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.

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.

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 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.

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.

An association between HIV-1 subtypes and mode of transmission in Cape Town, South Africa.

OBJECTIVES: To determine HIV-1 env and gag subtypes in male homosexual and heterosexual populations in Cape Town, South Africa. DESIGN: DNA was isolated from blood originating from 61 patients attending local clinics. Samples were divided according to presumed mode of transmission: male homosexual (n = 26), heterosexual/vertical (n = 32), blood transfusion (n = 1) and unknown (n = 2). METHODS: Proviral HIV-1 DNA was subtyped by heteroduplex mobility assay (HMA) based on the 799 base-pair V3-V5 region of the env gene (n = 47) or by sequence analysis of the p17 region of the gag gene (n = 33), or both. For HMA, reference plasmids were constructed containing the V1-V5 env region sequences (1.2-kb) representative of local subtypes. Subtype designation of reference subtypes was confirmed by sequence analysis of the V3-loop region. RESULTS: Analysis of the partial gag sequences and HMA of the V3-V5 env region identified three subtypes: B, C and D. A fourth env subtype, subtype E, was also identified by HMA. Subtypes were found to segregate according to mode of transmission, with subtype B viruses found in 96% (25 out of 26) of the male homosexual group and subtype C viruses found in 81% (26 out of 32) of the heterosexual/vertical transmission group. Subtype B viruses were also found in four heterosexual patients, one patient infected by blood transfusion and in two patients with unknown mode of transmission. Subtype D viruses were found in one male homosexual patient and one heterosexual patient. A subtype E virus was identified in a heterosexual patient. No discrepancy was found in subtype designation in samples analysed in both between the gag and env regions (n = 19). CONCLUSIONS: Subtype B viruses were associated with male homosexual transmission and subtype C viruses with heterosexual transmission, suggesting two independent epidemics. This data may have implications in the selection of appropriate vaccines for different risk groups in the country.

PIP: Investigations of the genetic heterogeneity of human immunodeficiency virus (HIV) -1 are important both to monitor the spread of the virus to new population groups and to the development of vaccines the efficacy of which could be influenced by virus variations. This study analyzed serum samples from 61 HIV-1 infected individuals recruited from clinics in Cape Town, South Africa. The mode of transmission was male homosexual in 26 cases, heterosexual/vertical in 32 cases, blood transfusion in 1 case, and unknown in 2 cases. Proviral HIV-1 DNA was subtyped by heteroduplex mobility assay (HMA) based on the 700 base-pair V3-V5 region of the env gene or by sequence analysis of the p17 region of the gag gene. This process identified 3 subtypes: B, C, and D. A fourth env subtype (E) was also identified by HMA. Subtypes were significantly (p 0.001) associated with the mode of HIV transmission. Subtype B viruses were found in 96% of male homosexual sera and subtype C viruses were identified in 81% of the heterosexual/vertical transmission group. Subtype B viruses were also identified in 4 heterosexuals, 1 person infected through blood transfusion, and the 2 cases where the mode of transmission was unknown. Subtype D viruses were found in 1 male homosexual and 1 heterosexual, while a subtype E virus was identified in a heterosexual patient. These findings imply that heterosexual and homosexual HIV-1 epidemics in South Africa were independent. Both epidemiological and molecular data suggest that the initial epidemic in South Africa was, in part, a result of the introduction of HIV-1 by homosexuals who had sexual contacts with men in the US or Europe. The second, heterosexual epidemic was most likely a result of regional spread.

Coat protein phylogeny and systematics of potyviruses.

The feasibility of applying molecular phylogenetic methods of analysis to aligned coat-protein sequences and other molecular data derived from coat proteins or genomic sequences of members of the proposed taxonomic family of Potyviridae, is discussed. We show that comparative sequence analysis of whole coat-protein sequences may be used reliably to differentiate between sequences of closely related strains, and to show groupings of more distantly related viruses; that coat proteins of putative Potyviridae cluster according to the proposed generic divisions, and, even if some are only very distantly related, the members of the family form a cluster distinct from coat proteins of other filamentous and rod-shaped viruses. Taxonomic revisions based on perceived evolutionary relationships, and the lack of feasibility of erecting higher taxa for these viruses, are discussed.

Enzyme-assisted immune detection of plant virus proteins electroblotted onto nitrocellulose paper.

A technique for the detection of plant virus coat proteins in plant sap is described. The method entails the electroblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis-fractionated plant extracts onto nitrocellulose paper, probing the paper with virus-specific rabbit antisera, and indirect detection of virus proteins with horseradish peroxidase-conjugated goat anti-rabbit globulins. The sensitivity and specificity of the technique were tested using brome mosaic and barley stripe mosaic viruses. As little as 1 ng per track of virus protein was detectable, either as pure virus or when mixed with plant sap. Distant serological relationships were detected amongst tobamoviruses, and amongst the bromoviruses, with single antisera. The uses of the technique in probing capsid configuration in a presumed aphid picornavirus, and in routine diagnostic practice, are described.

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.

A polymerase chain reaction method adapted for selective amplification and cloning of 3' sequences of potyviral genomes: application to dasheen mosaic virus.

'Universal' degenerate oligonucleotide primers were used to amplify cDNA sequences containing the 3' untranslated region (3' UTR) and a portion of the coat protein gene sequence of dasheen mosaic potyvirus (DMV). These primers were based on the conserved WCIEN and QMKAAA 'boxes' of the potyviral coat protein and the poly-A tail found at the 3' end of the genome. The forward genome-sense primers were designed taking into consideration the codon degeneracy of the WCIEN and QMKAAA residues for several potyviruses. The anti-sense reverse primer has 21 T residues followed by either A, C or G at the 3' end to ensure specific priming at the end of the 3' UTR and beginning of the poly-A tail. The specificity of amplification was verified using the known potyviruses (watermelon mosaic 2 and soybean mosaic viruses). To demonstrate the applicability of this method, the 3' UTR of the unsequenced DMV was amplified, cloned and sequenced. Sequence comparisons with other potyviral 3' UTRs revealed DMV to be quite distinct: nucleotide sequence similarities of only 34% to 44% were found with sequenced viruses indicating no close affinities with any other potyvirus. The potyvirus 3' sequence amplification procedure is simple and rapid, is potentially useful in developing virus specific probes and may be used to differentiate strains and species of potyviruses on the basis of the 3' UTR sequences.

Microcomputer-Based Quantification of Maize Streak Virus Symptoms in Zea mays.

ABSTRACT We investigated the use of computer-assisted image analysis techniques for the objective quantification of maize streak virus (MSV) symptoms in Zea mays. We compared independent duplicate evaluations of chlorotic lesion areas occurring on MSV-infected leaves using visual assessment, a commercial image analysis system, and a custom image analysis system employing software developed in our laboratory. Relative to visual assessments of disease severity, computer-assisted image analysis employing both the commercial and custom systems provided significant enhancements in the accuracy and precision of chlorotic area estimations. The commercial image analysis system afforded no significant improvement in precision or accuracy over the custom system. An important advantage of examining images using the custom-written software was that the software permitted a high degree of analysis automation. Digitized images of maize leaves could be automatically analyzed by the custom software five times faster than, and with the same precision and accuracy as, when the same images were analyzed with the commercial software. Because of the flexibility of the image analysis techniques described, they should be applicable to the measurement of symptom severity in other plant host-pathogen combinations.

Evaluation of Maize Streak Virus Pathogenicity in Differentially Resistant Zea mays Genotypes.

ABSTRACT We devised a rapid technique for the objective and precise assessment of both the pathogenicity of maize streak virus (MSV) isolates and the MSV resistance of maize genotypes. The technique involves the use of agroinoculation to infect maize seedlings and the objective symptom evaluation by quantification of infection rates, stunting, and chlorotic leaf areas. In assessing the MSV resistance of 19 maize genotypes, we describe how the use of differentially virulent virus isolates enables the analysis of MSV resistance phenotypes, ranging from extremely susceptible to completely immune. We further demonstrate how quantification of chlorotic leaf areas by image analysis permits differentiation between degrees of MSV resistance that are indistinguishable from one another using currently employed symptom assessment approaches. Using chlorotic area measurements, we quantify the virulence of a diverse group of 10 MSV isolates and, through agroinoculation of differentially susceptible maize genotypes, we demonstrate the use of our technique in evaluating the pathogenicity of these isolates.

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.

Improved Efficiency of Zea mays Agroinoculation with Maize streak virus.

Agroinoculation is a technique permitting the transmission of geminivirus genomes cloned in Agrobacterium tumefaciens into a wide variety of mono- and dicotyledonous host plants. Most geminiviruses are obligately transmitted by insect vector species under natural conditions; therefore, agroinoculation has greatly simplified the study of this group of viruses. In many cases, agroinoculation has replaced insect transmission, and has been used to compare virulence characteristics among viruses. Here we report on the discovery that, in agroinfectious Maize streak virus constructs, the orientation of cloned viral genomes relative to the Cauliflower mosaic virus 35S (CaMV35S) promoter of the binary cloning vector pBI121 can significantly affect agroinfectivity of the constructs. Rates at which plants became symptomatic were significantly higher when agroinoculating maize seedlings with constructs containing the CaMV35S promoter upstream of the viral replication-associated protein (Rep) gene than when the same viruses were cloned either in the opposite orientation or into a vector without a strong eukaryotic promoter sequence. Plants infected using the construct with Rep cloned downstream of the CaMV35S promoter also displayed more stunting and, in the early stages of the infection, more severe chlorotic streak symptoms.

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.

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.

The use of serological differentiation indices for the phylogenetic analysis of plant virus relationships.

The potential use of serological differentiation indices (SDIs) in the phylogenetic analysis of plant virus coat protein relationships is presented. Relationship dendrograms were constructed from SDI data for viruses in the tymovirus and Subgroup I geminivirus taxonomic groups, using distance or phenetic methods embodied in the computer programmes FITCH and NJTREE. Relationship dendrograms for geminiviruses agreed well with those constructed by others from sequence or other data; the dendrogram for tymoviruses was not in agreement with one calculated from amino acid composition data, or from other properties, but appears consistent with recently-shown sequence similarities between tymoviruses. Implications of these results for the classification and taxonomy of plant viruses are discussed.