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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Imported Fire Ant and Household Insects Research » Research » Publications at this Location » Publication #214260
Title: Detection and quantitation of Solenopsis invicta virus-2 genomic and intermediary replicating viral RNA in fire ant workers and larvae

Author
item Hashimoto, Yoshifumi
item Valles, Steven

Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/6/2008
Publication Date: 2/13/2008
Citation: Hashimoto, Y., Valles, S.M. 2008. Detection and quantitation of Solenopsis invicta virus-2 genomic and intermediary replicating viral RNA in fire ant workers and larvae. Journal of Invertebrate Pathology, 98:243-245.

Interpretive Summary: The red imported fire ant was introduced into the United States in the 1930s and currently infests about 300 million acres. It causes approximately $6 billion in damage annually in livestock and agricultural production and poses a serious threat to human health. USDA-ARS scientists at the Center for Medical, Agricultural and Veterinary Entomology (Gainesville, FL) have recently discovered a new RNA virus in the fire ant. Studies suggest that the virus (SINV-2) may be an effective microbial control agent for fire ants. In order to develop the virus as a control agent for fire ants, a more thorough understanding of the virus is necessary. To accomplish this goal, scientists must be able to quantify the level of infection and track its progression. Here the development of a real-time PCR method capable of quantifying the number of genome copies of the virus in individual fire ants is reported. This method will assist in epidemiological studies and aid in the development of the virus as a fire ant control agent.

Technical Abstract: A quantitative real-time PCR (QPCR) method was developed to detect and quantify Solenopsis invicta virus-2 (SINV-2) infecting individual ants of Solenopsis invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting an upstream region of the SINV-2 conserved domain of RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. SINV-2 RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV-2 RdRp plasmid constructs. A strong linear relationship [r2 = 0.999; y = (-3.38 ± 0.11) x + (39.27 ± 0.73)] between CT and SINV-2 RdRp copy number was observed within a dynamic range of 5 to 5 x 107 copies. The QPCR reaction was optimized for oligonucleotide primer and magnesium concentrations to achieve the highest sensitivity and reaction efficiency. Strand-specific cDNA synthesis oligonucleotide primers and RNase digestion after cDNA synthesis allowed quantification of positive (genomic) and negative (replicative) strands of the SINV-2 genome. Both strands were detected in adult workers and larval fire ants indicating that the virus was replicating within the ant. The ratio of plus to minus genome strand ranged from 199-fold in larvae to 479-fold in workers with an average ratio of 339-fold.