Pago de Tributos en Importaciones

Reverse genetics systems have played a major role in the analysis and development of protective, non-pathogenic, APV vaccine strains. APV A field strain #8544, an actual vaccine strain isolated from an outbreak in northern England in 1985, was found to protect turkeys from challenge with APV. However, it was also shown to produce post- vaccination disease in some cases, even following passage in Vero cells (Williams et al.,

1991a; Williams et al., 1991b). Naylor & Jones (1994) and Naylor et al. (2007)

determined that this was due to the presence of a sub-population of virulent virus. Plaque purification produced 12 viruses that were free of the disease-causing sub-populations. These isolates had varying degrees of sequence conservation and differing abilities to induce protective immunity. Complete sequencing of the original isolate (field strain #8544), of the attenuated (virus P20), and of selected plaque-purified isolates (viruses C, K, F, H, and L) revealed a set of point mutations which produced four phenotypes for the virus: protective against APV challenge but severely pathogenic (isolate #8544),

protective but some pathogenicity (virus P20), protective and non-pathogenic (viruses C and K), or non-protective and non-pathogenic (viruses F, H and L). Briefly, compared to the pathogenic progenitor strain isolate #8544, the P20 virus had nine base changes: one change in the F gene start sequence, two in the F gene itself (coding), one in the M2-1 gene (coding), one in the SH-G gene junction, one in the G gene (non-coding), and two in the L gene (non-coding). The protective viruses C and K were found to have the same sequence as the P20 virus. Virus L differed in sequence from P20 in having two bases that matched isolate #8544 (F gene start and L gene) and extensive mutation to the G gene (55 A to G mutations in the coding region). Viruses F and H differed from P20 by

one mutation each to the SH-G gene end sequence. The four SH-G gene junctions found upon sequencing the progenitor strain 8544 (IR mutant 1), the protective virus C (IR mutant 2), and the two non-protective viruses H and F (IR mutant 3 and IR mutant 4, respectively) are shown in figure 4.4. The IR mutant 1 represents the naturally occurring SH-G gene junction for virus strain 8544 and does not represent a sequence that is a mutation of the ‘wt’ virus. All other viruses were sequenced only across the 11 regions of mutation found between isolate #8544 and P20 and were found to have identical sequence to virus P20 (Naylor et al., 2007).

pCATLUC minigenome gene junction (GJ):

UAAUAGUUAUGAAAAAAUCGAUGGGACAAGUAACCAUG

IR mutant 1 (pathogenic, protectivestrain #8544 GJ):

UAAUGAAUUUUGACAACACUAGUGCCAAAUAAUAGGCAACAG UAUUAUUUAAUUAAAAAGAAAGGUCGGGACAAGUAUCUCUAUG

IR mutant 2 (non-pathogenic, protective viruses C, K and P20 GJ):

UAAUGAAUUUUGACAACACUAGUGCCAAAUGAUAGGCAACAG UAUUAUUUAAUUAAAAAGAAAGGUCGGGACAAGUAUCUCUAUG

IR mutant 3 (non-pathogenic,non-protective virus H GJ):

UAAUGAAUUUUGACAACACUAGUGCCAAAUGAUAGGCAACAG UAUUAUUUUAUUAAAAAGAAAGGUCGGGACAAGUAUCUCUAUG

IR mutant 4 (non-pathogenic,non-protective virus F GJ):

UAAUGAAUUUUGACAACACUAGUGCCAAAUGAUAGGCAACAG UAUUAAUUAAUUAAAAAGAAAGGUCGGGACAAGUAUCUCUAUG

Gene Junction Mutations

GS CAT gene GE IR GS LUC gene GE

Figure 4.4Dicistronic minigenome (mRNA sense) gene junction mutants. Shown here are the IR mutant 1-4 gene junction sequences (antigenome sense), with stop and start codons shown in blue, point mutation position in red, and the GE and GS sequences underlined.

93 As the only mutations in common in the non-protective (viruses F and H) and the

protective (viruses C, K and P20) genomes were single base changes in the SH-G gene junction, it was hypothesized that the differences in the ability of these viruses to elicit protection to APV challenge may be due to changes in the amounts of SH and G proteins expressed by the virus. The effect of the mutations on gene expression was investigated by replacing the existing dicistronic pCATLUC minigenome gene junction with the SH- G gene junction and carrying out point mutations to create the four desired gene junction sequences.

4.3.1.1 Generation of IR mutant 1

The mutation of the pCATLUC minigenome gene junction to the SH-G gene junction was carried out using a series of overlapping primers. As described in section 2.6.1, it is possible to insert large fragment into plasmid using a modified QuickChange site directed mutagenesis protocol. The first step was to create a large fragment that contained the desired SH-G gene junction of 150-200 nucleotides that matched the start of the LUC gene and the end of the CAT gene. To create the insertion fragment, a series of PCRs were carried out (shown diagrammatically in figures 4.5 to 4.7) using primers ranging from 19 to 84 bases (appendix A). PCRs 1 and 2 were carried out using the pCATLUC minigenome as the template DNA. A 20 base forward primer (primer DSMG IR F) which bound from 200bp upstream of the end of the CAT gene stop codon was used with a reverse primer (primer IntMut/DSMG R) containing the final 15 bases of the CAT gene sequence followed by a non-binding region of 69 bases. This PCR gave a fragment of 269bp which was named fragment A. For fragment B, a PCR was carried out using a 40 base forward primer (primer IntMut/DSMG F) which contained the final 31 bases of the replacement gene junction followed by the first 19 bases of the LUC gene, and a 19 base reverse primer (primer DSMG IR R) that complimented the LUC gene 186 bases

downstream of the LUC gene start codon (figure 4.5). PCR for fragment C was carried out using fragments A and B and the full length cloned APV CASA genome) (Naylor et al., 2004) as the template (figure 4.6). Fragment C contained the desired progenitor gene junction flanked by sequence complementary to the CAT and LUC genes.

Figure 4.5 Generation of fragments A and B. A) Using the pCATLUC

minigenome as the template for PCR, primers DSMG IR F and IntMut/DSMG R were used to introduce 69bp (in green) of SH-G gene junction sequence to the 3’ end of a 200bp fragment (in light green) of the pCATLUC plasmid (fragment A). For fragment B, the pCATLUC plasmid was again used as the template for PCR and primers IntMut/DSMG F and Primer DSMG IR R were used to introduce 31bp (in green) of SH-G gene junction region sequence to the 5’ end of an 179bp fragment (in light blue) of the pCATLUC minigenome. B) PCR product

fragments A (269bp) and B (210bp) were visualized on an agarose gel. 5’ TAATAGTTATGAAAAAATCGATGGGACAAGTAACCATG 3’

GS CAT gene GE IR GS LUC gene GE

Pt7.2 A PCRs 1 and 2 template: pCATLUC minigenome Primer: DSMG IR F Product:Fragment A Primer: IntMut/ DSMG R PCR 1 Product:Fragment B Primer: IntMut/ DSMG F Primer: DSMG IR R PCR 2 B _DNA ladder (bp) 1,000 600 400 200 ladder A B

GS SH Gene GE IR GS G gene GE

APV CASA

Product:Fragment C

Figure 4.6 Generation of fragment C. A) Fragments A and B were used in PCR with the APV full length cloned genome (APV CASA) to create fragment C. Fragment C has the SH-G intergenic flanked by pCATLUC minigenome

sequence. B) PCR product fragment C (458bp) was visualized on an agarose gel.

A