The Huck Institutes of the Life Sciences

Virus particles in insect gut

An example of using TEM to find barley yellow dwarf virus particles in aphid hindguts
Barley yellow dwarf virus particles in the lumen of the aphid hindgut (blue arrows) and in the basal lamina (red arrow). The particle in the basal lamina is labelled with ferretin. Bar = 200 nm. Photograph provided by Fred Gildow (Plant Pathology Dept.)

Barley yellow dwarf virus particles in the lumen of the aphid hindgut (blue arrows) and in the basal lamina (red arrow). The particle in the basal lamina is labelled with ferretin. Bar = 200 nm. Photograph provided by Fred Gildow (Plant Pathology Dept.)

Barley yellow dwarf virus is a single-stranded RNA virus that affects a variety of cereal crops, including barley, wheat and oats. It is transmitted by aphids.

Here, we used TEM to show barley yellow dwarf virus particles in the lumen of the aphid hindgut (blue arrows) and in the basal lamina.

Protocol

  1. Feed aphids on barley plants infected with Barley Yellow Dwarf Virus (BYDV) and allow to acquire virus
  2. Microinject aphids with rabbit anti-BYDV followed by goat anti-rabbit conjugated to ferretin
  3. Fix aphids overnight in 2% glutaraldehyde, 1% formaldehyde in 0.05 M cacodylate buffer, pH 7.2
  4. Wash 3 times in 0.1 M cacodylate buffer
  5. Incubate 1hr at rm temp in 2% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2
  6. Wash 1 time in 0.1 M cacodylate buffer, 2 times in water
  7. En bloc stain 1 hr in 2% aqueous uranyl acetate
  8. Dehydrate in acetone, then infiltate with Epon araldite resin
  9. Polymerize 24 hrs at 60C
  10. Ultrathin sections are cut and placed on formvar and carbon coated copper grids
  11. Contrast with uranyl acetate and lead citrate