The Investigation To Sequential Pathologic Changes Of Silkworm Larvae Infected By Bombyx Mori Bidensovirus

Bombyx mori Bidensovirus(BmBDV), is the type species of genus Bidensovirus, family Bidnaviridae and also the unique animal bipartite virus until now, which contains two segmented linear single-stranded DNA genome VD1(6.5 kb) and VD2(6.0 kb). BmBDV particles are icosahedral, no-enveloped virions with 20-24 nm in diameter. BmBDV is a pathogen of fatal densovirus disease in bombyx mori L( Lepidoptera, Insecta), resulting in a loss of sericulture. BmBDV multiplies only in midgut epithelium of silkworm larvae resulting hypertrophic nuclei of columnar and goblet cells. Symptoms of virus-infected larvae include flaccid body texture, lack of appetite, retarded growth and diarrhea. Although several pathobiological studies had been performed, these studies are fragmented and incomplete and sequential changes in BmBDV-infected silkworm larvae have not been fully examined yet.In this paper, detailed researches to sequential pathological features of BmBDV-infected larvae were performed by biostatistics, pathological section and RT-qPCR methods on, individual, tissue and molecular levels.On the individual level, The infection of BmBDV results in growth delay, prolonging of larvae instar and developmental dysplasia in silkworm larvae, and eventually led to the deaths of silkworm. The LT50 of BmBDV to larvae was about 21 days. Virus-infected silkworms excredet the bead-like dung and the color of its anterior and posterior midgut changed from green to pale yellow and its fold disappeared. The sensitivities of various stages larvae to BmBDV had no difference. The growth of 4th infected silkworm slowed down after 3 days post infection(dpi) and stopped after 10 dpi. Although BmBDV-infected 5th instar larvae could develop to pupa stage, the output of cocoon was reduced by 4.9%, the weight of pupa decreased 17.9% and pupal survival rate was reduced by 5%. We further verified the fact that BmBDV cannot vertical transmission to their offspring.On the tissue level, Nuclei of some columnar cells started to hypertrophy at 2 dpi. After molting(5 dpi), the hypertrophic phenomenon disappeared, however almost all of nuclei of the columnar epithelium cells hypertrophy at 8 dpi. A large number of hypertrophic columnar cells were peeled and were released into the midgut lumen at 4 and 9 dpi. Columnar cells disappeared and nuclei of goblet cell began to hypertrophy at 10 dpi. The most serious lesions in goblet cells occurred in 13 dpi and the secretory vesicle of goblet cell degenerated and the whole cell was occupied by hypertrophic nuclear, which showed that goblet cells also were infected by BmBDV at the late stage of infection. BmBDV multiplication led to nuclear hypertrophy, which had been confirmed by immunohistochemistry method.On the molecular level, BmBDV infection cycle was about 60 h. Transcriptional analysis showed that transcription ofviral genes began at 48 hours post-infection(hpi) and reached peak at 7 to 9 dpi. Afterwards, the virus transcription level suddenly reduced to the lowest level at 10 to 11 dpi. However there was a brief rise from 11 to 14 dpi, which indicated that BmBDV replicated in goblet cells at this stage.There are close relationship among the pathological changes of BmBDV-infected larvae at individual, tissue and molecular levels showed the close relation. The transcription of the BmBDV, caused the lesions of midgut tissue, which affected the growth and development of silkworm. BmBDV transcription began at 2 dpi, nuclei of midgut columnar cells started to hypertrophy at 2 dpi and the growth of silkworm slowed down after 3 dpi. After molting, pathological cells disappeared and the viral genes transcriptions were also reduced. Amost all of the columnar cells were peeled at 10 dpi, the virus transcription level suddenly reduced to the lowest level at 10 dpi and the growth of silkworm stopped after 10 dpi. Viral genes transcription rose in the goblet cells resulted in hypertrophic nuclei of goblet cells from 11 to 14 dpi. These results showed that the molecular pathological changes were the root cause of tissue pathological changes and the individual lesions and the time of molecular pathological changes was sooner than the tissue and individual pathological changes.Moreover, we also established a rapid molecular diagnostic system for detecting BmBDV. Silkworm excrement was used as the testing material to avoid the complicated process of extracting. The infection of BmBDV in silkwom can rapidly, simply and accurately be determined by detecting the virus in silkworm feaces.We adequately elaborated sequential pathologic changes of silkworm larvae infected by BmBDV. Our studies can provide a theoretical basis and technical support for BmBDV diagnosis and control in the sericultural production and lay a foundation for studying viral infection mechanism and the interaction between virus and host.