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CHAPTER V
72 The RCA-based sequence-independent approach is a powerful methodology that allows detecting new circular DNA structures, mainly viruses (Niel et al., 2005). This technique uses the bacteriophage phi29 DNA polymerase together with random hexamer primers to generate thousands of copies of circular DNA templates (Dean et al., 2001; Esteban et al., 1993; Rector et al., 2004; Stevens et al., 2010). The phi29 DNA polymerase is a high fidelity enzyme with strand displacement activity, proof-reading activity and high progression (Garmendia et al., 1992) and when combined with random primers there is no need for previous knowledge of the sequence to be amplified. In combination with RCA, a SISPA approach has been successfully applied to human plasma and cat saliva samples to identify nine members of the family
Anelloviridae (Biagini et al., 2007). The applied approach was based on the use of endonuclease restriction of target sequences previously converted to double-stranded DNA, followed by non-specific linker ligation and PCR amplification (Biagini et al., 2007; Allander et al., 2001). However, this technique is poorly discriminative if the objective is to detect specific viruses, since RCA by random primers is able to amplify whatever circular DNA present in a given sample.
In the present study we demonstrate that the combined RCA-SISPA is a useful approach also for swine species. Although the major part of fragments obtained corresponded to other genomes, 3 sequences related to TTSuV2 were detected. Although the filtration process combined with Benzonase-treatment should be probably improved to furtherly reduce endogenous DNA contamination, the results confirm that the RCA-SISPA approach works also in swine.
73 Furthermore, the study demonstrates that the optimized Anello-RCA combined with Anello-PCR technique is a reproducible tool for anellovirus detection in sera from swine and human species. The advantages of this novel assay are: a) the handling is very simple; b) no expensive devices are necessary; c) the costs per reaction are very low; and d) more importantly multiple copies of complete viral genome could be easily obtained.
According to quantitative PCR results obtained, the Anello-RCA technique amplifies target sequences more than 10000-fold, highlighting the efficiency of the PCR assay.
Furthermore, Anello-PCR applied on Anello-RCA products was successful in amplifying the full-length genomes of swine and human TTVs. Full-length genomes could be directly cloned and no further manipulation or amplification with specific primers was necessary. For identification purposes, only Anello-PCR products can be even sequenced without prior cloning. Due to the short nature of Anello-RCA and Anello-PCR primers, other templates including bacterial genome and host DNA sequences may be also amplified. Analysis of the different sequences belonging to the family Anelloviridae shows that multiple genomic locations are susceptible to be the targets for the short Anello-RCA primers, depending on the TTV isolate. Taking into account that also unexpected fragments were obtained from PCR, the Anello-RCA would not be so strictly specific to Anelloviridae genomes, as it happens for the standard RCA. However, the amplicons corresponding to other templates were always shorter than expected TTVs full-length genome and, therefore, easy to discriminate from subsequent analyses.
74 Considering that the expected PCR fragments were more clearly obtained from human sera (Fig. 13B; 13C; 14B), probably the Anello-RCA could be considered more extendable to human than swine TTV species. Taking into account the high variability of Anellovirus sequences and the frequency of co-infections of many genotypes in humans, the optimization of the Anello-PCR for each of them should be performed. By Anello-RCA and optimized genotype-specific Anello-PCRs, studies of genetic diversity could derive advantage.
In comparison with conventional PCR, the sensitivity of the presented technique is higher and complete viral genomes could be easily obtained. Indeed, members of the genus Iotatorquevirus and Alphatorquevirus were detected by this new developed technique in samples previously resulted negative for anelloviruses by conventional PCR. Theoretically, considering the location of the Anello-primers in the highly conserved region surrounding the TATA box in the UTR and the easiness of the method described here, whatever anelloviral sequence presenting no many mismatches should be amplified. Therefore, the Anello-RCA could be useful in detecting and cloning full-length anelloviral genomes from other species whose TTVs sequences have not been fully characterized as yet. To date, only partial TTVs sequences from animal species such as chicken, bovine and sheep, are present in GenBank database. Phylogenetic analysis of these partial sequences aligned with complete TTV genomes reveals the lacked characterization of untranslated region in these animal TTV sequences. However, since the primers that we have used for RCA are deduced from a UTR fragment highly conserved among all sequenced TTVs, we can not exclude the probability to detect anelloviral sequences from these animal species by using our
75 approach. Furthermore, also from birds species, whereof nowadays neither partial TTVs sequences have been detected, anelloviral sequences could be discovered by this technique. As in amplifying full-length TTSuV and TTV genomes Anello-PCRs were properly optimized, further different reaction conditions for detecting anellovirus in the other species should be tested.
At present, no positive TTV DNA detections were obtained from serum samples of various animal species examined for Anello-RCA/Anello-PCR at the described conditions (see Materials & Methods).
It would be also interesting to verify if anelloviral sequences can be detected in these animal species by using the RCA-SISPA approach.
BothAnello-RCA/PCR and RCA-SISPA methods described here, and the second
one also in early reports (Biagini et al., 2007), are applicable to detect members of the family Anelloviridae. It could seem quite surprising to compare a sequence-based (PCR) method with a sequence-independent single-primer amplification (SISPA), since it is known that the classical SISPA approach suffers sensibility (under 106 target copies/mL) (Allander et al., 2001; Biagini et al., 2007). Therefore, in the present study we simply show the application of two different optimized methods for detecting anelloviral sequences. Certainly the novel RCA followed by Anello-PCR results a faster and easier method that could increase the probability to obtain Anelloviridae genomes. The RCA-SISPA technique is more tedious and pricey than the Anello-RCA due to the high number of different size fragments to be analyzed. Indeed, the restriction enzymes (TaqI and Csp6I) used for RCA-SISPA theoretically cut several times all TTV sequences available in the GenBank database. This situation results in a
76 series of different size fragments seen on the agarose gel that need to be linked to corresponding adaptors for subsequent PCR amplification, cloning and sequencing. Since each digestion product represents a likely part of viral genome, no discrimination of the fragments can be done leading to intensive cloning and sequencing. Based on the current results, only few fragments generated using RCA-SISPA method represent anelloviral sequences. On the contrary, Anello-RCA combined with Anello-PCR allows selection of fragments to be analyzed prior to cloning and/or sequencing, thus making this method less tedious. While Anello-RCA is applicable to detect mainly anellovirus species, the more unspecific RCA-SISPA method can potentially detect any circular DNA virus present in a given sample. Considering that the primers used in Anello-RCA/PCR are located in the UTR and the easiness of the method described here, this novel technique could be applicable for the detection of members of the family Anelloviridae in other, yet unexamined animal species.
