The genetic relatedness of EHV-1 isolated from gazelle, zebra, and onager was investigated by PCR-RFLP analysis and comparing the nucleotide sequences of the 3.5-kbp region from a part of ORF 62 and ORF 63 with those of EHV-1 isolated from horse.
The whole genome of the examined isolates was amplified as 18 overlapping fragments by using 18 pairs of primers. The lengths of the amplified fragments were ranged from 5.6 to 14 kbp. The amplified products were digested with BamHI, BglII EcoRI, EcoRV, HindIII, PstI, and PvuII endonucleases.
All the amplified PCR products showed restriction site variations except the fragment 10F10A which was identical among the isolates examined. The restriction site variations were mapped in 23 ORFs and 5 non-coding regions in the UL region, 4 ORFs in the US region, and 6 ORFs and 4 non-coding regions in the inverted repeat. There was a degree of uniformity of the restriction maps of the gazelle, zebra, and onager isolates. Minor variations, however, were detected among those isolates.
PCR-RFLP analysis of the fragment 0F1R showed that the restriction patterns were different among all isolates examined. Moreover, this fragment in onager isolate was shorter than those of the other isolates, indicating a 1.6 kbp deletion. PCR-RFLP analysis of fragment 4F4R showed that the examined isolates are identical but distinct from horse isolates. On the contrary, the RFLP analysis of fragment 11F11R showed that gazelle isolate was identical to horse isolates but varied from onager and zebra isolates.
Moreover, mobility shift of restriction fragments were shown in all isolates examined. Therefore, the nucleotide sequences corresponding to the nucleotides from 108486 to 111984 of the Ab4p genome of onager zebra, gazelle, and 3 of horse isolates. The sequenced region included about 300 bp of ORF 62 5’-end, the 1600-bp corresponding to the intergenic region between ORF 62 and ORF 63, and the 1600-bp corresponding to the complete gene sequence of ORF 63. The results showed that the sequenced region consisted of 6 domains.
The first domain (D1) corresponds to the nucleotides from 108486 to 108962 of the Ab4p genome. The nucleotide sequences of this domain were almost conserved in onager zebra, gazelle isolates with exception of some point mutations. This domain was also completely conserved among horse isolates.
The second domain (D2) corresponds to the nucleotides from 108963 to 109022 of the Ab4p genome, which is the tandem sequence repeat domain. The sequence repeat unit is an 18-bp sequence of 5’-GCTAGCGCTAACGCTAGG-3’. The length of this domain varied among all isolates according to the differences in the copy number of the sequence repeat.
The third domain (D3) corresponds to the nucleotides from 109023 to 110735 of the Ab4p genome. The nucleotide sequences of this domain were almost conserved in onager and zebra isolates with exception of some point mutations. A 19-bp deletion was found in T-529 corresponding to the nucleotides from 109023 to 109041 of the Ab4p genome. This domain was conserved among gazelle and horse isolates with exception of some point mutations.
The fourth domain (D4) corresponds to the nucleotides from 110736 to 111058 in the Ab4p genome. In this domain, the nucleotide sequences of gazelle and onager isolates were almost identical with exception of some point mutations, but different from that of zebra isolate. However, the nucleotide sequences of onager, zebra, and gazelle isolates showed variation with horse isolates.
The fifth domain (D5) corresponds to the nucleotides from 111059 to 111707 in the Ab4p genome. In this domain, the nucleotide sequences of onager, zebra, and gazelle were almost identical with exception of some point mutations. The nucleotide sequences of the horse isolates were identical.
The six domain (D6) corresponds to the nucleotides from 111708 to 111984 in the Ab4p genome. This domain, which includes the N-terminal region of ICP0, was identical among all isolates.
The nucleotide sequences of D1, D3, D4 and D5 were used to construct four unrooted phylogenetic trees to examine the genetic relationship among isolates examined (Fig. 9). In the unrooted trees which were constructed from the nucleotide sequences of D1 and D5, I found that onager, zebra, and gazelle isolates were closely related to each other and constructed a new branch. In the unrooted tree which was constructed from the nucleotide sequences of D3, I found that gazelle isolate was genetically related to horse isolates. In the unrooted tree which was constructed from the nucleotide sequences of D4, the gazelle isolate was closer to onager and zebra isolates.
The amino acids of ORF 63 (ICP0) were highly conserved among onager, zebra, gazelle, and horse isolates in the N-terminal portion. However, the predicted amino acids in the C-terminal portion were conserved in onager, zebra, and gazelle isolates. The gazelle isolate was closer to onager isolate than zebra. Moreover, the gazelle isolate was conserved with Ab4p in the amino acids numbers 441 to 532.
PCR-RFLP analysis of the fragment 0F1R showed that this fragment in the isolate onager isolate was shorter than those of the other isolates. Therefore, nucleotide sequence of these isolate corresponding to nucleotides number from 961 to 3100 of the Ab4p genome were read by using primer set 18F-18R to identify the deletion point. The sequence includes ORF 1 upstream to ORF 3. Results showed that there is a deletion of 1598 bp. The deletion corresponds to the nucleotides from 1120 to 2717 in Ab4p. Deletion started from the ORF 1 upstream till middle of the non-coding region between ORF 2 and ORF 3. Therefore, ORF 1 and ORF 2 were completely lost in onager isolate.
These results indicate that gazelle isolate might be a resultant virus from a natural recombination between a progeny virus of onager and zebra isolates and that of horse. It is not known that whether the genomic variation of gazelle isolate was occurred in gazelle over long time or occurred in other intermediate host before transmitted to gazelle. The results showed that ORF 1 and ORF 2 in onager isolate were deleted.
Finally, we concluded that EHV-1 isolated from onager, zebra, and gazelle are a new subtype of EHV-1 that is genetically distinct from EHV-1 from horse. ORF 1 and ORF 2 were deleted in the onager. Moreover, we hypothesize that gazelle isolate is a natural recombinant virus which resulted from the recombination between a progeny virus of onager and zebra isolates and that of horse. This recombination might be occurred through the co-infection of one individual with both viruses. The co-infection indicated the contact between these animals, which should be caused by human activity including capturing, displaying and keeping wild animals in contact with domestic animals. Although it is not clear that the recombinant could be a threat to domestic horses but the potential threat of the recombinant should be considered. Further eco-virological investigations should be continued to predict the emergence of new virulent EHV-1. |
