Studies On BmDNV-6 Of Densovirus Of BomByx Mori.

Densoviruses(DNVs) belong to the subfamily Densoviridae, family Parvovidae. Under the subfamily Densoviridae, there are three genera, namely, Densovirus, Iteravirus and Brevidensovirus. They can infect Lepidoptera, Diptera, Orthoptera and Odonate as parasites. BmDNV belongs to the Iteravirus. Since three decades ago, more than 10 strains of BmDNV have been isolated from silkworm. Owing to the complexity of BmDNV nucleic acid, Bergoin et al proposed that BmDNVs were divided into 5 categories, namely, BmDNV-1, BmDNV-2, BmDNV-3, BmDNV-4, BrnDNV-5.The new DNV isolated from Bombyx mori gene library in Southwest Agricultural University was temporarily termed as BmDNV-6. The research focused on the classification, epidemiology as well as molecular biology of BmDNV-6.The results was as follows.1 Isolation and identification of BmDNV-61.1 The characters of BmDNV-6 nucleic acidSize After isolation and purification of BmDNV-6, the resultsof virus nucleic acid gelose electrophoresis showed that the size of BmDNV-6 nucleic acid was about 6 500nts, different from that of BmDNV-1(5 000nts) and BmDNV-2(6 600nts and 6 100nts). Distinct disparities existed among the three kinds of BmDNV nucleic acid,suggesting they were three different viruses (Fig.1).types BmDNV-6 virus nucleic acid was digested by DNase and RNase respectively, electrophoresis results showed BmDNV-6 nucleic acid could be digested by DNase not by RNase indicating BmDNV-6 nucleic acid was DNA, namely, BmDNV-6 was DNA virus.Single or double strand BmDNV-6 nucleic acid prepared through low-saline solution interacted with 1.8% formaldehyde for Oh, 24h respectively,results showed the absorbency at 260nm apparently increased, indicating BmDNV-6 was single-stranded virus.1.2.Iserology relationship between BmDNV-6 and BmDNV-1, BmDNV-2BmDNV-1, BmDNV-2 and BmDNV-6 hybridized by immunity blotting method respectively with BmDNV-1, BmDNV-2 and antibodies of IFV. The results showed BmDNV-1 and BmDNV-2 hybridized with their corresponding antibodies, giving birth to hybridization signals, while no signals existed when BmDNV-6 hybridized with BmDNV-1 BmDNV-2 and the antibodies of BmlFV. That was to say, no serology relationship existed among BmDNV-6, BmDNV-1 and BmDNV-2.Because BmDNV-6 was single-stranded DNA virus with a genome of 6 500nt, it multiplied ininfected tissues nucleus of sick silkworms, leading to swelling of nucleus. According to the rules of virus classification, it should belong to DNVs. However, there was no serology relationship between BmDNV-6 and BmDNV-1, BrnDNV-II which were discovered and isolated earlier. Therefore, BmDNV-6 was a kind of new DNV.2. Histopathology researches of BmDNV-62.1 Diagnostics of silkworm infected by BmDNV-6The diagnostics were as follows.Inappetent, languishing, the body color turning yellow, becoming transparent gradually, and the trachea could be seen. Dissect the body wall, we saw the midgut wall was very thin, and few mulberry leaves fragments in enterocele. Additionally, enterocele was full of canary gut- liquid. After Bombyx mori group was infected, they developed irregularly, body sizes were ragged, the great mass of chests were empty. Evacuate bead-like feces or brown sullage, and in the end, silkworms intenerated to die.2.2 Parasite tissues and pathological changes of BmDNV-6After staining paraffin slices with HE, results of observations showed that the pathological changes of silkworms infected by BmDNV-6 first appeared in nucleus of midgut cells, which exhibited as the swell of nucleus. When biggest, their sizes could reach about 3 times of normal one. As the infection went deteriorated, the damages virus towards intestine wall became severer, large numbers of nucleus swelled, cells breached so as that the intestine wall only left a piece of fundus membrane. Meanwhile, the infections of virus towards cells have not been limited to cylindraceous cells, cyathiform cells and stem cells any longer, even the fat bodies and dermal cells also been infected. Here, the whole bodies of silkworms intenerated, quickly met with their death.Results of FISH showed the pathological courses were consistent with results of HE dyeing. At the forepart of virus infection, a small quantity of FISH signals were found in midgut nucleus; when infected badly, not only all the cells of midgut (including the column cells and cyathiform cells) had FISH signals, but also other tissue cells including fat body, muscle and dermal cells, etc had FISH signals,. Results of FISH ulteriorly substantiated that the swell of infected nucleus was caused by the saturation of virus particles in nucleus. In addition, the results showed terminal BmDNV-6 could infect other tissue cells except for the round in shape cells in midgut, including cyathiform cells, fat bodies and so on. In other words, the infection of this virus towards Bombyx mori was systemic infection.2.3 Proliferation of BmDNV-6 in infected cellsIn initial stages of infection, chromatin in nucleus distributed symmetrically. The development of nucleolus was exceptional, changed from mono-nucleolus to double nucleoli, and ulteriorly rnulti-nucleoli appeared.These nucleoli continued developed, and connected into pieces in the end, became generant matrix of virus which included immature virus particles. The virus particles packaged and matured in generant matrix of virus. Initially virus particles distribution of conglobation could be seen. However, as the mature virus particles increased, they released from matrix to nucleus, and distributed far and wide in nucleus. Virus particles were global whose diameters were 22nm. In the courses of virus development, tumefaction of mitochondria, vacuolation and decrease of microvilli could also be observed.3. Molecular biology reseaches of BmDNV-6Through digesting the full genome of BmDNV-6, reclaiming the digested segments and products of plasmid PUC119/XbaI, after combining and transforming, DNA sequences of 3 positive clones (BmDNV-6 DNA-1, BmDNV-6 DNA-2 and BmDNV-6 DNA-3)were obtained.The full length of BmDNV-6 was 1575bp, GC content in this sequence was low, being 25.5%; GC mainly distributed at start bit of the sequence and lOOObp location.; BmDNV-6 DNA-1 had one digesting site, namely, PstI)'1410; 3 ORF were found aggregately. The plus strand had one big ORF, namely ,ORF1, which encoded 222 amino acids; minus strand had two smaller ORFs, namely, ORF2 and ORF3, encoded 70 and 28 amino acids respectively; in NCBI, through homology contrast of BmDNV-6 DNA-1 by Blastn, we found it had high homology with five sequences in BmDNV-2 VD2. In the five homologous sequences, three were located in minus strand, the other two were in plus chain. Sequences located in regions 964-114lbp,1275-132lbp,1441-1534bp in BmDNV-6 DNA-1 were not found homologues in NCBI. The analysis of BmDNV-6 DNA-1 homology suggested that the fragment might formed through the fusion of partial fragments in double-stranded DNA in BmDNV-2 VD2 (Fig.2).BmDNV-6 DNA-2 was 924bp long, with a low GC content of 27.3%; GC largely distributed between 350bp and 450bp. After looking for ORFs in both plus and minus strands in BmDNV-6 DNA-2, only one ORF was found, namely ORF1, which encoded 63 amino acids. In NCBI, Blastn was used to conduct homology contrast for BmDNV-6 DNA-2 and we found it was the same as the 3095-4018bp region in plus strand in BmDNV-2 VD2, suggesting BmDNV-6 DNA-2 is a part of BmDNV-2 VD2's plus strand (Fig.2).BmDNV-6 DNA-3 was 1299bp long, with a GC content of 27.3%. GC maily distributed between 408bp and 536bp. A HindUl incision site was located in 888bp. After looking for ORFs in both plus and minus strands in BmDNV-6 DNA-2, three ORFs were found. ORF1 and ORF2 were located inplus strand, ORFl was in 37-267bp, encoding 77 arnino acids, ORF2 was in 149-322bp,encoding 58 amino acids. ORF3 was situated in 297-1283bp of minus strand, encoding 199 amino acids. The three ORFs overlapped mutually. In NCBI, Blastn homology contrast showed it was the same as the 2646-3944bp region in plus strand in BmDNV-2 VDl, suggesting BmDNV-6 DNA-2 is a part of BmDNV-2 VDl's plus strand (Fig.2).4. Evolution of BmDNV-6The evolution tree of family Parvoviridae was constructed with nucleic acid sequence of six genera (Fig.3). The tree indicated that the subfamily Densoviridae and family Parvovidae were onthe two branches respectively. But BmDNV-6 and BmDNV-2 constructed a branch singleness,it suggested that the relation between BmDNV-6 and BmDNV-2 was close. So it was misgivings that BmDNV-1 and BmDNV-2 were classified one genera, BmDNV-2 would be disjoined with BmDNV-6 and found a new genera.5.Epidemiology features of BmDNV-65.1 Epidemic area of BmDNV-6Using immunodiffusion method, intenerate materials collected from Sichuan, Chongqing, Yunnan were detected with BmDNV-1, BmDNV-2 and antiserum of IFV. Results showed Bombyx mori intenerate disease prevalent in Sichuan, Chongqing, Yunnan was BmDNV-2 while BmDNV-6 was only found in Bombyx mori gene library, SWAU, falling into partial happening phase.5.2 Stability of BmDNV-6There were discrepancies in stability when BmDNV-2 and BmDNV-6 were under different physical and chemical treatments. The stability of BmDNV-6 in 100 °C steam and bleaching powder solution was similar to BmDNV-2, but apparent differences existed when under xerothermic 100°C and 70°C, BmDNV-6 had higher stability than BmDNV-2 when under xerothermic condition. Similarly, BmDNV-6 had higher stability than BmDNV-2 when in 2% formaldehyde and 0.5% calcareousness solution. Therefore, BmDNV-6 had higher stability than BmDNV-2 under different conditions, namely, BmDNV-6 had higher adaptability to circumstances than BmDNV-2.5.3 Pathogeny of BmDNV-6After BmDNV-6 and BmDNV-2 of different concentrations infected different breeds of the third instar, day-to-day and accumulative mortality indicated that different concentrations of BmDNV-6 were all highly pathogenic to different varieties of Bombyx mori, while the mortality caused by high concentration of BmDNV-2 apparently exceeded that caused by low concentration. Besides, when infected by 10"3 or 10"4 concentration, the evocable mortality showed differences between breeds, namely, fastness of BmDNV-2 to breeds exhibited as: crossbreed QingsongX Haoyue>Haoyue>Qingsong. The change trend of these two pathogenies accumulative mortality to bombyx mori were consistent with high concentration, the discrepancies were few. But as the concentration decreased, the discrepancies got more and more distinct. Pathogeny of BmDNV-6 was greatly higher than BmDNV-2, but after being diluted 1000 times, pathogenicity of the two pathogenies to Qingsong took on few discrepancies. Using Reed-Muench method, the LD50 of the three breeds to BmDNV-6 and BmDNV-2 respectively were worked out, which suggested that the fastness of Qingsong to BmDNV-2 was more than 10 times weaker than Qingsong XHaoyue while...