Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions

Background. A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses in the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. Results. Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. Conclusion. Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity. © 2008 van der Walt et al; licensee BioMed Central Ltd.

Replicative intermediates of maize streak virus found during leaf development

Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombinationdependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR. © 2010 SGM.

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa

Beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (Melopsittacus undulatus) from a breeding farm in South Africa. The isolates share >99% nucleotide sequence identity with each other and ~96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91. 6 and 86. 6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV. © 2010 Springer-Verlag.

Reconstructing the history of maize streak virus strain a dispersal to reveal diversification hot spots and its origin in southern Africa

Maize streak virus strain A (MSV-A), the causal agent of maize streak disease, is today one of the most serious biotic threats to African food security. Determining where MSV-A originated and how it spread transcontinentally could yield valuable insights into its historical emergence as a crop pathogen. Similarly, determining where the major extant MSV-A lineages arose could identify geographical hot spots of MSV evolution. Here, we use model-based phylogeographic analyses of 353 fully sequenced MSV-A isolates to reconstruct a plausible history of MSV-A movements over the past 150 years. We show that since the probable emergence of MSV-A in southern Africa around 1863, the virus spread transcontinentally at an average rate of 32.5 km/year (95% highest probability density interval, 15.6 to 51.6 km/year). Using distinctive patterns of nucleotide variation caused by 20 unique intra-MSV-A recombination events, we tentatively classified the MSV-A isolates into 24 easily discernible lineages. Despite many of these lineages displaying distinct geographical distributions, it is apparent that almost all have emerged within the past 4 decades from either southern or east-central Africa. Collectively, our results suggest that regular analysis of MSV-A genomes within these diversification hot spots could be used to monitor the emergence of future MSV-A lineages that could affect maize cultivation in Africa. © 2011, American Society for Microbiology.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Global genetic diversity and geographical and host-species distribution of beak and feather disease virus isolates

Psittacine beak and feather disease (PBFD) has a broad host range and is widespread in wild and captive psittacine populations in Asia, Africa, the Americas, Europe and Australasia. Beak and feather disease circovirus (BFDV) is the causative agent. BFDV has an ~2 kb single stranded circular DNA genome encoding just two proteins (Rep and CP). In this study we provide support for demarcation of BFDV strains by phylogenetic analysis of 65 complete genomes from databases and 22 new BFDV sequences isolated from infected psittacines in South Africa. We propose 94% genome-wide sequence identity as a strain demarcation threshold, with isolates sharing > 94% identity belonging to the same strain, and strain subtypes sharing> 98% identity. Currently, BFDV diversity falls within 14 strains, with five highly divergent isolates from budgerigars probably representing a new species of circovirus with three strains (budgerigar circovirus; BCV-A, -B and -C). The geographical distribution of BFDV and BCV strains is strongly linked to the international trade in exotic birds; strains with more than one host are generally located in the same geographical area. Lastly, we examined BFDV and BCV sequences for evidence of recombination, and determined that recombination had occurred in most BFDV and BCV strains. We established that there were two globally significant recombination hotspots in the viral genome: the first is along the entire intergenic region and the second is in the C-terminal portion of the CP ORF. The implications of our results for the taxonomy and classification of circoviruses are discussed. © 2011 SGM.

A proposal to change existing virus species names to non-Latinized binomials

A proposal has been posted on the ICTV website (2011. 001aG. N. v1. binomial_sp_names) to replace virus species names by non-Latinized binomial names consisting of the current italicized species name with the terminal word "virus" replaced by the italicized and non-capitalized genus name to which the species belongs. If implemented, the current italicized species name Measles virus, for instance, would become Measles morbillivirus while the current virus name measles virus and its abbreviation MeV would remain unchanged. The rationale for the proposed change is presented. © 2010 Springer-Verlag.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

Centre for High-Performance Computing, Rosebank, Cape Town, South Africa Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicativeform-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepDI662) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepDI662 inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepDI662 inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic. © 2011 SGM.

Spatial and temporal analysis of Barmah Forest virus disease in Queensland, Australia

Barmah Forest virus (BFV) disease is one of the most widespread mosquito-borne diseases in Australia. The number of outbreaks and the incidence rate of BFV in Australia have attracted growing concerns about the spatio-temporal complexity and underlying risk factors of BFV disease. A large number of notifications has been recorded continuously in Queensland since 1992. Yet, little is known about the spatial and temporal characteristics of the disease. I aim to use notification data to better understand the effects of climatic, demographic, socio-economic and ecological risk factors on the spatial epidemiology of BFV disease transmission, develop predictive risk models and forecast future disease risks under climate change scenarios. Computerised data files of daily notifications of BFV disease and climatic variables in Queensland during 1992-2008 were obtained from Queensland Health and Australian Bureau of Meteorology, respectively. Projections on climate data for years 2025, 2050 and 2100 were obtained from Council of Scientific Industrial Research Organisation. Data on socio-economic, demographic and ecological factors were also obtained from relevant government departments as follows: 1) socio-economic and demographic data from Australian Bureau of Statistics; 2) wetlands data from Department of Environment and Resource Management and 3) tidal readings from Queensland Department of Transport and Main roads. Disease notifications were geocoded and spatial and temporal patterns of disease were investigated using geostatistics. Visualisation of BFV disease incidence rates through mapping reveals the presence of substantial spatio-temporal variation at statistical local areas (SLA) over time. Results reveal high incidence rates of BFV disease along coastal areas compared to the whole area of Queensland. A Mantel-Haenszel Chi-square analysis for trend reveals a statistically significant relationship between BFV disease incidence rates and age groups (ƒÓ2 = 7587, p<0.01). Semi-variogram analysis and smoothed maps created from interpolation techniques indicate that the pattern of spatial autocorrelation was not homogeneous across the state. A cluster analysis was used to detect the hot spots/clusters of BFV disease at a SLA level. Most likely spatial and space-time clusters are detected at the same locations across coastal Queensland (p<0.05). The study demonstrates heterogeneity of disease risk at a SLA level and reveals the spatial and temporal clustering of BFV disease in Queensland. Discriminant analysis was employed to establish a link between wetland classes, climate zones and BFV disease. This is because the importance of wetlands in the transmission of BFV disease remains unclear. The multivariable discriminant modelling analyses demonstrate that wetland types of saline 1, riverine and saline tidal influence were the most significant risk factors for BFV disease in all climate and buffer zones, while lacustrine, palustrine, estuarine and saline 2 and saline 3 wetlands were less important. The model accuracies were 76%, 98% and 100% for BFV risk in subtropical, tropical and temperate climate zones, respectively. This study demonstrates that BFV disease risk varied with wetland class and climate zone. The study suggests that wetlands may act as potential breeding habitats for BFV vectors. Multivariable spatial regression models were applied to assess the impact of spatial climatic, socio-economic and tidal factors on the BFV disease in Queensland. Spatial regression models were developed to account for spatial effects. Spatial regression models generated superior estimates over a traditional regression model. In the spatial regression models, BFV disease incidence shows an inverse relationship with minimum temperature, low tide and distance to coast, and positive relationship with rainfall in coastal areas whereas in whole Queensland the disease shows an inverse relationship with minimum temperature and high tide and positive relationship with rainfall. This study determines the most significant spatial risk factors for BFV disease across Queensland. Empirical models were developed to forecast the future risk of BFV disease outbreaks in coastal Queensland using existing climatic, socio-economic and tidal conditions under climate change scenarios. Logistic regression models were developed using BFV disease outbreak data for the existing period (2000-2008). The most parsimonious model had high sensitivity, specificity and accuracy and this model was used to estimate and forecast BFV disease outbreaks for years 2025, 2050 and 2100 under climate change scenarios for Australia. Important contributions arising from this research are that: (i) it is innovative to identify high-risk coastal areas by creating buffers based on grid-centroid and the use of fine-grained spatial units, i.e., mesh blocks; (ii) a spatial regression method was used to account for spatial dependence and heterogeneity of data in the study area; (iii) it determined a range of potential spatial risk factors for BFV disease; and (iv) it predicted the future risk of BFV disease outbreaks under climate change scenarios in Queensland, Australia. In conclusion, the thesis demonstrates that the distribution of BFV disease exhibits a distinct spatial and temporal variation. Such variation is influenced by a range of spatial risk factors including climatic, demographic, socio-economic, ecological and tidal variables. The thesis demonstrates that spatial regression method can be applied to better understand the transmission dynamics of BFV disease and its risk factors. The research findings show that disease notification data can be integrated with multi-factorial risk factor data to develop build-up models and forecast future potential disease risks under climate change scenarios. This thesis may have implications in BFV disease control and prevention programs in Queensland.

Surveillance of dengue fever virus: a review of epidemiological models and early warning systems

Dengue fever is one of the world’s most important vector-borne diseases. The transmission area of this disease continues to expand due to many factors including urban sprawl, increased travel and global warming. Current preventative techniques are primarily based on controlling mosquito vectors as other prophylactic measures, such as a tetravalent vaccine are unlikely to be available in the foreseeable future. However, the continually increasing dengue incidence suggests that this strategy alone is not sufficient. Epidemiological models attempt to predict future outbreaks using information on the risk factors of the disease. Through a systematic literature review, this paper aims at analyzing the different modeling methods and their outputs in terms of accurately predicting disease outbreaks. We found that many previous studies have not sufficiently accounted for the spatio-temporal features of the disease in the modeling process. Yet with advances in technology, the ability to incorporate such information as well as the socio-environmental aspect allowed for its use as an early warning system, albeit limited geographically to a local scale.

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly

Cryo-electron tomography of Marburg Virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.