Aims & Objectives
40
CHAPTER II
Aims & Objectives
41 The identification of previously uncharacterized viruses in humans and animals represents an important objective in virology. The optimization of molecular tools for this purpose, including representational difference analysis, immunoreactive cDNA expression library screening, DNA microarrays, and use of degenerate PCR primers, has resulted in the detection and characterization of hepatitis C virus (HCV) (Chaoo et al., 1989), human herpesvirus 8 (HSV) (Chang et al., 1994), the severe acute respiratory syndrome (SARS) coronavirus (Wang et al., 2002, 2003), and TTV (Mushahwar et al., 1999; Nishizawa et al., 1997). However, these methods are labor-intensive and require weeks to analyze each sample. Access to a preinfection as well as an acute phase sample may also be required. These limitations inhibit analysis of multiple serum samples and thereby reduce the chance of identifying viral agents. Main objective of the current study was to develop innovative and highly sensitive methods for the detection and characterization of members of the family Anelloviridae.
The rolling-circle amplification (RCA) is a novel technique used to amplify complete circular DNA genomes (Dean et al., 2001; Niel et al., 2005). In this method, a random hexamer primer anneals to multiple sites on a DNA template; these sites are isothermally extended by Phi29 DNA polymerase (Garmendia et al., 1992; Esteban et al., 1993), therefore producing multiple copies of the complete genome, without the need for prior knowledge of their sequences. When combining the RCA technique with the SISPA approach, the potential of SISPA is increased (Biagini et al., 2007). This approach is based on the use of endonuclease restriction of target sequences
Aims & Objectives
42 previously converted to double-stranded DNA, followed by nonspecific linker ligation and PCR amplification (Reyes et Kim., 1991; Allander et al., 2001). Considering that the enzymes used cut several time all TTV isolates, each fragment obtained could represent a new partial TTV sequence. The recent characterization of novel anelloviruses in cat (Biagini et al., 2007) highlighted the potential of the RCA-SISPA method for detecting species-specific anelloviruses in yet unexamined animals.
Furthermore, to increase the probability to discover new anelloviral sequences, a specific RCA followed by PCR amplification was also optimized.
Therefore, in the present study we aimed to: a) perform in swine serum sample the combined RCA-SISPA approach previously set up in other species (Biagini et al., 2007); b) optimize in pig and human sera a novel RCA assay based on Anellovirus-specific primers (Anello-RCA) followed by inverse PCR (Anello-PCR) to obtain the full-length genomes; c) apply the optimized Anello-RCA/PCR in other animal species whose TTVs have not been fully characterized yet.
In addition, the efficiencies of the two techniques RCA-SISPA and Anello-RCA/PCR were compared, highlighting their advantages and limitations in obtaining our purpose.
