Project Title: Molecular methods for virus detection in fruit plants
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Table 6. Number of samples in which apple stem pitting virus was detected in extracts from apple and pear bark by PCR in December 1998 and February 1999 and in leaf extracts in May 1999.
Failure of PCR to detect ASPV in all these trees was probably not due to a lack of sensitivity. Tests on two trees taken at random, 1 apple and 1 pear, gave positive results with extract dilutions of 10-4 and PCR is more sensitive than ISEM. Despite the good performance of the IC-RT-PCR test there is obviously still a requirement for more genome information to be able to detect all the isolates of ASPV that we have in woody plants. We attempted to obtain such information from isolate 7/40 as it was not possible to detect ASPV by PCR using any of the primers either in samples from the pear tree 7/40, or in samples from this isolate that had been transferred to N. occidentalis. This isolate is of particular interest as tree 7/40 has symptoms of pear stony pit for which ASPV is thought to be the cause. Obtaining nucleotide sequence data for ASPV isolate 7/40ASPV isolate 7/40 shows typical symptoms in the herbaceous host Nicotiana occidentalis and there was no indication of contamination by another virus because inoculation to other herbaceous plants (Chenopodium quinoa, C.amaranticolor, Nicotiana clevlandi, and Cucumis sativus) failed to produce symptoms. Virus particles of 7/40 were similar in appearance to other ASPV isolates in ISEM and particles were heavily decorated with ASPV antiserum (Yanase). The same antiserum was used in western blot analysis. A faint cross reaction was observed to a protein from 7/40-infected N. occidentalis that migrated similarly to the standard ASPV isolate VYA, indicating that the coat protein of 7/40 is of the correct size for ASPV. cDNA library experiments Attempts were made to obtain cDNA clones of ASPV from viral RNA in two ways. In one, virus was trapped on plates coated with ASPV (Yanase) antiserum, to concentrate the virus. In the other, virus was concentrated centrifugally and reverse transcription primed with oligodT. Concentration of the cDNA was attempted by PCR with random primers and oligodT. None of the resulting clones in pUC18 proved to be virus related. PCR experiments In further PCR experiments, various sources of template were used. cDNA was made from immunocaptured ASPV isolates 7/40 and VYA and also from total RNA extracted from VYA and 7/40-infected N.occidentalis leaves (RNeasy plant kit, Qiagen). An oligodT-anchor primer was used in the reverse transcription procedure and PCR carried out using a variety of primers (Table 7). In each case a product of the expected size was amplified with VYA cDNA but nothing was seen in the 7/40 samples. There was the possibility that 7/40 did not have a polyA tail, but amplification using primers ASPV5 & 1 and ASPVF1& ASPVR5 from cDNA primed with random primers also failed to produce products for 7/40. Table 7. Primer combinations used for attempts to amplify ASPV isolates 7/40 and VYA
Cloning from dsRNAAn alternative route to provide viral RNA template for cDNA synthesis is to use purified double stranded RNA (dsRNA) replication intermediates isolated from infected N.occidentalis (Jelkmann et al., 1992). Double stranded RNA is extremely stable and has proved to be the best target for sensitive, reproducible PCR detection of ASGV (Kummert et al., 1998). DsRNA was isolated from healthy and 7/40 infected N.occidentalis plants (Fig. 1). No dsRNA was isolated from healthy material, so N.occidentalis does not contain non-viral dsRNAs. This meant that the dsRNA observed from 7/40 infected plants is of viral origin. Very little ASPV dsRNA was isolated from infected leaf material and the optimum time for harvesting for maximum dsRNA presence is still unknown. Dodds (1993) observed that the host and season have effects on dsRNA quantity and quality and that there is a poor correlation between relative dsRNA levels and relative virus concentration. The small quantity of dsRNA (200ng) isolated from 7/40 infected herbaceous host plants is not sufficient for standard cloning techniques which have been reported (Asamizu et al., 1985; Antoniw et al., 1986). However, sequence data has been obtained from several nanograms of dsRNA for two viruses that infect cherry (Jelkmann 1995; Keim-Konrad and Jelkmann 1996) but this technique involves the use of a very toxic compound, methyl mercuric hydroxide. Heat denaturation and reverse transcription of dsRNA is reported to be relatively inefficient (Jelkmann et al., 1989) but was successful in our hands for construction of a PCR amplifiable cDNA library immobilised on oligo(dT)25 beads (Dynal). PCR amplification was necessary due to the small amount of first strand cDNA produced so several forward primers were tried in combination with oligodT-anchor. A product approximately 440bp was obtained with the primer combination ASPV5- adapter (lane 3, fig. 2). This PCR product was cloned and sequence analysis confirmed the viral origin (fig. 3). The sequence of 7/40 does not fully explain why this isolate was not detected by the IC/RT-PCR test, although it must be noted that there are several mismatches within the 7/40 sequence for the diagnostic primers. It is possible that the lack of detection of 7/40 in previous PCR experiments was due to improper annealing of the reverse primer ASPV1. It is possible to amplify 7/40 using ASPV5 and oligo(dT)-adapter primers from dsRNA so PCR amplification experiments were carried out on total RNA extracted from healthy, and both VYA and 7/40 ASPV isolates in N.occidentalis. As for the diagnostic IC/RT-PCR test the cDNA was primed using an oligo(dT)-anchor primer but amplified using the primer combination ASPV5 and adapter. A product of the expected size was amplified from VYA infected plant material but nothing was amplified from the healthy or 7/40 infected plants (data not shown). This is surprising because we have used this primer combination for successful amplification from 7/40 dsRNA. Discussion and proposed future work The immunocapture PCR procedure using primers ASPV1 and ASPV5 was successful in amplifying many but not all isolates of ASPV. Sequence was obtained of an isolate (7/40) from pear that was consistently not detected in either the immunocapture PCR or in PCR from total RNA extracts from pear and from N. occidentalis. The sequence indicated that differences at the ASPV1 site might partially account for the failure to amplify this isolate, however, further sequence is needed for a detailed comparison of 7/40 with other isolates of ASPV. The novel technique that was used to obtain the cDNA library from very small amounts of dsRNA is promising, particularly as it used heat rather than the extremely toxic methyl mercuric hydroxide to denature the dsRNA. Antiserum is essential to the immunocapture PCR protocol and is unavailable commercially. It should be possible to utilise the protein expressed in objective 3 in E. coli for this purpose. This will probably not be satisfactory for ELISA but the results with ISEM emphasised the importance of considering serology as a test for this virus and indicated that it may be more successful than a nucleic acid based method because of the variability of the genome. A small amount of development of the results obtained here should further improve the PCR assay for ASPV and should also facilitate investigation of the relationship between pear stony pit and ASPV. 
H 7/40 M 
1613 2036 3054 4072 5090 Figure 1. dsRNA isolated from healthy (H) and 7/40 infected N.occidentalis (7/40) loaded onto a 0.8% agarose gel along with 1Kb (M) markers (Gibco BRL). 
Figure 2. PCR amplification products generated from the immoblised 7/40 cDNA library using the oligo(dT)-adapter and various ASPV forward primers in lanes: (1) ASPV 7956 (2) ASPV F1 (3) ASPV 5 (4) ASPV 3 (5) random primers oligodT-adapter primer with either (1) ASPV7956, (2) ASPVF1, (3)ASPV5; (4) ASPV3, or (5) random primers
Figure 3. The sequence of the ASPV (isolate 7/40) PCR product with the positions and sequence of the diagnostic primers ASPV5, ASPV3 and ASPV1 (in lower case). ASPV5 atgtctggaa cctcatgctg c ATGTCTGGAA CCTCATGCTG CAGACTCAGA GCCCCCCAGC GAACTGGGTT
ASPV3 ctcttgaacc agctgatggc CGGAGTTGAA AGCTCTGCAG CACTAGAACC TGCTGATGGG CTCGTGAGGC
aacct aaccggggcg gctat ASPV1 CATGCTTTAG CTTATTTTTG TTTTAACTAG ATTTTCAAAA |