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Characterization of cypovirus isolates from tropical and temperate Indian saturniidae silkworms
K R Qanungo1, S C Kundu, A K Ghosh
1Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India.
Abstract:
Cytoplasmic polyhedrosis virus (cypovirus, CPV) isolates from larvae of saturniidae silkworms Antheraea mylitta, Antheraea proylei, and Antheraea assamensis were obtained and characterized. Scanning electron microscopic (SEM) studies revealed the presence of hexahedral (rhombic dodecahedron) polyhedral bodies of various size (1-4 microns) in individual hosts while transmission electron microscopic (TEM) studies of isolated virions showed their uniform (icosahedral) shape and size (approximately 55 nm). Genomes of these isolates were obtained from purified polyhedral bodies and analyzed by agarose and polyacrylamide gel electrophoresis. Eleven bands ranging from about 350 bp to 3.9 kb (total of 25.35 kb) were detected. These bands were completely degraded after treatment of the genome preparations with RNase A at low salt but not at high salt concentration. The banding pattern of the genomes remained unaffected by treatment of the respective preparations with DNase I, indicating that the genomes were composed of 11 double-stranded (ds) RNA segments. The RNA banding patterns of the CPV isolates from the saturniidae silkworms were similar to each other. These characteristics were similar to that of Antheraea mylitta cypovirus 4 (AmCPV-4) but different from that of Bombyx mori cypovirus 1 (BmCPV-1). In comparison to AmCPV-4 the isolates contained an additional, eleventh RNA segment and the size of the RNA segments was also different. Dot blot hybridization showed hybridization of the cloned 9th and 10th segment cDNAs from the A. mylitta CPV (AmCPV) isolate with RNAs from the A. proylei and A. assamensis CPV (ApCPV, AaCPV) isolates but not with RNA from BmCPV-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) of polyhedral proteins from the AmCPV isolate showed the presence of 3 major and 5 minor bands of M(r) from 29.4 K to 163 K, but BmCPV-1 polyhedral bodies showed different protein banding pattern. Two heterologous cell lines infected with the AmCPV isolate showed very slow virus growth with the accumulation of polyhedral bodies in the cytoplasm of infected cells indicating a restricted host range of this CPV isolate.
Insights
This study characterized cytoplasmic polyhedrosis virus (CPV) from Saturniidae silkworms. The CPV isolates showed distinct genomic and protein profiles, differing from Bombyx mori cypovirus 1 and indicating a restricted host range.
Area of Science:
- Virology
- Insect Pathology
- Molecular Biology
Background:
- Cytoplasmic polyhedrosis viruses (CPV) are significant pathogens affecting various insect species, including commercially important silkworms.
- Understanding the genetic diversity and characteristics of CPV isolates is crucial for developing effective pest management strategies in sericulture.
Purpose of the Study:
- To isolate and characterize Cytoplasmic polyhedrosis virus (CPV) from larvae of Saturniidae silkworms: Antheraea mylitta, Antheraea proylei, and Antheraea assamensis.
- To compare the genomic and protein profiles of these CPV isolates with known CPV strains, such as Antheraea mylitta cypovirus 4 (AmCPV-4) and Bombyx mori cypovirus 1 (BmCPV-1).
- To investigate the host range of the characterized CPV isolates.
Main Methods:
- Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for morphological analysis of polyhedral bodies and virions.
- Agarose and polyacrylamide gel electrophoresis for analyzing the double-stranded RNA (dsRNA) genome segments.
- RNase A and DNase I treatment to confirm the nature of the viral genome.
- Dot blot hybridization using cloned cDNAs for genetic relatedness analysis.
- SDS-PAGE for characterizing polyhedral proteins.
- Infection of heterologous cell lines to assess virus growth and host range.
Main Results:
- SEM revealed hexahedral polyhedral bodies (1-4 microns), while TEM showed uniform icosahedral virions (approx. 55 nm).
- Genomic analysis identified 11 double-stranded RNA (dsRNA) segments (totaling 25.35 kb) in the CPV isolates.
- The RNA banding patterns were similar among the Saturniidae silkworm CPV isolates but distinct from BmCPV-1, with an additional eleventh RNA segment compared to AmCPV-4.
- Dot blot hybridization confirmed genetic relatedness between the AmCPV isolate and the A. proylei and A. assamensis CPV isolates, but not with BmCPV-1.
- SDS-PAGE revealed distinct protein profiles for the AmCPV isolate compared to BmCPV-1.
- Infection of heterologous cell lines resulted in slow virus growth and polyhedral body accumulation, suggesting a restricted host range.
Conclusions:
- The CPV isolates from Antheraea spp. silkworms represent distinct strains with unique genomic and protein characteristics.
- These isolates show closer genetic relatedness to AmCPV-4 than to BmCPV-1, highlighting variations within the genus.
- The observed restricted host range of the AmCPV isolate has implications for its epizootiology and potential use as a biopesticide.
