Characterization of Heliothis Virescens Ascovirus 3h orf21 that Encodes a Virion Protein

Abstract

Homologues of Heliothis virescens ascovirus 3h (HvAV-3h) orf21 are found in 9 completely sequenced members of the ascoviruses, but so far their functions are unknown.Here, orf21 (3h-21) was cloned in-frame into a pET-28a bacterial expression vector. The fusion protein produced by this construct was used for the preparation of a polyclonal antiserum. RT-PCR analysis showed a single transcript of 3h-21 of approximately 0.7kb was transcribed beginning at 24h post-infection in infected Helicoverpa armigera larvae.Western blot analysis of extracts from HvAv-3h-infected Helicoverpa armigera larvae detected a 25.6 kDa protein late in infection. This antiserum also reacted with a 25.6 kDa protein in purified virions of HvAv-3h. The protein was not extensively modified post-translation. Immunoelectron microscopy confirmed that the 3H-21 is associated with the structure of HvAV-3h virions.

Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affect pathogenicity against different host larvae species

“…Proteins extracted from the purified HvAV-3h virions that were produced by different host larval species, as described above, were separated using 12% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, USA). After blocking with defatted milk powder [5% milk powder in Tris buffered saline with Tween (TBST) buffer], the prepared polyclonal antiserum against 3H-43, 3H-107, 3H-135, and 3H-171, as well as the previously prepared polyclonal antiserum against 3H-21 ( 54 ), 3H-38 ( 55 ), 3H-53 ( 56 ), 3H-117 ( 51 ), and 3H-122 ( 57 ), were used as primary antibodies (dilute ratio 1:3,000). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000) (Millipore, Billerica, MA, USA) was used as a secondary antibody.…”