Genetic Variation in Field Populations of Baculoviruses: Mechanisms for Generating Variation and Its Potential Role in Baculovirus Epizootiology

Baculoviridae is a family of insect-specific DNA viruses that have been used as biological control agents for insect pest control. In most cases these baculovirus control agents are natural field isolates that have been selected based on their infectivity and virulence. The advent of molecular tools such as restriction endonucleases, targeted polymerase chain reaction and new DNA sequencing strategies have allowed for efficient detection and characterization of genotypic variants within and among geographic and temporal isolates of baculovirus species. It has become evident that multiple genotypic variants occur even within individual infected larvae. Clonal strains of baculovirus species derived either by in vitro or in vivo approaches have been shown to vary with respect to infectivity and virulence. Many of the cell culture derived plague-purified strains have deletions that interrupt egt expression leading to virus strains that kill infected hosts more quickly. As well, in vitro clones often involve larger genomic deletions with the loss of pif gene function, resulting in strains deficient for oral infectivity. There are an increasing number of baculovirus species for which complete genome sequences are available for more than one strain or field isolate. Results of comparative analysis of these strains indicated that hr regions and bro genes often mark "hot spots" of genetic variability between strains and of potential recombination events. In addition, the degree of nucleotide polymorphisms between and within strains and their role in amino acid substitutions within ORFs and changes in promoter motifs is also beginning to be appreciated. In this short review the potential mechanisms that generate and maintain this genetic diversity within baculovirus populations is discussed, as is the potential role of genetic variation in host-pathogen interactions.

Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration

Objective： To construct the recombinant baculovirus Ac-cytomegalovirus （CMV）-hSox9 for gene therapy of intervertebral disc degeneration. Methods： Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDul-green fluorescene protein （ GFP ）-CMV （pgGC）-hSox9. The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani （LB） agarose culture medium containing isopropyl-β-D-thiogalactoside （IPTG）, X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9 Bacmid DNA was extracted. After verification, recombinant baculovirns Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells. The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining. Results： Polymerase chain reaction （ PCR ） confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein. Western blotting and indicated that exogenous hSox9 disc cells. staining analysis Conclusions： The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.