The studied groups consisted of 15 confirmed HTLV-1 infected adults whom clinically diagnosed with HTLV-1 associated disease, it was possible to
amplify a fragment of the expected size of 767 bp with designated primers LTR-A
and LTR-B. Almost HTLV-1 LTRs of all DNA specimens were completely sequenced.
Sequence analysis – We therefore purified a 767 bp segment (Fig. 1) from all samples.
The amplified fragment from these samples was cloned into the pTZ57R/T cloning
vector and one clone per amplification products were sequenced in both directions by
Macrogen (www.macrogen.com). Obtained
sequences from the samples showed the trustworthy of amplified fragments with homogeneous
population of PCR products, and the variations observed were as a result of
alternations in viral genomic sequences linked to the geographic origins. Thus, most of the diversity in
local strains were not known to inhibit viral expression or replication. In order to obtain information concerning LTR nucleotide
diversities, all the collected sequences were compared with the equivalent sequenced
derived from different regions of Khorasan province and other endemic areas. Sequence
analysis revealed that the 13 nucleotide sequences from patients were exactly
identical and showed no significant differences within studied group. The exceptions to this general
pattern were two alternations in HAM/TSP85 samples (A285 to
C, A541 to G) and one
alternation was in an ATL 90 case at position C696 to T, which
is located in the R and U5 regions, respectively. LTR alignment from isolates showed 99.7-100% similarity
amongst the sequences. These nucleotide variations may be primarily a result
from their low fidelity related to the reverse transcriptase enzyme.
Nucleotide alignment were carried out among sequences
and compared with the sequences of the HTLV-1 strains from various geographic distant
regions including Japan (ATK- 1, H5, TSP-1, MT-2; Seiki et al. 1983, Gray et
al. 1990), the Caribbean area (HS-35, CH; Malik et al. 1988, Ratner et al.
1991), Brazil (pt-8; Schultz et al. 1991), Romania (H990; Schultz et al. 1991),
Melanesia (MEL-1; Gessain et al. 1993), the United States (SP; Paine et al.
1991), a variant from Gabon (GeneBank Accession number L33266; Moynet et al.
1995), Chile (ST; Dekaban et al. 1992) and Zaire (EL; Paine et al. 1991, Ratner
et al. 1991), as well as the sequences of human T-cell lymphotropic virus type
I from Torbat-e-Heydarieh and Neyshabur from Khorasan provinces (Watanabe et
al. 1985) and sequences of HTLV-1 strain Mo from the United States (Shimotohono
et al. 1985).
Alignment of the sequences of the 767bp segment of
the Japanese HTLV-1 strain ATK-1 (Fig. 3), with sequences from this study exhibited
a 1.5-2.4% dissimilarity. The variability among the
sequences obtained from the Mashhadi strains was 0.5%. The nucleotide sequences
for this fragment showed that there are alternations in nucleotide G36 to
A, C37 to
G, G172 to
A, A241 to G, represented
a distinctive feature of the local isolates when compared to the ATK-1 strain. Despite this overall
low degree of genomic variability in nucleotide sequence, no nucleotide variations were observed in the poly(A) signal
(AATAAA), the TATA box and the CAP site, comprising nucleotide 88 to 98 on the LTR gene, was totally conserved between the HTLV-
1 Mashhadi strain sequences Office1 examined here.
Phylogenetic analysis – The
phylogenetic trees constructed were very similar by two different methods (UPGMA, NJ) using the 767 bp LTR gene.
Although similar, we selected
the NJ method because it provided the most illustrative phylogenetic tree. As
shown in Fig. 4, three main groups were clearly identified. In the
first group appeared strain ptM3 of HTLV-1 that represents an early ancestor
from which the Melanesian and cosmopolitan strains diverged, whereas in the
second group appeared the Melanesian MEL-1 strain. The third group or
cosmopolitan subgroup appeared clearly separated from the African and
Melanesian groups and contained the Mashhadi strains described here and distinguishable from reference sequences for groups B, C, D, E, F,
and G. Office2
Our results suggest that these Mashhadi isolates can be clustered
with the same group as other Khorasan province and Iran isolates such as Mashhadi
jew and Mashhadi immigrant patient to German with the same clade with cosmopolitan (a) subtype. Although the analysis shows that
the strains from Japan and Africa seem not to be in the same branch as the
strains described here Mashhad, they do not form a completely separate
were submitted to Gen Bank after analysis: BankIt1824992 HTLV-1_LTR (Torbat-e Heydarieh __Iran)
migi nigah kardam hich taghiri dar u3 ya felan ya felan moshahgede nakardam
ba Mehran sohbat kon
Mehran kamel chek kon