Molecular Epidemiological Analysis of the Origin and Transmission Dynamics of the HIV-1 CRF01_AE Sub-Epidemic in Bulgaria

Abstract

HIV-1 subtype CRF01_AE is the second most predominant strain in Bulgaria, yet little is known about the molecular epidemiology of its origin and transmissibility. We used a phylodynamics approach to better understand this sub-epidemic by analyzing 270 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1995 and 2019. Using network analyses at a 1.5% genetic distance threshold (d), we found a large 154-member outbreak cluster composed mostly of persons who inject drugs (PWID) that were predominantly men. At d = 0.5%, which was used to identify more recent transmission, the large cluster dissociated into three clusters of 18, 12, and 7 members, respectively, five dyads, and 107 singletons. Phylogenetic analysis of the Bulgarian sequences with publicly available global sequences showed that CRF01_AE likely originated from multiple Asian countries, with Vietnam as the likely source of the outbreak cluster between 1988 and 1990. Our findings indicate that CRF01_AE was introduced into Bulgaria multiple times since 1988, and infections then rapidly spread among PWID locally with bridging to other risk groups and countries. CRF01_AE continues to spread in Bulgaria as evidenced by the more recent large clusters identified at d = 0.5%, highlighting the importance of public health prevention efforts in the PWID communities.

Keywords: HIV-1; circulating recombinant forms; drug resistance; molecular epidemiology; prevention; transmission clusters; transmission dynamics.

Figure 1

Noncumulative epidemiologic curve of HIV-1 CRF01_AE diagnoses in Bulgaria from 1995 to 2019 by transmission category. HET, heterosexual; MSM, men who have sex with men; PWID, persons who inject drugs; MSM/PWID (persons who reported both MSM and PWID); MTC, mother-to-child.

Figure 2

Inference of subtype CRF01-AE clusters in Bulgaria using MicrobeTrace. (A) Six clusters were identified using a genetic distance (d) of 1.5% compared to (B) ten clusters at d = 0.5%. Sex is indicated by circles (male) and addition sign (female). (C) Transmission network at d = 3.5%. Transmission category is indicated with color (red, men who have sex with men (MSM); green, persons who inject drugs (PWID); blue, heterosexual (HET); gold, persons reporting both MSM and PWID; purple, mother-to-child (MTC). Cluster totals by node (members) and total number of links in the transmission network is provided.

Figure 3

Nextstrain maximum-likelihood (ML) analysis of global HIV-1 CRF01_AE sequences. (A) The ML tree was constructed using 270 sequences from Bulgaria and 1458 global CRF01_AE sequences, including two from Bulgaria from a different study, 7 from Africa (Cameroon = 5, the Central African Republic = 2), 544 from China, 1 from Hong Kong, 3 from Taiwan, 20 from Europe (Belgium = 2, Czech Republic = 2, Finland = 1, France = 1, Slovenia = 1, Sweden = 7, the United Kingdom = 6), 276 from Vietnam, 49 from Laos, 362 from Thailand, 19 from the Philippines, 2 from Singapore, 3 from Indonesia, 2 from Myanmar, 6 from the United States, and two from the Middle East (Afghanistan = 1, Iran = 1). Three reference subtype J sequences were used as the outgroup. Tree branches are colored by country of birth as provided in the key. The arrows show the Bulgarian outbreak cluster showing inferred dating of the most recent common ancestor (CI, confidence interval). (B) Inferred geographical transmission routes with potential origins indicated by colored lines connecting countries, as provided in the (A).