NSW Arbovirus Surveillance & Vector Monitoring Program
Laboratory Methods

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Mosquito Collections and Identification

Mosquito trapSorting mosquitoes on a cold tableMosquitoes are collected overnight in dry-ice baited mosquito traps. They are then sent live in cool humid eskies via overnight couriers to the Department of Medical Entomology at Westmead Hospital for identification and processing for arbovirus isolation. The mosquitoes are identified according to keys and illustrations in "Mosquitoes and Mosquito-borne Disease in Southeastern Australia" by Russell (1993), "A Colour Photo Atlas of Mosquitoes of Southeastern Australia" by Russell (1996), "The Mosquitoes of Victoria" by Dobrotworsky (1965) and the twelve volume series "The Culicidae of the Australasian Region" by Lee et al. (1980 - 1989) and Debenham et al. (1989).


Arbovirus Isolation Methodology

Mosquitoes are sorted into pools of  up to 25 mosquitoes, according to locality, site, date of capture, species, sex and (for females) whether blood-engorged or not. Each Grinding tubespool of mosquitoes is placed into separate 5cm plastic sample tubes, which contains 5 sterile 5mm glass beads and 2ml of media. Each sample tube with the mosquitoes is then placed for 20MOSAVEX minutes into MOSAVEX (MOSquito ArboVirus EXtractor), a mechanical device developed at Westmead Hospital to grind mosquitoes. Once the mosquitoes are ground, the tubes are centrifuged for 20 minutes at 4,000 rpm and 40C. Aliquots (50ul) of supernatant are taken from each of the centrifuged mosquito pools and inoculated onto C6/36 mosquito cells in two adjacent wells, in one 96-well tissue culture plate.

A 96 well plateThe cells are allowed to grow for 3-4 days and then 50ml of the cells' supernatant fluid is transferred to fresh Baby Hamster Kidney (BHK) cells and a further line of C6/36 cells, to increase the chances of isolating arboviruses. The plate containing the C6/36 cells that were initially inoculated with the mosquito supernatant is "fixed" with acetone. After a further 3-7 days, the BHK cells are examined for any cell damage, i.e. cytopathic effects (CPE), caused by virus. Cell Innoculation ProcedureFifty microlitres of the C6/36 cells' supernatant fluid from the second passage is transferred to porcine stable - equine kidney (PSEK) cells and a further line of BHK cells and the C6/36 plate 'fixed' with acetone. The PSEK and BHK cells are examined for CPE 3-7 days later. Any wells found to show evidence of CPE indicating possible virus growth are then repassaged onto fresh BHK or PSEK cells (depending in which cell line the CPE occurred), and grown for a further 3-7 days. Specimens from the final cell culture wells thought to have virus are propagated and inoculated onto 96-well tissue culture plates seeded with C6/36 cells for identification. They are also divided into aliquots and stored at -80oC as reference specimens within the Department of Medical Entomology, Westmead Hospital.

Arbovirus Identification Methodology

The fixed C6/36 plates are screened for the presence of flaviviruses (Murray Valley encephalitis, Kunjin, Edge Hill, Stratford, Alfuy and Kokobera viruses) using a broadly reacting flavivirus monoclonal antibody (4G2) via a fixed cell Enzyme-Linked Immunosorbent Assay (ELISA). One aliquot of the presumptive viral isolates is tested by ELISA using specific monoclonal antibodies for the presence of alphaviruses (Barmah Forest, Ross River and Sindbis viruses), as well as screened for flaviviruses using 4G2. Any specimens from the initial screen and any presumptive isolates positive for 4G2 are identified by ELISA using specific flavivirus monoclonal antibodies.

Sentinel Chicken Surveillance

The Chicken Sentinel component of the NSW Arbovirus Program involves testing flocks in selected areas in southwestern NSW for flavivirus antibody over a six-month period (November-April). Flocks of 15 birds each are located at Bourke, Condobolin, Deniliquin, Forbes, Griffith, Lake Cargelligo, Leeton, Menindee, Warren and Wentworth. The flocks are bled prior to deployment to the test sites and then at weekly intervals until the end of the season.

All samples are submitted to the Arbovirus & Emerging Diseases Unit at ICPMR for testing by a competitive flavivirus group specific ELISA. This test was developed in-house in 1992 and has been shown to detect antibody to Murray Valley encephalitis, Kunjin, Kokobera, Stratford, Alfuy, Edge Hill, Dengue serotypes 1-4 and Japanese encephalitis virus. Any positive samples detected by this method are confirmed by neutralisation.


References

Lee D.J., Hicks M.M., Griffiths M., Russell R.C. and Marks E.N. 1980 - 1984. The Culicidae. of the Australian Region. Vols. 1 - 3. Australian Government Publishing Service, Canberra.

Lee D.J., Hicks M.M., Griffiths M., Debenham M.L., Bryan J.H., Russell R.C., Geary M. and Marks E.N. 1987 - 1988. The Culicidae. of the Australian Region. Vols. 4 - 6. Australian Government Publishing Service, Canberra.

Lee D.J., Hicks M.M., Debenham M.L., Griffiths M., Marks E.N., Bryan, J.H., M., and Russell R.C., 1989. The Culicidae. of the Australian Region. Vol. 7. Australian Government Publishing Service, Canberra.

Lee D.J., Hicks M.M., Debenham M.L., Griffiths M., Marks E.N., and Bryan J.H., 1988 -1989. The Culicidae. of the Australian Region. Vols. 8 - 10. Australian Government Publishing Service, Canberra.

Lee D.J., Hicks M.M., Griffiths M., Debenham M.L., Marks E.N., Bryan J.H., and Russell R.C., 1989. The Culicidae. of the Australian Region. Vol. 11. Australian Government Publishing Service, Canberra.

Russell R.C. 1991. Arbovirus surveillance, New South Wales, 1989-91. Commun. Dis. Intell. 15:229-32.

Russell R.C. 1993. Mosquitoes and mosquito-borne disease in southeastern Australia. Department of Medical Entomology, Westmead, NSW. 310pp.

Russell R.C. 1996. A Colour Photo Atlas of Mosquitoes of Southeastern Australia. Department of Medical Entomology, Westmead, NSW. 193pp.

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