Apart from the usage of leaky UAG amber stop codons, it has been reported that pI:charge can affect the production of viable recombinant virus [15] . The pI of the epitope is thought to be an important factor as the hybrid coat protein pI:charge value can affect epitope presentation. It was also reported that TMV was more tolerant to positively charged epitopes on its surface. Thus, it was initially speculated that the failure in expression of the foreign peptide was possibly due to the pI:charge value of recombinant CGMMV CP which was different from the wild-type CGMMV CP pI:charge value ( Table 2) . Table 2 shows the isoelectric point (pI) and charge of the wild-type CGMMV CP, the read-through recombinant CGMMV CP, and the EB4 insert. The charge of the EB4 insert is positive and potentially suitable for expression on the surface of the CGMMV CP [2] . Hence, the inserted peptide is speculated to be expressed if the pI:charge value of modified virus CP resembles the pI:charge value of unmodified virus CP. Earlier transcripts (data not shown) generated from fusion clones without a read-through sequence, where their pI values deviated significantly (>6.0) from the wildtype CP (5.08), were not able to cause infection in inoculated plants leading to the suggestion initially that pI:charge value Journal of Biomedicine and Biotechnology 410  420  430  440  450  460  470  480  490  500   510  520  530  540  550  560  570  580  590  600   610  620  630  640  650  660  670  680  690  700 Sequence not determined Deleted sequence Gaps introduced for alignment Figure 6 : Sequence analyses of RT-PCR-amplified products from putative chimeric CGMMV RNA at different days postinoculation (dpi). The sequenced alignment shows that EB4 sequence was truncated and not complete after 21 dpi. The putative chimeric CGMMV produced did not express the EB4 and its genome resembled the wild-type CGMMV. Introduced read-through sequences and extra codons are underlined. Complete EB4 sequence is aligned accordingly with the other sequences.