- Volume 90, Issue 1, 2009

The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14–22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD50 was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.

Post-mortem host degradation by infection of Bombyx mori nucleopolyhedrovirus (BmNPV) requires the synergistic activation of two virus-encoded genes, cathepsin (v-cath) and chitinase (v-chiA). Previous studies have suggested that V-CHIA is essential for the proper folding of the nascent V-CATH polypeptide in the endoplasmic reticulum, and that the putative V-CHIA–V-CATH interaction might be mediated by N-linked glycans of V-CATH. Sequence analysis shows that BmNPV V-CATH includes three consensus N-linked glycosylation sites (asparagine 38, 65 and 158). To clarify the role of N-linked glycans of V-CATH in its biological activity, we generated three recombinant BmNPVs expressing mutant V-CATHs, and found that the two residues, asparagine 38 and 65, which are localized in the pro-region of V-CATH, are the glycosylation sites of BmNPV V-CATH. Western blot analysis also showed that removal of N-linked glycans from BmNPV V-CATH resulted in production of the insoluble forms of V-CATH and V-CHIA. These results demonstrate that N-linked glycans located in the pro-region of BmNPV V-CATH are essential for the proper folding of V-CATH and V-CHIA.

DNA β is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA β contains a single gene, βC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the β-glucuronidase reporter gene driven by a βC1 promoter-deletion series of the DNA β associated with cotton leaf curl Multan virus identified a 68 nt region (between −139 and −207) which is important for βC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the βC1 start codon. Mutation of the G-box resulted in a significant reduction in βC1 promoter activity and DNA β replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA β.

We examined therapeutic in vitro and in vivo approaches using lentivirus-based packaging of small interfering RNAs (siRNAs) targeting the non-integrin laminin receptor mRNA for treatment and prevention of prion disorders. Transfection of N2aSc+ cells with recombinant plasmids expressing three different siRNAs, significantly reduced both the LRP (laminin receptor precursor) and PrPSc levels by approximately 40–60 %. Stereotactic intracerebral microinjection of recombinant lentiviral vectors LVsiRNA-LRP 7 and 9 into the cortex of C57BL/6 wild-type mice resulted in a significant reduction of the LR levels in the cortex 15 days post-injection by 62 and 82 %, respectively. Intracerebral RML inoculation of C57BL/6 mice after microinjection with recombinant lentiviral vector LVsiRNA-LRP 7 into the hippocampus resulted in a significant reduction of both LRP and PrPSc levels by 36 and 41 %, respectively, concomitant with a significant prolongation of the pre-clinical phase. Lentiviral vectors expressing siRNAs targeting LRP mRNA represent a novel delivery system for the treatment of transmissible spongiform encephalopathies.

Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml−1 (PrPTSE : MnO2=1 : 110) decreased PrPTSE levels by ≥4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals.

A distinctive feature of the cytomegaloviruses is their wide tissue tropism, demonstrated by the infection of many organs and cell types in an active infection. However, in experimental models of systemic infection, the earliest stages of infection are not well characterized, and it is unclear whether only certain cells are initially infected. Using a recombinant murine cytomegalovirus (MCMV) expressing green fluorescent protein (GFP), we tracked viral infection after systemic administration via intraperitoneal injection and showed that specific cells are infected within the first hours. We provide evidence that MCMV traffics as free virus from the peritoneal cavity into the mediastinal lymphatics, providing access to the bloodstream. We demonstrate that MCMV productively infected CD169+ subcapsular sinus macrophages in the mediastinal lymph nodes, ER-TR7+ CD29+ reticular fibroblasts in the spleen and hepatocytes. Infection in the spleen followed a distinctive pattern, beginning in the marginal zone at 6 h and spreading into the red pulp by 17 h. By 48 h after infection, there was widespread infection in the spleen and liver with degeneration of infected cells. In addition, infected dendritic cells appeared in the white pulp of the spleen at 48 h post-infection. On the other hand, cowpox virus showed a different pattern of infectivity in the spleen and liver. Thus, early MCMV infection produces a distinct pattern of infection of selective cells.

Herpesviruses have previously been isolated from African and South-American bats. Recently, herpesviruses detected from European insectivorous bats (family Vespertilionidae) were classified molecularly as betaherpesviruses and gammaherpesviruses. In the current study, we performed PCR analyses targeting the UL30 catalytic subunit region of the DNA polymerase gene of the African and South American herpesviruses and new Malagasy and Cambodian herpesviruses isolated from bats, especially frugivorous bats from the families Pteropodidae and Phyllostomidae. The sequences obtained from the amplified products indicated that these isolates belonged to the genus Simplexvirus of the subfamily Alphaherpesvirinae. These results extend the taxonomic range of bat herpesviruses with the description of four members in the subfamily Alphaherpesvirinae. Furthermore, these data confirm and extend the geographical distribution of herpesvirus in bats to three more continents (Africa, South America and Asia) and indicate the presence of these viruses in frugivorous bats of the families Pteropodidae and Phyllostomidae.

Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1ΔE1E2-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1W529A was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.