- Volume 88, Issue 12, 2007

Group II nucleopolyhedroviruses (NPVs), e.g. Helicoverpa armigera (Hear) NPV and Spodoptera exigua (Se) MNPV (multiple NPV), lack a GP64-like protein that is present in group I NPVs, e.g. Autographa californica (Ac)MNPV, but have an unrelated envelope fusion protein named F. Three AcMNPV viruses were constructed by introducing AcMNPV gp64, HearNPV f or SeMNPV f genes, respectively, into a gp64-negative AcMNPV bacmid. Sf21 cells were incubated with different amounts of inactivated budded virus to occupy receptors and were subsequently infected with a fixed amount of infectious virus to compete for attachment. The results suggest that GP64 and F act on their own and use different receptors, while the two different F proteins exploit the same receptor. Additionally, gp64-null AcMNPV pseudotyped with baculovirus F was, in contrast to GP64, unable to transduce mammalian cells, indicating that mammalian cells do not possess baculovirus F protein receptors despite the structural similarity of baculovirus F to vertebrate viral fusion proteins.

The open reading frame Ha107 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) encodes a putative protein of 51 kDa with homologues in a few group II NPVs and a granulovirus. Ha107 was transcribed as polyadenylated transcripts in infected HzAM1 insect cells. The transcripts were initiated at two distinct locations, one upstream of Ha106 (superoxide dismutase gene, sod) and the second upstream of Ha107. By Western blot analysis, two forms of the HA107 protein were detected in infected cells, a major polypeptide of 48 kDa and a minor one of 51 kDa. Western blot and immunoelectron microscopy analyses further showed that the HA107 protein was associated with the nucleocapsids of both budded virions (BVs) and occlusion-derived virions. A Ha107 knockout virus expressing enhanced green fluorescent protein and polyhedrin was constructed using bacmid technology. A one-step virus growth curve indicated that the BV titre of the knockout virus was significantly higher than that of the parental virus and a Ha107 repair virus. Bioassays indicated that the knockout virus was able to infect third-instar H. armigera larvae; however, its median lethal dose (LD50) was significantly higher than those of the parental virus and Ha107 repair virus. These data indicate that Ha107 encodes a non-essential structural protein of HearNPV virions and that deletion of this gene increases the BV titre and LD50 of the occluded virus.

Cotesia vestalis is an endoparasitoid of larval stages of Plutella xylostella, the diamondback moth. For successful parasitization, this parasitoid injects a polydnavirus into its host during oviposition. Here we isolated two genes, which we named CvBV1 and CvBV2. CvBV1 was located on segment CvBV-S5 with a size of 790 bp, while CvBV2 was located on segment CvBV-S51 with a size of 459 bp. A gene copy of CvBV2 was found on segment CvBV-S48, which we name CvBV2’. Gene duplication occurred in both genes, tandem gene duplication for CvBV1 and segmental duplication for CvBV2. Gene transcripts of the two genes were detected in hosts as early as 0.5 h post-parasitization (p.p.) and continued to be detected for six days, and tissue-specific expression patterns showed that they could be detected in the haemolymph and brain at 2 h p.p., suggesting that they could participate in early protection of parasitoid eggs from host cellular encapsulation.

The cucumber mosaic virus (CMV)-encoded 3a movement protein (MP) is indispensable for CMV movement in plants. We have previously shown that MP interacts directly with the CMV-encoded 2a polymerase protein in vitro. Here, we further dissected this interaction and determined the amino acid sequences that are responsible for the MP and 2a polymerase protein interaction. Both the N-terminal 21 amino acids and the central GDD motif of the 2a polymerase protein were important for interacting with the MP. Although each of the regions alone was sufficient for the interaction with MP, quantitative yeast two-hybrid analyses showed that they acted synergistically to enhance the binding affinity. The MP N-terminal 20 amino acids were sufficient for interacting with the 2a polymerase protein, and the serine residue at position 14 played a critical role in the interaction. Multiple sequence alignment showed that the 2a protein interacting regions and the serine at position 14 in the MP are highly conserved among subgroup I and II CMV isolates.

Like many plant RNA viruses, infection by potato spindle tuber viroid (PSTVd) is known to lead to RNA silencing and a marked reduction in visible disease. To examine the relationship between RNA silencing and this recovery phenomenon in greater detail, we have carried out time-course analyses of viroid-specific small RNA accumulation using several viroid–host combinations. These analyses revealed the presence of two size classes of viroid-specific small RNAs in infected plants, and sequence analysis subsequently demonstrated the presence of a previously undescribed cluster of small RNAs derived primarily from negative-strand PSTVd RNA. Although the clustering patterns were similar, the size distribution of PSTVd small RNAs isolated from symptomatic leaf tissue became more heterogeneous with time. The process by which viroid-specific small RNAs are generated appears to be more complicated than previously believed, possibly involving multiple DICER-LIKE activities, viroid RNA substrates and subcellular compartments.

During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.

Prions, the putative causative agents of transmissible spongiform encephalopathies, are neurotropic pathogens that spread to the central nervous system via synaptically linked neural conduits upon peripheral infection. Axons and their transport processes have been suggested as mediators of nerve-associated prion dissemination. However, the exact cellular components and molecular mechanisms underlying neural spread are unknown. This study used an established hamster scrapie model to pursue a novel experimental approach using nerve conduits containing segments devoid of neurites generated by incomplete nerve regeneration following Wallerian degeneration to probe the necessity of axons for the neural propagation of prions. For this purpose, animals were subjected to unilateral sciatic neurectomy 4 weeks before footpad inoculation with scrapie agent. The results showed that the regional nerve is the prime conduit for cerebral neuroinvasion and revealed, as evidenced by the accumulation of pathological prion protein PrPTSE, that prions can proceed along segments of peripheral neural projections without detectable axonal structures.

Following the bovine spongiform encephalopathy (BSE) crisis, the European Union has introduced policies for eradicating transmissible spongiform encephalopathies (TSEs), including scrapie, from large ruminants. However, recent European Union surveillance has identified a novel prion disease, ‘atypical’ scrapie, substantially different from classical scrapie. It is unknown whether atypical scrapie is naturally transmissible or zoonotic, like BSE. Furthermore, cases have occurred in scrapie-resistant genotypes that are targets for selection in legislated selective breeding programmes. Here, the first epidemiological study of British cases of atypical scrapie is described, focusing on the demographics and trading patterns of farms and using databases of recorded livestock movements. Triplet comparisons found that farms with atypical scrapie stock more sheep than those of the general, non-affected population. They also move larger numbers of animals than control farms, but similar numbers to farms reporting classical scrapie. Whilst there is weak evidence of association through sheep trading of farms reporting classical scrapie, atypical scrapie shows no such evidence, being well-distributed across regions of Great Britain and through the sheep-trading network. Thus, although cases are few in number so far, our study suggests that, should natural transmission of atypical scrapie be occurring at all, it is doing so slowly.

In human cytomegalovirus (HCMV)-infected cells, the 86 kDa immediate-early (IE) 2 protein plays a key role in transactivating downstream viral genes. Recently, IE2 has been shown to interact with histone deacetylase 1 (HDAC1) and HDAC3. HDAC1 recruited by IE2 was required for IE2-mediated autorepression of the major IE (MIE) promoter, whereas IE2–HDAC3 interaction was suggested to relieve the repressive effect of HDAC3 on viral early promoters. However, whether IE2 indeed inhibits HDAC's deacetylation activity on viral promoters and interacts with other HDACs remains unclear. Here, we provide evidence that IE2 functionally interacts with HDAC2 and negates its repressive effect on the viral polymerase promoter. IE2 interacted with HDAC2 in both virus-infected cells and in vitro, and required the conserved C-terminal half for HDAC2 binding. The subcellular localization of HDAC2 was changed in virus-infected cells, showing colocalization with IE2 in viral transcription and replication sites. The overall HDAC2 protein levels and its deacetylation activity slightly increased during the late stages of infection and the IE2-associated deacetylation activity was still sensitive to an HDAC inhibitor, trichostatin A. In transfection assays, however, histone acetylation of the viral polymerase promoter was suppressed by HDAC2, and this was relieved by IE2 binding. Therefore, our data demonstrate that IE2 functionally interacts with HDAC2 and modulates its deacetylation activity on the viral polymerase promoter. Our results also support the idea that interactions of IE2 with several HDACs to modulate the host epigenetic regulation on viral MIE and early promoters are important events in the process of productive infection.