‘wnoxoor 208, 195-146 (1905) Biological and Molecular Characterization of Subtype D, G, and A/D Recombinant HIV-1 ‘Transmissions in Sweden THOMAS LEITNER,“! DAVID ESCANILLA,* SILVIA MARQUINA"? JOHAN WAHLBERG,t CHRISTINA BROSTROM,+ HANS BERTIL HANSSON,§ MATHIAS UHLEN.t and JAN ALBERT* “Deparment of Cline! Violgy, Sedsh lite for Infecous Disease Convoiearoinska insti, Stockho, Sweden: apanmant of Biochemistry and Biotechnology, Foy Insitute of Technology. Stockholm, Sweden: ‘Division of infectcus Diseases, Dor ‘Microbiology, Patotogy ard Infgcvous Diseases. Keralnsha Inet, Huddinge University Hospi). Stockholm Sweden, {nd §Rogional Cantor fer infacbous Disease Conta, Unversity Hospi Mimo, Sweden Received January 17, 1896; escapted Febwvary 17, 1995 HIV-1 can be subdivided into at last nine genetic subtypes (A through H and O}, but in Europe and the United States ‘there is an almost complate dominance of subtype B. In this stay three Swedish HIV-t Wanemission choine of subtypes ‘othor than subiype B have been biological and molecularly characterized. The three index easas ware Alrican men. The BIT gag and nv Va regions of te HIV-1 genome wara dractly sequenced from uncultured lymphocytes, Prylogenatic ‘analyses showed thatthe HIV-1 variants within each wansmission ercup were gensticaly closely related, supporting the ’pidemipiogcal information. The inciiduals i transmission groups | {n = 9) and in = 2} certied subtype G and D vius, ‘eepectively.Inoreatingy. all fur indvidvale In transmission group Il gaplayod 9 recombinant gonetype with eubiype D 1817 ong soauence and svblypo A V3 sequenca The biological phenotype of virus isolates (rapi/high. syneyiurrinaiing ‘0F alow ow, pon-syreytanricg) correlated withthe clinical stage ofthe infaced indivduel The study aloo suggestes thatthe corelation between biological phonotype and VO genotype that has boon established for subtype B HIV-! variants may be vali az for ther subtypes This study demonstrates tht HIV-t variant of eubtypes other than B Including 2 iien of umurcoay, subiype A/D recombinant INTRODUCTION Human immunodeticiency vitus type 1 (HIV-1) displays: fan unusually high degree of genetic variability in vivo. Analysis of HIV-1 env genas of virus strains from different geogrephic locales has revealed that HIV-1 can be di Vided into two major groups, M (major and O (outlier) (Chemeau et al, 1994), HIV-1 group M isolates can be further subdivided into at least eight distinct genetic eub- types (A-H) (Myers et af, 1993; Louwagie et al, 1993: Salminen et a1, 1993; Janssens et af, 1994). Phylogenetic tree analyses have shown that all subtypes have an aver- age nucleotide distence of 1010 13% toa common ances: tral node in the envelope gene (Myers et al, 1993). This suggests that the sublypoe may have arisen from a com mon ancestral virus (Myers er a, 1993) HiV-1 of subtype B predominates in tine United States, Europe, and South America, whereas subtypes A and D ‘are mainly found in Cential Africa (Myers et af, 1993). The other subtypes show a more patchy distribution; sub- "To whom conespondence and reprin requests should be ox ‘eox99¢ at Departmant of Clinica Veale, Swett Inetite fringe tious Diesaee ConolKaroineka theta. S-108 21 Stockholm, Se: on. Fan int+46-8-720 2248 Ema tisbiochan tins, * ncdakonat adatess: Depatinent of Miczoniogy, School of Mad lng, Universit of Suenos Ales, Ovenoe Aes, Azgontna 42.6820105 $6.00, {eon ay Recess be 0 being transmitted Europe. 01 type © has been found in Eastern and Southern Africa, 28 well as in India; subtype F in Central African Republic, Thailand, and India; and subtype F in Zaire, Brazil, and Romania. In many countries two or mare subtypes coex- ist; subtypes A and D have been found in Uganda, sub- types B and E in Thailand, and sublypes @ and F in Brazil, Consequently, the situation in the United States and most European counties with an almost complete predomi nance of one subtype, i. subtype B, is not typical for mast other parts of the world. In Sweden the predomi- nance of subtype B variants is not likely ta be permanent, since a large proportion of newly diagnosed HIV fected persons are of Aitican origin. Almost 45% (170 of 384) of the new HIV-1 cases reported in Swedan in 1993, ‘ete of African origin (M. Ameborn, personal communi: cation). The Identification of gonetic subtypes of HIV-t raises: important questions about the possible existence of sud- \ype-specilic differences in immunogenicity, transmissi bility, and pathogenicity, However, so far very litle data fon these Important issues are available. Another i= portant question Is if the corrolation between biological phenotype and disease progression that has been estab. lished for subtype 8 viruses will hold true also for other subtypes, Thus, subtype B virus isolates can be divided Into two groups; rapiaihigh (Asjo ot ai, 1986; Fenyo erCHARACTERZATION OF HIV! TRANSMISSIONS sar al, 1888), syncytium-inducing isolates (Tersmette et af, 1988), and slon/low As et el, 1986; Fenyt etal, 1988), non-synoytium-inducing isolates (Tersmette et af, 1986). Rapid/high isolates are characterized by their ability to replicate in established tumor cell ines (Asi et ef, 1986; Feny6 et al, 1988). They also grow rapidly to high thers and induce syncytia in primary lymphocytes (Asié et ef, 1986; Fenyé et af, 1988; Tersmnette et al, 1988). In con trast, slow/low virus isolates are unable to replicate In tumor cell lines, they grow more slowly and to lower titers in primary iymohooytes, and they induce no or only small syncytia (Asj® et af, 1986; Feny® et 21, 1988; Ters- mette et al, 1988). Patients carrying repicstigh, syncy- tiuminducing virus show accelerated rete of disease progression (Koot et al, 1993; Kerisson et af, 1994) {in this study we have biologically and molecularly characterized three separate chains of transmission in Sweden of virus belonging to subtypes othor than sub ‘ype 8. The investigations were initisted because the three male index cases were suspected ot having ne glected to inform their fomale sexual partners that they were HIV-1 infocted ang thereby of having deliberately exposed these females to the risk of becoming HIV-t infected, Thus, a primary goal was to establish if the HIV- 1 strains carried by the index cases and the respective ‘female sexval partners were genetically closely related However, the study also gave us an cpportunity to docu: ment and characterize transmission of nonsubtype B vi- rus in Sweden, MATERIALS AND METHODS Patients Taree separate transmission groups (ll) were in- vestigated (Table 1). Each group consisted of a mate index case and one (groups I and Il) oF two (group |) female sexual partners. Transmission group til also included 2 child and @ second mele sexuel partner of ine femaie. The index cases were AMrican immigrants wino had been Infected with HIV-1 in their country of ‘origi, whereas the female soxual partners became in- fected in Sweden. Duplicate heparinized blood samples {20 mi) were obtained on separate days trom all subjects in the throo transmission groups, except the child in transmission group Ill from whom only one sample was obtained. Peripheral blood mononuclear celis (PBMC) wore obtained by centrifugation on Ficoll~Paque (Phar- macia, Sweden) and used for virus isolations end POR as described below. Virus isolation and biological characterization HIV-1 was Isolated from PBMC by cocultvation with phytohemagglutinin (PHA)-stimulated PBMC. trom healthy seronegative blood donors as previously de- scribed (Albert ef af, 1987; Fiore etal, 1994), The biologi- cal phenotype of the isolates was determined by testing the replicative capacity in the MT-2, HUT-78, and C-8166 T-cell lines (Fiore et ai, 1994). For this purpose PHA: stimulated blood donor PBMC were infected with frst or ‘second passage virus and when these cultures became Virus positive @ cocultvation was carried out with 1 10" infected PBMC and 3X 10° MT-2, HUT-78, and C- £8166 colls, respectively, The Isolates were also tested for the ability o replicate in monaeyte-detived macrophages (MOM) as described (Valentin ef af, 1994}, All cultures were carried for at least 21 days and supematants were tested twice weekly for presence of p24 antigen (Sund- vist ef al, 1988). MT-2 ceil cultures were also checked for the appearance of syncytia. Cultures were considered virus positive if increasing p24 antigen levels were de- tected in two consecutive samples of culture superna- tant. However, the MT-2 cell cullures were considered virus positive only it they also showed typical synoytia Polymerase chain reaction (PCR) and HIV-1 DNA load determinations Regions of the HIV-1 gag, pol, and eny genes corre- sponding tothe entire p17 gag protein, the emino-termi- hal half of the reverse transcriptase (RT), and the V3 domain of the envelope wore ampitied by nested PCR from uncultured pationt PBMC #8 previously doseribed (Alber and Fenyd, 1980; Scarlatti et a/, 1983; Albert et 41, 1984) The RT and V3 primers have been described previously (Scariatt ot af, 1993; Albert et al, 1994), The nested primers for amplification of p17 gag, in transmis- sion groups I! and Il, were as follows (their positions in the HIV-1 MN strain are given in parentheses): the outer pair was JAI62, 5" ATCTCTAGCAGTGGCGCCOGAA- GAG-3" (624); and JA155, 5-CTGATAATGCTGAAAACA- TGGGTAT-3" (1300); and the inner primers were JA163, S'CTCTOGACGCAGGACTOGGCTTGCT:-3” (679): and JAI64, §/-biotin-COCATGCATTCAAAGTTCTAGGTGAS! (1237), The p17 gag primers used in group | were as previously described (Albert et af, 1994). Cycling profiles were 84° for 80 sec, 60° for 30 sec, and 72° for 60 sec for p17 gag and AT amplifications and 94° for 30 sec, 50° for 80 sec, end 72° for 60 sec for V3. The samples ‘were fiat amplified with outer primers for 30 cycles and subsequently 3 of the product was further amplified for 30 cyclos with Inner primers, Presence of amplified mate- Tia! was verified by electrophoresis on 1.5% agarose gels, stained by ethidium bromide. The viral DNA copy numbers in the samples were determined by limiting dilution as previously described (Binchmann etal, 1981). Briefly, the PBMC lysates were serially diluted in fivefold steps, then § to 10 PCR reac- tions were performed on each dilution. Since the nested PCR can reproducibly detect a single DNA copy (Brinch:198 LENTNER ET AL, man et al, 1991; Albert et al, 1994), he DNA copy num- ors could be calculates using Roed-Muench calculation ‘and the Poisson distribution formula (Reed and Muench, 41938; Brinchmann et ai, 1991). DNA sequencing ‘The virus populations in the PBMC samples wore di- rectly sequenced on both strands by solic-phase ONA sequencing as described (Hultman et al, 1989, 1991; Leitner et al, 1993). DNA fragments obtained by amplifi- cation with inner primers were purified by immabitization fon steptavidin-coated magnetic beads (Dynabeads M280-streptavidin; Dynal AS, Norway). Single-stranded DNA was obtained by denaturation with sodium hydrox- ide. The sequencing reaction was performed with fluo- rescelmiabeled primers according to the Sanger mothod using @ commercial kit (AutoRead, Pharmacia). The se- quencing primers for RT, V3, and p17 gag in transmission group i have been described previously (Scarlett et af, 1983; Albert et af, 1994) and the sequencing primers: for p17 gag in groups II and Il were JAI17, 8/-FITC- GCATTTAAAGTICTAGTTGAS' (1237); and JAISSF, 6'- FITO-CTCTCGACGCAGGACTOGGCTTGCT-3’ (678). The sequence fragments were analyzed on 6% polyacryl- amide gels in an automated laser fluorescent sequenc- ing apparatus (ALF; Pharmacia LKB, Sweden). The se- quencing method allows accurate identification and ‘uantitication of polymorphic nucleotide positions within the virus population in a sample (Leitner ef af, 1993). nucleotide was scored as polymorphic only if at least 18% of the virus population was reproducibly estimated. to be mutant. Polymorphic positions were indicated by IUPAC codes, To ensure that no sampling artifacts were introduced during PCR, the ONA copy number in each sample was determined as described above. Finally a ‘consensus based on the sequences from two indepen- ent samples obtained within a fow days of each other was constructed for each individual Phylogenetic analyses ‘The DNA sequences were aligned manually with the aid of the sequence editor in the GDE (Genetic Data Environment) package (Smith, 1992). Amino acid transla- tions were also made in GE, Phylogenetic inference was done using the PHYLIP package, version 362 (Felsenstein, 1993), and PAUP 3.1.1 (Swatford, 1981), to- gether with MacClade 3,04 (Maddison and Maddison, 1992). The PHYLIP programs DNAML {maximum likeli- hood), DNAPARS (maximum parsimony], and DNADIST, together with NEIGHBOA (Jukes-Cantor plus neighbor joining) were used to craata traes, SEQBOOT was used to bootstrap data infiles, in which 1000 sets were made, and CONSENSE to compute consensus trees. PAUP was sod to produce unweighted parsimony trees, then Mac Clade was used to calculate a weighting matrix based Con the infile and the unweighted tree to finelly calculate weighted trees in PAUP (Hillis et ai, 1994; Korber et al, 1984; Maddison and Madison, 1992). To visuatize and graphically edit the tees the program TREETOOL, ver ‘sion 1.0, in GDE was used. Sequences classified as sub- types A through H were obtained from the Los Alamos database and used as controls. The subtype of the se- ‘quences from each transmission group was first deter- mined by comparison with selected reference sequences {rom all subtypes. Then a detailed phylogenetic analysis wes carried out using all available sequences of the sublype in question. Finally trees for presentation were ‘generated using the sequences from all tree transmis: sion groups and selected reference sequences. ‘Sequence identification and accession numbers. The sequences have been given names according to the WHO Globai Programme on AIDS proposal for stan- dardization of HIV sequence nomenclature (Korber er al 1994 In the figures of this paper both the codes for each patient and the sequence names, composed of country code and sample number, are given.” RESULTS Epidemiological and clinical characteristics ‘Three seperate transmission groups (I, I, 10} were in- vestigated (Table 1). Each group consisted of a male index case and one (group Il and Ill) or two (group I) female sexual partners, Transmission group Ill also in- cluded a child and @ second mate sexual partner of the female. The index cases were African immigrants who. had become infected with HIV-1 In their country of origin, whereas the temele sexual partners became infected in ‘Sweden, Transmission group | consisted of one HIV-1-seroposi: tive male index case (0) and two HIV-1-seropositive female sexual partners (Io and i-2) (Table 1), The index case was a man from central Africe, who had tested ositive for HIV-1 antibodies by ELISA and Western olot- ting in Sweden 18 months prior to the estimated time. point for transmission. Approximately 1 year after the transmission he had had low CD4* lymphocyte counts: (35 X 10"fiter) and dermatologic problems, but no AIDS- defining iliness, Thus, he was classified as category BS. ‘according 10 tho 1983 revised classification systom for, HIV infection of the Centers for Disease Control (CDC) (1992), The first female sexual partner (I-1) had a short (a few weeks) sexval relationship with the index case. * Nuclstde sequence dae nore deposited with ell WHO names in the EMBL, Genfank, and DDB) Nucleotide Sequence Daiabeses under Aooeesion Numbers L40745- 40763CHARACTERIZATION OF H-Y TRANSMISSIONS 138 Tague 1 Epidemiological and Cfnical Charactorists of Individuals in Three Separate HIV-1 Transmission Groups Previous antibody Time for sampieg (c08* temphoote Sujet (code) niin status (mons [rrone) Symptems counts (ero%Aie) Transmission group 1 Male index case (0) Cental Aiea ——Serepos. (18) 48 Symptomatic 2% Female sexual parser (1) No 7 Asymptomatc 100 Formate sosual partner (12) Seroneg.(-#) 48 ‘Symptomatic a0 ‘Transmission group I Male index cage (ILO) Upande Serpe. (96) 2048 Asymataravic a0 Formate sexual partner (-1) Seronag.(=80), 210 48 ‘Asymoromatc 0 “Teanamission group Il Male index e580} Ugande Seropos. (12) a Asympzomate 11000 Fomale sexual paner(H-1) ‘Soronea.|=7) an Asmpiomsic 620 Chi erate (2) ‘Seroneg.|=7) +10 Asynpromac 3700 Second mls somal peter of ND 0 Asimptamaic 520 Female (3) ‘Note Tim for sempling is given in respect to astimated time pont of ansission "TaN antibody stats prior to ransmnision. She stated that the Index case was the first sexual con- tact of her lite. No earlier serum samples were available for serological testing, Approximately 3 woeks after the ‘estimated time point for transmission she experienced {an acute febrile iiness, which retrospectively was con- sidered as a probable seroconversion illness. Seven months after transmission female |-1 already had se- verely reduced CD4" lymphocyte counts (100 x 10%, but no AIDS-defining illness (CDC category A8). The sec cond female sexual partner (1-2) had 8 short sexual rela tionship with the index ease shortly after the first sexual partner. Female 2 had tested negative for HIV antibod ies 4 montng earlier. Female 2 also experienced 2 prot- able seroconversion liness 2 fewr weeks after the trans- mission, Six months fier transmission she had low D4" lymphocyte counts (80 X 10iter), but no AIDS- detining illness (CDC category B3). Duplicate bload sar- ples for PCR and virus isolation were obtained from the three subjects 6 and 7 months after the suspected time points for the transmissions to female It and F2, respac- Waly Transmission group Il consisted of one HIV-1-seropos- itive male index case (\-0) and one KIV-t-seropasitive female sexual partner (I-1) (Table 1). The index case was a man trom Uganda, who was documented to have been Hiv-t-infected for atleast 8 years, He was asymptomatic and his CD4* lymphocyte counts were slightly sup pressed (#20 x 10%niten, but stil within the normal range of our laboratory, placing him in COC catagory AZ. The forale sexual partner (I+) had a sexual relationship with the index case that lasted § months. She had tested negative for HIV antibodies 5 years earlier, but more recent serum samples were not available. When the blood samples for POR and virus isolation were obtained 2 to 6 months after transmiesion she was asymptomatic. with normal OD4* lymphocyte counts (620 x 10%iter) (CDC category At), Transmission group Ill consisted of one HIV-1-ser0- positive male index case (II-0), one HIV-1-seropositive female sexual partner (Il-1), the female's HIV-1 seropost tive child (l-2), end @ second HiV-1-seropositive male senual partner ofthe female (II/3) (Table 1). The index cease of transmission group Ill (subject I-0} was 3 man from Uganda, who had been found HIV-1 seropositive pproximately 12 months before the suspected time point for transmission, Eleven months after the transmission he was asymptomatic with normal OD” lymphocyte counts (1000 x 10*fiter) (CDC category At). The female sexual partner (+1) hed # sexual reletionship with the index case for 6 months. She had been tested and found negative for HIV-1 antibodies 18 and & months prior to the probable time point for transmission. She experienced a probable seroconversion illness approximately 1 month after rensmission, Eleven months after wansmission she was asymptomatic with narmal CD4" lymphocyte counts (620 x 10%fiter) (CDG category AN). The female gave birth to a child (II-2) approximately & months prior to transmission. This child was negative for HIV-1 antibod- ies in chord blood at birth, Thus, the female was not HIV- 1 infected at this time point. The child probably became Infected through breastfeeding at 8 months of age. Ten ‘months ater tansmission the child was seropositive, but ‘asymptomatic with normal GD4" lymphocyte counts. The second man ill) had a sexval relationship with the female staring shorly before she experienced her pr: mary HIV-1 infection. No earlier serum samples were0 LETINER ET AL TaBle 2 Virological Cnoractorstic of Indviduala in Theos Separate HIV"! Transmission Groupe iver DNA load ius 'olation Subject (code) (copies/to? P2Mo) {ne, postve/no, stem] Bolosicel ohenowne Transmission group 1 Male Index cae (0) 19 1“ Ropiaigh Female sexual parner (+1 8 a8 Regis Formate eoxval pares (2) 7 mw Raping “Tiensmission group I Male index case (-0) oss oe ND Female soxval parer it) ans on ND “Tensiiasion grou I Male ina case (0) 028 we Slowiow Female ooxval partner (ht) ND on ND Chi of mete ih) aie ND ND Second male senusl poner of 23 ue Slowiow female (3) available from this man. Ten months after transmission he was asymptomatic with normal CD4* tymphooyte counts (520 X 10%fter) (CDC category At). The study showed that subject III probably becarne infected by the female during her primary infection (see below). Virus load and biological phenotype of virus isolates ‘The HIv-1 ONA load in PBMC was determined by per- forming multiple PCR analyses on serially diluted PMC. lysates and calculations according to the Poisson distr- bution formula (Brinchmann et af, 1991). In transmission ‘group I all subjects tad e high virus DNA load (Table 2). All virus isolation attempts were successful in these three individuals. Furthermore, these virus isolates repli- cated and induced syncytia in several established tumor cell ines (HUTT, MT-2, C8166) and thus displayed @ rapid/high, syncytiui-inducing phenotype. In contrast, the subjects in transmission groups Il and Ill, who re- mained immunologically intact, had lower virus DNA load in PBMC. Only 2 of 10 virus isolation attempts from these Individuals were successful and these two virus isolates displayed a slow/low phenotype. All isolates, including those with rapid/high phenotype, replicated efficiently in macrophages (date not shown). Env, gag, and pol gene sequences A.341-bp fragment corresponding to the V3 domain of the HIV-1 envelope was sequenced directly from uncul- tured P8MC from all individuals, The deduced amino acid sequences are shown in Fig. 1. Itshould be stressed that all sequences presented in this study describe the virus populations within the infected individuals rather than single clones. DNA corresponding to 100,000 PBMC. was amplified and directly sequenced without any clon- ing step. Since the DNA copy number in the PBMC sam- ples was determined, we know thet between 12 and 1700 HIN-1 malecules went into each PCR (Table 2), The sequencing method allows nucleotide positions which differ between Individual molecules in a sample (poly- _momphic positions) 10 be detected and roughly quantified ‘as long as the miner variant is present in at least 15% cf the totel population (Leitner et al, 1993), Consequently, ‘each sequence is an average (ar consensus} of the popu- lation with a resolution dawn t0 16% Within each vansmission group the V3 sequences were closely related, but between the groups significant differences were evident. In HIV-1 of sublype B it has been shown that the biological phenotype of a virus can be predicted by the V3 loop sequence (De Jong et al, 1992; Foucher et al, 1992). In particular, positively ‘charged amino acids at position 311 or 328 have been found to be highly predictive of rapidihigh, synoytium- inducing phenotype (De Jong ef af, 1992). The same correlation was found in our data set. Thus, the subjects, belonging to transmission groups Il and ilf who carried virus with slow/low phenotype had sequences with neu- ‘ral or negatively charged amino acids at these two post tions. In contrast, the sequences from the subjects in transmission group |, who carried rapidmigh virus, had ‘a positively charged amino acid at position 311 (arginine) and a neutral amino acid at position 328 (alanine). How- ever, there were only minor differences in tne overall charge of the V3 loops between the three transmission ‘groups; the charge ranged from +3 10 +4, The degree of intrapationt V3 sequence variability was relatively high in the index cages of transmission groups Il and Ill, but low in the index cese of transmission group |, as well as in all recipients. Figure 2 shows the p17 gag sequences obtained fromCHARACTERIZATION OF HIV-1 TRANSMISSIONS “ FIG. 1. ligament of ans ‘amine acid sequences tor the HIV-1 anvloze Vi domain from three anamission groups. The sequences are _stgned agains ine irdexcese in aach tanstrssion group. dendicaion ofthe individua's and sarees is given nett the eavespondig sequence. “Tne V3 loop i indicated athe top, and postions corresponcing to HIV-1 MIM are gwen fr the fet ene las! mina aod. A cach derctae ority to ncex ease ana dos inceate gaps invocuces to aig> the sequences. Pestons in which the wus populaian of an mavcusl dsplayea sequAnC® obmorphism are Indicated by assigning tha two amino acids i quaston with one underneath the chee. A codon was scored a5 polymorphic ‘nly ifthe minor polvrarphic nucleotide vatiant existed in at east 16% of the population Sites wih synoryrnous polymorphisms ae indicated as Uundertned leter in index case saquences and equal signs in cients: Number indicates polymorphic codons with more than to posable smin> 364s, scoorang to ie flowing code: 1 = G, & and 0 ‘he subjects in the three transmission groups. The p17 secuences within each transmission group were closely releted, but distinct between the groups. The sequences from trensmission group Il showed only two postions with polymorphism, one synonymous mutation in 87498 (I-2) ‘and one nonsynonymous rmutation in SE6986 (li-3}, Those ‘sequences may seom surprisingly similar, but it should be remembered that subjects I, lir2, and II-3 were analyzed vwthin 1010 11 months after transmission and that subjocts U2 and (3 were infected by the female (ll-1) when she experienced her primary HIV“ infection, The individuals in transmission group | were found 10 corty subtype G HIV-1 variants (see below). We decided to determine pol gene sequences in these individuals, since very few aubtype G viruses have been cescribed at all and no subtype G po! gene sequences have pre- viously been published (Fig. 3). The sequences from the individuals in transmission group | wore closely related also in pot. Furthermore, they were found to be distinct from previously published po! gene sequences, further supporting that these sequences represent the first de soribed subtype G po! sequences (data not shown). The sequences did not carry any mutations that have been reported to be linked to drug resistance. Phylogenetic analyses Phylogenetic tree and bootstrap analysas ware used to compare the sequences from the three transmission {groups to each other and to a set of reference sequences from the Los Alamos Database. In the analyses available HIV-1 subtype A through H sequences were included, selected as described under Materials and Methods. Fig- Ure 4 shows trees genorated by the maximum liketinood algorithm, but trees generated using other algorithms (parsimony, weighted parsimony, and neighbor joining) gave similar results Figure 4A shows the tree generated using sequences {rom the p17 gag coding region. In the tree itis easy 10 identity seven approximately equidistant sequence clus- ters that correspond to HIV-1 subtypes A through H (sub- type E gag sequences have not been described). It should be noted that subtypes D and B are not clearly ‘separated in the tree. Thus, subtype D is divided into two. sequence clusters which are separated by the subtype 8 cluster. In trees derived with other methods subtypes 8 and D always wera very close, but the two subtype D clusters were held together. The bootstrap values for the subtypes range from 78% for subtype A to 99% for sud- type C. For reasons described above it is not possible to give a bootstrap value for subtype D in this tree, but In parsimony analyses the value was between 34 and 81%. The exact intorpretation of results from bootstrap analysis is unclear, but Hillis and Bull (1993) have shown that bootstrap proportions of >70% in most situations cortespond to a probability of >95% that the correspond- Ing clade is real. Thus, according to this interpretation ‘the low bootstrap value for the sublype D and the archi- tecture of the tree indicate that this subtype may not be. significantly distinct from subtype B in the p17 gag coding region. The clusters cortesponding to the other subtypes all had bootstrap values exceeding 70%wae LEITNER ET AL. Pion Serle Somes esse oer FIG. 2 Alonmert of renslatec amino ac sequences for he HIV-1 p17 gag fragment fem thvee transmission groups. The sequarces are aligned ‘aginst the index case in esch Wrensmission gf0up, Kletilcation ofthe ndviduals are samples ate given next tothe corresponding sequence. ‘Te slat of te p17 cosing sequence ard the laavage site botwaen pI7 and p24 ar indicate. Th fist ond lest amino 6ids an each alignment a0 are gen cortesponding o postiens Inthe HIV-1 MN Isola; nol tat the sequences ara iteleaved. Symbols are aa in Fig. 1. Numbers Inicate polymorph codon with more than two fossible amino selds, according o the folowing code: 1 =, €, and C:2= N.S, and Ki K Rend O14 =G, K R,and E FIG. 9, Alignment of tanslated amino acid sequences for the fragment in the HIV" pol gare ftom tarsmission group |. The sequences aro ‘igned seainst the index case. The cleavago site Seton th protease ard pBB and pSt ooding regions atte ster he ragment is inckeated ‘and numbers are given coresponsing fo postions in RT (reverse wanecriprsce) ofthe HIV-1 MN isclate; note tht the sequances are Ineeaved Symbols areas in Fig.(CHARACTERIZATION OF HIV-t TRANSMISSIONS 13 FIG. 4 Unrocted maximum likened tees based on (A) pt? gag and (8) anv V2. The sequences from the thee trensmission gloups were anatyaedtogeter wih known subiype A through H sequences, Tho tvee vansmigsion Groups tI and Il ae inglatac. The evoloray stance bewoun mo sequences can bo derived by adng the connecing branches and using th icles Seale below each Wed. Imponart boots alas, In percentages of 1000 re ‘The phylogenetic ee analysis showed that the pt7 gag sequences derived from transmission group | were most closely related to the VI191 and LBV217 sequences in the Los Alamos Database, These two sequences have been considered members of an as yet poorly character- ized new subtype of HIV-1 (subtype G), The fact that the clade consisting of VI191, LBV217, and the sequences from transmission group | nad a bootstrap value of 80% suggests that all these sequences are members of 2 distinct subtype of HIV-1 (subtype G). The high bootstrap value for the cluster consisting of the sequences from transmission group | (100%) as well as the close similar ties between the sequences strongly indicated that they Indeed wore closely linked epidomioiogicelly, Simiterty, the phylogenetic analysis supported the hypothesis that the sequences from transmission groups Il and Ill, re- spectively, were closely linked enideriologically. Even though both groups of sequences were of subtype D, they were clearly separate trom each other as well as from other subtype D p17 gag sequences. The high boot- slrap values (81 and 98%, respectively) supported that they represented monophyletic clades. Figure 4B shows the tree obtained using the V3 ee- quences as well as selected bootstrap values. The tree resembled the p17 gag tree; the cifferent subtypes that have been described for the env gene (subtypes A through H) were easily identified, However, subtypes A G, and H ‘were found to be closely related. The cluster of sublype Ahad taxa joining close to the canter of the tree, indicating melngs, calculated wih maximum parsimony algo, are indicated inthe ees. that this subtype is less well defined and more divergent than the others (B, C, D, E, and F) in this env gene frag- ment. Therefore, no bootstrap values are presented for sublypes A, G, and H (they ranged from 35 to 40%). The two subtype H sequences did not cluster together in this twee, but did so with the neighbor joining method. ‘The V3 sequences from transmission group | clustered with two other subtype G sequences, as they did in p17 _gag, The V3 sequences from the individuals in ansmis- sion group Il formed a distinct clade within aubtype D, similar to what they did in the p17 gag tree. Interestingly, the V3 sequences from the individuals in transmission group lll clustered together with subtype A sequences, despite the fact that they clearly clustered with subtype D sequences in p17 gag. This observation stvongly indi cated that all individuals in transmission group Il were Infected with @ subtype A/D recombinant virus. Oetelled analysis of the sequences indicated that the site of re- combination was not within the two sequenced frag: ‘ments, The tnonophyletic grouping of the three tansris- sion groups was supported by very high bootstrap values (100% of groups I and III, 98% of group II) for all three groups. It is also interesting that the direction of the transmissions is correctly indicated by the tree analysis, since the sequences of index cases aro found closor to the center of the tree. DISCUSSION In this study we have biologically and molecularly characterized three Swedish HIV-1 transmission chains.14 LeEITNeR EF AL Phylogenetic analyses showed that the individuals in ‘transmission groups I-Ill were infected with HIV-1 of subtype G, D, and A/D recombinant viruses, respectively. HIV-1 subtype B viruses predominate in Europe and the United States, and only a few individuals infected with virus belonging to other subtypes have Deen reported in these countries (Salminen ef al, 1993; Charneau er al, 1994; Loussert-Ajaka et al, 1994). As a result of this our knowledge of the biology of HIV-1 is largely based on studies of subtype B virus. From a global perspective it is obvious that this concentration on subtype 8 is inap- propriate and recent initiatives by the WHO and others, is likely o give important new information (WHO Network for Isolation and Characterization, 1994; Ichimura et al, 1994; Dumitrescu et 8), 1994). Our study ilustrates also that in some European countries the predominance of subtype 8 virus may be changing; aporoximately 50% of newly detected HIV-1 cases in Sweden have been in- fected outside of Europe and this study demonstrates that secondary transmissions are now occurring, The virus strains carried by the individuals within each of the three transmission groups were genetically closely related to each other, but they were distinct from those of the other transmission groups as well as from previously published HIV-1 p17 gag and env V3 sequences. This stfongly indicated that the epidemiological information was correct, Le., that the virus strains within each trans- mission group were epidemiotogically closely inked. The trees even suggested the transmission direction; the in- dex case virus sequence was located earlier on the ‘group specific branch, ie. the recipient virus sequence ‘eppeared to have evolved from that of the index case. ‘Qu; investigations were initiatec because the three male index cases were suspected of having neglected to in form their female sexual pariners that they were HIV-1 infected and thereby of having deliberately exposed these females to the risk of becoming HIV-1 infected. We have previously performed a similar forensic investiga tion (Albert et a1, 1994). These snd ather studies demon- strate that DNA sequencing can be a very powerful too! for reconstructing transmission patterns (Ou et af, 1992; Holmes et al, 1993; Albert et al, 1994). The biology of the virus isolates obtained from the individuals in the three transmission groups differed markedly. Thus, the virus ieolates in transmission chain | were of rapid/high, syncytium-inducing phenotype, whereas the isolates in transmission chain Il and Ill were of slow/low phonotype. For individuals Infected with sub- ‘ype B virus it has been clearly shown that presence or development of rapidiigh virus is strongly correlated to an increased risk of rapid disease progression (Koot et 21, 1993; Karlsson et al, 1994), Our study suggests that the same correlation exists also for other subtypes. The Individuals who carried virus with rapid/high phenotype had sovere immunodeficiency and high virus load, whereas individuals infected with slow/low virus gener ally had no oF moderate immunodeficiency and low vitus load. It Is especialy interesting to note that the two fe- males in transmission chain |, who became infected with rapid/high, subtype G virus variants, already had very low .CD4" lymphocyte counts 6 10 7 months after infection. In agreement with this we have recently shown thet primary infection with rapidfhigh virus 's a poor prognostic sign (Fiore et at, 1994). Thus, the subtype G viruses included in this study appeared to be highly virulent, but this should not be interpreted as a general feature of subiype G HIV-1 variants, Clearly the issue of possibte biological ditferences between diferent subtypes of HIV-1 cannot bbe determined by case reports, but only by carefully con ducted epidemiological studies. This is illustrated by the fact that a recent study within the WHO framework nas suggested that rapidihigh phenotype may be overrepre- sented armong subtype D viruses. However, in the pres ent study it was not possible to isolate virus trom the Individuals infected with subtype D virus and the DNA sequences revealed a slow/low, non-syncytiuminducing genoype. From this, it Is clear that there exist subtype D virus strains with slow/lowr phenotype. It is also inter esting 20 note that among the virus isolates characterized inthis study, the correletion between biological pheno type and V3 loop genotype was very sirong. This indi cates that the pattern described for subtype B virus, in wich positively charged amino acids at positions 311 ‘end/or 328 strongly correlates to rapidhigh, syncytium inducing phenotype (De Jong etal, 1982; Fouchier eta, 1992), may be valid elso for several other subtypes, ‘The virus trenemitted from the index ease in transmis sion group I! to female il-1 and then to the child I-2 and male II3 is clearly a recombinant virus with a subtype D #17 gag gene and a subtype A env gene, The precise site for recombination could not be established, since there were no signs of recombination within the se quenced regions. Furthermore, there were no indications ‘of the presence of botn subtype A and D genomes in the index case. Therefore, itis likely that the recombination ‘occurred earlier in the transmission chain, Nevertheless, the recombinant appeared to be stable and fully rans: rmissible. Recently, @ subtype 8/F recombinant (Sabino et al, 1994) and a possible subtype G/H recombinant Uanssons ot af, 1994) have been published and in addi tion the HIV-1 MAL isolate appears to be a subtype A/D recombinant, Furthermore, it has been suggested that subtype E viruses in Thailand, which cluster with subtype A sequences in gag. but are distinet from subtype A in env, may represent subtype VE recombinants (Myers et al, 1993}. Recombination betwoon subtypes requires that ‘one individual is infected with two virus variants belong- ing to different subtypes. Whether this double infection Js the result of two consecutive infections (superintec- tion) or @ double primary infection (Le, infection with theCHARACTERIZATION OF HiV-1 TRANSMISSIONS. us second strain before seroconversion 10 the fret) is not clear. For vaccine development it would be important 10 Gistinguish between these two possibilities. Fither way ‘exposure to two different subtypes shoulc be less com mon than multiple exposure to different strains of the same subtype. Thus, the increasing rumber of reported recombinants between subtypes indicates that infection ‘with multipfe strains can occur and that recombination js not unusual. There are at least three studies which indicate that recombination within subtype can also os- cur (Delassus et al, 1991; Simmonds et al, 1991; Vartan Jan et af, 1992). Furthermore, recombination Is consié- red to occur at a high rate in retroviral replication (Hu and Temin, 1980) and recently recombination as a result Of superinfection with feline immunodeficiency virus has been reported (Kyaw-Tanner et al, 1994). In conclusion, this study demonstrates that nonsub- ‘ype B HiV-1 variants are being transmitted in at least fone European country. 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