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Diversity of HIV-1 subtype C strains isolated in Romania
Infection, Genetics and Evolution 11 (2011) 270–275
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Diversity of HIV-1 subtype C strains isolated in Romania§,§§ Simona Paraschiv a,*, Brian Foley b, Dan Otelea a a b
Molecular Diagnostics Laboratory, ‘Prof. Dr. Matei Bals’ National Institute for Infectious Diseases, Str. Calistrat Grozovici, nr. 1, Sector 2, 021105 Bucharest, Romania HIV Databases, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 March 2010 Received in revised form 19 May 2010 Accepted 2 July 2010 Available online 8 July 2010
Two unique aspects particularities of the HIV-1 epidemics in Romania are the high prevalence of subtype F1 strains and the large pediatric population infected in the late 1980s and early 1990s. During recent years, more infections with other subtypes have been seen in newly diagnosed patients. After subtype B, subtype C was the most frequent one. This subtype is prevalent in countries from sub-Saharan Africa and India, being responsible for half of the total HIV-1 infections in the world. We have identified 37 patients infected with subtype C, sequenced the reverse transcriptase and protease regions of their pol genes, and applied phylogenetic analyses to the sequences. We have also included 20 subtype F1 strains isolated from both teenagers (children at the time of diagnosis) and adults. The phylogenetic analysis was performed by using the PhyML method, the GTR (general time reversible) model of evolution and gamma distribution of variability of rates between sites, empirically calculated from the data. The epidemiological data indicates that the main route of transmission for the adult subjects was by heterosexual contact and a relatively small number of patients were possibly infected abroad. In three cases, blood transfusion prior to 1989 or surgical procedures at early ages were suspected to be the cause of the HIV infection and three other patients were most probably parenterally infected. The phylogenetic analyses showed that the Romanian C strains are very diverse overall, clustered in several groups characterized by common transmission route (transfusion/surgical procedures) or local geographical relatedness. The HIV-1 epidemics in Romania apparently followed different patterns for subtypes F and C. While subtype F1 seems to have been monoclonally introduced and extensively spread in the 80s, the subtype C strains, although present in the late 80s, failed to spread to the same extent. ß 2010 Elsevier B.V. All rights reserved.
Keywords: HIV-1 Romanian epidemic Subtype C Phylogenetic analysis
1. Introduction The genetic diversity and evolution of the human immunodeficiency viruses are important factors that shape the global HIV epidemic, with implications in diagnosis, pathogenesis, drug treatment and vaccine development (Peeters et al., 2003; Martı´nez-Cajas et al., 2008). The HIV-1 strains from patients worldwide have been classified in 4 distinct genetic groups: M (major), O (outlier), N (non-M, non-O) and the newly discovered P group (Plantier et al., 2009). The global pandemic is mainly caused by strains belonging to the M group, which includes nine subtypes (A, B, C, D, F, G, H, J and K), up to six sub-subtypes (A1–A4 and F1– F2), inter-subtype circulating recombinant forms (CRFs) and
§ This paper has been contributed through the 15th International Bioinformatics Workshop on Virus Evolution and Molecular Epidemiology, Rotterdam, The Netherlands, September 7–11, 2009. §§ Note: Nucleotide sequences reported in this paper are available in the GenBank. For subtype C, the accession numbers are HM191521–HM191557 and for subtype F1 are HM191558–HM191577. * Corresponding author. Tel.: +40 212010980x3085; fax: +40 213186116. E-mail address: [email protected] (S. Paraschiv).
1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.07.002
unique recombinant forms (URFs) (Robertson et al., 2000; Paraskevis and Hatzakis, 1999). Subtype C is estimated to be responsible for more than half of all the HIV-1 infections reported worldwide, being prevalent in countries that account for more than 80% of all global HIV-1 infections (India and sub-Saharan Africa) (Hemelaar et al., 2006; Buonaguro et al., 2007). This subtype was also described in countries from South America (Brazil, Argentina, Uruguay), Israel and some European countries such as Sweden, Denmark and United Kingdom (Soares et al., 2003; MacHuca et al., 2001; Thomson and Najera, 2007). In recent years, HIV infections with non-B subtypes are increasingly registered in Europe, associated with immigration and heterosexual transmission (Snoeck et al., 2004; Vercauteren et al., 2009). In Romania, it has been shown that the subtype F1 is highly prevalent, being responsible almost exclusively for infections in earlier diagnosed patients (Apetrei et al., 1998a; Paraschiv et al., 2009). In addition to the high prevalence of subtype F1 strains, another particularity of the HIV-1 epidemics in Romania is the large pediatric population infected in the late 1980s and early 1990s (10,000 HIV infections from a total of 15,000; http://www.cnlas.ro/ images/doc/date_romania_31_decembrie_2008_english.pdf), most of them being abandoned children living in public institutions. These children were most probably infected by use of improperly sterilized
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needles contaminated with blood products (Hersh et al., 1993; Op de Coul et al., 2000). There were also reported cases of transfusions with unscreened blood prior to 1990, the year when Romania started testing blood for HIV (Hersh et al., 1991). The nosocomial nature of Romanian pediatric epidemic was documented in the early 90s, immediately after the HIV surveillance system was set up (Hersh et al., 1991, 1993) and then confirmed by phylogenetic analysis (Apetrei et al., 1997, 1998b; Op de Coul et al., 2000; Paraschiv et al., 2007). After 1992, a relatively constant number of newly diagnosed patients have been reported in Romania, most of them adult (24–35 years) heterosexuals. During recent years, more infections with other subtypes, including CRFs and URFs, have been seen in newly diagnosed patients (Florea et al., 2010). Subtype C was the second most prevalent one after subtype B (Paraschiv et al., 2008). The purpose of the present study was to assess the phylogenetic relatedness of the newly sequenced Romanian subtype C strains with sequences of HIV-1 strains isolated in different other parts of the world (Europe, Africa, Asia, South-America), in comparison to previously sequenced Romanian subtype F1 strains. 2. Materials and methods 2.1. Study population We studied 37 patients infected with subtype C strains out of more than 1500 HIV-1 strains genotyped between 2003 and 2009 in Romania, as a part of the public program for drug resistance testing using genotypic assays; the samples have been obtained from Romanian naı¨ve (n = 9) and treated patients (n = 28). Five of
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the antiretroviral naı¨ve patients were diagnosed between 2007 and 2008 (Table 1). For these 37 patients, we generated a total of 47 subtype C sequences, but only one sequence per patient was used in the analyses in this report. Epidemiological data (year of birth, way of transmission, travels abroad) have been collected for most of the patients. Additionally, 20 subtype F1 strains isolated from both drug-naı¨ve teenagers (children at the time of diagnosis) and adults were also included in this analysis. These F1 sequences have been previously analysed for the presence of drug resistance mutations (Paraschiv et al., 2007). The study has been conducted according to the current local ethical regulations. 2.2. HIV sequencing RNA samples were extracted from 500 ml of plasma using the Sample extraction module of the commercial kit ViroseqTM HIV-1 Genotyping System (Celera Diagnostics, Alameda, CA), according to the manufacturer’s recommendations. The RT-PCR was performed on a GeneAmp System 9700 (Applied Biosystems) thermal cycler. The 1.8 kb product, representing the first two thirds of the pol gene, was purified using MicroCon YM-100 concentrators and then sequenced bidirectionally on an ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems) using Big Dye Terminator chemistry and six different primers. The raw analysis of the sequences was made using Sequencing Analysis Software Version 3.7 (Applied Biosystems); they were then assembled with ViroSeq 2.5/2.7/2.8 HIV-1 Genotyping System Software (Celera Diagnostics, Alameda, CA). The correctness of each electropherogram interpretation was validated by the operator and the sequences were saved in Fasta
Table 1 Epidemiological data for the studied patients. Sequence name
Year of birth
Mode of transmission
Travel outside of country
Other epidemiological information
08Rtm3032 05Rb465 06Ris1868 06Rag4223 06Rag4224 05Rdb3581 08Rph1009 05Rph3772 05Rcl7143 09Rb540 09Rif2046 05Rb7041 05Rb1063 05Rdb4524 03Rcl3025 08Rb1014 05Rvn4513 07Rct886 09Rif2413 05Rb7139 08Rct1663 05Rb7680 08Rct5524 03Rb562 03Rb1961 06Rph4288 07Ris1767 07Ril744 03Ril3330 08Rb326 05Rbz1529 05Rdb7504 05Rgl3596 07Rb2851 05Rdb4089 03Rb2273 05Rb6615
1985 1967 1955 1970 1972 1989 1961 1985 1961 1961 1972 1957 1948 1988 1971 1977 1963 1974 1979 1969 1979 1970 1969 1977 1971 1987 1977 1969 1975 1967 1985 1971 1975 1970 1987 1970 1962
Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Surgical procedure as infant Heterosexual Parenteral, in childhood (1987) Heterosexual Heterosexual Heterosexual Heterosexual Transfusion or heterosexual Surgical procedure as infant Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Transfusion in 1989 Heterosexual Heterosexual Heterosexual Heterosexual Parenteral, in childhood Heterosexual Heterosexual Heterosexual Parenteral, in childhood Heterosexual Heterosexual
Y/Germany Y/many counties NA No No
ARV naive
a
Newly diagnosed at the time when the blood sample was drawn.
No No No No Y/Turkey No
No No NA Y/Turkey Y/Ukraine, Moldova No NA No Y/Sudan, Thailand No Y/SUA, Spain No No No No No
Partner of 06Rag4224, ARV naive Partner of 06Rag4223, ARV naive
Many partners
Sex worker
Many partners
ARV naivea ARV naive Partner sex worker from Japan From husband Arabia Many partners Many partners, sailor From husband Israel Partner of 03Rb2273 ARV naivea ARV naı¨ve, from sex workera ARV naı¨vea
No No NA
Many partners Infected in Bucharest Many partners ARV naı¨vea
No No
Partner of 03Rb1961 Many partners
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format. The Fasta files were used for subtyping purposes by comparing them with reference sequences of all known HIV-1 subtypes, using the publicly available algorithm REGA HIV-1&2 Automated subtyping tool version 2.0 (www.jose.med.kuleuven.be/genotypetool/html/indexhiv.html). 2.3. Reference strains In addition to the HIV-1M group subtype reference set sequences provided by the HIV Sequence Databases at Los Alamos National Lab (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html) several sequences from subtypes C and F1 were selected using both Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST) and the geographic distribution of HIV-1 sequences tool available at the Los Alamos HIV Sequence Database (www.hiv.lanl.gov/components/sequence/HIV/geo/geo.comp). 2.4. Phylogenetic and recombination analyses The alignment of the analyzed sequences (subtypes C and F1) along with the reference sequences of HIV-1 group M, subtype A (A1, A2), B, C, D, F (F1, F2), G, K, J and group O was performed using CLUSTAL W multiple-sequence alignment software as implemented in BioEdit software version 7.0 (www.mbio.ncsu.edu/BioEdit/ bioedit.html). To eliminate any possible clustering due to the adaptive convergence forced by drug resistance mutations, we built phylogenetic trees both before and after eliminating from the alignment all codons known to be associated with antiretroviral drug resistance. The phylogenetic analyses were performed using the maximum likelihood method of PhyML and the neighbor joining method of PAUP, the GTR (general time reversible) as the model of evolution and gamma distribution of variability of rates between sites, calculated empirically from the data with 8 categories of rates. For bootstrap values, a phylogenetic analysis was performed by using the Neighbor-Joining method with Tamura-Nei nucleotide substitution model and gamma distributed rates among sites, using the Mega 4 software, and 1000 bootstrap replicates of the data. For all phylogenies, the sequence of HIV-1 group O (O.SN.AJ302646) was used as outgroup. Our sample names are according to the WHO/ UNAIDS nomenclature which indicates the year of isolation, the country origin (R) and the isolate number. For epidemiological purposes, we have added to the names of the subtype C sequences the code for the residence county (two letter) of each patient. The F1 subtype sequences analysed were distinctly marked by adding the suffix ‘a’ for the strains isolated from adults and ‘c’ for the ones coming from children at the time of diagnosis. Phylogenetic trees were visualized using the FigTree version 1.3.1 program (http:// tree.bio.ed.ac.uk/software/figtree/). All sequences were screened for hypermutation using the Hypermut 2.0 (http://www.hiv.lanl.gov/ content/sequence/HYPERMUT/hypermut.html). One sequence that fell outside the subtype C crown group in all phylogenetic trees was screened for inter-subtype recombination using RIP (http:// www.hiv.lanl.gov/content/sequence/RIP/RIP.html) with a window size of 100, and also using the NCBI virus genotyping tool (http:// www.ncbi.nlm.nih.gov/projects/genotyping/formpagex.cgi) with a window of 90 and step size of 30. 3. Results The available epidemiological data showed that the main route of transmission for the subtype C infected patients we evaluated was by heterosexual contact (Table 1). Fifteen of these patients are currently living in the Bucharest area, the rest residing in scattered regions of the country. A relatively small number of the patients reported that they could have been infected abroad, possibly in
Turkey, Germany, Spain, Sudan and Ukraine (see Table 1). In three cases (06Rph4288, 05Rdb4524 and 05Rdb3581) transfusion or surgical intervention as new-borns are suspected to have been the cause of the infection. These patients were diagnosed early in childhood and the transfusion and surgical events were reported to have happened in 1989. The phylogenetic tree (Fig. 1) illustrates that the HIV-1 subtype C sequences from Romanian patients are rather diverse, with 4 sub-clades within subtype C, each characterized by common transmission route (transfusion, heterosexual contact) and/or local geographical relatedness. Likewise, the sub-subtype F1 sequences from the early nosocomial infected children form another sub-clade within the global distribution of sub-subtype F1. The first sub-clade is the group of sequences of which 2 originated from a heterosexual couple (06Rag4224, 06Rag4223), these sequences seems to be related with one sequences from Bostwana. The second sub-clade (distinctly marked in Fig. 1 with ‘Heterosexuals’) is a transmission route group and also a local geographical relatedness cluster which included 12 subtype C sequences; these strains were mostly isolated from the patients living in the Bucharest area (8 of 12), all heterosexuals and also these seems to be related with the C.BW.AF110967 sequence. Another sub-clade (marked in Fig. 1 with ‘1985–1987 Nosocomial’) is a transmission route group composed of 5 sequences: 05Rdb4524, 05Rph3772, 05Rdb4089, 05Rbz1529 and 06Rph4288. 06Rph4288 was isolated from the patient with reported blood transfusion in 1989, while the strain 05Rdb4524 originates from a patient with a surgical intervention as an infant. The corresponding patients of other three sequences (05Rph3772, 05Rdb4089, 05Rbz1529) have in common the fact that they were all of the same age and most probably horizontally infected during childhood (see Table 1). This group of sequences seems to be more related to C sequences originating from South-America (Brazil and Argentina), as well as some of the other studied sequences (05Rb465, 05Rb1063, 08Rb326, 05Rvn4513, 05Rcl7143). In contrast with the present data on subtype C in Romania, in South-America there is strong evidence of the monophyletic origin of this subtype, the HIV-1 strain/s of this particular subtype being introduced from Eastern Africa (Ethiopia) to Brazil (Fontella et al., 2008; Bello et al., 2008). The last sub-clade (label with ‘Heterosexuals Born in late 1960s’) is composed of 5 sequences: (08Rph1009, 05Rb7680, 05Rb6615, 05Rb7139 and 05Rb7041). The corresponding strains were isolated from patients, all born in the late 1960s, that have reported to have contracted the infection by heterosexual contact in the same geographical location. The Romanian sequences are distinctly marked in bold font in Fig. 1. A number of subtype C sequences did not cluster within the groups described above. The sequence 05Rdb3581 that fell outside the subtype C group is a C/F1 recombinant, also indicated in the tree. The phylogenetic analysis of the F1 subtype sequences isolated from Romanian drug-naı¨ve patients, adults and children at the time of diagnosis revealed two major aspects (Fig. 1). First, the F1 Romanian group clustered together with sequences originating from Angola, re-confirming the common origin of the HIV-1 epidemic in these two countries (Guimara˜es et al., 2009). The second observation was that the all the F1 sequences isolated from nosocomial infected children (c) and half of the sequences from heterosexual adults (a) fell into a monophyletic cluster (marked in the tree with ‘Nosocomial and heterosexuals’) together with the F1 reference sequence from Romania (F1.RO.AB485659) and another F1 Romanian strain isolated in Spain (F1.ES.DQ979023). Several sequences from adults (F1.04R2307a, F1.04R634a, F1.04R2093a, F1.04R304a, F1.04R2152a) fell outside the nosocomial transmission, as had previously been observed (Paraschiv et al., 2007). Two other sequences isolated in Spain also fell in the F1 Romanian/ Angolan group, both of them from patients of Romanian origin.
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Fig. 1. Phylogenetic analysis of HIV-1 subtype C sequences. The tree was generated as described under Section 2. After removing the drug resistance associated codons, the size of the nucleotide sequence used to generate the tree was1139 nt. The reference sequence name contains information about the isolate: subtype, country, GenBank accession number. The sequences from this study were named as follows: subtype, year of isolation, one-letter code for Romania (R), the residence county code (two letter) and the isolate number. The Romanian sequences are distinctly marked in bold font. The identified subtype C and F1 groups were marked in the tree. Numbers at nodes indicate the bootstrap values as percentages (only those >70% are shown).
4. Discussion Genotyping of HIV strains from patient plasma samples serves at least two purposes. One is to detect the mutations responsible for resistance to antiretroviral treatment. Second use is subtyping of the infective strain, empowering the epidemiological surveil-
lance in different geographical regions and/or populations. The HIV subtype distribution worldwide is important not only from an epidemiological point of view, since it also may influence the clinical outcome (virological failure and/or CD4 response). There are already several published studies on the role of HIV-1 genetic diversity in resistance and cross-resistance to antiretroviral drugs,
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some of them on subtype C. The existing data suggest that the sequence differences between subtype C and subtype B strains are translated in alterations of the response to treatment and the emergence of resistance (Armstrong et al., 2009, Soares et al., 2007). For instance, subtype C strains display higher IC50 for atazanavir at the baseline (Fleury et al., 2006). It has been reported that some mutations in particular have significance in subtype C strains: V106 M confers NNRTI cross-resistance, the association of N83T and M89L in the protease restore the replicative capacity only in subtype C (Gonzalez et al., 2004; Abecasis et al., 2005; Brenner et al., 2003; Marconi et al., 2008). Furthermore, I93L that has been characterized as a secondary resistance mutation in the subtype B strains, seems to induce hypersusceptibility to protease inhibitors in subtype C (Gonzalez et al., 2003). Other virologic and biochemical studies suggested that K65R, involved in conferring variable degrees of phenotypic resistance to all NRTIs except AZT and d4T seems more likely to emerge in subtype C strains (Invernizzi et al., 2009; Doualla-Bell et al., 2006). These observations enforce the conclusion that HIV drug resistance genotyping should be accompanied by subtyping due to the clinical implications of the subtype differences. Unfortunately, the number of subtype C strains in this study was rather low and the treatment history too diverse to provide a basis for meaningful correlations between resistance profiles and this particular subtype. An unexpected epidemiological situation was described in Romania in the mid-90s when a high prevalence of serotype F strains was detected (Apetrei et al., 1997). Strains of the F subtype had been previously reported mainly in central Africa and South America. Subsequent studies have confirmed that the prevalence of the F1 subtype was unusually high (Apetrei et al., 1998a,b). The first reported subtype C strain was isolated from a Romanian patient in 2000 (Apetrei et al., 2003). The analysis of the HIV-1 sequences generated through the drug resistance genotyping program in Romania (samples tested during 2003–2009) revealed that the majority (92.5%) of the patients are infected with F1 subtype strains, whereas infections with subtype B and C are much lower (3.3% and 2.3% respectively). The proportion of non-F1 strains is a few times bigger in newly diagnosed and/or newly infected patients: 10.7% subtype B, 5.3% subtype C, 2.3% subtype A and 2.9% CRFs (Florea et al., 2010). The present data suggest that the main route of transmission of HIV-1 subtype C strains was by heterosexual contact. A relatively small number of patients were possibly infected outside the country. In three cases transfusion/ surgical procedures, performed before screening was implemented in Romania, are suspected to be the cause of infection. The phylogenetic analysis revealed that the HIV-1 subtype C in Romanian patients partitioned into several sub-epidemics, characterized by common transmission route (transfusion/surgical procedures) or local geographical relatedness. In contrast, subtype F1 infections are predominantly associated with one outbreak. The Romanian F1 strains clustered with sequences from Angola, and distinct from the F1 sequences originating from other geographic regions such as Brazil and Finland. The HIV-1 epidemics in Romania apparently followed different patterns for subtypes F and C. The close relatedness of subtype F1 strains suggests that they were monoclonally introduced and extensively spread in the 80s, accounting in the late 80s and early 90s for the quasitotality of HIV infections. This hypothesis is sustained by the results of several previous studies (Apetrei et al., 1997, 1998b; Op de Coul et al., 2000). Subtype C strains were also present in the late 80s (confirmed transfusion event in 1989) but they failed to spread to the same extent. Most of the subtype C strains were introduced later and through multiple import events leading to limited outbursts in specific risk groups and in circumscribed regions of the country. More accurate dating of the two patterns of spreading
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Diversity of HIV-1 subtype C strains isolated in Romania§,§§ Simona Paraschiv a,*, Brian Foley b, Dan Otelea a a b
Molecular Diagnostics Laboratory, ‘Prof. Dr. Matei Bals’ National Institute for Infectious Diseases, Str. Calistrat Grozovici, nr. 1, Sector 2, 021105 Bucharest, Romania HIV Databases, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 March 2010 Received in revised form 19 May 2010 Accepted 2 July 2010 Available online 8 July 2010
Two unique aspects particularities of the HIV-1 epidemics in Romania are the high prevalence of subtype F1 strains and the large pediatric population infected in the late 1980s and early 1990s. During recent years, more infections with other subtypes have been seen in newly diagnosed patients. After subtype B, subtype C was the most frequent one. This subtype is prevalent in countries from sub-Saharan Africa and India, being responsible for half of the total HIV-1 infections in the world. We have identified 37 patients infected with subtype C, sequenced the reverse transcriptase and protease regions of their pol genes, and applied phylogenetic analyses to the sequences. We have also included 20 subtype F1 strains isolated from both teenagers (children at the time of diagnosis) and adults. The phylogenetic analysis was performed by using the PhyML method, the GTR (general time reversible) model of evolution and gamma distribution of variability of rates between sites, empirically calculated from the data. The epidemiological data indicates that the main route of transmission for the adult subjects was by heterosexual contact and a relatively small number of patients were possibly infected abroad. In three cases, blood transfusion prior to 1989 or surgical procedures at early ages were suspected to be the cause of the HIV infection and three other patients were most probably parenterally infected. The phylogenetic analyses showed that the Romanian C strains are very diverse overall, clustered in several groups characterized by common transmission route (transfusion/surgical procedures) or local geographical relatedness. The HIV-1 epidemics in Romania apparently followed different patterns for subtypes F and C. While subtype F1 seems to have been monoclonally introduced and extensively spread in the 80s, the subtype C strains, although present in the late 80s, failed to spread to the same extent. ß 2010 Elsevier B.V. All rights reserved.
Keywords: HIV-1 Romanian epidemic Subtype C Phylogenetic analysis
1. Introduction The genetic diversity and evolution of the human immunodeficiency viruses are important factors that shape the global HIV epidemic, with implications in diagnosis, pathogenesis, drug treatment and vaccine development (Peeters et al., 2003; Martı´nez-Cajas et al., 2008). The HIV-1 strains from patients worldwide have been classified in 4 distinct genetic groups: M (major), O (outlier), N (non-M, non-O) and the newly discovered P group (Plantier et al., 2009). The global pandemic is mainly caused by strains belonging to the M group, which includes nine subtypes (A, B, C, D, F, G, H, J and K), up to six sub-subtypes (A1–A4 and F1– F2), inter-subtype circulating recombinant forms (CRFs) and
§ This paper has been contributed through the 15th International Bioinformatics Workshop on Virus Evolution and Molecular Epidemiology, Rotterdam, The Netherlands, September 7–11, 2009. §§ Note: Nucleotide sequences reported in this paper are available in the GenBank. For subtype C, the accession numbers are HM191521–HM191557 and for subtype F1 are HM191558–HM191577. * Corresponding author. Tel.: +40 212010980x3085; fax: +40 213186116. E-mail address: [email protected] (S. Paraschiv).
1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.07.002
unique recombinant forms (URFs) (Robertson et al., 2000; Paraskevis and Hatzakis, 1999). Subtype C is estimated to be responsible for more than half of all the HIV-1 infections reported worldwide, being prevalent in countries that account for more than 80% of all global HIV-1 infections (India and sub-Saharan Africa) (Hemelaar et al., 2006; Buonaguro et al., 2007). This subtype was also described in countries from South America (Brazil, Argentina, Uruguay), Israel and some European countries such as Sweden, Denmark and United Kingdom (Soares et al., 2003; MacHuca et al., 2001; Thomson and Najera, 2007). In recent years, HIV infections with non-B subtypes are increasingly registered in Europe, associated with immigration and heterosexual transmission (Snoeck et al., 2004; Vercauteren et al., 2009). In Romania, it has been shown that the subtype F1 is highly prevalent, being responsible almost exclusively for infections in earlier diagnosed patients (Apetrei et al., 1998a; Paraschiv et al., 2009). In addition to the high prevalence of subtype F1 strains, another particularity of the HIV-1 epidemics in Romania is the large pediatric population infected in the late 1980s and early 1990s (10,000 HIV infections from a total of 15,000; http://www.cnlas.ro/ images/doc/date_romania_31_decembrie_2008_english.pdf), most of them being abandoned children living in public institutions. These children were most probably infected by use of improperly sterilized
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needles contaminated with blood products (Hersh et al., 1993; Op de Coul et al., 2000). There were also reported cases of transfusions with unscreened blood prior to 1990, the year when Romania started testing blood for HIV (Hersh et al., 1991). The nosocomial nature of Romanian pediatric epidemic was documented in the early 90s, immediately after the HIV surveillance system was set up (Hersh et al., 1991, 1993) and then confirmed by phylogenetic analysis (Apetrei et al., 1997, 1998b; Op de Coul et al., 2000; Paraschiv et al., 2007). After 1992, a relatively constant number of newly diagnosed patients have been reported in Romania, most of them adult (24–35 years) heterosexuals. During recent years, more infections with other subtypes, including CRFs and URFs, have been seen in newly diagnosed patients (Florea et al., 2010). Subtype C was the second most prevalent one after subtype B (Paraschiv et al., 2008). The purpose of the present study was to assess the phylogenetic relatedness of the newly sequenced Romanian subtype C strains with sequences of HIV-1 strains isolated in different other parts of the world (Europe, Africa, Asia, South-America), in comparison to previously sequenced Romanian subtype F1 strains. 2. Materials and methods 2.1. Study population We studied 37 patients infected with subtype C strains out of more than 1500 HIV-1 strains genotyped between 2003 and 2009 in Romania, as a part of the public program for drug resistance testing using genotypic assays; the samples have been obtained from Romanian naı¨ve (n = 9) and treated patients (n = 28). Five of
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the antiretroviral naı¨ve patients were diagnosed between 2007 and 2008 (Table 1). For these 37 patients, we generated a total of 47 subtype C sequences, but only one sequence per patient was used in the analyses in this report. Epidemiological data (year of birth, way of transmission, travels abroad) have been collected for most of the patients. Additionally, 20 subtype F1 strains isolated from both drug-naı¨ve teenagers (children at the time of diagnosis) and adults were also included in this analysis. These F1 sequences have been previously analysed for the presence of drug resistance mutations (Paraschiv et al., 2007). The study has been conducted according to the current local ethical regulations. 2.2. HIV sequencing RNA samples were extracted from 500 ml of plasma using the Sample extraction module of the commercial kit ViroseqTM HIV-1 Genotyping System (Celera Diagnostics, Alameda, CA), according to the manufacturer’s recommendations. The RT-PCR was performed on a GeneAmp System 9700 (Applied Biosystems) thermal cycler. The 1.8 kb product, representing the first two thirds of the pol gene, was purified using MicroCon YM-100 concentrators and then sequenced bidirectionally on an ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems) using Big Dye Terminator chemistry and six different primers. The raw analysis of the sequences was made using Sequencing Analysis Software Version 3.7 (Applied Biosystems); they were then assembled with ViroSeq 2.5/2.7/2.8 HIV-1 Genotyping System Software (Celera Diagnostics, Alameda, CA). The correctness of each electropherogram interpretation was validated by the operator and the sequences were saved in Fasta
Table 1 Epidemiological data for the studied patients. Sequence name
Year of birth
Mode of transmission
Travel outside of country
Other epidemiological information
08Rtm3032 05Rb465 06Ris1868 06Rag4223 06Rag4224 05Rdb3581 08Rph1009 05Rph3772 05Rcl7143 09Rb540 09Rif2046 05Rb7041 05Rb1063 05Rdb4524 03Rcl3025 08Rb1014 05Rvn4513 07Rct886 09Rif2413 05Rb7139 08Rct1663 05Rb7680 08Rct5524 03Rb562 03Rb1961 06Rph4288 07Ris1767 07Ril744 03Ril3330 08Rb326 05Rbz1529 05Rdb7504 05Rgl3596 07Rb2851 05Rdb4089 03Rb2273 05Rb6615
1985 1967 1955 1970 1972 1989 1961 1985 1961 1961 1972 1957 1948 1988 1971 1977 1963 1974 1979 1969 1979 1970 1969 1977 1971 1987 1977 1969 1975 1967 1985 1971 1975 1970 1987 1970 1962
Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Surgical procedure as infant Heterosexual Parenteral, in childhood (1987) Heterosexual Heterosexual Heterosexual Heterosexual Transfusion or heterosexual Surgical procedure as infant Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Heterosexual Transfusion in 1989 Heterosexual Heterosexual Heterosexual Heterosexual Parenteral, in childhood Heterosexual Heterosexual Heterosexual Parenteral, in childhood Heterosexual Heterosexual
Y/Germany Y/many counties NA No No
ARV naive
a
Newly diagnosed at the time when the blood sample was drawn.
No No No No Y/Turkey No
No No NA Y/Turkey Y/Ukraine, Moldova No NA No Y/Sudan, Thailand No Y/SUA, Spain No No No No No
Partner of 06Rag4224, ARV naive Partner of 06Rag4223, ARV naive
Many partners
Sex worker
Many partners
ARV naivea ARV naive Partner sex worker from Japan From husband Arabia Many partners Many partners, sailor From husband Israel Partner of 03Rb2273 ARV naivea ARV naı¨ve, from sex workera ARV naı¨vea
No No NA
Many partners Infected in Bucharest Many partners ARV naı¨vea
No No
Partner of 03Rb1961 Many partners
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format. The Fasta files were used for subtyping purposes by comparing them with reference sequences of all known HIV-1 subtypes, using the publicly available algorithm REGA HIV-1&2 Automated subtyping tool version 2.0 (www.jose.med.kuleuven.be/genotypetool/html/indexhiv.html). 2.3. Reference strains In addition to the HIV-1M group subtype reference set sequences provided by the HIV Sequence Databases at Los Alamos National Lab (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html) several sequences from subtypes C and F1 were selected using both Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST) and the geographic distribution of HIV-1 sequences tool available at the Los Alamos HIV Sequence Database (www.hiv.lanl.gov/components/sequence/HIV/geo/geo.comp). 2.4. Phylogenetic and recombination analyses The alignment of the analyzed sequences (subtypes C and F1) along with the reference sequences of HIV-1 group M, subtype A (A1, A2), B, C, D, F (F1, F2), G, K, J and group O was performed using CLUSTAL W multiple-sequence alignment software as implemented in BioEdit software version 7.0 (www.mbio.ncsu.edu/BioEdit/ bioedit.html). To eliminate any possible clustering due to the adaptive convergence forced by drug resistance mutations, we built phylogenetic trees both before and after eliminating from the alignment all codons known to be associated with antiretroviral drug resistance. The phylogenetic analyses were performed using the maximum likelihood method of PhyML and the neighbor joining method of PAUP, the GTR (general time reversible) as the model of evolution and gamma distribution of variability of rates between sites, calculated empirically from the data with 8 categories of rates. For bootstrap values, a phylogenetic analysis was performed by using the Neighbor-Joining method with Tamura-Nei nucleotide substitution model and gamma distributed rates among sites, using the Mega 4 software, and 1000 bootstrap replicates of the data. For all phylogenies, the sequence of HIV-1 group O (O.SN.AJ302646) was used as outgroup. Our sample names are according to the WHO/ UNAIDS nomenclature which indicates the year of isolation, the country origin (R) and the isolate number. For epidemiological purposes, we have added to the names of the subtype C sequences the code for the residence county (two letter) of each patient. The F1 subtype sequences analysed were distinctly marked by adding the suffix ‘a’ for the strains isolated from adults and ‘c’ for the ones coming from children at the time of diagnosis. Phylogenetic trees were visualized using the FigTree version 1.3.1 program (http:// tree.bio.ed.ac.uk/software/figtree/). All sequences were screened for hypermutation using the Hypermut 2.0 (http://www.hiv.lanl.gov/ content/sequence/HYPERMUT/hypermut.html). One sequence that fell outside the subtype C crown group in all phylogenetic trees was screened for inter-subtype recombination using RIP (http:// www.hiv.lanl.gov/content/sequence/RIP/RIP.html) with a window size of 100, and also using the NCBI virus genotyping tool (http:// www.ncbi.nlm.nih.gov/projects/genotyping/formpagex.cgi) with a window of 90 and step size of 30. 3. Results The available epidemiological data showed that the main route of transmission for the subtype C infected patients we evaluated was by heterosexual contact (Table 1). Fifteen of these patients are currently living in the Bucharest area, the rest residing in scattered regions of the country. A relatively small number of the patients reported that they could have been infected abroad, possibly in
Turkey, Germany, Spain, Sudan and Ukraine (see Table 1). In three cases (06Rph4288, 05Rdb4524 and 05Rdb3581) transfusion or surgical intervention as new-borns are suspected to have been the cause of the infection. These patients were diagnosed early in childhood and the transfusion and surgical events were reported to have happened in 1989. The phylogenetic tree (Fig. 1) illustrates that the HIV-1 subtype C sequences from Romanian patients are rather diverse, with 4 sub-clades within subtype C, each characterized by common transmission route (transfusion, heterosexual contact) and/or local geographical relatedness. Likewise, the sub-subtype F1 sequences from the early nosocomial infected children form another sub-clade within the global distribution of sub-subtype F1. The first sub-clade is the group of sequences of which 2 originated from a heterosexual couple (06Rag4224, 06Rag4223), these sequences seems to be related with one sequences from Bostwana. The second sub-clade (distinctly marked in Fig. 1 with ‘Heterosexuals’) is a transmission route group and also a local geographical relatedness cluster which included 12 subtype C sequences; these strains were mostly isolated from the patients living in the Bucharest area (8 of 12), all heterosexuals and also these seems to be related with the C.BW.AF110967 sequence. Another sub-clade (marked in Fig. 1 with ‘1985–1987 Nosocomial’) is a transmission route group composed of 5 sequences: 05Rdb4524, 05Rph3772, 05Rdb4089, 05Rbz1529 and 06Rph4288. 06Rph4288 was isolated from the patient with reported blood transfusion in 1989, while the strain 05Rdb4524 originates from a patient with a surgical intervention as an infant. The corresponding patients of other three sequences (05Rph3772, 05Rdb4089, 05Rbz1529) have in common the fact that they were all of the same age and most probably horizontally infected during childhood (see Table 1). This group of sequences seems to be more related to C sequences originating from South-America (Brazil and Argentina), as well as some of the other studied sequences (05Rb465, 05Rb1063, 08Rb326, 05Rvn4513, 05Rcl7143). In contrast with the present data on subtype C in Romania, in South-America there is strong evidence of the monophyletic origin of this subtype, the HIV-1 strain/s of this particular subtype being introduced from Eastern Africa (Ethiopia) to Brazil (Fontella et al., 2008; Bello et al., 2008). The last sub-clade (label with ‘Heterosexuals Born in late 1960s’) is composed of 5 sequences: (08Rph1009, 05Rb7680, 05Rb6615, 05Rb7139 and 05Rb7041). The corresponding strains were isolated from patients, all born in the late 1960s, that have reported to have contracted the infection by heterosexual contact in the same geographical location. The Romanian sequences are distinctly marked in bold font in Fig. 1. A number of subtype C sequences did not cluster within the groups described above. The sequence 05Rdb3581 that fell outside the subtype C group is a C/F1 recombinant, also indicated in the tree. The phylogenetic analysis of the F1 subtype sequences isolated from Romanian drug-naı¨ve patients, adults and children at the time of diagnosis revealed two major aspects (Fig. 1). First, the F1 Romanian group clustered together with sequences originating from Angola, re-confirming the common origin of the HIV-1 epidemic in these two countries (Guimara˜es et al., 2009). The second observation was that the all the F1 sequences isolated from nosocomial infected children (c) and half of the sequences from heterosexual adults (a) fell into a monophyletic cluster (marked in the tree with ‘Nosocomial and heterosexuals’) together with the F1 reference sequence from Romania (F1.RO.AB485659) and another F1 Romanian strain isolated in Spain (F1.ES.DQ979023). Several sequences from adults (F1.04R2307a, F1.04R634a, F1.04R2093a, F1.04R304a, F1.04R2152a) fell outside the nosocomial transmission, as had previously been observed (Paraschiv et al., 2007). Two other sequences isolated in Spain also fell in the F1 Romanian/ Angolan group, both of them from patients of Romanian origin.
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Fig. 1. Phylogenetic analysis of HIV-1 subtype C sequences. The tree was generated as described under Section 2. After removing the drug resistance associated codons, the size of the nucleotide sequence used to generate the tree was1139 nt. The reference sequence name contains information about the isolate: subtype, country, GenBank accession number. The sequences from this study were named as follows: subtype, year of isolation, one-letter code for Romania (R), the residence county code (two letter) and the isolate number. The Romanian sequences are distinctly marked in bold font. The identified subtype C and F1 groups were marked in the tree. Numbers at nodes indicate the bootstrap values as percentages (only those >70% are shown).
4. Discussion Genotyping of HIV strains from patient plasma samples serves at least two purposes. One is to detect the mutations responsible for resistance to antiretroviral treatment. Second use is subtyping of the infective strain, empowering the epidemiological surveil-
lance in different geographical regions and/or populations. The HIV subtype distribution worldwide is important not only from an epidemiological point of view, since it also may influence the clinical outcome (virological failure and/or CD4 response). There are already several published studies on the role of HIV-1 genetic diversity in resistance and cross-resistance to antiretroviral drugs,
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some of them on subtype C. The existing data suggest that the sequence differences between subtype C and subtype B strains are translated in alterations of the response to treatment and the emergence of resistance (Armstrong et al., 2009, Soares et al., 2007). For instance, subtype C strains display higher IC50 for atazanavir at the baseline (Fleury et al., 2006). It has been reported that some mutations in particular have significance in subtype C strains: V106 M confers NNRTI cross-resistance, the association of N83T and M89L in the protease restore the replicative capacity only in subtype C (Gonzalez et al., 2004; Abecasis et al., 2005; Brenner et al., 2003; Marconi et al., 2008). Furthermore, I93L that has been characterized as a secondary resistance mutation in the subtype B strains, seems to induce hypersusceptibility to protease inhibitors in subtype C (Gonzalez et al., 2003). Other virologic and biochemical studies suggested that K65R, involved in conferring variable degrees of phenotypic resistance to all NRTIs except AZT and d4T seems more likely to emerge in subtype C strains (Invernizzi et al., 2009; Doualla-Bell et al., 2006). These observations enforce the conclusion that HIV drug resistance genotyping should be accompanied by subtyping due to the clinical implications of the subtype differences. Unfortunately, the number of subtype C strains in this study was rather low and the treatment history too diverse to provide a basis for meaningful correlations between resistance profiles and this particular subtype. An unexpected epidemiological situation was described in Romania in the mid-90s when a high prevalence of serotype F strains was detected (Apetrei et al., 1997). Strains of the F subtype had been previously reported mainly in central Africa and South America. Subsequent studies have confirmed that the prevalence of the F1 subtype was unusually high (Apetrei et al., 1998a,b). The first reported subtype C strain was isolated from a Romanian patient in 2000 (Apetrei et al., 2003). The analysis of the HIV-1 sequences generated through the drug resistance genotyping program in Romania (samples tested during 2003–2009) revealed that the majority (92.5%) of the patients are infected with F1 subtype strains, whereas infections with subtype B and C are much lower (3.3% and 2.3% respectively). The proportion of non-F1 strains is a few times bigger in newly diagnosed and/or newly infected patients: 10.7% subtype B, 5.3% subtype C, 2.3% subtype A and 2.9% CRFs (Florea et al., 2010). The present data suggest that the main route of transmission of HIV-1 subtype C strains was by heterosexual contact. A relatively small number of patients were possibly infected outside the country. In three cases transfusion/ surgical procedures, performed before screening was implemented in Romania, are suspected to be the cause of infection. The phylogenetic analysis revealed that the HIV-1 subtype C in Romanian patients partitioned into several sub-epidemics, characterized by common transmission route (transfusion/surgical procedures) or local geographical relatedness. In contrast, subtype F1 infections are predominantly associated with one outbreak. The Romanian F1 strains clustered with sequences from Angola, and distinct from the F1 sequences originating from other geographic regions such as Brazil and Finland. The HIV-1 epidemics in Romania apparently followed different patterns for subtypes F and C. The close relatedness of subtype F1 strains suggests that they were monoclonally introduced and extensively spread in the 80s, accounting in the late 80s and early 90s for the quasitotality of HIV infections. This hypothesis is sustained by the results of several previous studies (Apetrei et al., 1997, 1998b; Op de Coul et al., 2000). Subtype C strains were also present in the late 80s (confirmed transfusion event in 1989) but they failed to spread to the same extent. Most of the subtype C strains were introduced later and through multiple import events leading to limited outbursts in specific risk groups and in circumscribed regions of the country. More accurate dating of the two patterns of spreading
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