CHAPTER 1: LITERATURE REVIEW
1.7 Detection and diagnosis of tospoviruses
1.7.2 Diagnosis
Several tospoviral diagnostic techniques are available, and more continue to be developed.
Basically, the techniques can be classified into symptomatology, host indexing, serology, electron microscopy and molecular techniques.
Host indexing
Indexing is any test that reproducibly assesses the presence or absence of transmissible pathogens, or identifies a disease on the basis of reaction induced on specific indicator plants. Tospoviruses can be detected on the basis of symptom production on indicator plants. The indicator plants must be virus-free and easy to grow. They must react rapidly and specifically to a particular virus species, exhibit diagnostic symptoms under different conditions, and be readily available during the growing seasons (Jan et al., 2012). There are a number of indicator plants for tospoviruses (Table 1.2). Though it has high sensitivity, indexing is time-consuming and does not identify the actual virus.
22 Table 1.2 Some indicator hosts for tospoviruses
Botanical name Common name Family
Arachis hypogaea Groundnut Fabaceae
Capsicum annuum Pepper Solanaceae
Chenopodium album Fat hen Amaranthaceae
Chenopodium quinoa Quinoa Amaranthaceae
Cucumis sativus Cucumber Cucurbitaceae
Datura stramonium Apple of Peru Solanaceae
Impatiens Snapweed Balsaminaceae
Solanum lycopersicum Tomato Solanaceae
Spinacia oleracea Spinach Amaranthaceae
Nicotiana tabacum Common tobacco Solanaceae
Nicotiana glutinosa Tobacco Solanaceae
Nicotiana clevelandii Cleveland’s tobacco Solanaceae
Nicotiana benthamiana Tobacco Solanaceae
Petunia hybrida Petunia Solanaceae
Phaseolus vulgaris Field bean Fabaceae
Pisum sativum Peas Fabaceae
Vigna unguiculata Cowpea Fabaceae
23 Symptomatology
Symptoms assessment is the initial step in tospovirus diagnosis. While some tospoviral diseases are readily diagnosed by visual examination of host symptoms, other hosts though infected, are often symptomless and require further diagnostic tests. Sometimes, different viruses can cause similar symptoms in a plant (Webster et al., 2004). In other instances, different strains of the same virus may induce different symptoms in the same host. Some symptoms are cultivar-dependent and also vary with environmental conditions (Naidu and Hughes, 2003).
Electron microscopy
The electron microscope uses a beam of highly energetic electrons to illuminate objects on a very fine scale to yield the topography, morphology, composition and crystallographic information. For viruses to be visualized, their concentration must be at least 105-6 particles per ml (Biel et al., 2004). Compared to other techniques, electron microscopy is expensive, and requires highly technical and experienced staff for operation. It cannot detect viruses with much higher sensitivity and specificity. However, as the only spherical and enveloped plant viruses, tospoviruses are readily identified by electron microscopy (German et al., 1992; Sivparsad and Gubba, 2008).
Serology
Serological detection, or immunochemical diagnostics, is based on the use of monoclonal or polyclonal antibodies for virus detection. The antibodies are capable of binding to specific virus antigens (Strange, 2003). The most commonly used serological method is enzyme-linked immunosorbent assay (ELISA). It has two main variants, namely double antibody sandwich ELISA (DAS-ELISA) and triple antibody sandwich ELISA (TAS-ELISA) (Naidu and Hughes, 2003; Strange, 2003).
ELISA can be used to test multiple plants for a single tospovirus using a well per plant sample.
Also, a single plant can be simultaneously tested for many tospoviruses on a single plate with different antibodies coated to each well in duplicate or triplicate. It is accurate, cheap, highly
24 sensitive, simple, and can quantify pathogens. A major limitation of ELISA is that it requires monoclonal or polyclonal antibody sera specific for each virus of interest that does not cross-react with plant proteins. As such, one must to know the virus to be detected. It often fails if virus titre in the test sample is low (Webster et al., 2004; Jan et al., 2012). Despite advances made in virus diagnostics, serology remains very important in tospovirus identification.
Molecular diagnostics
They are also called nucleic-acid based methods. They rely on specific complementary association of the different bases that make up the nucleic acid molecules. Molecular assays are better alternatives to the other methods in terms of sensitivity, rapidity, specificity or in situations where no suitable serological tests are available. Specificity is directly related both to the design of primers and amplification protocols (Strange, 2003; Olmos et al., 2007). Commonly used molecular techniques are the reverse transcription polymerase chain reaction (RT-PCR) and next generation sequencing (NGS).
RT-PCR involves an initial step of reverse transcription that converts single strand RNA to complementary DNA (cDNA). This is accomplished by using the reverse transcriptase enzyme.
Subsequently, the newly synthesized cDNA is amplified using conventional PCR. RT-PCR is very sensitive and requires minimum skill to perform. The efficiency of virus detection depends on quality of template nucleic acid, polymerase enzyme, buffer composition, stability, purity and concentration of deoxynucleotide triphosphates (dNTPs) and cycling parameters (Jan et al., 2012).
Real-time RT-PCR, also called quantitative RT-PCR, is a quantitative and highly sensitive method for tospovirus detection. It does not need downstream analysis of PCR amplicons by gel electrophoresis, Southern blotting or sequencing as is the case with routine PCR assays (Vidhyasekaran, 2007).
25 Next-Generation Sequencing (NGS)
NGS, also called high-throughput sequencing, is a term used to describe a number of different modern sequencing technologies that allow the sequencing of nucleic acids much more quickly, accurately and cheaply than Sanger sequencing. This is accomplished by fragmenting entire genomes into small pieces and then ligating those small pieces to designated adapters for random read during DNA synthesis (Grada and Weinbrecht, 2013). Massive amounts of data, usually tens or hundreds of gigabytes per single run or terabytes per experiment, are generated in NGS (Xuan et al., 2013). Rare variant sequences can be identified (Reis-Filho, 2009) and complete genome sequences are identified in an unbiased fashion (Kreuze et al., 2009).
Several NGS platforms are available including; Roche GS-FLX 454, Illumina Genome Analyzer, ABI SOLiD Analyzer, Polonator and Helicos HeliScope. These platforms generate different base read lengths, error rates, and error profiles relative to Sanger sequencing and to each other. The average read length is 35-500 continous base-pair reads, compared to 1000-1200 bp for Sanger sequencing (Zhang et al. 2011). Since NGS technologies produce different short reads, coverage is very important if accurate genomic assembies are to be obtained.
The massive data generated needs to be properly stored and managed to successfully enjoy the benefits of NGS. Many software tools are available for NGS data analysis (Magi et al., 2010;
Zhang et al., 2013). The functions of the data analysis tools fit into several categories of alignment, assembly, base calling, genome annotation and data analysis utilities. Some of the software packages provide a user-friendly interface, easy-to-use data input and output formats, and integrated multiple computing programs into one software package. Examples of such end-user packages are the CLC Genomics Workbench, NextGENe and SeqMan Ngen (Zhang et al., 2011).