Susceptibility to Ranavirus Through Frogs and Salamanders Using q-PCR For Detection and Quantification Thomas Brigman Department of Biology, York College of Pennsylvania Introduction  Amphibian populations is declining globally and the die-offs seem to be linked to infectious diseases (Gary et al. 2009, Blaustein and Kiesecker 2002). Ranaviruses is the highest reported reason for mortality in amphibians (Green et al. 2002).  It has been reported that 43% of the reported die-offs of amphibians from 2000 to 2005 are from the Ranaviruses (Gary et al. 2009).  Ranavirus is in the family Iridoviridae, which are large, double-stranded DNA viruses, with a noticeable icosahedral shape that is mostly noticeable in the cytoplasm of infected cells (Chinchar 2002 and Green et al. 2002).  The major capsid protein (MCP) is highly conserved in the Ranavirus, and is commonly sequenced at 500bp rejoin at the 5’ to identify Ranavirus (Gary et al and Chinchar 2002).  Because of the potentially devastating effect of Ranavirus infections among susceptible amphibians species, a lot of effort has been put into early and rapid detection of the virus (Chinchar 2002).  A strain of Ranavirus, FV3, which is known to effect common frogs, toads and salamanders, has been sequenced. FV3 replication is rapid and can be detected within 2 hours post infection (Chinchar 2002 ).  Since Ranavirus is known to effect frogs and salamanders, determining which species is more susceptible to infection could play an important role in early detection of infection within a environment. Objectives  To prepare a new rapid technique to detect for Ranavirus within frogs and salamanders.  To determine if salamanders or frogs, within a vernal pool located in York, Pennsylvania, is more susceptible to Ranavirus. Figure 1. A 1% agarose gel. lane one is 100 bp ladder. Lane 2 is water and primers PCR. Lane 3 is plasmid and primers PCR. (576bp ) Conclusions  Ranavirus can be detected using PCR and q-PCR protocol that was developed and can help in rapid detection.  It could not be proven that Ranavirus was more susceptible in frogs or salamanders. (Fisher's exact test p=0.0909). This could be due to a low sample size. Acknowledgements I would like to give a special thanks to my mentors Dr. Meda Higa and Dr. Bridgette Hagerty for the guidance. Also I would like to thank Victor Chinchar for the supply of the MCP plasmid and Carrie Reall for the DNA samples. Methods Extract MCP plasmid Collect frogs(n=6) and salamanders(n=6) DNA Run PCR with MCP plasmid Develop Standard Curve Test with positive sample Test with DNA samples q-PCR with MCP plasmid and DNA Figure 2. C T value of MCP plasmid dilutions(n=5) at known quantity (ug). Line represents liner regression.. Standard Curve of MCP Plasmid Results Effective MCP Primers DNA SampleC T MeanC T Std a AM b AM AM AM AM AM RS c RS RS RS RS RS RS Table 1. C T Values of DNA Samples a is Standard Deviation b is Abystoma maculatum (Spotted Salamander) c is Rana sylvatica ( Wood Frog) Figure 3. Amount of amplification of MCP primers of known infected frog DNA sample( 3 replicates), on Log scale over a period of cycles Amplification Amount of Positive Frog Sample Amplification Log Scale Cycles Quantity(ug ) C T Value Future Studies  Consider increasing sample size over a long period of time instead of a short period of time.  Toads should also be studied with regards to susceptibility to Ranavirus.  Coming up with a proper way of testing the water for Ranavirus could also be beneficial for early detection.