1. 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. 2009 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 40033.532.12 AM 40124.520.94 AM 40240.000.61 AM 40332.302.65 AM 40426.000.12 AM 40529.191.73 RS c 40036.191.52 RS 40134.461.12 RS 40230.250.56 RS 40334.543.26 RS 40440.000.10 RS 40532.840.79 RS 40630.810.26 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.