Susceptibility to Ranavirus Through Frogs and Salamanders Using q-PCR For Detection and QuantificationThomas 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.

AcknowledgementsI 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 StandardCurve

Test with positive sample

Test with DNA samples

q-PCR with MCP plasmid and DNA

Figure 2. CT 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 Sample CT Mean CT Stda

AMb 400 33.53 2.12

AM 401 24.52 0.94

AM 402 40.00 0.61

AM 403 32.30 2.65

AM 404 26.00 0.12

AM 405 29.19 1.73

RSc 400 36.19 1.52

RS 401 34.46 1.12

RS 402 30.25 0.56

RS 403 34.54 3.26

RS 404 40.00 0.10

RS 405 32.84 0.79

RS 406 30.81 0.26

Table 1. CT 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

Am

plif

icat

ion

Log

Sca

le

Cycles

Quantity(ug)

CT V

alue

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.