Abstract in Mashhad, Iran. The sequences wereAbstract in Mashhad, Iran. The sequences were


Sequence and Phylogenetic
Analysis of LTR region of Human T Cell Lymphotropic Virus Type 1 from Mashhad,

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Seyed Ali Mirhosseini1, Mehran
Moharreri1, Seyed Rohollah Arab1, Baratali Mashkani2, Seyed
Abdolrahim Rezaee3, Reza Farid Hosseini4 and Houshang Rafatpanah1 

1Inflammation and Inflammatory Diseases
Research Center, School of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran.

2Biochemistry and Nutrition Research
Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad,

3Immunology Research Center, School of
Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.?

4Allergy Research Center, School of
Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.?

T-cell lymphotropic virus type 1 (HTLV-1) is widespread around the globe, with
endemic areas in Southern part of Japan, Africa, the Caribbean, South America and Northeast
of Iran. The virus is responsible for causing adult T-cell leukemia (ATL) and
the neurological disorder HTLV-1 associated myelopathy or tropical spastic
paraparesis (HAM/TSP) in less than 5% of the infected individuals. Variations
in the provirus sequence of HTLV-1 have been used as a tool for molecular
epidemiology investigation of HTLV-1 infection.

Methods: In current
study, the amplified LTR region was purified, cloned and sequenced from 15
infected individuals lived in Mashhad, Iran. The sequences were compared with
the sequences from Neyshabur, Torbat-e Heydarieh and Sabzevar. It was also
compared with a reference sequence obtained from GenBank (Accession No. J02029)
from Japan.

LTR sequence in the samples belonged to the transcontinental subgroup A. There
was no mismatch in the LTR sequences in 13 out of 15 samples collected from
Mashhad with those from Neyshabur, Torbat-e Heydarieh, and Sabzevar. However,
these LTR sequences were difference in 1 % of the nucleotides from the J02029.

results indicates that HTLV-1 infection in the Northeast of Iran had a common
origin. However, further investigation is required to identify the source of
small differences were observed in those two samples. 

Keywords: human T-cell lymphotropic virus type 1
– HTLV-1 associated myelopathy/tropical spastic paraparesis – HTLV-1 phylogeny
– Mashhad




The development of TSP/HAM
in HTLV-1 infected individuals remains unknown, being that

few infected individuals
have this disease outcome, and it is probably related to environmental,
host and viral factors,
such as proviral load, the methylation profile of proviral DNA,
the integration site of
HTLV-1 into the host genome and viral mutations6,7,8.
Recent studies have showed
trough Sanger sequencing and real-time PCR that
mutations in the LTR region
may increase the HTLV-1 proviral load9,10 and it could consecutively
affect the TSP/HAM outcome.





Human T-lymphotropic virus type 1(HTLV-1)
a complex type C retrovirus belonging to the
genus deltaretrovirus. In addition to adult T-cell leukemia (ATL) and HTLV-I-associated
myelopathy/tropical spastic paraparesis (HAM/TSP), the virus is considered to be
contribute in various inflammatory diseases, most notably
uveitis, arthritis, myositis and alveolitis (2-4). Although the
distribution of HTLV-1 is global but the higher rates of infected individuals
have been observed in Southern part of Japan, Africa, the Caribbean, South
America (5,6). The first infected adults were
detected by Farid in 1993 with 2.1% prevalence in
Mashhad, a populous
city in Northeastern of Iran with more than 25 million pilgrims and visitors annually. In these regions, between 0.5 and 20% of the general population have specific
antibodies against HTLV-1 and are considered to be healthy carriers(7). Most of HTLV-1 infected individuals
remain Asymptomatic carriers (ACs) through their lives and in a few infected subjects, ATL
or HAM/TSP will develop(8).

 Although genome similarities among
HTLV-1 strains is very high, a few nucleotide variation seems to be specific of
the geographical origin of patients. Moreover, disease-specific sequences
have not been found from patients with ATL or HAM/TSP, instead the genomic
variability of HTLV-1 appears to depend on geographic origin more than the
pathology. The development rate of HTLV-1 is very low (particularly compared
to HIV). Currently, based on LTR sequence variation 7 subtypes have been characterized
for this virus; the Cosmopolitan subtype (1a) that has distributed globally
and is further divided into subgroups (A–E), 4 African subtypes (1b, 1d, 1e, 1f)
and a Melanesian/Australian subtype (1c).

In 2005 the Cosmopolitan subtype
(1a) was identified in Torbat-e Heydarieh, Sabzevar and Neyshabur by Rafatpanah
et al. based on obtained sequences from infected adults 7.

To examine the molecular epidemiology and identify the features of the local HTLV-1 strains that circulating in Mashhad community, we performed Polymerase chain reaction (PCR) to amplify the LTR region, then the fragments were purified and cloned into TA cloning vector. Furthermore, all specimens were sequenced and phylogenetic analyses describe HTLV-1 subtype a, mostly affecting the individuals from Mashhad., which is the first time this subtype has been reported in HTLV-1-associated diseases.  


HTLV-1 infected samples whom clinical diagnose of HAM/TSP, ATL and ACs were participated in this study. These
specimens were chosen from volunteers who were referred to Department of
Neurology at the Ghaem Clinic after confirmation tests. All ACs were diagnosed
as HTLV-1 carriers at the time of blood sampling. Serum samples were tested for detection of anti-HTLV-1 and 2 antibodies
using ELISA method, then positive results were confirmed by PCR using the
primer targeted Tax gene. Moreover, we explained
all the goals and methods of the study briefly for our patients and came to an
agreement to obtain written informed consent. Briefly, 6 ml blood
samples were obtained from 15 infected individuals living in Mashhad. Peripheral blood mononuclear cells (PBMCs) were separated from
blood samples by using ficoll method. DNA was extracted from PBMCs using the DNA
Genomic Purification Kit (Blood mini kit, Qiagen, Germany) according to protocols recommended
by manufacturer and samples were stored at ?20 ?C.

Thereafter, Polymerase chain
reaction (PCR) was performed on seventy nanograms of DNA to amplify complete LTR region approximately 780bp long and
corresponding to position 60 to 830 in ATK-1, the prototype Japanese HTLV-1 strain, using one set of overlapping oligonucleotide
primers designed by Geneious software 9.0.5. LTR-A
Assembling all reactions components for PCR amplification was carried out on
ice and quickly transferred reactions to a thermocycler preheated to the
denaturation temperature 95°C. The procedure was done in a 25 ul volume that
contained 25M MgCL2 -1.5ul, 10M dNTPs -0.5ul, 10× Reaction Buffer-2.5, 0.5ul
Prime Accupol™ DNA Polymerase, template DNA 2ul, 0.5 ul of each specific
primer, 17 ul dH2O. The PCR machine (Astec PC700, Kyoto, Japan) using an
initial denaturation step at 95°C for 5 minutes, followed by 30 cycles of 95°C
for 30 seconds and 60°C for 30 seconds, 72°C 1 minute and a final extension of
72°C for 5 minutes. Gel was stained by ethidium bromide, then electrophorese on
2% agarose gel with molecular-weight size marker and a sample (5 ul) of each amplified
product were conducted in TAE buffer (Tris-acetate EDTA) at 70V for 30 minutes
and visualized under UV light.




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
subgroup. Office3 

The sequences were submitted to Gen Bank after analysis: BankIt1824992 HTLV-1_LTR
(Torbat-e Heydarieh __Iran) KR819400



Fig. 1. Alignment of the nucleotide sequence of the LTR from
different HTLV-I isolates. Only those changes observed with other published
sequence are mentioned. Nucleotide positions are numbered according to the
ATK-1 prototype sequence starting from the first LTR nucleotide (14). Points represent homologies. 13 HTLV-1 –LTR local isolates
were identical. HAM/TSP85 and ATL90 were the exceptional.


There is close relationship between the phylogenetic HTLV-1 genotypes and
the geographical origin of infected- individuals, although up to date the
significant correlation exists between diversities observed in geographical
origins and clinically pathology outcomes are not well identified. HTLV-1
phylogenetic studies of different ethnic groups are helpful because it assists
to identify the probable time of introducing and spreading the different HTLV-1
strains into countries and investigate the possible origins of HTLV-1 in
endemic areas during mobility of human populations. These proofs confirm the
nucleotide heterodiversities observed in different HTLV-1 strains. Here, we define
the full-length sequence of LTR region of infected individuals lived in
Mashhad, a populous town where
the most Caucasians residents have different economic,
social, health and cultural background in northeast of Iran. Mashhad has higher level of HTLV-1 antibodies in blood
donors as compared to that blood donors from different provinces of Iran and
the prevalence of HTLV-1 in one of the highest in Asia.Office4  Seroprevalence
studies conducted between 2005 to 2013 indicated low prevalence of
HTLV-1infection in different provinces including Office5 Azerbaijan, Ilam, Hormozgan, and Bushehr, the
percentages of blood donors with the virus were 0.34, 0.21, 0.18, and 0.01% respectively,
suggesting an unseen threat for the public health. So, it is necessary to educate and
increased awareness concerning the sexually/blood transmitted diseases or transition pathways in general
population to decreased the number of new


From 15 confirmed samples who had been clinically
diagnosed HTLV-associated diseases, 5 patients were ACs, 5 patients were ATL
and 5 infected adults were HAM/TSP that were recognized as HTLV-1 positive by
two certain different serology methods. Analysis of sequences indicated that
all sequenced LTR showed an identical pattern in nucleotide sequences among samples
from Torbat-e-heydariyeh. The nucleotide pattern observed among samples
permitted us to primarily assume the presence of variation in the sequence
which was specific of certain geographical origin as compared to the isolate
ATK-1 (GeneBank accession numbers J02029).


the phylogenetic studies and ascertain genotype, the LTR region was selected because
of highest variability rather than other regions of the virus (14) while other regions of HTLV-1 genome
have more constant region. Office6 For this purpose the current study focused on 767 base
pair fragment of the LTR gene, to analyze the genetic similarities in fifteen
of the isolates specimens since such a gene initiates transcription, polyadenylation and
splicing. We, and other ( Rafaat panah) have observed G36 to
A, C37 to
G, G172 to
A, A241 to G,
when compared to the ATK-1 isolate as a reference. By aligning the sequences of
the reference strains and sequences from isolates of current study with other
HTLV-1 sequences of geographically distant strains, they indicated considerable
similarities in nucleotide sequences when compared to the ATK-1 prototype
strain. Nucleotide similarity was: T (Chile) 0.8%, pt-8 (Brazil) 0.8%, ATK-1,
MT- 2, TSP-1, H-5 (Japan) 1.2– 2.3% and CH (Caribbean) 1.4%. However, Low evolutionary, constant genome
and high similarities in sequences have been reported for each HTLV-1-patient
by molecular epidemiology, but up to date no specific-disease nucleotide
variations recommended to develop ATL or HAM/TSP from virus carriers. So,
geographical origin, transmission routs, host genetics seem to associate with
clinical symptoms. Along with previously studies, on the molecular level,
HTLV-1 have shown low drift of diversities in nucleotide sequences amongst
HTLV-1 isolates in Sabzevar and other endemic populations. Thus, the diversities observe in nucleotide among HTLV-1 strains representing the close
correlation with geographical origin of the HTLV-I strain than the pathology.

validate the trustworthiness of the topology of the phylogenetic tree
constructed, different methods such as UPGMA and NJ were used. Data analysis
indicated that it is likely to observe local isolates are closely related to
isolate from Torbat-e-heydariyeh, Sabzevar and Neyshabur and all the strains
studies here belong to the Cosmopolitan group. In this examination is also
possible to notice that the Japanese
strains MT-2, TSP-1 as well as strains EL from Zaire, a variant from Gabon, and
HS-35 from the Caribbean area cluster with the different group. This finding is in
agreement with other studies conducted in different geographical suggested that
the Cosmopolitan subtype was found to be widespread. Similarly, Cosmopolitan
type was observed in the Reunion Island, Iranian-born Mashhadi Jews and Kuwaiti
HTLV-I isolates, strongly suggesting the common origin of all these isolates.

lines of proof here suggested that spreading of HTLV-1 infection through
American continent initiated with the first human population movement. in
contrast, other phylogenetic analysis revealed that the origin of all HTLV-1
subtype is Africa, according to the absence of HTLV-1 among Amerindians and the
high prevalence of infection among individuals of African descendent in the
Caribbean area and in South America. It is possible that HTLV-1 was brought to
Mashhad by African slave immigrants as suggested by felani. These researchers
propose that HTLV-1 originated in region called an Indian Malayan, from there
apparently distribute to Africa by marines thousands of years ago, and that
later due to slave trade in the XVI century, it spread to the New World and
Iran. An analysis of the LTR region of HTLV-1 isolates from different ethnic groups of endemic regions have
suggested that the virus may have been introduced into Iran on a number of
occasions from several African heredities, probably during and after the slave trade
or through recent Mongoloid
invasion when the city served as an important crossroad and trade center (Van Dooren et al. 1998). HTLV-1
is predominantly frequent among descendants of African slaves.
Amongst these hypothesis, Slavery seems more likely to have introduced the
virus to these cities, because it has been commonly performed even before the
rise of Islam in these cities. As a consequence of increased human mobility, HTLV-1a infection has also reached different Iran’s neighbors, such as
Pakistan, turkey, Saudi Arabia, whereas HTLV-1 is still uncommon in the general
population. Furthermore, the social
interaction among countries such as international trade, tourism, and
pilgrimage seems to be playing a central role in virus dissemination. Phylogenetic
analyses of the LTR region in the virus isolates examined here as well as in
strains from different ethnic groups in Iran’s neighbors will make it possible to confirm the
origin of the HTLV-1 strains in these populations and to determine the exact time
of introduction of viral strains into Iran especially Mashhad.













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