Most HIV-1 genetic subtypes have entered Sweden

Objective: The aim of this study was to document which genetic subtypes of HIV-1 have entered Sweden and to study transmission patterns of these virus variants.

Patients: All HIV-1-infected individuals at Danderyds Hospital, Stockholm, Sweden, who were suspected of carrying a virus of African origin were prospectively included in the study. The study subjects originated from 15 different African countries.

Methods: The V3 domain of the HIV-1 envelope was directly sequenced from uncultured peripheral blood mononuclear cells from 75 individuals included in the study. Phylogenetic analyses were used to determine genetic subtype and to study transmission patterns.

Results: The virus strains carried by the study subjects belonged to six established subtypes of HIV-1 (27A, 4B, 18C, 18D, 2G, 2H). Two individuals from Zaire carried a subtype, which had not been classified previously, provisionally named subtype J. Eleven transmissions of non-subtype B strains in Sweden were documented.

Conclusions: This study shows that most genetic HIV-1 subtypes have entered Sweden despite the relatively low prevalence of HIV infection in the country. Thus, the complete dominance of subtype-B infections which was seen during the early phase of the HIV-1 epidemic in Europe and the US has been broken in Sweden.

Introduction

HIV-1 is characterized by an unusually high degree of genetic variation. A majority of HIV-1 sequences cluster into one large group called M (major), whereas a few are clearly distinct, group O (outlier) [¹]. Group M sequences have been further subdivided into eight genetic subtypes, A through H [¹]. Recently, sequences which appear to cluster into two additional new group M subtypes (provisionally called subtypes I and J) were described [^2,3]. All established subtypes have been found in Central Africa, whereas subtype B is most prevalent in Western Europe and the US [¹]. At present, it is not known if subtype-specific differences exist in biological properties of HIV-1, but it has been suggested that subtype-E variants may be more effectively transmitted via the heterosexual route than subtype-B variants [⁴].

Sweden has a relatively low prevalence of HIV infection (4200 officially reported cases), but the proportion of heterosexually infected individuals is as large as 33%. [⁵]. Epidemiological investigations indicate that a majority of these cases (61%) carry virus strains of African origin [⁵]. Thus, at least 20% of Swedish HIV cases could be suspected of carrying non-subtype-B HIV-1 variants. The aim of this study was to document which HIV-1 subtypes have entered Sweden and to study transmission patterns of these virus variants in Sweden.

Material and methods

Subjects

All HIV-1-infected individuals attending the outpatient clinic for HIV-infected patients at Danderyds Hospital, Stockholm, Sweden who were known or suspected to be infected with an HIV-1 strain of African origin were able to participate in the study. Such individuals were identified by a thorough epidemiological investigation, which is part of the routine evaluation of every newly diagnosed HIV case at the clinic. Eligible individuals have been prospectively included and followed up since September 1994. Informed consent was obtained from all participants and the study was approved by the local ethics committee.

DNA sequencing and phylogenetic analyses

The V3 domain of the HIV-1 env gene was amplified directly from crude lysates of uncultured peripheral blood mononuclear cells by polymerase chain reaction (PCR) with nested primers (JA167-JA170) and directly sequenced as described elsewhere [⁶].

Phylogenetic trees were constructed using the PHYLIP programs DNAML (maximum-likelihood, using empirical base frequencies and a transition/transversion ratio of 1.42) and DNADIST (using empirical base frequencies and a transition/transversion ratio of 1.42) together with NEIGHBOR (neighbour-joining) [⁷]. Bootstrap analysis (1000 resamplings) was performed with the neighbour-joining method. Prototype sequences from subtypes A through J were obtained from the Los Alamos database and used as controls [¹]. The sequences have been deposited in the EMBL/GenBank database under accession numbers U76114-U76186 and L41176-L41179.

Results

Approximately 270 HIV-infected individuals are followed at the Department of Infectious Diseases at Danderyds Hospital. The epidemiological investigations indicated that 80 of these individuals carried an HIV-1 strain of African origin and blood samples were available from 75 of these individuals. There were 33 men and 42 women all of whom were likely to have been heterosexually infected.

The subtype of the HIV-1 variants carried by the study subjects was determined by direct sequencing of the V3 domain of the HIV-1 envelope and phylogenetic tree analyses. Figure 1 shows a representative phylogenetic tree. From two individuals (SE6486 and SE7195) we were unable to obtain V3 sequences. These virus variants were shown to belong to subtype D and G, respectively, by analysis of p17gag sequences [⁸] (data not shown). Among the 75 subjects, we documented infections with six established HIV-1 subtypes (subtypes A, B, C, D, G and H). Two individuals from Zaire carried virus variants (SE7022 and SE7887) that could not be classified into previously reported subtypes. These strains have been described in detail in a recent separate report [³] and have provisionally been named subtype J by the Los Alamos database [¹]. Two additional individuals carried virus variants (SE8603 and SE9340) which were differently classified by different phylogenetic methods. Both sequences clustered in subtype D in the maximum-likelihood tree (Fig. 1), whereas they were excluded from subtype D with a bootstrap value of 70% in the neighbour-joining tree (data not shown). Therefore these two sequences were regarded as unclassified (U).

Fig. 1

The HIV-1 variants carried by the 75 subjects originated from 15 different African countries (Fig. 2). From four of these countries (Botswana, Eritrea, Mauritania, and Mozambique) there have been no previous reports of which genetic subtypes are prevalent among HIV-1 infected individuals. Subtypes A and D dominated in Uganda, whereas subtype C dominated in north-eastern and southern Africa.

Fig. 2

Four individuals were shown to carry subtype B HIV-1 variants. Since this subtype dominates in Europe, it was of interest to evaluate if these individuals were likely to have been infected before or after their arrival in Sweden. Three of these individuals were Africans and one was a Swedish woman who had worked in Kenya for one year. All were found to be HIV-1-seropositive shortly after arrival or return to Sweden. The epidemiological investigations revealed that all individuals had had sexual contacts in Africa and had failed to identify possible sources of infection in Sweden.

The epidemiological investigations also indicated that 10 of the 75 subjects included in the study were persons of Swedish origin who had been infected through sexual contact with persons of African origin. Four of these transmissions had occurred in Sweden, whereas six had occurred in Africa. In addition, seven individuals of African origin had become infected in Sweden through sexual contact with persons of African origin. All these seven individuals had tested negative for HIV antibodies on at least one occasion after their arrival in Sweden. Thus, we have documented 11 transmissions of non-subtype-B HIV-1 variants in Sweden. Finally, six pairs of samples were obtained from individuals who were suspected of having infected each other; in all cases the suspected transmission history was supported by phylogenetic and bootstrap analyses (Fig. 1).

Discussion

In this study of 75 residents in Sweden infected with HIV-1 variants of African origin, we have documented infections with subtypes A, B, C, D, G, H, and J. In addition to the HIV-1 variants described here, we have found a few cases of subtype E infections among Swedish individuals infected in Thailand (unpublished), consequently subtypes F, I and O are the only genetic subtypes that have not yet been found in Sweden. Thus, we have shown that most HIV-1 subtypes have entered Sweden, despite a comparably low incidence and prevalence of HIV infection. This means that the complete dominance of subtype B infections, which was seen during the early phase of the HIV-1 epidemic in Europe and the Americas, has been broken in Sweden. Recent studies indicate that a similar change may be occurring in other European countries [^9-12]. Thus, it is becoming increasingly important to verify that serological and genetic assays for the detection and quantification of HIV-1 detect all genetic subtypes with equal efficiency. Furthermore, a future HIV-1 vaccine must be effective against all subtypes if it is going to be successful in Europe. Finally, the introduction of new subtypes could possibly alter the clinical picture of the disease as well as the rate of transmission [⁴].

The influx of heterosexually infected HIV-1 carriers to Sweden does not appear to have caused a dramatic change in the number of heterosexually acquired infections in Sweden. The number of such infections has remained relatively stable over the last ten years [⁵]. Sporadic secondary infections do occur, however. In our study, 11 individuals became infected with nonsubtype B variants in Sweden. Interestingly, a majority of these individuals (seven out of 11) were of African origin, suggesting that transmissions may preferentially occur within ethnic minority groups.

The HIV-1 variants that were obtained in this study originated from 15 different African countries and from four of these countries (Botswana, Eritrea, Mauritania, and Mozambique) we are not aware of any previous reports on HIV-1 subtyping. We found that four individuals originating from four different countries carried subtype B virus variants, and the epidemiological investigations indicated that they all had been infected in Africa. Thus, even though subtype B is rare in Africa, it appears to be present in several different parts of the continent.

In conclusion, we have shown that most genetic subtypes of HIV-1 have entered Sweden. A few of these non-subtype-B infections are the result of transmissions in Sweden, but a majority of individuals carrying such variants are immigrants of African origin who were infected before moving to Sweden.

Acknowledgements

We thank S. Marquina, P. Schipper, E. Lilja and A. L. Andersson for expert technical assistance.