Molecular Characterization of Human T Cell Leukemia Virus Type 1 Subtypes in a Group of Infected Individuals Diagnosed in Portugal and Spain

Abstract

Over the past decade, Portugal and Spain received large numbers of immigrants from HTLV-1 endemic areas. Our aim was to investigate the diversity of subtypes circulating in these two countries and the introduction of new variants. We performed a molecular analysis of HTLV-1 strains in patients diagnosed since 1998. LTR and env proviral sequences from 26 individuals were analyzed to generate phylogenetic trees along with reference HTLV-1 subtypes from several geographic origins. Epidemiological and clinical data were recorded. Most subjects were immigrants (57.7%) from South America and Africa. All isolates belonged to the cosmopolitan A subtype. Most carried the transcontinental subgroup A, but five subjects carried subgroup D and one carried subgroup C, previously unreported in Europe. HTLV strains showed separate clusters linked to the patients' geographic origin. Although subjects with HTLV-1 infection tend not to be engaged in high-risk practices, silent dissemination of a broad diversity of HTLV-1 viruses may still occur.

H uman T cell lymphotropic virus type 1 (HTLV-1) was the first human retrovirus isolated in 1979 from a patient with T cell malignancy.¹ It is estimated that 10–20 million people worldwide are infected,² and although the majority of HTLV-1 carriers remain asymptomatic, the virus is associated with exceptionally severe diseases, such as adult T cell leukemia/lymphoma (ATLL) and an inflammatory syndrome of the central nervous system named HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Different HTLV-1 subtypes have now been identified over the world, which exhibit a fairly well-defined geographic distribution. HTLV-1 strains have been classified into six different subtypes³: the cosmopolitan subtype A, the Central African subtype B, the Australo-Melanesian subtype C, and subtypes D, E, and F. Furthermore, the cosmopolitan subtype A has been divided in five subgroups⁴: the transcontinental subgroup A, the Japanese subgroup B, the West Africa/Caribbean subgroup C, the North Africa subgroup D, and the black Peruvian subgroup E. The study of the genetic heterogeneity of HTLV-1 is important for several reasons: it contributes to our knowledge about the origin of the virus and the migratory movements of ancient populations and provides a laboratory tool to study genetic regions that can be responsible for pathogenicity and to monitor viral transmission.

HTLV-1 seroprevalence in the Iberian Peninsula (Portugal and Spain) is very low,⁵ affecting mainly immigrants from endemic areas and natives with links to those areas. Contrary to the measures implemented in Spain, the screening of HTLV antibodies in blood donors has been mandatory in Portugal since 1995. Although surveillance studies in different risk populations and HTLV-1-associated diseases have been developed in both countries,^6,7 very few have focused on the genetic circulating subtypes.

The objective of the present study was to perform a molecular analysis of HTLV-1 infection in Portugal and Spain so as to assess the subtype diversity of these viruses circulating in the population. Both countries are in close geographic-socioeconomic proximity but with differences in immigration patterns. The population living in Portugal is characterized by a high proportion of immigrants, mostly as a result of the strong historic and ongoing links with several (mainly West) African countries. However, in more recent years, immigrants from Eastern European and Southern American (mainly Brazil) regions have been contributing for the ethnics and cultural heterogeneity of the population. Immigration is a more recent phenomenon in Spain. This country has received a great flow of immigrants within the past 10 years, mainly from South American countries with which it shares historic links and to a lesser extent from North and sub-Saharan Africa.⁸

We have analyzed the genetic subtypes in 26 HTLV-1-infected individuals (16 from Portugal and 10 from Spain). Blood samples were taken from the infected individuals recruited between 1998 and 2008. Epidemiological data, namely country of origin, probable route of transmission, HIV coinfection, and clinical status, were collected for each case enrolled in the study.

DNA was extracted from whole blood with the midi spin columns from the QIAamp DNA blood Midi kit (Qiagen) using the procedure recommended by the manufacturer. Two sets of nested polymerase chain reaction (PCR) oligonucleotide primer pairs were used to amplify genomic regions within the long terminal repeat (LTR) and the env gene of the HTLV-1 genome. The LTR region (616 bp) was amplified using primers and conditions described elsewhere.⁹ The env gene (1450 bp), including the coding regions of gp46 and p21 viral envelope glycoproteins, was also amplified as described elsewhere.¹⁰ The PCR products were purified with QIAquick columns (Qiagen, Studio City, CA) prior to automated DNA sequencing using the PCR inner primers and the ABI PRISM Big Dye Terminator Kit (Applied Biosystems, Foster City, CA).

Phylogenetic analysis was performed based on the LTR and env sequences obtained. Trees were generated from the multiple aligned sequences using the neighbor-joining method to infer the Kimura two-parameter distance matrix. Reliability of the tree was estimated from 1000 bootstrap replicates. The analysis was performed along with reference sequences from each HTLV-1 subtype obtained from the GenBank database and another two Spanish isolates reported elsewhere¹¹ [isolate ES02-ECM from an injecting drug user (IDU) patient coinfected with HIV-1 and isolate ES02-JCP from a patient who acquired the infection through heterosexual contact. Only LTR sequences were available for these patients]. These later sequences had been classified as cosmopolitan subtype A.

The majority of the analyzed patients (57.7%) were immigrants from South America and Africa. Among 10 Portuguese and Spanish native subjects, five of them had acquired the infection without traveling to endemic areas (three by transplantation in Spain, one who had lived in France for many years, and another who was identified as a Portuguese IDU with high-risk behavior and who was coinfected with HIV-1). Patient PtAd had not traveled to endemic areas, but her husband reported travels to Uruguay and South Africa and most likely acquired the infection in those areas. For the remaining native patients data were not available. Thirteen out of 26 patients (50%) had developed HAM/TSP, one patient developed B cell lymphoma, one developed an ATLL, another patient developed strongyloidiasis, and the rest remained asymptomatic (Table 1).

Table 1.

Epidemiological and Clinical Data of HTLV-1-Infected Individuals Living in the Iberian Peninsula ^a

Individuals	Age (years)	Gender	Country of residence	Geographic origin	Traveling contacts	Clinical status	Acquisition mode
PtAd	51	F	Portugal	Portugal	Partner traveled to Uruguay and South Africa	HAM/TSP	Heterosexual
PtDj	NA	M	Portugal	Africa	NA	Strongyloidiasis	NA
PtEm	46	F	Portugal	Portugal	NA	HAM/TSP	Heterosexual
PtFd	9	M	Portugal	Africa	NA	HAM/TSP	Vertical
PtIs	23	F	Portugal	Portugal	NA	NA	NA
PtLc	46	F	Portugal	Mozambique	Mozambique	B cell lymphoma	Vertical
PtNs^b	32	M	Portugal	Portugal	No traveling	Asymptomatic	IDU
PtOs	42	M	Portugal	Brazil	Brazil	Asymptomatic	Heterosexual
PtCr^b	42	M	Portugal	Guinea-Bissau	Spain	Asymptomatic	IDU
PtDk1/2^b	41	M	Portugal	Liberia	African countries	Asymptomatic	Heterosexual
PtEr	55	F	Portugal	São Tomé e Principe	São Tomé e Principe	HAM/TSP	Heterosexual
PtGr	72	F	Portugal	Portugal	France	HAM/TSP	Heterosexual
PtJq	NA	M	Portugal	Portugal	NA	Asymptomatic	NA
PtMm	24	M	Portugal	Guinea	Senegal	ATLL	Heterosexual
PtOf	63	F	Portugal	Mozambique	Mozambique	HAM/TSP	Heterosexual
PtPd	33	M	Portugal	NA	NA	NA	NA
ES-P1	51	F	Spain	Perú	Perú	HAM/TSP	NA
ES-P2	52	M	Spain	Spain	Spain	HAM/TSP	Transplant
ES-P3	48	F	Spain	Brazil	Brazil	HAM/TSP	Heterosexual
ES-P4	49	F	Spain	Perú	Perú	HAM/TSP	Transfusion
ES-P5	NA	M	Spain	Spain	NA	HAM/TSP	Heterosexual
ES-P6	43	F	Spain	NA	NA	NA	NA
ES-P7	63	F	Spain	Colombia	Colombia	Asymptomatic	NA
ES-P8	35	F	Spain	Perú	Perú	Asymptomatic	Vertical
ES-P9	53	F	Spain	Spain	Spain	HAM/TSP	Transplant
ES-P10	55	M	Spain	Spain	Spain	HAM/TSP	Transplant

NA, not available; F, female; M, male; HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesis; ATLL, adult T cell leukemia/lymphoma; IDU, injecting drug user.

Coinfected with HIV-1.

Both LTR and env phylogenetic trees reveal that all HTLV-1 isolates from the studied patients belong to cosmopolitan subtype A, but they group in differentiated clusters with a clear geographic origin (Figs. 1 and 2). The tree topology was also confirmed by maximum parsimony analysis. In infected individuals living in Portugal we observed three different clusters: one patient from Liberia coinfected with HIV-1 and from whom we have taken two blood samples and generated two sequences, PtDk1 and PtDk2, respectively, which fall within West African/Caribbean subgroup C; five patients (PtCr, PtMm, PtPD, PtFd, and PtDj) from the Africa cluster fall into North Africa subgroup D; and the other 10 individuals fall into transcontinental subgroup A. However, all infected individuals living in Spain gather in transcontinental subgroup A and six of them within the Latin American cluster. Curiously, within this cluster we also observed all sequences obtained from Portuguese native patients together with one patient born in Brazil and one in São Tomé e Principe. By tree topology we see that sequences collected in 1998 and the most recent sequences mix together with no differences in their genetic distances; in addition, we had not detected recombination of different HTLV subtypes/subgroups when analyzing two regions (LTR, env), which is a very rare phenomenon.

FIG. 1.

Phylogenetic analysis of HTLV-1 strains based on alignment of LTR sequences from studied patients (in bold) and GenBank sequences. Numbers at nodes indicate bootstrap support level from 1000 replicates (only nodes above 70% are labeled).

FIG. 2.

Phylogenetic analysis of HTLV-1 strains based on alignment of env sequences from studied patients (in bold) and GenBank sequences. Numbers at nodes indicate bootstrap support level from 1000 replicates (only nodes above 70% are labeled).

This study shows that HTLV-1 transcontinental subgroup A was the predominant variant in our population; however, subjects carrying subgroups D and C were also detected. To our knowledge, this is the first time these subgroups have been reported in Europe, which confirms the introduction of new variants in our continent. HTLV-1 subgroup D was detected in subjects from Guinea-Bissau and Guinea in West Africa. This is in agreement with a recent study that describes the existence of two different clades within this subgroup, one from North Africa and the other from West Africa, indicating a more extensive geographic distribution than previously reported.¹² The subject carrying HTLV-1 A subgroup C came from Liberia where it has not been reported so far. However, this country neighbors Côte d'Ivoire and Ghana where this variant is endemic.¹³

In addition to its epidemiological interest, the introduction of new variants in our population may have a prognostic value. Furukawa and colleagues found that subjects infected with HTLV-1 transcontinental subgroup A have a higher risk of developing disease compared with HTLV-1 subgroup B.⁹ In accordance with this, it has been reported that the lifetime risk of HAM/TSP in the United Kingdom is about 3% in contrast to 0.25% in Japan.¹⁴ So far, there are no data about the relative risk of HTLV-1-related disease development in subjects infected with other subgroups such as D or C. In our study, of the four subjects with subgroup D for whom clinical data were available, three had developed HTLV-1-related diseases. Furthermore, one of them was a 9-year-old child with HAM/TSP. This is a chronic myelopathy of adults and a very rare event in children. More studies are needed to determine the true role in disease development of these new imported HTLV variants.

In our study population, subtypes found in immigrants grouped in clusters related to their geographic origin. Most subjects had acquired the infection by heterosexual intercourse or vertical transmission. It is noteworthy that only one immigrant patient was an IDU. Despite the limited size of the study population, these data are in agreement with more extensive epidemiological studies in which the use of intravenous drugs among immigrant populations from Latin America and Africa is not extended.⁸ This would favor a silent dissemination of HTLV-1 subtypes in the resident population rather than a rapid dissemination similar to what has been observed in its counterpart, HTLV-2, which has been extended among IDU natives between Portugal and Spain.^6,15

Three cases of HAM/TSP after transplantation were included in our study. All were detected in native Spaniards. Two patients had been infected by the same organ donor in 2000 (Patients ES-P9 and ES-P10)⁶ and the other was reported in 2008 (Dr. José M. Miró, personal communication). It is well known that both transfusion and transplant transmission are related to a higher risk of HAM/TSP development. In blood donors and transplants, universal screening is mandatory in several countries of the European Union (Portugal), but not in others (Spain). The increase in immigration and the dissemination of these viruses could modify the policy in countries in which screening is not mandatory.

In conclusion, HTLV-1 subtype A transcontinental subgroup A was the most prevalent in the Portuguese–Spanish populations, but new HTLV-1 subtype A viral variants are now circulating due to a rise in immigration. There seems to be a silent dissemination of these variants, with no burst-like spread due to the almost complete absence of IDUs in immigrants. However, this is a vulnerable collective group that can be involved in high-risk practices acquired in the reception country. Therefore, this low but continued presence requires periodic surveillance studies in risk populations, especially those susceptible to rapidly developing disease (e.g., transplants). Likewise, molecular analysis results in a better understanding of the circulating subtypes and the ability to detect possible pattern changes.

Sequence Data

GenBank accession numbers for sequences generated in this study were GU126474–GU126483 (LTR, Spain), GU126484–GU126490 (env, Spain), AY739051–AY739064 (LTR, Portugal), and DQ663633–DQ663648 (env, Portugal).

Footnotes

Acknowledgments

We thank Dr. V. Soriano and Dr. J. Gonzalez-Lahoz for their support. We also thank the clinicians of the HTLV Spanish Study Group who provided samples for the study.

Author Disclosure Statement

No competing financial interests exist.

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