D. P. Martin

Brazilian Begomovirus Populations Are Highly Recombinant, Rapidly Evolving, and Segregated Based on Geographical Location

ABSTRACT The incidence of begomovirus infections in crop plants sharply increased in Brazil during the 1990s following the introduction of the invasive B biotype of the whitefly vector, Bemisia tabaci. It is believed that this biotype transmitted begomoviruses from noncultivated plants to crop species with greater efficiency than indigenous B. tabaci biotypes. Either through rapid host adaptation or selection pressure in genetically diverse populations of noncultivated hosts, over the past 20 years various previously unknown begomovirus species have became progressively more prevalent in cultivated species such as tomato. Here we assess the genetic structure of begomovirus populations infecting tomatoes and noncultivated hosts in southeastern Brazil. Between 2005 and 2010, we sampled and sequenced 126 DNA-A and 58 DNA-B full-length begomovirus components. We detected nine begomovirus species in tomatoes and eight in the noncultivated host samples, with four species common to both tomatoes and noncultivated hosts. Like many begomoviruses, most species are obvious interspecies recombinants. Furthermore, species identified in tomato have probable parental viruses from noncultivated hosts. While the population structures of five well-sampled viral species all displayed geographical subdivision, a noncultivated host-infecting virus was more genetically variable than the four predominantly tomato-infecting viruses.

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.

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.

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.

Characterisation of Potato virus Y nnp strain inducing veinal necrosis in pepper: A naturally occurring recombinant strain of PVY

Summary.The full-length genome of Potato virus Y (PVY) nnp strain, recovered from pepper showing veinal necrosis of leaves, was cloned and sequenced, finding an organisation typical for PVY species. It consists of 9699 nucleotides (nt) excluding the 3′ terminal poly(A) tail and contains an open reading frame of 9186 nt, encoding the putative polyprotein of 3061 amino acids.In ELISA, the isolate reacted with a monoclonal antibody specific for PVYC but not with antibodies against PVYN or PVYO. Sequence analysis strongly suggests that PVY-nnp originated from a recombination event involving a virus of the PVYO type and another parental virus, maybe resembling the PVYNP isolates, given the reasonably high similarity shared by PVY-nnp and Lye84.2 and Son41 isolates. The recombination event involved a breakpoint near the middle of the P1 gene, around position 603 of the viral genome. Proof for the existence of such a recombination comes from several lines of evidence, including similarity analysis, recombination analysis using six different methods and the different locations of nnp within phylogenetic trees constructed from genomic regions on either side of the identified recombination breakpoint.

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

Summary. 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.

Coat protein gene diversity among Chrysanthemum virus B isolates from India

Summary.The complete coat protein (CP) sequences from 29 Indian isolates of Chrysanthemum virus B (CVB) were determined and analysed in relation to other previously characterized carlaviruses. The CP genes of the Indian CVB isolates were highly heterogeneous, sharing nucleotide sequence identities of 74–98%. Based on phylogenetic analyses, the isolates formed three groups potentially representing either two or three major CVB strain groupings. Recombination analysis revealed at least one definite recombination event involving the exchange of sequences between members of different groups. To our knowledge this is the first reported evidence of homologous recombination in carlaviruses.

Virological and Immunological Factors Associated with HIV-1 Differential Disease Progression in HLA-B*58:01 Positive Individuals

ABSTRACT Molecular epidemiology studies have identified HLA-B*58:01 as a protective HIV allele. However, not all B*58:01-expressing persons exhibit slow HIV disease progression. We followed six HLA-B*58:01-positive, HIV subtype C-infected individuals for up to 31 months from the onset of infection and observed substantial variability in their clinical progression despite comparable total breadths of T cell responses. We therefore investigated additional immunological and virological factors that could explain their different disease trajectories. Cytotoxic T-lymphocyte (CTL) responses during acute infection predominantly targeted the TW10 and KF9 epitopes in p24Gag and Nef, respectively. Failure to target the TW10 epitope in one B*58:01-positive individual was associated with low CD4+ counts and rapid disease progression. Among those targeting TW10, escape mutations arose within 2 to 15 weeks of infection. Rapid escape was associated with preexisting compensatory mutations in the transmitted viruses, which were present at a high frequency (69%) in the study population. At 1 year postinfection, B*58:01-positive individuals who targeted and developed escape mutations in the TW10 epitope (n = 5) retained significantly higher CD4+ counts (P = 0.04), but not lower viral loads, than non-B*58:01-positive individuals (n = 17). The high population-level frequency of these compensatory mutations may be limiting the protective effect of the B*58:01 allele.

Virus-Derived ssDNA Vectors for the Expression of Foreign Proteins in Plants

Plant viruses with ssRNA genomes provide a unique opportunity for generating expression vehicles for biopharming in plants, as constructs containing only the replication origin, with the replication-associated protein (Rep) gene provided in cis or in trans, can be replicationally amplified in vivo by several orders of magnitude, with significant accompanying increases in transcription and expression of gene(s) of interest. Appropriate replicating vectors or replicons may be derived from several different generic geminiviruses (family Geminiviridae) or nanoviruses (family Nanoviridae), for potential expression of a wide range of single or even multiple products in a wide range of plant families. The use of vacuum or other infiltration of whole plants by Agrobacterium tumefaciens suspensions has allowed the development of a set of expression vectors that rival the deconstructed RNA virus vectors in their yield and application, with some potential advantages over the latter that still need to be explored. Several modern applications of ssDNA plant vectors and their future potential will be discussed.

Genomic sequence analysis of four new chrysanthemum virus B isolates: evidence of RNA recombination

Chrysanthemums worldwide suffer from a high incidence of infection with chrysanthemum virus B (CVB), a member of the genus Carlavirus, family Betaflexiviridae. Three major lineages or strains of this virus have been found in India, but none have been characterized beyond the genetic variation they display in their coat protein genes. Here, we describe the analysis of four near-complete genome sequences (from the three lineages) representing the genetic diversity of these strains. Ranging in size from 8815 to 8855 nucleotides (excluding the polyA tail), these four isolates have a genome organization very similar to that of the recently reported Japanese isolate of CVB, with which they share between 70 and 73% genome-wide sequence identity. We present further evidence that recombination may feature quite prominently in the evolution of CVB.