Evidence of Multiple Introductions and Autochthonous Transmission of the HIV Type 1 CRF02_AG Clade in Brazil

Abstract HIV-1 CRF02_AG is the most prevalent intersubtype recombinant form worldwide. Six HIV-1 samples from patients living in Rio de Janeiro, Brazil, were subtyped as CRF02_AG at the pol gene between 2004 and 2011. To trace the origin of these viruses, they were compared with 793 CRF02_AG pol sequences of African origin and another four Brazilian CRF02_AG pol sequences previously described. Phylogenetic analysis reveals that there have been at least four introductions of the CRF02_AG clade in Brazil, as signified by the presence of four phylogenetically distinct lineages, probably originated from western African countries (Benin, Ghana, and Guinea-Bissau). At least two CRF02_AG Brazilian lineages were successful in getting established and disseminated throughout the Rio de Janeiro state, with evidence of both horizontal and vertical transmission. Continuous epidemiological surveillance of HIV-1 strains circulating in Brazil is of paramount importance to the early detection of newly emerging viral lineages.     

Infectious DNA clone of HIV type 1 A/G recombinant (CRF02_AG) replicable in peripheral blood mononuclear cells

We constructed an infectious DNA clone of the HIV-1 A/G recombinant 97GH-AG2, which was isolated in Ghana in 1997 and was classified originally as subtype A. By phylogenetic and recombination breakpoint analyses, p97GH-AG2 was grouped in the circulating form of A/G recombinants (CRF02_AG) and was found to contain the least amount of subtype G-derived region among the known CRF02_AG HIV-1 DNAs. This result suggests that CRF02_AG may be a predominant form in Ghana. Virions produced by transfection of p97GH-AG2 into 293T cells grew in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs). 97GH-AG2 also replicated efficiently in CCR5-expressing HeLa cells, MAGIC5, but only weakly in the parent MAGI cells, indicating that 97GH-AG2 uses mostly CCR5 as a coreceptor. Isolation of the first HIV-1 (CRF02_AG) DNA clone that replicates in PBMCs will accelerate the molecular analysis of this subtype.     

HIV type 1 circulating recombinant form CRF09_cpx from west Africa combines subtypes A, F, G, and may share ancestors with CRF02_AG and Z321

Two HIV-1 intersubtype recombinant forms are circulating widely in populations and have become important strains in the pandemic: CRF01_AE in Southeast Asia and CRF02_AG in West and West Central Africa, respectively. Several other circulating recombinant forms (CRF) have also been identified, but with fewer numbers of infections and/or more limited geographic spread. Here we expand knowledge of HIV-1 CRF using clinical samples, principally from West Africa, that were difficult to classify by partial genome sequencing. DNA was extracted from primary patient peripheral blood mononuclear cells (PBMC). The virtually complete HIV-1 genome was amplified by polymerase chain reaction (PCR) and directly sequenced. Additional strains were characterized by partial envelope sequencing. Phylogenetic analysis was used to identify and map intersubtype recombination breakpoints. Four virtually complete genome sequences and two partial envelope sequences represent CRF09_cpx, a newly identified complex recombinant HIV-1 whose principal focus seems to be in West Africa. This recombinant includes segments of subtypes A, F, G, and unclassified genetic material. It shares unique unclassified regions with the early Zaire strain Z321. There are similarities in structure, but considerable genetic distances, between CRF09_cpx and CRF02_AG IbNG. In conclusion, it is possible that this CRF shared common ancestors with both Z321 and CRF02_AG in the course of the pandemic, perhaps arising by recombination between earlier forms of these strains. Although newly identified, at least one infection with CRF09_cpx has already occurred outside of Africa.     

Characterization of protease resistance-associated mutations in HIV type 1 drug-naive patients following the increasing prevalence of the CRF02_AG strain in Morocco

The purpose of this study was to investigate the amino acid substitutions in the protease of HIV-1 B and non-B subtypes and evaluate whether the emergence of resistance-associated mutations (RAMs) could have a significant correlation with the increasing prevalence of CRF02_AG strains in Morocco. A total of 162 protease gene sequences were successfully amplified from drug-naive HIV-1-infected individuals. We identified eight (sub)subtypes and CRFs: B(66%), A1(3.7%), C(1.2%), F1(0.6%), F2(0.6%), G(1.2%), CRF02_AG(25.3%), and CRF01_AE(1.2%). Phylogenetic analysis of CRF02_AG strains showed that 9.8% of isolates had a closer connection with reference strains from Morocco and 15.4% clustered with reference strains from eight West African and three European countries. When compared to the B subtype, patients with the CRF02_AG strain had a significantly higher prevalence of mutations associated with resistance to some antiprotease drugs, mainly tipranavir (TPV): H69K (97% vs. 5%; p<0.0001), L89M (95% vs. 1%; p<0.0001), and M36I/L (93% vs. 44%; p<0.0001). Most of the CRF02_AG strains (97%) significantly showed at least two TPV-RAMs (p=0.002) compared to the B subtype (7%). Multivariate analysis revealed that CRF02_AG infection was the only factor highly associated with the occurrence of more than two TPV-RAMs (C=0.42; p<0.0001). These results support the importance of transmitted drug resistance mutations (M36I/L, H69K, and L89M) in the protease gene of HIV-1 CRF02_AG isolates. This HIV drug resistance transmission before protease inhibitor (PI) exposure raises concern about its influence on the susceptibility of CRF02_AG strains to some PIs, especially tipranavir, which will soon be introduced as part of the second line therapeutic regimens in Morocco.     

Development and application of a high-throughput HIV type 1 genotyping assay to identify CRF02_AG in West/West Central Africa

In West/West Central Africa, CRF02_AG is the most prevalent HIV-1 strain and circulates in the milieu of rare subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs). The molecular complexity of HIV-1 epidemics in this region and the need to extensively sample large populations, such as in the case of vaccine trials, pose seemingly conflicting requirements between full-genome sequencing and high-throughput low-resolution assays. Here we describe the development and evaluation of a multiregion hybridization assay (MHAcrf02) for the efficient genotyping of CRF02_AG in West/West Central Africa. Subtype A, G, and CRF02_AG-specific fluorescent probes were designed flanking five recombination breakpoints in CRF02_AG and were used in real-time PCRs. A panel representing West/West Central African HIV-1 genetic diversity was evaluated by MHAcrf02. The sample set, previously characterized by full-genome sequencing, included CRF02_AG and CRF02_AG-containing recombinants (n = 28), other subtypes, CRFs, and URFs (n = 34). DNA from peripheral blood mononuclear cells, cocultures, and plasmids was used as template. When the patterns of probe reactivity were evaluated. CRF02_AG was identified with a 100% specificity and sensitivity. In conclusion, MHAcrf02 will permit more efficient characterization of HIV-1 in West/West Central Africa, where CRF02_AG is an important strain. Together with other regional genotyping assays MHAcrf02 will contribute to the development of a global picture of HIV-1 diversity and geographic distribution, providing a strong foundation for intervention, including vaccine development.     

No difference in clinical progression between patients infected with the predominant human immunodeficiency virus type 1 circulating recombinant form (CRF) 02_AG strain and patients not infected with CRF02_AG,in Western and West-Central Africa: a four-year prospective multicenter study

To compare human immunodeficiency virus (HIV) type 1 disease progression in patients infected by the predominant strain circulating recombinant form (CRF) 02_AG in western and west-central Africa and in patients infected by other strains, a prospective multicenter cohort study was conducted in Cameroon and Senegal. Among the 335 patients, a broad HIV-1 group M subtype diversity was observed in the envelope V3-V5 region, but strain CRF02_AG predominated in both Cameroon and Senegal (61.2% and 62.9%, respectively; P<.8). Multivariate analyses showed no difference between patients infected by CRF02 strains and those infected by other strains in terms of survival (adjusted hazards ratio [HR], 1.16; 95% confidence interval [CI], 0.76-1.78; P=.5), clinical disease progression (HR, 0.79; 95% CI, 0.50-1.25; P=.3), or square root CD4 cell decline (regression coefficient, -0.01; 95% CI, -0.82 to 0.81; P=.9). This study suggests that the predominance of HIV-1 CRF02_AG strain in western and west-central Africa should have no major clinical consequences.     

HIV-1 CRF09_cpx circulates in the North West Province of Cameroon where CRF02_AG infections predominate and recombinant strains are common

This study describes the HIV-1 genetic diversity that currently circulates in Bamenda, the provincial capital of the North West province of Cameroon. Phylogenetic analysis of the protease (pro) gene of 20 HIV-1-seropositive individuals identified 11 (55%) CRF02_AG, one D, one F2, one J, and four (20%) unclassifiable strains. Interestingly, the remaining two (10%) samples, 02CMNYU3072 and 03CMNYU3224, originating from epidemiologically unlinked individuals, were classified as CRF09_cpx, representing the first reported cases of this complex circulating recombinant form (CRF) in Cameroon. Additional analysis of the C2V5 portion of the envelope (env) gene confirmed the CRF09_cpx identity of these isolates and classified the remaining isolates as CRF02_AG (n = 12, 63%), subtype D (n = 2, 11%), subtype F2 (n = 2, 11%), and subtype A1 (n = 1). In combination, the pro and env subtyping results revealed three (16%) isolates with discordant subtypes including J( pro )CRF02_AG( env ), CRF02_AG( pro )D( env ), and CRF02_AG( pro )F2( env ). In conclusion, this study highlights the presence of HIV-1 CRF09_cpx in Cameroon and identifies three possible intersubtype recombinants (ISRs) containing CRF02_AG in a town where CRF02_AG infections predominate, and stresses the commonness of HIV-1 recombinant strains in a region where broad genetic diversity exists.     

Comparison of the Mechanisms of Drug Resistance among HIV, Hepatitis B, and Hepatitis C

Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most prevalent deadly chronic viral diseases. HIV is treated by small molecule inhibitors. HBV is treated by immunomodulation and small molecule inhibitors. HCV is currently treated primarily by immunomodulation but many small molecules are in clinical development. Although HIV is a retrovirus, HBV is a double-stranded DNA virus, and HCV is a single-stranded RNA virus, antiviral drug resistance complicates the development of drugs and the successful treatment of each of these viruses. Although their replication cycles, therapeutic targets, and evolutionary mechanisms are different, the fundamental approaches to identifying and characterizing HIV, HBV, and HCV drug resistance are similar. This review describes the evolution of HIV, HBV, and HCV within individuals and populations and the genetic mechanisms associated with drug resistance to each of the antiviral drug classes used for their treatment.     

Identification of a Novel HIV Type 1 Circulating Recombinant Form (CRF52_01B) in Southeast Asia

Abstract Thirty HIV-1 URF_01AE/ B' complete or nearly full-length genome sequences sampled within Southeast Asia were obtained from the Los Alamos HIV Sequence Database. Phylogenetic and recombinant analyses revealed that three sequences indeed displayed the identical recombinant structure. Of note, the three subjects, harboring novel CRF01_AE/B recombinants, did not have apparent epidemiological linkage. They fulfilled the criteria for the designation of a new circulating recombinant form (CRF) and constituted the 52nd CRF identified in the worldwide HIV-1 pandemic. In this chimera, two short subtype B segments were inserted into a backbone of CRF_01AE. The breakpoints corresponded to HXB2 nucleotide positions 2930, 3251, 8521, and 9004 approximately. This CRF is the first one identified by neatening and analyzing the sequences already presented in the Los Alamos HIV Sequence Database. This indicates that we should pay attention not only to explicit subtype sequences but also to those classified as a unique recombinant form (URF) so far.     

Emergence of complex and diverse CRF02-AG/CRF06-cpx recombinant HIV type 1 strains in Niger,West Africa

On the basis of partial env and gag subtyping, we documented that the majority of HIV-1 strains circulating in Niger were CRF02-AG (54.3%) or CRF06-cpx (18.1%) and that 9% of the samples were possible recombinants between CRF02 and CRF06. To determine in more detail the precise structure of these viruses we sequenced the full-length genomes for three such strains (97NE-003, 00NE-036, and 00NE-095). From the bootscan and phylogenetic tree analysis it is evident that the new viruses are the result of recombination events between CRF02-AG and CRF06-cpx strains. Importantly, each virus had a different complex recombinant structure with multiple breakpoints, leading to viruses with complex mosaic patterns.     

Isolation and characterization of a full-length molecular DNA clone of Ghanaian HIV type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.     

Circulation of novel HIV type 1 A, B/C, and F subtypes in Argentina

The Argentine HIV-1 epidemic is considered to be represented mainly by subtype B and diverse B/F recombinants, with apparent absence of pure subtype F. In this study we describe three novel HIV-1 variants isolated from four infants born in different and distant provinces of Argentina. Partial analysis of different gene fragments spanning 18.5-40.8% of the HIV-1 complete genome revealed two subtype A HIV-1 strains in siblings, a B/C recombinant with a novel mosaic structure, and a putative subtype F. Characteristic patterns of genomic and amino acid sequences of the newly reported subtype F isolate suggest a closer genetic relationship to Argentine B/F recombinants than any other subtype F strain described so far, while the A and B/C subtypes found correspond to unusual genotypes in Argentina. Understanding the origin, diversity, and spread of HIV-1 strains worldwide will be necessary for the development of an effective vaccine approach.     

Subtypes A,C, G,and recombinant HIV type 1 are circulating in Bangladesh

We analyzed the genetic diversity of HIV-1 circulating in Bangladesh by direct sequencing and subsequent phylogenetic analysis of the V3 region of the env gene and p17 fragment of the gag gene from nine unrelated patients. The sequences from one sample grouped into subtype A, five samples grouped into subtype C, and one grouped into subtype G. In addition, two patients appeared to be infected with different recombinant viruses consisting of subtype A and unclassifiable viral sequences. Epidemiological analysis revealed heterosexual transmission in the majority of cases. Furthermore, most subjects had a history of traveling, either to India or to the Arabian Peninsula. This study shows that several HIV-1 subtypes are circulating in Bangladesh, and we conclude that there must have been several introductions of HIV-1 into the Bangladeshi population.     

Identification of a novel circulating recombinant form (CRF) 36_cpx in Cameroon that combines two CRFs (01_AE and 02_AG) with ancestral lineages of subtypes A and G

An array of CRFs have been identified in Cameroon, the most notable being CRF02_AG. HIV-1 in the East Province of Cameroon is particularly diverse: in a recent study, we found a high proportion of unique recombinant forms (URFs). Herein we describe the analysis of the full-length sequences of two of these URFs, which, after preliminary analysis of gag, pol, and env fragments, appeared to be a novel CRF. This novel strain, CRF36_cpx, contains fragments that can be assigned to the CRF01_AE, CRF02_AG, and subtype A and G radiations. Forty percent of the genome can be classified as CRF02_AG, including regions in gag, pol, env, and the accessory genes. Twenty-seven percent is CRF01_AE, comprising the majority of gag, the beginning of env, and the end of env into the 3' LTR. Twenty percent of the genome can be assigned to subtype A, with segments in pol and env. The remaining 13% of the sequence is classifiable as subtype G, in pol and vpu. The subtype A and G lineages formed by the CRF36_cpx sequences are unique and appear ancestral in nature. CRF36_cpx is both the first to combine more than one CRF and the first to include fragments of CRF02_AG. The ancestral sequences present in CRF36_cpx represent a link to extinct strains, and, potentially, insight into the evolution of HIV-1.     

Prevalence of Hepatitis B,C, HIV and syphilis markers among refugees in Bari,Italy

Background  

The aim of this study was to assess the prevalence of Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) serological markers and the prevalence of VDRL positive subjects in a population of refugees of various nationalities, living in the Asylum Seeker Centre in Bari Palese, Southern Italy.     

Comparative Analysis of Within-Host Mutation Patterns and Diversity of Hepatitis C Virus Subtypes 1a, 1b,and 3a

Understanding within-host evolution is critical for predicting viral evolutionary outcomes, yet such studies are currently lacking due to difficulty involving human subjects. Hepatitis C virus (HCV) is an RNA virus with high mutation rates. Its complex evolutionary dynamics and extensive genetic diversity are demonstrated in over 67 known subtypes. In this study, we analyzed within-host mutation frequency patterns of three HCV subtypes, using a large number of samples obtained from treatment-naïve participants by next-generation sequencing. We report that overall mutation frequency patterns are similar among subtypes, yet subtype 3a consistently had lower mutation frequencies and nucleotide diversity, while subtype 1a had the highest. We found that about 50% of genomic sites are highly conserved across subtypes, which are likely under strong purifying selection. We also compared within-host and between-host selective pressures, which revealed that Hyper Variable Region 1 within hosts was under positive selection, but was under slightly negative selection between hosts, which indicates that many mutations created within hosts are removed during the transmission bottleneck. Examining the natural prevalence of known resistance-associated variants showed their consistent existence in the treatment-naïve participants. These results provide insights into the differences and similarities among HCV subtypes that may be used to develop and improve HCV therapies.     

Comparative Analysis of Glycoprotein B (gB) of Equine Herpesvirus Type 1 and Type 4 (EHV-1 and EHV-4) in Cellular Tropism and Cell-to-Cell Transmission

Glycoprotein B (gB) plays an important role in alphaherpesvirus cellular entry and acts in concert with gD and the gH/gL complex. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry rivaling no significant differences when compared to parental virus. We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses. The reduction in virus growth may be attributable to the loss of functional interaction between gB and the other envelope proteins involved in virus entry, including gD and gH/gL. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1. In conclusion, our results show that the exchange of gB between EHV-1 and EHV-4 is possible, but results in a significant attenuation of virus growth in the case of EHV-4_gB1. The generation of stable recombinant viruses is a valuable tool to address viral entry in a comparative fashion and investigate this aspect of virus replication further.