Supplementary File 1

Supplementary Figure 1

The total number of the Middle East and North Africa (MENA) HIV-1 sequences in Los

Alamos HIV Database in relation to HxB2 (GenBank accession number K03455) position.

Abbreviations: gag: group antigen gene, pol: polymerase gene, env: envelope gene. Analysis

was done by screening the number of sequences per a window of HIV-1 genome with a size

of 100 bases each.

Supplementary references

List of the publications with Middle East and North Africa (MENA) HIV-1 sequences

submitted to Los Alamos HIV database (LADB) and that were used in the study. Besides

that, direct sequence submissions were used and can be found in LADB through the

geography search tool (https://www.hiv.lanl.gov/components/sequence/HIV/geo/geo.comp).

Algeria: (Abdellaziz et al., 2016; Bouzeghoub et al., 2008; Bouzeghoub et al., 2006), Egypt:

(El Sayed et al., 2000), Iran: (Baesi et al., 2014; Hamkar et al., 2010; Jahanbakhsh et al.,

2013; Memarnejadian et al., 2015; Naderi et al., 2006; Sarrami-Forooshani et al., 2006;

Soheilli et al., 2009), Saudi Arabia: (Badreddine et al., 2007), Kuwait: (Chehadeh et al.,

2015; Voevodin et al., 1996), Lebanon: (Pieniazek et al., 1998), Libya: (de Oliveira et al.,

2006; Visco-Comandini et al., 2002; Yerly et al., 2001), Morocco: (Akrim et al., 2012; Annaz

et al., 2011; El Annaz et al., 2012; Miri et al., 2012), Sudan: (Hierholzer et al., 2002),

Tunisia: (Ben Halima et al., 2001; El Moussi et al., 2017; Jlizi et al., 2008), Yemen: (Saad et

al., 2005).

References:

Abdellaziz, A., Papuchon, J., Khaled, S., Ouerdane, D., Fleury, H., Recordon-Pinson, P.,

2016. Predominance of CRF06_cpx and Transmitted HIV Resistance in Algeria: Update

2013-2014. AIDS Res Hum Retroviruses 32, 370-372.

Akrim, M., Lemrabet, S., Elharti, E., Gray, R.R., Tardy, J.C., Cook, R.L., et al., 2012. HIV-1

Subtype distribution in morocco based on national sentinel surveillance data 2004-2005.

AIDS Res Ther 9, 5.

Annaz, H.E., Recordon-Pinson, P., Baba, N., Sedrati, O., Mrani, S., Fleury, H., 2011.

Presence of drug resistance mutations among drug-naive patients in Morocco. AIDS Res

Hum Retroviruses 27, 917-920.

Badreddine, S., Smith, K., van Zyl, H., Bodelle, P., Yamaguchi, J., Swanson, P., et al., 2007.

Identification and characterization of HIV type 1 subtypes present in the Kingdom of Saudi

Arabia: high level of genetic diversity found. AIDS Res Hum Retroviruses 23, 667-674.

Baesi, K., Moallemi, S., Farrokhi, M., Alinaghi, S.A., Truong, H.M., 2014. Subtype

classification of Iranian HIV-1 sequences registered in the HIV databases, 2006-2013. PLoS

One 9, e105098.

Ben Halima, M., Pasquier, C., Slim, A., Ben Chaabane, T., Arrouji, Z., Puel, J., et al., 2001.

First molecular characterization of HIV-1 Tunisian strains. J Acquir Immune Defic Syndr 28,

94-96.

Bouzeghoub, S., Jauvin, V., Pinson, P., Schrive, M.H., Jeannot, A.C., Amrane, A., et al.,

2008. First observation of HIV type 1 drug resistance mutations in Algeria. AIDS Res Hum

Retroviruses 24, 1467-1473.

Bouzeghoub, S., Jauvin, V., Recordon-Pinson, P., Garrigue, I., Amrane, A., Belabbes el, H.,

et al., 2006. High diversity of HIV type 1 in Algeria. AIDS Res Hum Retroviruses 22, 367-

372.

Chehadeh, W., Albaksami, O., Altawalah, H., Ahmad, S., Madi, N., John, S.E., et al., 2015.

Phylogenetic analysis of HIV-1 subtypes and drug resistance profile among treatment-naive

people in Kuwait. J Med Virol 87, 1521-1526.

de Oliveira, T., Pybus, O.G., Rambaut, A., Salemi, M., Cassol, S., Ciccozzi, M., et al., 2006.

Molecular epidemiology: HIV-1 and HCV sequences from Libyan outbreak. Nature 444,

836-837.

El Annaz, H., Recordon-Pinson, P., Tagajdid, R., Doblali, T., Belefquih, B., Oumakhir, S., et

al., 2012. Drug resistance mutations in HIV type 1 isolates from patients failing antiretroviral

therapy in Morocco. AIDS Res Hum Retroviruses 28, 944-948.

El Moussi, A., Thomson, M.M., Delgado, E., Cuevas, M.T., Nasr, M., Abid, S., et al., 2017.

Genetic Diversity of HIV-1 in Tunisia. AIDS Res Hum Retroviruses 33, 77-81.

El Sayed, N.M., Gomatos, P.J., Beck-Sague, C.M., Dietrich, U., von Briesen, H., Osmanov,

S., et al., 2000. Epidemic transmission of human immunodeficiency virus in renal dialysis

centers in Egypt. J Infect Dis 181, 91-97.

Hamkar, R., Mohraz, M., Lorestani, S., Aghakhani, A., Truong, H.M., McFarland, W., et al.,

2010. Assessing subtype and drug-resistance-associated mutations among antiretroviral-

treated HIV-infected patients. AIDS 24 Suppl 2, S85-91.

Hierholzer, M., Graham, R.R., El Khidir, I., Tasker, S., Darwish, M., Chapman, G.D., et al.,

2002. HIV type 1 strains from East and West Africa are intermixed in Sudan. AIDS Res Hum

Retroviruses 18, 1163-1166.

Jahanbakhsh, F., Hattori, J., Matsuda, M., Ibe, S., Monavari, S.H., Memarnejadian, A., et al.,

2013. Prevalence of transmitted HIV drug resistance in Iran between 2010 and 2011. PLoS

One 8, e61864.

Jlizi, A., Ben Ammar El Gaaied, A., Slim, A., Tebourski, F., Ben Mamou, M., Ben

Chaabane, T., et al., 2008. Profile of drug resistance mutations among HIV-1-infected

Tunisian subjects failing antiretroviral therapy. Arch Virol 153, 1103-1108.

Memarnejadian, A., Menbari, S., Mansouri, S.A., Sadeghi, L., Vahabpour, R., Aghasadeghi,

M.R., et al., 2015. Transmitted Drug Resistance Mutations in Antiretroviral-Naive Injection

Drug Users with Chronic HIV-1 Infection in Iran. PLoS One 10, e0126955.

Miri, L., Ouladlahsen, A., Kettani, A., Bensghir, R., Marhoum Elfilali, K., Wakrim, L., 2012.

Characterization of protease resistance-associated mutations in HIV type 1 drug-naive

patients following the increasing prevalence of the CRF02_AG strain in Morocco. AIDS Res

Hum Retroviruses 28, 571-577.

Naderi, H.R., Tagliamonte, M., Tornesello, M.L., Ciccozzi, M., Rezza, G., Farid, R., et al.,

2006. Molecular and phylogenetic analysis of HIV-1 variants circulating among injecting

drug users in Mashhad-Iran. Infect Agent Cancer 1, 4.

Pieniazek, D., Baggs, J., Hu, D.J., Matar, G.M., Abdelnoor, A.M., Mokhbat, J.E., et al., 1998.

Introduction of HIV-2 and multiple HIV-1 subtypes to Lebanon. Emerg Infect Dis 4, 649-

656.

Saad, M.D., Al-Jaufy, A., Grahan, R.R., Nadai, Y., Earhart, K.C., Sanchez, J.L., et al., 2005.

HIV type 1 strains common in Europe, Africa, and Asia cocirculate in Yemen. AIDS Res

Hum Retroviruses 21, 644-648.

Sarrami-Forooshani, R., Das, S.R., Sabahi, F., Adeli, A., Esmaeili, R., Wahren, B., et al.,

2006. Molecular analysis and phylogenetic characterization of HIV in Iran. J Med Virol 78,

853-863.

Soheilli, Z.S., Ataiee, Z., Tootian, S., Zadsar, M., Amini, S., Abadi, K., et al., 2009. Presence

of HIV-1 CRF35_AD in Iran. AIDS Res Hum Retroviruses 25, 123-124.

Visco-Comandini, U., Cappiello, G., Liuzzi, G., Tozzi, V., Anzidei, G., Abbate, I., et al.,

2002. Monophyletic HIV type 1 CRF02-AG in a nosocomial outbreak in Benghazi, Libya.

AIDS Res Hum Retroviruses 18, 727-732.

Voevodin, A., Crandall, K.A., Seth, P., al Mufti, S., 1996. HIV type 1 subtypes B and C from

new regions of India and Indian and Ethiopian expatriates in Kuwait. AIDS Res Hum

Retroviruses 12, 641-643.

Yerly, S., Quadri, R., Negro, F., Barbe, K.P., Cheseaux, J.J., Burgisser, P., et al., 2001.

Nosocomial outbreak of multiple bloodborne viral infections. J Infect Dis 184, 369-372.

Supplementary Table 1

The Middle East and North Africa (MENA) partial pol alignments in relation to strain HxB2,

Genbank accession number K03455. These alignments were used to reconstruct the

phylogeny of the dominant subtypes/CRFs in the region.

Subtype/CRF1 HxB2 start HxB2 endRT2-codons

removed

CRF01_AE 2271 3290 1 to 37

CRF02_AG 2253 3223 1 to 29

CRF06_cpx 2253 3266 1 to 23

CRF35_AD 2253 3263 -

A1 2262 3266 1 to 37

B 2262 3266 1 to 37

C 2262 3263 1 to 37

D 2253 3281 -

1CRF: Circulating recombinant forms, 2RT: Reverse transcriptase

Supplementary Figure 2a

The temporal distribution of the Middle East and North Africa (MENA) sequences used to

assess genetic diversity of HIV-1 in the MENA. Out of 2036 MENA sequence, 45 had

unknown year of collection and were not included in the figure. These sequence were

collected in Iran (n=35), Djibouti (n=6), Somalia (n=2), Egypt (n=1) and Tunisia (n=1).

Supplementary Figure 2b

The temporal distribution of the Middle East and North Africa (MENA) partial pol sequences

used to infer domestic transmission.

Phylogenetic subtyping of partial pol MENA sequences

Maximum likelihood (ML) trees used for phylogenetic subtyping of the Middle East and

North Africa (MENA) partial pol sequences (n=712).

The ML phylogenetic trees of the MENA sequences and reference sequences downloaded

from Los Alamos HIV database [LADB] (n=158, Year: 2010) were constructed using

GARLI v2.0 software by applying the GTR+I+Γ nucleotide substitution model and with

statistical support determined using approximate likelihood ratio test Shimodaira-Hasegawa

(aLRT SH-like) branch support in PhyML v3.1. An aLRT-SH value of more than or equal

0.90 was considered significant. The trees were viewed using Figtree v1.4.2 software. The

software is available at the following website: http://tree.bio.ed.ac.uk/software/figtree/.

Colour code for terminal branches of the ML trees is as follows: Algeria, Djibouti, Iran,

Kuwait, Libya, Morocco, Somalia, Sudan, Tunisia and Yemen. Significant branches

(aLRT SH-like ≥ 0.90) are shown in black colour. LADB reference sequences are shown in

grey colour.

Figure 1. The subtyping ML tree of CRF01_AE with the highest Garli likelihood score.

Figure 2. The subtyping ML tree of CRF02_AG with the highest Garli likelihood score.

Figure 3. The subtyping ML tree of CRF06_cpx with the highest Garli likelihood score.

Figure 4. The subtyping ML tree of CRF35_AD with the highest Garli likelihood score.

Figure 5. The subtyping ML tree of sub-subtype A1 with the highest Garli likelihood score.

Two sequences were excluded from subsequent analysis as they were found to belong to non-

A1 clade.

Figure 6. The subtyping ML tree of subtype B with the highest Garli likelihood score. Two

sequences were excluded from subsequent analysis as they were found to belong to non-B

clade.

Figure 7. The subtyping ML tree of subtype C with the highest Garli likelihood score.

Figure 8. The subtyping ML tree of subtype D with the highest Garli likelihood score.

Maximum likelihood trees used for identification of MENA transmission

clusters.

Maximum likelihood (ML) phylogenetic trees of the major MENA subtypes/CRFs (A1, B, C,

D, CRF01_AE, CRF02_AG, CRF06_cpx, CRF35_AD) were constructed using GARLI v2.0

software by applying the GTR+I+Γ nucleotide substitution model and with statistical support

determined using approximate likelihood ratio test Shimodaira-Hasegawa (aLRT SH-like)

branch support in PhyML v3.1. An aLRT-SH value of more than or equal 0.90 was

considered significant. The trees were viewed using Figtree v1.4.2 software. The software is

available at the following website: http://tree.bio.ed.ac.uk/software/figtree/.

Colour code for terminal branches of the ML trees is as follows: Algeria, Djibouti, Iran,

Kuwait, Libya, Morocco, Somalia, Sudan, Tunisia and Yemen. Significant branches

(aLRT SH-like ≥ 0.90) are shown in black colour. GenBank reference sequences are shown

in grey colour. MENA transmission clusters are highlighted in grey-bordered boxes. Mixed

MENA clusters are highlighted in grey colour.

Figure 1. The ML tree of CRF01_AE with the highest Garli likelihood score.

Figure 2. The ML tree of CRF02_AG with the highest Garli likelihood score. The Libyan

nosocomial outbreak is highlighted in the collapsed light blue triangle.

Figure 3. The ML tree of CRF06_cpx with the highest Garli likelihood score.

Figure 4. The ML tree of CRF35_AD with the highest Garli likelihood score.

Figure 5. The ML tree of sub-subtype A1 with the highest Garli likelihood score.

Figure 6. The ML tree of subtype B with the highest Garli likelihood score.

Figure 7. The ML tree of subtype C with the highest Garli likelihood score.

Figure 8. The ML tree of subtype D with the highest Garli likelihood score.