls were a group of 14 patients undergoing open resection for similar pathology. Primary outcomes were costs. Secondary outcomes were complications, conversions, blood loss, length of operation, and length of hospital stay. RESULTS: The laparoscopic approach for hepatic left lateral resection (bisegmentectomy 2 and 3) was performed in fourteen patients (group I, median age 54 [range 26-82] years). In the open group, fourteen patients from a prospectively collected database with the same type of resection were selected (group II, median age 64 [range 29-76] years). Costs of theatre usage in the laparoscopic group were significantly lower (p=0.031). No significant differences in costs of disposable instruments, ward stay and total costs were observed between the two groups. There were three complications in the laparoscopic group compared with two complications in the open group. In the laparoscopic group there were 2 conversions (14%). Median blood loss was significantly lower in the laparoscopic group (50 mls [range 0-750], (p=0.001) versus the open group (500 mls [range 150-750]). Furthermore, operation time was also significantly lower in the laparoscopic group (116 [range 85-261] minutes) versus the open group (165 [range 96-217] minutes, p=0.016). Median length of stay was 6 [range 4-11] days in group I versus 6 [range 5-13] days in group II (p=0.508). CONCLUSION: Costs of laparoscopic liver resections proved to be equivalent to open surgery. Furthermore, implementation of a laparoscopic liver resection programme seems feasible and safe with reduced blood loss and operation time and comparable morbidity and length of stay.

Preliminary studies on the prevalence of Mycoplasma bovis mastitis in dairy in cattle in Australia.

A highly sensitive and specific PCR (MB-PCR) was used in preliminary studies to detect M. bovis in milk samples to investigate its association with high somatic cell count (SCC), an indicator of subclinical mastitis and one of the factors in down grading the quality of milk. A total of 186 and 167 herds were tested with 43% and 62% of herds positive for M. bovis in Victoria and North Queensland, respectively. The quarter milks from 52 cows with persistently high SCC were tested by MB-PCR and culture to investigate the association of M. bovis with major mastitis pathogens (MMP). M. Bovis was detected in 77% of cows of which 19% alone had M. bovis without any other bacteria, 17% had M. bovis in combination with major mastitis pathogens and 40% had M. bovis in combination with non-major mastitis pathogens. We believe that M. bovis is widespread in dairy cattle and has the potential to produce disease alone or to predispose the udder to disease caused by major mastitis and environmental pathogens. These studies have revealed a hitherto unrecognised high prevalence of M. bovis in dairy cattle in North Queensland and Victoria in Australia. These initial studies also give a clear association between M. bovis and elevated somatic cell counts.

Geographic distribution and evolution of Sindbis virus in Australia.

The molecular epidemiology and evolution of Sindbis (SIN) virus in Australia was examined. Several SIN virus strains isolated from other countries were also included in the analysis. Two regions of the virus genome were sequenced including a 418 bp region of the E2 gene and a 484 bp region containing part of the junction region and the 5' end of the C gene. Analysis of the nucleotide and deduced amino acid sequence data from 40 SIN virus isolates clearly separated the Paleoarctic/Ethiopian and Oriental/Australian genetic types of SIN virus. Examination of the Australian strains showed a temporal rather than geographic relationship. This is consistent with the virus having migratory birds as the major vertebrate host, as it allows for movement of virus over vast areas of the continent over a relatively short period of time. The results suggest that the virus is being periodically redistributed over the continent from an enzootic focus of evolving SIN virus. However, SIN virus strains isolated from mosquitoes collected in the south-west of Australia appear to represent a new SIN virus lineage, which is distinct from the Paleoarctic/Ethiopian and Oriental/Australian lineages. Given the widespread geographic dispersal of the Paleoarctic/Ethiopian and Oriental/Australian lineages, it is surprising that the South-west genetic type is so restricted in its area of circulation. Nucleotide sequence data from the C gene of the prototype strain of the alphavirus Whataroa were also determined. This virus was found to be genetically distinct from the SIN virus isolates included in the present study; however, it is clearly SIN-like and appears to have evolved from a SIN-like ancestral virus.

Development of a specific DNA probe and PCR for the detection of Mycoplasma bovis.

Mycoplasma bovis is responsible for several production diseases in cattle, including mastitis, arthritis, pneumonia, abortion and infertility. Current methodologies for detecting and identifying M. bovis are time consuming and difficult. Tests which rely on antigen or antibody detection have poor sensitivity and specificity. In this paper associated protocols for the development of a hybridization probe and PCR are described. A genomic library (SauIIIA digested) was prepared from M. bovis DNA (Colindale Reference Strain: NC10131:02) and cloned into pUC19. Colony hybridization, using a probe preparation made from purified M. bovis DNA, was used to identify colonies of interest. M. bovis DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridization analysis with immobilized DNA from M. bovis (two strains), M. dispar, M. agalactiae, M. bovigenitalium (two strains), M. ovipneumoniae, a Group 7 strain, M. arginini and bacteria belonging to different genera. Four probes were found to hybridize only with M. bovis and M. ovipneumoniae DNA, whereas one probe reacted with genomic DNA from only one of the two M. bovis strains. The level of sensitivity of the dot blot hybridization assay was 200 CFU (colony forming units)/mL. To enhance the sensitivity further, an M. bovis-specific PCR assay was developed. The primers were designed using sequences obtained from the probe DNA which discriminated M. bovis from all other Mycoplasma DNA tested. The minimum amount of target DNA that could be detected by the PCR assay was that isolated from 10-20 CFU/mL. The PCR assay was therefore 10 times more sensitive than dot blot hybridization.

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.

Geographic distribution and evolution of Ross River virus in Australia and the Pacific Islands.

We examined the molecular epidemiology and evolution of Ross River (RR) virus in Australia and the Pacific Islands. Nucleotide sequences of the E2 and E3 genes of five RR virus strains revealed remarkable conservation between 1959 and 1989 with a maximum divergence of only 3.3%. Sequence data from a 505-base pair fragment of the E2 gene from 51 additional strains showed that RR virus has diverged genetically into three separate groups although at least 95% sequence homology was still maintained between all 56 strains. Each genetic type predominates in a particular geographic region of Australia and can be broadly defined as occurring in the western, northeastern, and southeastern regions of Australia. However, some RR virus strains did not follow this pattern of geographic distribution indicating movement of virus by the travel of viremic humans or livestock across the continent. The Pacific Islands isolates all belong to the southeastern genotype. These findings suggest genetic divergence and independent evolution of RR virus within geographically isolated enzootic foci; however, selective pressures maintain high nucleotide conservation in nature.

Nucleotide sequence analysis of Jembrana disease virus: a bovine lentivirus associated with an acute disease syndrome.

The complete nucleotide sequence of the RNA genome of Jembrana disease virus (JDV), a lentivirus that causes an acute disease syndrome in Bali cattle (Bos javanicus), is reported. In addition to the gag, pol and env genes and flanking long terminal repeats (LTRs) that characterize all retroviruses, a number of accessory genes represented by small multiply spliced ORFs in the central and 3'-terminal regions of the genome, including tat and rev that are typical of lentiviruses, were identified. The genome of JDV was 7732 bp in length, 750 bp smaller than the genome of bovine immunodeficiency virus (BIV) strain BIV127. A striking feature of the genome was the many deletions relative to BIV127, the largest of which were 471 bp from the env gene and 157 bp from the U3 (promoter) region in the LTR. There were also several insertions of up to 33 bp in the JDV genome relative to BIV127 found in the env gene and small ORFs that overlap env. Other significant genomic differences between JDV and BIV127 included changes to cis-acting sequences throughout the genome such as promoter and enhancer sequences in the LTR, the trans-activation response region, splice sites and frameshift sequences; alterations to the gag precursor protein cleavage sites and thus the processed products; loss of the vpw and vpy ORFs; and amino acid changes in all coding regions. The significance of these changes is discussed in relation to the differences in pathogenicity between JDV and BIV.

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.

Glycosylation and antigenic variation among Kunjin virus isolates.

Previous studies have found Kunjin (KUN) virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation.

Genomic sequence analysis identifies Jembrana disease virus as a new bovine lentivirus.

Jembrana disease virus, the cause of an acute, severe disease in Bali (Bos javanicus) cattle in Indonesia was recently identified as a retrovirus, and possibly a lentivirus. We have produced sequence data representing 598 bp of the pol gene, amplified by PCR from viral cDNA using broadly reactive universal primers for retroviruses and more specific genus-reactive primers for lentiviruses. When the sequence data were compared with that of known lentiviruses and other bovine retroviruses, the closest alignment was with bovine immunodeficiency-like lentivirus (BIV), showing 74% nucleotide sequence identity. This confirmed that JDV is a lentivirus and that it is distinguishable from BIV. The pathogenesis of Jembrana disease is most unusual for a lentivirus infection and differs markedly from that reported for BIV infection.

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.

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.

Characteristics of a retrovirus associated with Jembrana disease in Bali cattle.

A virus causing Jembrana disease in Bali cattle (Bos javanicus) was demonstrated to have characteristics of a retrovirus. Reverse transcriptase activity was detected in virus purified by sucrose gradient centrifugation. Electron microscopic examination of tissue from the affected cattle indicated that the virus matured by C-type budding through the plasma membrane and into intracytoplasmic vacuoles of cells in lymphoid tissue, with the formation of circular enveloped virus particles ranging in diameter from 96 to 124 nm with an eccentric nucleoid. Western immunoblotting using sera from recovered animals demonstrated virus proteins of M(r) 100K, 45K, 42K, 33K, 26K, 16K and 14K. The 26K protein of Jembrana disease virus cross-reacted in Western blots with the 26K capsid protein of bovine immunodeficiency virus (BIV). The apparent morphogenesis, protein structure and antigenic relationship with BIV suggested the virus was a lentivirus.

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.

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.