| Nucleopolyhedroviruses (NPVs) as a kind of microorganism infected insect pests, are ideal biological insecticides, because of their rich resources, special action mechanism on target insect pests and environmental safety. However, compared to numbers of species of NPVs found, the application of NPVs as microbial pesticides, has not meet their potential to control insect pests. How to expand the host range and improve their virulence is very important to expand the usage of NPVs. In nature, recombination between NPVs are common, recombination in NPVs with different host ranges can generate NPVs with expanded host range. Besides that, as the virulence of NPV is closely related with the host, serial passage in new host can significantly improve the virulence of NPV against the host, but at the same time, its infectivity on former host decrease. It is unknown whether when NPV propagated in two hosts alternatively, it will evolve high virulence against both of the hosts or not. Therefore, it is meanful to study the recombination of NPVs and its experimental evolution during alterantive passages in two hosts, these will not only clarify the evolution rules of NPVs, but also provide new biological resources with improved host range and virulence. In this paper, we studied the recombination of Spodoptera liutra nucleopolyhedrovirus (S1NPV) and S. exigua nucleopolyhedrovirus (SeNPV) which could only infect its native host, and after obtaining NPV with expanded host range, we further investigated the experimental evolution of the NPV when propagated in two hosts for20generations. Based on these, we also investigated the variation in ultrastructure of polyhedra and the genetic variation of NPVs related with co-infection and alternate passages. The main results are as follows:1Host range expansion of NPV after co-infection and virulence evolution of NPV during alternate passages in two hostsTwo NPVs was selected to co-infect in vivo, One was S. litura NPV (S1NPV) Japanese Fukuoka isolate which could not successfully infect its closely related host-S. exigua, and the other was S. exigua NPV(SeNPV) American isolate which also could not infect5. litura. After oral co-infection of S1NPV and SeNPV in S. exigua, a new NPV isolate which could infect both S. litura and S. exigua was isolated. The results showed that, compared with S1NPV before co-infection, the biological activity of the new isolate against S. litura was not changed, and the lethal concentration of50percent individuals (LC50) was not significantly different from that of S1NPV before co-infection. But at the same time, the new variant also showed biological activity against S. exigua, howbeit the LC50was still significantly lower than that of SeNPV before co-infection. The new virus isolate with expanded host range was then propagated in S. litura and S. exigua alternately by four kinds of mode for20generations. Two modes were passages beginning with S. exigua or S. litura and strictly propagating in two hosts alternately, and the other two modes were passages beginning with S. exigua or S. litura and unstrictly propagating in two hosts alternately. The evolution of virulence of NPV during alternate passages was investigated. It was found that virulence of NPV against S. exigua was significantly improved during alternate passages, but only in one mode which the passage began with S. litura and passed in two hosts strictly, the improvement in virulence of NPV positively related with the generations during20passages. And in the other three modes, virulence didn’t not keep on increasing during20passages, after14passages, the virulence against S. exigua became decrease slightly. Although the virulence against S. exigua improved, their virulence against S. litura didn’t change significantly. All these indicated that NPVs could enhance their adaptability to a host and keep their adaptability to the other host during alternate passages in the two hosts.2Variation in ultrastructure of polyhedrin after co-infection and alternate passagesThe ultrastructure of the polyhedron after co-infection and alternative passages were observed under Transmission Electronic Microscope (TEM). The results showed that, all the observed viruses were multicapsid nucleopolyhedrovirus, co-infection did not cause changes in the size of the viral polyhedra, and diameter of the polyhedrin was not significantly different between S1NPV before co-infection and S1NPV after co-infection. But after strictly alternate passages which began in S. litura or serial passage in S. litura, the diameter of the viral polyhedra significantly reduced. It was also found co-infection and alternate passages did not result in the changes of the mean number of occlusion-derived virus (ODV) per polyhedra. The average number of ODV per polyhedra in all the observed isolates had no significant difference, but it caused changes in the number of nuclecapsids per ODV. After co-infection and one alternate passage in S. exigua and S. litura, the number of nucleocapsid per ODV was significantly lower than the initial S1NPV Fukuoka isolate, after20generations, it was still significantly lower than that of Fukuoka isolate. While the number of nucleocapsid per ODV did not significantly change after serial passages in S. litura. In addition, distribution of ODV with different number of nucleocapsid was compared. Nucleocapsid were evenly distributed in SeNPV,62.5%viruses have three nucleocapsids per ODV. Whereas distribution of nucleocapsid in ODV were largely differentiated in the remaining isolates, the number of nucleocapsid per ODV ranged from1to12after20alternate passages. All the results indicated that co-infection and alternate passages caused changes in the assembly of polyhedra which resulted in the ultrastructural changes of polyhedra.3Variation in polh and iap gene of NPV with expanded host range after co-infectionThrough genome digestion and gene clone, we found genetic variation with co-infection. Virus genomic DNA restriction digested results showed that genomic DNA restriction map of NPV with expanded host range after co-infection did not change compared with that of S1NPV Fukuoka isolate. DNA were digested by restriction enzyme Pst I, Hind III, EcoR I and Xba I, and all the restriction maps of DNA from S1NPV before and after co-infection with the same enzyme were the same. Further virus polyhedrin gene (polh) analysis showed that after mixed infection, the polh gene sequences was changed compared with S1NPV Fukuoka isolate, there was one base mutation loci281, the amino acid sequence consequently changed. Another inhibitor of apoptosis gene (iap) was cloned from NPV with expanded host range, and it only had80%homology with the reported iap gene from S1NPV Chinese isolate G2and Japanese isolate.4Analysis of variation in complete genome of NPV with co-infection and alternate passageComplete genome of three NPV isolates related with co-infection and alternate passage was analyzed. It was found that, compred to S1NPV Fukuoka isolate, the size of whole genome of NPV with expanded host range was smaller, and the number of gene decreased, and the length of all the genes increased. And after alternate passages, the length of all the genes further increased. Besides that, the ratio of gene length and whole genome was improved with co-infection and alternate passages. No obvious variation in GC content was found. Further sequence analysis showed that there was a point mutation in lef-8(late expression factor) gene, and there was a255bp fragment insertion in NPV after co-infection, and the fragment was kept in NPV after20alterante passages. |
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