Chimaeric plant-produced bluetongue virus particles as potential vaccine candidates.

Bluetongue virus (BTV) causes bluetongue disease in ruminants and sheep. The current live attenuated and inactivated vaccines available for prevention pose several risks, and there is thus a need for vaccines that are safer, economically viable, and effective against multiple circulating serotypes. This work describes the development of recombinant virus-like particle (VLP) vaccine candidates in plants, which are assembled by co-expression of the four BTV serotype 8 major structural proteins. We show that substitution of a neutralising tip domain of BTV8 VP2 with that of BTV1 VP2 resulted in the assembly of VLPs that stimulated serotype-specific antibodies as well as virus-specific neutralising antibodies.

Viruses, variants and vaccines.

The current SARS-CoV-2 pandemic has brought a number of major global clinical, sociological and economic issues into sharp focus. We address some of these issues, focusing on short-term factors such as virus mutations and vaccine efficacy, and also considering the longer-term implications of the current pandemic. We discuss societal responses to the presence of a pathogen that will probably remain in circulation for decades or longer, and to future new emergent viruses.

First Report of a Potyvirus Infecting Albuca rautanenii in the Namib Desert.

Chlorotic, streak-like symptoms were observed in April 2013 on a single specimen of Albuca rautanenii (Schinz) J.C.Manning & Goldblatt (Family: Hyacinthaceae) found among other plants near Homeb in the Namib Desert, Namibia. No potential insect vectors (e.g., aphids) were observed on or around the infected plant. An extract from symptomatic leaves was assessed by transmission electron microscopy (leaf dip method) to ascertain if the symptoms were viral in origin. Long, flexuous threadlike particles 687 to 825 nm in length and 12.5 nm in diameter were observed. The morphology and size of the particles were indicative of a putative member of the taxonomic family Potyviridae. To confirm this, RT-PCR using universal potyvirus primers which amplify part of the nuclear inclusion b gene (NIb) was conducted (1) on total RNA extracted from leaf tissue (Qiagen RNeasy Plant Mini Kit). The triplicated reaction yielded amplicons of the expected size (~350 bp), which were cloned into the pJET 1.2 vector (Thermo Scientific, Waltham, MA) according to manufacturer's instructions. The sequences of 10 clones were trimmed to remove vector and primer ends and were deposited in the EBI database under the accession numbers LK995422 to LK995431. Curated sequences were used to search the GenBank database using BLASTn and tBLASTx, as well as for phylogenetic analysis. Intra-clonal nucleotide sequence similarity ranged from 97.99 to 99.72%. BLASTn searches showed all clones were 72% identical to Papaya ringspot virus isolate 1 accession JQ314105.1 (87% coverage), followed by Bean yellow mosaic virus clone Brn167 accession JF707769.1 (72% identity with 86% sequence coverage). The translated peptide fragment was most similar to Sugarcane mosaic virus isolate Beijing (AY042184.1), with a query cover of 98% and a similarity of 81%. Phylogenetic analysis was performed with a set of 57 reference potyvirus genomes, with their NIb regions aligned with the cloned nucleotide sequences according to the parameters used previously (1). The clones formed a distinct cluster, at a node with Cocksfoot streak virus (CSV) (NC_003742.1). An identity matrix of the aligned NIb clones and CSV showed a nucleotide identity range of 68.79 to 70.23%. These results suggest that the virus isolate belongs to the family Potyviridae, genus Potyvirus, supported by the characteristic morphological features of the virion and its relatedness to CSV. Moreover, the clustering of all sequences at a single node suggests a homogeneous viral population, without significant strain variation. Genetic distance inferred by phylogenetic analysis further suggests that the isolate is a novel species within the genus, which we tentatively name Albuca mosaic virus, AlbMV. To our knowledge, this is the first report of any plant virus infection in the native Namib Desert ecosystem. This is particularly relevant due to the scarcity and uniqueness of plant life in this hyperarid desert environment, and additional monitoring of this virus infection and other desert plant species is encouraged. Reference: (1) L. Zheng et al. Plant Pathol. 59:211, 2010.

Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan.

Most mastreviruses (family Geminiviridae) infect monocotyledonous hosts and are transmitted by leafhopper vectors. Only two mastrevirus species, Tobacco yellow dwarf virus from Australia and Bean yellow dwarf virus (BeYDV) from South Africa, have been identified whose members infect dicotyledonous plants. We have identified two distinct mastreviruses in chickpea stunt disease (CSD)-affected chickpea originating from Pakistan. The first is an isolate of BeYDV, previously only known to occur in South Africa. The second is a member of a new species with the BeYDV isolates as its closest relatives. A PCR-based diagnostic test was developed to differentiate these two virus species. Our results show that BeYDV plays no role in the etiology of CSD in Pakistan, while the second virus occurs widely in chickpea across Pakistan. A genomic clone of the new virus was infectious to chickpea (Cicer arietinum L.) and induced symptoms typical of CSD. We propose the use of the name Chickpea chlorotic dwarf Pakistan virus for the new species. The significance of these findings with respect to our understanding of the evolution, origin and geographic spread of dicot-infecting mastreviruses is discussed.

First Report of Maize streak virus Field Infection of Sugarcane in South Africa.

Prior to the introduction of highly resistant sugarcane varieties, Sugarcane streak virus (SSV) caused serious sugar yield losses in southern Africa. Recently, sugarcane plants with streak symptoms have been identified across South Africa. Unlike the characteristic fine stippling and streaking of SSV, the symptoms resembled the broader, elongated chlorotic lesions commonly observed in wild grasses infected with the related Maize streak virus (MSV). Importantly, these symptoms have been reported on a newly released South African sugarcane cultivar, N44 (resistant to SSV). Following a first report from southern KwaZulu-Natal, South Africa in February 2006, a survey in May 2007 identified numerous plants with identical symptoms in fields of cvs. N44, N27, and N36 across the entire South African sugarcane-growing region. Between 0.04 and 1.6% of the plants in infected fields had streak symptoms. Wild grass species with similar streaking symptoms were observed adjacent to one of these fields. Potted stalks collected from infected N44 plants germinated in a glasshouse exhibited streak symptoms within 10 days. Virus genomes were isolated and sequenced from a symptomatic N44 and Urochloa plantaginea plants collected from one of the surveyed fields (1). Phylogenetic analysis determined that while viruses from both plants closely resembled the South African maize-adapted MSV strain, MSV-A4 (>98.5% genome-wide sequence identity), they were only very distantly related to SSV (~65% identity; MSV-Sasri_S: EU152254; MSV-Sasri_G: EU152255). To our knowledge, this is the first confirmed report of maize-adapted MSV variants in sugarcane. In the 1980s, "MSV strains" were serologically identified in sugarcane plants exhibiting streak symptoms in Reunion and Mauritius, but these were not genetically characterized (2,3). There have been no subsequent reports on the impact of such MSV infections on sugarcane cultivation on these islands. Also, at least five MSV strains have now been described, only one of which, MSV-A, causes significant disease in maize and it is unknown which strain was responsible for sugarcane diseases on these islands in the 1980s (2,3). MSV-A infections could have serious implications for the South African sugar industry. Besides yield losses in infected plants due to stunting and reduced photosynthesis, the virus could be considerably more difficult to control than it is in maize because sugarcane is vegetatively propagated and individual plants remain within fields for years rather than months. Moreover, there is a large MSV-A reservoir in maize and other grasses everywhere sugarcane is grown in southern Africa. References: (1) B. E. Owor et al. J Virol. Methods 140:100, 2007. (2) M. S. Pinner and P. G. Markham. J. Gen. Virol. 71:1635, 1990. (3) M. S. Pinner et al. Plant Pathol. 37:74, 1998.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization.

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge: a proof-of-concept study.

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.

Strategies for the prevention of cervical cancer by human papillomavirus vaccination.

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed.

Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. Xanthi.

The production of vaccine antigens in plants is a safe and potentially very cost-effective alternative to traditional expression systems. We investigated the possibility of transgenic plant expression of the Human papillomavirus (HPV) type 16 L1 major capsid protein, with and without nuclear localisation signals, in Nicotiana tabacum cv. Xanthi plants. The genes were stably integrated into the N. tabacum genome, and both the expressed proteins were capable of assembling into capsomers and virus-like particles. The proteins in concentrated leaf extracts (L1(Tr)) were tested for antigenicity using a panel of characterised monoclonal antibodies (Mabs). Neutralising and conformation-specific Mabs (H16:V5 and H16:E70) were shown to bind to both types of the plant-produced particles. We estimated the L1(Tr) product yield to be 2-4 microg per kg of fresh leaf material. Rabbits immunised with small doses of plant-produced particles elicited a weak anti-HPV-16 L1 immune response. Our results support the feasibility of using transgenic plants for the production of HPV vaccines.

Investigation of Maize streak virus pathogenicity determinants using chimaeric genomes.

Genes and intergenic regions were reciprocally exchanged between a highly pathogenic Maize streak virus (MSV) isolate (MSV-MatA) and three less pathogenic isolates (MSV-Kom, MSV-R2, and MSV-VW) to determine the contribution of individual genome constituents to MSV pathogenicity in maize. Comparison of disease symptoms produced by the 54 resulting chimaeras and parental viruses enabled identification of genome constituents that are primarily responsible for the heightened pathogenicity of MSV-MatA in maize. Whereas pathogenicity determinants were detected in all of the MSV genomic regions examined, generally only chimaeras containing the MSV-MatA long intergenic region, coat protein gene, and/or movement protein gene were more pathogenic than the milder MSV isolates from which most of their genomes were derived. The pathogenicity of chimeras was strongly influenced by the relatedness of their parental viruses and evidence was found of nucleotide sequence-dependent interactions between both coding and intergenic regions.

Biological and Genomic Sequence Characterization of Maize streak virus Isolates from Wheat.

ABSTRACT Maize streak virus (MSV) is best known as the causal agent of maize streak disease. However, only a genetically uniform subset of the viruses within this diverse species is actually capable of producing severe symptoms in maize. Whereas these "maize-type" viruses all share greater than 95% sequence identity, MSV strains isolated from grasses may share as little as 79% sequence identity with the maize-type viruses. Here, we present the complete genome sequences and biological characterization of two MSV isolates from wheat that share approximately 89% sequence identity with the maize-type viruses. Clonal populations of these two isolates, named MSV-Tas and MSV-VW, were leafhopper-transmitted to Digitaria sanguinalis and a range of maize, wheat, and barley genotypes. Whereas the two viruses showed some differences in their pathogenicity in maize, they were both equally pathogenic in D. sanguinalis and the various wheat and barley genotypes tested. Phylogenetic analyses involving the genome sequences of MSV-Tas and MSV-VW, a new maize-type virus also fully sequenced in this study (MSV-VM), and all other available African streak virus sequences, indicated that MSV-Tas and MSV-VW are close relatives that together represent a distinct MSV strain. Sequence analyses revealed that MSV-VM has a recombinant genome containing MSV-Tas/VW-like sequences within its movement protein gene.

Forced recombination between distinct strains of Maize streak virus.

Recombination between divergent virus genomes is believed to be a major mechanism for generation of novel virus genotypes. We have examined the recombination process in geminiviruses by forcing recombination between two distinct isolates of Maize streak virus (MSV), MSV-Kom and MSV-Set. Heterodimeric agroinfectious constructs containing tandemly cloned mixtures of complete or partial MSV-Set and MSV-Kom genomes were used to simulate a circular dimeric form similar to that which would be expected to occur following a single intermolecular crossing-over event between MSV-Set and MSV-Kom replicative form DNAs at the long intergenic region (LIR)-movement protein gene (MP) interface. We isolated, analysed and biologically characterized many of the recombinant MSV genomes that were generated from the constructs in planta. Apart from having the same simulated breakpoint at the LIR-MP interface, all the genomes examined had a second breakpoint that had been generated through either intramolecular homologous recombination or a replicational release mechanism. The pathogenicities of six predominantly MSV-Kom-like recombinants were tested in maize. While all were capable of producing a symptomatic infection in this host, none was more virulent than MSV-Kom and only two were more virulent than MSV-Set. The two most virulent recombinants were leafhopper transmitted to a range of differentially MSV-resistant maize, wheat and barley genotypes and both were found to have unique biological properties.

Sequence diversity and virulence in Zea mays of Maize streak virus isolates.

Full genomic sequences were determined for 12 Maize streak virus (MSV) isolates obtained from Zea mays and wild grass species. These and 10 other publicly available full-length sequences were used to classify a total of 66 additional MSV isolates that had been characterized by PCR-restriction fragment length polymorphism and/or partial nucleotide sequence analysis. A description is given of the host and geographical distribution of the MSV strain and subtype groupings identified. The relationship between the genotypes of 21 fully sequenced virus isolates and their virulence in differentially MSV-resistant Z. mays genotypes was examined. Within the only MSV strain grouping that produced severe symptoms in maize, highly virulent and widely distributed genotypes were identified that are likely to pose the most serious threat to maize production in Africa. Evidence is presented that certain of the isolates investigated may be the products of either intra- or interspecific recombination.

The relative infectivities and genomic characterisation of three distinct mastreviruses from South Africa.

The genomic nucleotide sequences of the cloned agroinfectious genomes of three South African mastreviruses obtained from Zea mays, a Setaria sp., and Panicum maximum (designated MSV-Kom, MSV-Set, and PanSV-Kar respectively), were determined. Additionally, their relative infectivities and virulence were analysed in a range of differentially susceptible wheat, maize, and barley genotypes. MSV-Kom produced moderate to severe streak symptoms in all maize genotypes tested, but only moderate to very mild symptoms in the wheat and barley genotypes. MSV-Set infected only the susceptible to tolerant maize genotypes, but was generally more severe in the barley and wheat genotypes than MSV-Kom. PanSV-Kar was incapable of infecting any of the wheat and barley genotypes and only produced very mild symptoms on the three most sensitive maize genotypes. Genomic characteristics in common with related mastreviruses were identified. Phylogenetic analysis indicated that while MSV-Kom was closely related to previously sequenced MSV isolates, MSV-Set and PanSV-Kar represented distinctly novel strains of MSV and PanSV respectively. In the case of MSV-Set, this is the most distantly related MSV strain yet characterised.

Investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants.

There has been a great deal of interest in the possibility that geminiviruses might be used as infectious gene vectors for expression of foreign proteins in plants. However, generic mastreviruses such as Maize streak virus (MSV) have no sequences which are dispensable for systemic infection of plants, and there is a strict limitation on the size of viral DNA which can be moved systemically. We attempted to complement the movement functions deleted from a wild-type-sized, replication-proficient gene replacement vector, by co-infecting plants with it and either wild type MSV, or a replication-deficient but putatively movement-proficient viral construct. While ssDNA formation by the gene replacement vector could be complemented in trans by co-transfected wild type virus, true systemic movement of either the vector, or of co-complementing constructs, did not occur. However, recombination between the two complementing viral constructs frequently occurred to generate wild-type virus genomes. The results therefore suggest that formation of ssDNA and size of the viral replicon are not the sole determinants of whether the MSV movement proteins can mobilise viral sequences and move them systemically in plants.

Analysis of the diversity of African streak mastreviruses using PCR-generated RFLPs and partial sequence data.

Maize streak virus (MSV) is the most economically significant member of a diverse group of African grass-infecting Mastrevirus species in the family Geminiviridae. We designed a single set of degenerate primers which enables the PCR amplification of an approximately 1300 bp DNA fragment spanning both conserved (the RepA gene) and variable (the long intergenic region and MP gene) portions of these viruses' genomes. Using restriction fragment length polymorphism (RFLP) analysis of PCR products obtained from 39 MSV, one SSV, and two PanSV isolates, it was possible to both identify the different virus species, which differ in nucleotide sequence by up to 40%, and to differentiate between MSV isolates sharing up to 99% sequence identity. The reliability of the RFLP data for typing the MSV isolates was verified by the phylogenetic analysis of the partial genomic nucleotide sequences of a representative subset of the MSV isolates. Based on both the RFLP and sequence data, the MSV isolates could be clearly differentiated into the four groups: these were a group of predominantly maize-infecting isolates, and three groups containing grass/wheat-infecting isolates. RFLP analysis also revealed a number of mixed virus infections in which, in certain instances, it was possible to identify individual population members.

Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses.

Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.