Development of molecular methods for epidemiology of insect viruses

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We developed a diagnosis system for virus diseases in silkworms and wild insects based on comparative studies on DNA sequences in the virus and host insects. A forecast method of virus disease expansion in insect populations was established by using molecular epidemiological techniques.1.The polyhedrin genes and DNA helicase genes were cloned and sequences from Samia cynthia ricini nuclear polyhedrosis virus (SrNPV) and Antheraea yamamainuclear polyhedrosis virus (AyNPV).2.An immunological sensor was developed using an monoclonal antibody against Bombyx moricytoplasmic polyhedrosis virus (BmCPV) and a quartz oscilator to detect BmCPV rapidly and easily.3.Quantitative changes of two genomic DNAs in the Bombyx moridensonuclear virus type II (DNV2) were investigated in the midgut cells infected with DNV2, using the competitive PCR method.4.We developed a very simple method to detect all of known viruses in Bombyx morisimultaneously in one reaction. The condition of PCR and primer designs were optimized.5.We set a model rearing shelf containing healthy silkworm larvae and a few nuclear polyhedrosis ones. Expansion of the virus was monitored in the larvae, feces, diet, etc., on the shelf, using the PCR diagnosis system.6.Random amplified polymorphic DNAs (RAPDs) were found to be closely linked to the nonsusceptibility gene to DNV2 (nsd-2). A linkage map was constructed based on recombination values among the RAPDs and nsd-2.