Genomic sequencing, discovery, and characterization of viral pathogens in Glassy-winged Sharpshooters (Homalodisca vitripennis: Hemiptera: Cicadellidae)

Authors: Hunter, Wayne

Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 10/23/2007
Publication Date: 12/10/2007
Citation: Hunter, W.B. 2007. Genomic sequencing, discovery, and characterization of viral pathogens in Glassy-winged Sharpshooters (Homalodisca vitripennis: Hemiptera: Cicadellidae). In: Proceedings of the 2007 Pierce's Disease Research Symposium, October 12-14, 2007, San Diego, California. p. 17-21.

Interpretive Summary: A new viral pathogen of the glassy-winged sharpshooter, GWSS, (Homalodisca vitripennis, aka H. coagulata) was discovered and characterized. Few pathogens of leafhoppers have been discovered which have potential for use as a biological control agent. To identify new pathogens of GWSS we used a genomic approach to isolate, sequence, and identify expressed sequence tags, ESTs, produced from field collected GWSS populations. Viral sequences were identified out of the initial 9,620 ESTs generated from single-pass 5’ end sequencing of the GWSS expression library. Using these sequences as a starting point, it was possible to completely sequence the full virus genome, and the virus was named HoCV-1. Further analyses and characterization of HoCV-1 demonstrated that it infected and crossed the midgut barrier of GWSS. The virus was classified as a member of the family Dicistroviridae, which are single-stranded RNA viruses which do not have a DNA stage. Two other viruses were also identified which are currently being characterized and which were taxonomically unrelated to HoCV-1. These leafhopper viral pathogens appear to induce increased mortality, 40% or more, during the nymphal stages of leafhopper development and may have further applications as biological control agents in the management of leafhopper pests in the reduction of the spread of Pierce’s disease of grapes.

Technical Abstract: A new viral pathogen, HoCV-1, discovered in the glassy-winged sharpshooter, GWSS, (Homalodisca vitripennis, aka H. coagulata) was examined for the mode of entry into the leafhopper. Few viral pathogens of leafhoppers have been discovered which have potential for use as a biological control agent. To identify new pathogens of GWSS we used a genomic approach to isolate, sequence, and identify expressed sequence tags, ESTs, produced from field collected GWSS populations. Viral sequences were identified out of the initial 9,620 ESTs generated from single-pass 5’ end sequencing of the GWSS expression library. Processing produced 8,795 ESTs which had lengths greater than 100 nucleotides post quality and vector trimming. The ESTs had an average read length of 689 bp, and an average inset size of 899 bp. Sequences shorter than 200 contiguous bases were removed from analyses. After assembly there were 3,008 sequences, 799 contigs with an average length of 1,113 bp, and 2,209 singlets. Using these sequences to identify a starting point, it was possible to completely sequence the full virus genome, and the virus was labeled, HoCV-1. Further in silico analysis revealed a genome containing 9,321-polyadenylated nucleotides encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5'-proximal ORF (ORF1, nt 420–5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other Dicistroviruses. These viruses are often reported to be in association with increased mortality of their infected host, as with fire ants, and honey bees. Virus analysis and detection from salivary gland (Sg) and midgut (Mg) tissues of GWSS adults were tested for HoCV-1, using rtPCR. Both types of tissues from individual insects were dissected and analyzed in a pairwise fashion. Only midgut tissues were shown to test positively for virus presence. Tissues were then prepared for examination by transmission electron microscopy. Virions were observed to be present in high numbers within midgut tissues of GWSS which tested positive for HoCV-1, and absent when GWSS tested negatively. The virions appeared to be taken up by the microvilli of the midgut and to propagate within the basal laminae. Examinations using TEM demonstrated that it infected and crossed the midgut barrier of GWSS. The virus was classified as a member of the family Dicistroviridae, which are positive-sense, ssRNA viruses which do not have a DNA stage. Two other viruses were also identified which are currently being characterized and which were taxonomically unrelated to HoCV-1. These leafhopper viral pathogens appear to induce increased mortality, 40% or more, during the nymphal stages of leafhopper development and may have further applications in the management of leafhopper pests to reduce the spread of Pierce’s disease of grapes.