Intrasubtype Diversity Research Articles

Intra- and inter-subtype HIV diversity between 1994 and 2018 in southern Uganda: a longitudinal population-based study.

There is limited data on human immunodeficiency virus (HIV) evolutionary trends in African populations. We evaluated changes in HIV viral diversity and genetic divergence in southern Uganda over a 24-year period spanning the introduction and scale-up of HIV prevention and treatment programs using HIV sequence and survey data from the Rakai Community Cohort Study, an open longitudinal population-based HIV surveillance cohort. Gag (p24) and env (gp41) HIV data were generated from people living with HIV (PLHIV) in 31 inland semi-urban trading and agrarian communities (1994-2018) and four hyperendemic Lake Victoria fishing communities (2011-2018) under continuous surveillance. HIV subtype was assigned using the Recombination Identification Program with phylogenetic confirmation. Inter-subtype diversity was evaluated using the Shannon diversity index, and intra-subtype diversity with the nucleotide diversity and pairwise TN93 genetic distance. Genetic divergence was measured using root-to-tip distance and pairwise TN93 genetic distance analyses. Demographic history of HIV was inferred using a coalescent-based Bayesian Skygrid model. Evolutionary dynamics were assessed among demographic and behavioral population subgroups, including by migration status. 9931 HIV sequences were available from 4999 PLHIV, including 3060 and 1939 persons residing in inland and fishing communities, respectively. In inland communities, subtype A1 viruses proportionately increased from 14.3% in 1995 to 25.9% in 2017 (P < .001), while those of subtype D declined from 73.2% in 1995 to 28.2% in 2017 (P < .001). The proportion of viruses classified as recombinants significantly increased by nearly four-fold from 12.2% in 1995 to 44.8% in 2017. Inter-subtype HIV diversity has generally increased. While intra-subtype p24 genetic diversity and divergence leveled off after 2014, intra-subtype gp41 diversity, effective population size, and divergence increased through 2017. Intra- and inter-subtype viral diversity increased across all demographic and behavioral population subgroups, including among individuals with no recent migration history or extra-community sexual partners. This study provides insights into population-level HIV evolutionary dynamics following the scale-up of HIV prevention and treatment programs. Continued molecular surveillance may provide a better understanding of the dynamics driving population HIV evolution and yield important insights for epidemic control and vaccine development.

Unveiling Blastocystis epidemiology in Morocco: subtype diversity among clinical patients with and without gastrointestinal manifestations in the Meknes region.

Blastocystis is an intestinal protist frequently identified in humans and other animals, though its clinical significance remains controversial. This study aimed to determine the prevalence and genetic diversity of Blastocystis in faecal samples from symptomatic (n = 55) and asymptomatic (n = 50) individuals seeking medical care in Meknes, Morocco. Detection of the protist was accomplished through coproparasitological examination and culture in Jones medium. Culture-positive samples were subjected to molecular analyses (PCR and Sanger sequencing) based on sequences of the small subunit ribosomal RNA gene. Epidemiological questionnaires on demographics and potential risk factors were collected from participating patients. The overall Blastocystis infection rate was 51.4% (54/105), with no differences between symptomatic (52.7%, 29/55) and asymptomatic (50.0%, 25/50) individuals. Sequence analyses identified three Blastocystis subtypes, with ST3 being the most prevalent (42.0%), followed by ST1 (34.0%), and ST2 (12.0%). Regarding intra-subtype diversity, allele 4 was found within ST1; alleles 11/12 and alleles 34/36 (alone or in combination) were identified within ST2 and ST3 respectively. Allele 34 in ST3 (40.8%) and allele 4 in ST1 (34.7%) were the most common genetic variants circulating in the surveyed clinical population. A statistically significant association between ST2 and the presence of flatulence was observed. This is the first study assessing the epidemiology and genetic diversity of Blastocystis sp. in the Meknes region, Morocco.

Subtype–host patterns and genetic differentiation of Blastocystis sp. in the Philippines

Blastocystis sp. is a gastrointestinal protozoan commonly encountered in humans and animals. Specificity to certain hosts may be associated with 38 known subtypes (STs) and 8 nonmammalian and avian STs (NMASTs). This can be determined by analyzing ST–host associations, ST–allele data, genetic variability analyses, and fixation index (FST) with sufficient data present. Thus, newly acquired and previously published data on Blastocystis sp. STs and NMASTs from the Philippines were compiled to determine the following: (1) ST–host associations, (2) ST–allele diversity per ST in certain hosts/sources, (3) intrasubtype diversity of certain STs found in different hosts using genetic variability analysis, and (4) comparison of similarities between specific ST populations to determine if these are the same circulating populations using FST. A total of 448 samples subtyped using both sequence-tagged site primers and the 600-bp barcoding region of the Blastocystis sp. SSU rRNA gene were analyzed in this study. Patterns of association for the Philippine samples were similar to those from neighboring Southeast Asian countries and around the world: ST1–ST4 were found in humans but ST3 was the most common, ST5 were found in pigs, and ST6 and ST7 were found in poultry. Blastocystis sp. from humans are mostly the same ST alleles (ST3 allele 34 and ST1 allele 4) while 3–5 ST alleles were found in the most common STs in pigs, macaques, and poultry. Also, ST1, ST3, ST5, and NMAST I are undergoing population expansion according to genetic variability analyses through possible addition of new alleles based on ST–allele diversity. Moreover, FST shows the same circulating population of ST1 in humans, pigs, and water indicating a possible waterborne route of cross-transmission. In contrast, ST3 found in humans possibly come from the same circulating population and is genetically distinct from those in nonhuman sources.

Further insight into the genetic diversity of Entamoeba coli and Entamoeba hartmanni.

Despite the species' wide distribution, studies of the genetic diversity within Entamoeba coli and Entamoeba hartmanni remain limited. In the present study, we provide further insight into the genetic diversity of both species based on analysis of partial nuclear small subunit ribosomal DNA sequences generated from human fecal DNAs from samples collected in Africa, South America, and Europe. Reinforcing the previous recognition that E. coli is a species complex, our data confirm the existence of the two subtypes, ST1 and ST2, previously identified plus, potentially, a new subtype, ST3. While ST1 appears to be genetically quite homogenous, ST2 shows a substantial degree of intrasubtype diversity. ST2 was more common in samples collected outside Europe, whereas ST1 showed no geographical restriction. The potentially novel subtype is represented to date exclusively by sequences from South American and African samples. In contrast to previous reports, our new data also indicate substantial variation in E. hartmanni that could also support the establishment of subtypes within this species. Here, however, no links were identified between subtype and geographical origin.

Divergent HIV-1 strains (CRF92_C2U and CRF93_cpx) co-circulating in the Democratic Republic of the Congo: Phylogenetic insights on the early evolutionary history of subtype C.

Molecular epidemiological studies revealed that the epicenter of the HIV pandemic was Kinshasa, the capital city of the Democratic Republic of the Congo (DRC) in Central Africa. All known subtypes and numerous complex recombinant strains co-circulate in the DRC. Moreover, high intra-subtype diversity has been also documented. During two previous surveys on HIV-1 antiretroviral drug resistance in the DRC, we identified two divergent subtype C lineages in the protease and partial reverse transcriptase gene regions. We sequenced eight near full-length genomes and classified them using bootscanning and likelihood-based phylogenetic analyses. Four strains are more closely related to subtype C although within the range of inter sub-subtype distances. However, these strains also have small unclassified fragments and thus were named CRF92_C2U. Another strain is a unique recombinant of CRF92_C2U with an additional small unclassified fragment and a small divergent subtype A fragment. The three remaining strains represent a complex mosaic named CRF93_cpx. CRF93_cpx have two fragments of divergent subtype C sequences, which are not conventional subtype C nor the above described C2, and multiple divergent subtype A-like fragments. We then inferred the time-scaled evolutionary history of subtype C following a Bayesian approach and a partitioned analysis using major genomic regions. CRF92_C2U and CRF93_cpx had the most recent common ancestor with conventional subtype C around 1932 and 1928, respectively. A Bayesian demographic reconstruction corroborated that the subtype C transition to a faster phase of exponential growth occurred during the 1950s. Our analysis showed considerable differences between the newly discovered early-divergent strains and the conventional subtype C and therefore suggested that this virus has been diverging in humans for several decades before the HIV/M diversity boom in the 1950s.

TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.

Study on the association between genetic variation of gag gene and HLA restricted CTL response in HIV-1 B' infected Chinese people

To identify the genetic variation of Gag gene in HIV-1 B' subtype infected Chinese people and to evaluate the association between the positive selection sites and HLA restricted CTL response. We downloaded all HIV-1 B' subtype gag gene sampled from China from the LANL HIV database. The sequences were grouped by years of collection and the mean intra-subtype diversity and divergence were examined by MEGA 5.0 software. The sites under positive selection were estimated by using Hyphy software. Then analyzed the association between positively selected sites and HLA restricted CTL epitopes. A set of 122 B' subtype HIV-1 gag sequences were collected from Henan, Hubei, Liaoning and Yunnan provinces, grouped by sampling year as < 2001, 2001 - 2003, 2004 - 2006 and 2007 - 2010. The mean intra-subtype diversity and divergence increased over time (r(1) = 0.497, P1 < 0.001; r(2) = 0.593, P2 < 0.001). This analysis revealed 34 positively selected sites and 97.1% of these sites were located within 47 known CTL epitopes. The most frequent HLA was HLA-A*02 (21.6%) followed by A*11(9.8%), B*35(9.8%) and B*57(5.9%). The percentages of HLA-A/B/C limited CTL epitopes were 43.1%, 49.0% and 7.9%, respectively. The mean intra-subtype diversity and divergence of B' subtype gag sequences increased over time. B' subtype HIV-1 virus has accumulated adaptations to HLA restricted CTL responses in HIV-1 infected Chinese.

Natural variation of HIV-1 group M integrase: implications for a new class of antiretroviral inhibitors.

HIV-1 integrase is the third enzymatic target of antiretroviral (ARV) therapy. However, few data have been published on the distribution of naturally occurring amino acid variation in this enzyme. We therefore characterized the distribution of integrase variants among more than 1,800 published group M HIV-1 isolates from more than 1,500 integrase inhibitor (INI)-naïve individuals. Polymorphism rates equal or above 0.5% were found for 34% of the central core domain positions, 42% of the C-terminal domain positions, and 50% of the N-terminal domain positions. Among 727 ARV-naïve individuals in whom the complete pol gene was sequenced, integrase displayed significantly decreased inter- and intra-subtype diversity and a lower Shannon's entropy than protease or RT. All primary INI-resistance mutations with the exception of E157Q – which was present in 1.1% of sequences – were nonpolymorphic. Several accessory INI-resistance mutations including L74M, T97A, V151I, G163R, and S230N were also polymorphic with polymorphism rates ranging between 0.5% to 2.0%.

HIV Type 1 Genetic Variability in Central Brazil

This study analyzed the genes pol and env to determine the genetic variability of HIV-1 in Central Brazil. Forty-one isolates of HIV-1-infected individuals had protease, reverse transcriptase, and C2C3/ env amplified by nested PCR and sequenced. The subtype was determined by the program REGA and phylogenetic analyses. The samples identified as putative recombinant forms were analyzed by SimPlot. A high prevalence of subtype B (95.1%) was observed, followed by mosaic viruses B/F (4.9%). The amino acid sequences from 30 HIV-1 isolates were analyzed for the antigenic intrasubtype diversity. The most prevalent gp120 V3 loop motif was the GPGR (United States/Europe) (43.3%), described in B and F subtypes, followed by the GPGK tetrapeptide (10%). The Brazilian variant B" (GWGR), GFGR, and GLGR tetrapeptides were found in 6.7%. Other V3 variants were found in eight isolates (26.7%). Phylogenetic tree analysis was also performed in order to verify the relationship of the HIV-1 samples from Central Brazil with other HIV-1 sequences that circulate in Brazil. The subtype B sequences from Central Brazil formed a polyphyletic cluster in the tree, indicating that these strains are similar to those from other geographic regions. These results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates.

Increasing genetic distance to HIV-1 subtype B and F1 consensus sequences in the Brazilian epidemic: A challenge for vaccine strategies based on central immunogens?

It has been postulated that the non-synonymous divergence (distance to the subtype consensus sequence) observed in several HIV-1 subtype populations during 1990s attained the maximum limit that is compatible with viral fitness or survival, at least in the V3 env gene domain. To test this hypothesis, 145 subtype B and 64 subtype F env V3 sequences isolated from Brazilian HIV-1 positive patients between 1989 and 2004 were analyzed. HIV-1 env V3 sequences were grouped by year of collection and the mean intra-subtype diversity and divergence were examined at synonymous, non- synonymous, and amino acid level. The analyses clearly show that the mean intra-subtype divergence constantly increases in both subtype populations in the last 15 years, and more importantly, this trend was not only driven by a significant increase of the synonymous distance but also by a significant increase of the non- synonymous and amino acid distances between Brazilian circulating viruses and subtype consensus sequences. These results clearly disagree with the notion that the non- synonymous distance to the HIV-1 subtype consensus observed at population level had already attained the maximum limit, and suggest that the likelihood for success of vaccines based on “central” immunogens, as those based on any other empirically selected viral sequence, could be continuously diminishing over time.

Genetic diversity and high proportion of intersubtype recombinants of HIV-1 in urban Cameroon

Results All the samples were infected with HIV-1 group M. No group N and O viruses were identified in the samples studied although they are endemic to Cameroon. HIV-1 infections were dominated by circulating recombinant form CRF02_AG (59%) accompanied by additional group M subtypes or CRFs: A, D, F2, G, CRF01_AE, CRF13_cpx and CRF22_01A1. A high percentage (26%) of the samples had discordant subtype designations between gag and env, and appear to be unique intersubtype recombinant forms (URF) with the majority (91%) involving recombination with either CRF01_AE or/and CRF02_AG. URFs containing CRF19 or CRF22 as well Fgag-Genv and Cgag-Aenv are reported. Some samples were not consistently detected by current HIV-1 testing assays. Conclusion Our study confirmed the existence of high number of cocirculating HIV-1 subtypes, the high intrasubtype diversity, and the high numbers of possible new recombinant viruses in urban Cameroon. These results highlight the epidemiologic importance of HIV-1 CRFs, especially CRF02_AG. from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006

Avian Influenza Virus Exhibits Rapid Evolutionary Dynamics

Influenza A viruses from wild aquatic birds, their natural reservoir species, are thought to have reached a form of stasis, characterized by low rates of evolutionary change. We tested this hypothesis by estimating rates of nucleotide substitution in a diverse array of avian influenza viruses (AIV) and allowing for rate variation among lineages. The rates observed were extremely high, at >10(-3) substitutions per site, per year, with little difference among wild and domestic host species or viral subtypes and were similar to those seen in mammalian influenza A viruses. Influenza A virus therefore exhibits rapid evolutionary dynamics across its host range, consistent with a high background mutation rate and rapid replication. Using the same approach, we also estimated that the common ancestors of the hemagglutinin and neuraminidase sequences of AIV arose within the last 3,000 years, with most intrasubtype diversity emerging within the last 100 years and suggestive of a continual selective turnover.

HIV-1 subtypes and recombinants in the Republic of Congo

To document the actual genetic diversity of HIV-1 strains in the Republic of Congo, 114 HIV-1 positives persons were sampled in 2003 and 2004 after their informed consent. They were attending the teaching hospital, the reference health center in Makelekele, Brazzaville and the regional hospital centers in Pointe-Noire, Gamboma and Ouesso. A total of 104 samples were genetically characterized by direct sequencing of the p24 gag region and 80 were also subtyped in the V3–V5 env region. The genetic subtype distribution of the Congolese strains showed the predominance of subtype A (36.5% and 32.5% in gag and env, respectively) and G (30.8% and 21.25%), whereas subtype D strains represented 12.5% and 15%. Subtypes C, F, H, J, K and the CRFs-01, -02, -05 -06, and also the recently characterized CRF18 were seen at lower rates. Finally, 4.8% ( gag) and 6.25% ( env) of the strains could not be classified. Moreover, a high intra-subtype diversity was observed in our study. Among 70 strains which have been characterized in the two genomic regions, 14 (20%) appeared to be unique recombinants. These data show a high genetic variability in the Republic of Congo, where all the subtypes have been documented together with certain subsubtypes and several CRFs.

Identification and Molecular Characterization of Subsubtype A4 in Central Africa

Phylogenetic analysis on partial env sequences of HIV-1-positive samples obtained from sentinel population groups in HIV serosurveillance studies in 1997 and 2002 in the Democratic Republic of Congo (DRC) revealed a high genetic diversity in the number of cocirculating subtypes/CRFs as well as intrasubtype diversity. A cluster of three strains that formed a distinct and well-separated group within the subtype A radiation was observed. To determine a more precise structure of these viruses, the full-length genomes were sequenced. Phylogenetic tree and bootscan analysis showed that all three newly characterized viruses formed a new nonrecombinant lineage that was more closely related to subtype A. The SUDI (Subtype Distance) program showed that the distances of the newly derived HIV-1 sequences to subtype A references fell in the range of distances previously characterized for subsubtypes. According to current nomenclature rules, we have thus designated this new lineage as subsubtype A4. Analysis of viral sequences from other African countries suggests that the A4 strains seem restricted to DRC where they already circulated at the onset of the HIV/AIDS epidemic.

Distribution of HIV-1 Variants in the Democratic Republic of Congo Suggests Increase of Subtype C in Kinshasa Between 1997 and 2002

The Democratic Republic of Congo (DRC) is characterized by low and stable HIV prevalences and high HIV-1 genetic diversity and is most probably the epicenter of HIV-1 group M. Our major goal was to study the distribution of HIV-1 variants over a 5-year period against a background of political instability and civil war. A total of 288 HIV-1-positive samples collected in 2002 from sentinel population groups in an HIV serosurveillance study performed in 4 cities (Kinshasa [capital city], Mbuji-Mayi [south], Lubumbashi [southeast], and Kisangani [northeast]) were genetically characterized by sequencing and phylogenetic analysis of the V3-V5 env region. The results were compared with those obtained in 1997. Similarly, as in 1997, an extremely high genetic diversity of HIV-1 strains overall and a heterogeneous geographic distribution were seen in 2002. All subtypes and several circulating recombinant forms were present, high intrasubtype diversity was observed, and 5.6% of the samples could not be classified. In each geographic region of the DRC, the genetic diversity was significantly higher than in neighboring countries. Comparison of subtype distribution in similar population groups in Kinshasa in 1997 and 2002 revealed an overall increase of subtype C in Kinshasa from 2.1% to 9.7% and, more precisely, from 0% to 18.9% in female sex workers (P = 0.013). Genetic characterization of HIV-positive samples from sentinel surveys adds significant additional information on new trends in the HIV epidemic. These changes could have implications regarding the spread of HIV infection in the DRC as well on vaccine and/or treatment strategies.

HIV/host interactions: new lessons from the Red Queen's country.

HIV/host interactions: new lessons from the Red Queen's country.

Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa.

The purpose of this study was to document the genetic diversity of human immunodeficiency virus type 1 (HIV-1) in the Democratic Republic of Congo (DRC; formerly Zaire). A total of 247 HIV-1-positive samples, collected during an epidemiologic survey conducted in 1997 in three regions (Kinshasa [the capital], Bwamanda [in the north], and Mbuyi-Maya [in the south]), were genetically characterized in the env V3-V5 region. All known subtypes were found to cocirculate, and for 6% of the samples the subtype could not be identified. Subtype A is predominant, with prevalences decreasing from north to south (69% in the north, 53% in the capital city, and 46% in the south). Subtype C, D, G, and H prevalences range from 7 to 9%, whereas subtype F, J, K, and CRF01-AE strains represent 2 to 4% of the samples; only one subtype B strain was identified. The highest prevalence (25%) of subtype C was in the south, and CRF01-AE was seen mainly in the north. The high intersubtype variability among the V3-V5 sequences is the most probable reason for the low (45%) efficiency of subtype A-specific PCR and HMA (heteroduplex mobility assay). Eighteen (29%) of 62 samples had discordant subtype designations between env and gag. Sequence analysis of the entire envelope from 13 samples confirmed the high degree of diversity and complexity of HIV-1 strains in the DRC; 9 had a complex recombinant structure in gp160, involving fragments of known and unknown subtypes. Interestingly, the unknown fragments from the different strains did not cluster together. Overall, the high number of HIV-1 subtypes cocirculating, the high intrasubtype diversity, and the high numbers of possible recombinant viruses as well as different unclassified strains are all in agreement with an old and mature epidemic in the DRC, suggesting that this region is the epicenter of HIV-1 group M.

Genetic subtypes of HIV type 1 in Hungary.

We examined the diversity of HIV-1 subtypes in 11 adults from Hungary, using the heteroduplex mobility assay (HMA) and DNA sequencing. HMA results showed that HIV-1 gp120 sequences from 10 patients were of subtype B, whereas 1 patient, infected in Africa, carried a subtype C strain. DNA sequencing confirmed the HMA results and revealed a high intrasubtype diversity in the C2V3 region of env in different clade B isolates, which suggests multiple introduction of subtype B to Hungary. This study shows that subtype B is the predominant HIV-1 clade in Hungary.

HIV type 1 diversity and the reliability of the heteroduplex mobility assay.

We investigated HIV-1 diversity by means of heteroduplex mobility assay (HMA) genotyping. We studied 199 samples from patients originating from 26 countries and living in France. The HMA successfully genotyped 182 (91%) of these samples, as follows: 77 (42%) subtype A, 57 (31%) subtype B, 5 (3%) subtype C, 5 (3%) subtype D, 8 (4%) subtype E, 22 (12%) subtype F, 5 (3%) subtype G, and 3 (2%) subtype H. We were not able to genotype 12 samples by means of the HMA. These latter strains were sequenced, and phylogenetic analyses revealed that they were highly divergent subtype A-, D-, or G-related strains. Eight (of 12) subtype D strains were indeterminate by HMA, owing to the broad intrasubtype diversity, suggesting that new reference subtype D plasmids are required, as previously proposed. Thirty-seven strains belonging to the different subtypes were sequenced, and the results showed perfect concordance with the HMA results. Interlaboratory quality controls confirmed the reliability of the HMA for HIV-1 subtyping, despite the extensive viral variability. However, plasmid selection must be continuously revised to cover viral diversification.