Genetic variation in vitro and in vivo of an attenuated Lassa vaccine candidate.

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.

Characterization of acute HIV-1 infection in high-risk Nigerian populations.

BACKGROUND: Acute phase of human immunodeficiency virus (HIV) infection (AHI) may account for a significant proportion of HIV-1 transmission. We identified and characterized individuals in Nigeria with AHI. METHODS: Individuals were tested using a combination of rapid HIV testing in mobile units and laboratory-based specimen pooling for nucleic acid amplification testing. Genome sequences were characterized. A linear segmented regression model was fit to serial viral load (VL) measurements to characterize early VL profiles. RESULTS: Sixteen AHIs were identified from 28 655 persons screened. Specimens were genotyped: 7 (43.8%) were CRF02_AG, 6 (37.5%) were subtype G, 1 (6.3%) was CRF06_cpx, and 2 (12.5%) were unique recombinant forms. No antiretroviral resistance mutations were detected. The mean duration of high VL burden from peak to nadir was 76 days (95% confidence interval [CI], 58-93 days), and the mean rate of viremic control was -0.66 log(10) VL per month. The mean VL at set-point was 4.5 log(10) copies/mL (95% CI, 3.9-5.1 log(10) copies/mL). CONCLUSIONS: This study is the first to characterize AHI among Nigerians identified as HIV infected before seroconversion who would be otherwise missed by conventional HIV testing. Infections by HIV subtypes in Nigeria exhibit long periods of high viral burden, which can contribute to increased transmissibility.

Detection of HIV-1 dual infections in highly exposed treated patients.

BACKGROUND: Genetic characterization of HIV-1 in Argentina has shown that BF recombinants predominate among heterosexuals and injecting drug users, while in men who have sex with men the most prevalent form is subtype B. OBJECTIVES: The aim of this work was to investigate the presence of HIV dual infections in HIV-infected individuals with high probability of reinfection STUDY DESIGN: Blood samples were collected from 23 HIV positive patients with the risk of reinfection from Buenos Aires. A fragment of the HIV gene pol was amplified and phylogenetic analyses were performed. Antiretroviral drug resistance patterns of all the sequences were analyzed. RESULTS: Five dual infections were detected with four patients coinfected with subtype B and BF recombinants and one patient was coinfected with two BF recombinants presenting different recombination patterns. Prolonged infection with a stable clinical condition was observed in the five individuals. Resistance mutation patterns were different between the predominant and the minority strains. CONCLUSIONS: Our results show that HIV dual infection can occur with closely related subtypes, and even with different variants of the same recombinant form in certain populations. Clinical observations showed neither aggressive disease progression nor impact on the resistance patterns in the dually-infected patients.

Changing epidemiology of human parvovirus 4 infection in sub-Saharan Africa.

Human parvovirus 4 infections are primarily associated with parenteral exposure in western countries. By ELISA, we demonstrate frequent seropositivity for antibody to parvovirus 4 viral protein 2 among adult populations throughout sub-Saharan Africa (Burkina Faso, 37%; Cameroon, 25%; Democratic Republic of the Congo, 35%; South Africa, 20%), which implies existence of alternative transmission routes.

HIV-1 recombinants with multiple parental strains in low-prevalence, remote regions of Cameroon: evolutionary relics?

BACKGROUND: The HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial pol amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%. RESULTS: Virus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02_AG. The second most common genetic form (9/164) was the recently described CRF22_01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more. CONCLUSIONS: These results show that while CRF02_AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIVcpz and the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.

Simian T-lymphotropic virus diversity among nonhuman primates, Cameroon.

Cross-species transmission of retroviruses is common in Cameroon. To determine risk for simian T-cell lymphotropic virus (STLV) transmission from nonhuman primates to hunters, we examined 170 hunter-collected dried blood spots (DBS) from 12 species for STLV. PCR with generic tax and group-specific long terminal repeat primers showed that 12 (7%) specimens from 4 nonhuman primate species were infected with STLV. Phylogenetic analyses showed broad diversity of STLV, including novel STLV-1 and STLV-3 sequences and a highly divergent STLV-3 subtype found in Cercopithecus mona and C. nictitans monkeys. Screening of peripheral blood mononuclear cell DNA from 63 HTLV-seroreactive, PCR-negative hunters did not identify human infections with this divergent STLV-3. Therefore, hunter-collected DBS can effectively capture STLV diversity at the point where pathogen spillover occurs. Broad screening using this relatively easy collection strategy has potential for large-scale monitoring of retrovirus cross-species transmission among highly exposed human populations.

Human immunodeficiency virus type 1 intersubtype recombinants predominate in the AIDS epidemic in Cameroon.

A broad and rapidly changing HIV Type 1 (HIV-1) diversity has been reported from different populations in Cameroon since the early epidemic. Our understanding of HIV-1 dynamics can be improved by a systematic surveillance in Cameroon as accessibility and use of antiretroviral drugs increase. To contribute to this, we genotyped 30 samples by sequencing the protease and reverse transcriptase (proRT) genes of HIV-1. Phylogenetic analysis of the HIV-1 proRT sequences using the MEGA3 software showed that 26 (86.7%) were recombinant forms which included 20 (66.7%) circulating recombinant forms: CRF02_AG, (50%), CRF06_cpx (3.3%), CRF11 _cpx (10%) and CRF37_cpx (3.3%), and 6 unique recombinant forms (URF, 20%). Two of the six URFs were second generation recombinants and 4 contained unclassified segments. HIV-1 subtypes A1 (3.3%), C (3.3%) and D (6.7%) were also identified. Although partial sequences of HIV-1 genome were analysed, our results indicate that recombinant HIV-1 variants predominate in the AIDS epidemic in Cameroon. With the widespread use of antiretroviral drugs in Cameroon and the circulation of several HIV-1 variants within this population, the emergence of recombinants with unknown diagnostic and clinical consequences is a concern.

Human immunodeficiency virus and tuberculosis in Argentina: prevalence, genotypes and risk factors.

The objective of this study was to determine the prevalence and genetic variability of human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted infections (STIs) among 205 patients with clinical diagnosis of tuberculosis (TB) in Buenos Aires in 2001. Infections with hepatitis B virus (HBV), HIV-1, hepatitis C virus (HCV), Treponema pallidum and human T-cell lymphotropic virus types I/II were diagnosed in 37/187 (19.8 %), 35/205 (17.1 %), 22/187 (11.8 %), 13/187 (7.0 %) and 4/181 (2.2 %) patients, respectively. Almost one in three participants (33.1 %) presented at least one infection in addition to TB. Multiresistance to TB drugs (isoniazid plus rifampicin) was detected in the isolates recovered from three patients. Injecting drug use was detected as the main risk factor for HIV, HBV and HCV infections. Of ten patients who died, eight were infected with HIV. HIV genetic characterization showed the presence of two different subtypes. Env subtype F was found in 13/24 samples (54.2 %) and subtype B in 11/24 samples (45.8 %) by heteroduplex mobility assay. Sequencing of the protease/RT region was performed in ten samples: three were characterized as subtype B and seven as B/F recombinants by bootscanning analysis. Phylogenetic analysis of four full-length sequences showed that three were the circulating recombinant form CRF12_BF. The results of this study suggest an urgent need to detect HIV infection in high-risk groups to prevent future HIV transmission as well as morbidity and mortality associated with TB by providing highly active antiretroviral therapy (HAART) and/or TB treatment. Collaboration between TB and HIV programmes seems to be the best approach to decrease the incidence of these diseases, especially in high-prevalence HIV settings.

Epidemiological and molecular characteristics of HIV-1 infection among female commercial sex workers, men who have sex with men and people living with AIDS in Paraguay.

An HIV seroprevalence and molecular study was conducted among 935 subjects: 723 female commercial sex workers, 92 men who have sex with men and 120 HIV-positive volunteers. The reported injection drug use rates were 0.7% in female commercial sex workers and 3% in men who have sex with men. Sexually transmitted infections were reported in 265 (37%) of the female commercial sex workers and 38 (41%) of the men who have sex with men. A total of 20 (2.8%) female commercial sex workers and 12 (13%) men who have sex with men became HIV infected during the study period. A history of sexually transmitted infection increased the risk of subsequent HIV infection twofold (adjusted odds ratio of 2.5) among the female commercial sex workers, while cocaine use had an adjusted odds ratios of 6.61 among men who have sex with men. From 130 samples, and based on heteroduplex mobility assaying for the env gene, with sequencing of part of pol and/or full genomes, subtype B was the predominant subtype identified (66%); followed by subtype F (22%) and subtype C (4%). Recombinant CRF12-BF strains were identified in 6% and CRF17_BF was identified in 2%.

Frequency of CCR5 variants among rural populations with low HIV-1 prevalence in Cameroon.

Among rural populations in Cameroon, HIV-1 prevalence is low and the genetic diversity broad. An unusual population-level genetic background may modulate this pattern of HIV infection. We examined HIV-1 prevalence, CCR5Delta32 and CCR5 promoter -2459 G genotype frequency among 1390 rural inhabitants. No individual was identified with the CCR5Delta32 allele, but homozygotes for the CCR5 promoter variant -2459G (27.5%) were relatively common. A seroprevalence of 3.1% of HIV-1 was reported.

Exposure to wild primates among HIV-infected persons.

HIV-1 is an immunosuppressive pathogen. Our behavioral data for 191 HIV-1-infected rural Cameroonians show frequent exposure to nonhuman primates through activities such as hunting and butchering. Immunosuppression among persons exposed to body fluids of wild nonhuman primates could favor the process of adaptation and subsequent emergence of zoonotic pathogens.

High genetic variability of HIV-1 in female sex workers from Argentina.

BACKGROUND: A cross-sectional study on 625 Female Sex Workers (FSWs) was conducted between 2000 and 2002 in 6 cities in Argentina. This study describes the genetic diversity and the resistance profile of the HIV-infected subjects. RESULTS: Seventeen samples from HIV positive FSWs were genotyped by env HMA, showing the presence of 9 subtype F, 6 subtype B and 2 subtype C. Sequence analysis of the protease/RT region on 16 of these showed that 10 were BF recombinants, three were subtype B, two were subtype C, and one sample presented a dual infection with subtype B and a BF recombinant. Full-length genomes of five of the protease/RT BF recombinants were also sequenced, showing that three of them were CRF12_BF. One FSW had a dual HIV-1 infection with subtype B and a BF recombinant. The B sections of the BF recombinant clustered closely with the pure B sequence isolated from the same patient. Major resistance mutations to antiretroviral drugs were found in 3 of 16 (18.8%) strains. CONCLUSION: The genetic diversity of HIV strains among FSWs in Argentina was extensive; about three-quarters of the samples were infected with diverse BF recombinants, near twenty percent had primary ART resistance and one sample presented a dual infection. Heterosexual transmission of genetically diverse, drug resistant strains among FSWs and their clients represents an important and underestimated threat, in Argentina.

Buenos Aires cohort of men who have sex with men: prevalence, incidence, risk factors, and molecular genotyping of HIV type 1.

The prevalence, incidence, risk factors, and molecular genotyping of HIV-1 infection among men who have sex with men (MSM) were assessed through a prospective cohort study. The study was conducted in Buenos Aires from February 2003 to December 2004. Sociodemographic, sexual risk behavior data, and blood samples for HIV testing were collected at baseline and at 6 and 12 months. Cox regression analysis was applied to determine risk factors associated with HIV seroconversion. HIV-positive samples were analyzed by partial (pro/RT) and full-length genome sequencing. Of 811 HIV-negative participants evaluated at baseline, 327 volunteers that fulfilled the inclusion criteria were enrolled. Retention rates at 6 and 12 months were 97.2% and 91.5%, respectively. Twelve MSM seroconverted for HIV infection [incidence rate = 3.9 (95% CI = 2.0-6.7) per 100 person-years]. HIV seroconversion was associated with a greater number of different sexual contacts in the preceding 6 months (> or =10, hazard ratio = 3.3, 95% CI: 1.1-10.4). By partial pro/RT genotyping analysis, 83% HIV-positive samples were subtype B and 17% samples were BF recombinants, most of these being unique recombinant forms. This study describes for the first time the recruitment and follow-up of a cohort of MSM in Argentina. Retention rates and HIV incidence rate were high. These data should be considered as a promising potential population for HIV vaccine trials.

Genomic characterization and molecular evolution analysis of subtype B and BF recombinant HIV-1 strains among Argentinean men who have sex with men reveal a complex scenario.

Currently, data on HIV-1 circulating strains among men who have sex with men (MSM) in Argentina is scarce. In South America, the distribution and the prevalence of BF recombinants are dissimilar and exhibit an underappreciated heterogeneity of recombinant structures. Here, we studied for the first time the genetic diversity of HIV-1 BF recombinants and their evolution over time through in-depth phylogenetic analysis and multiple recombination detection methods involving 337 HIV-1 nucleotide sequences (25 near full-length (NFL) and 312 partial pol gene) obtained from Argentinean MSM. The recombination profiles were studied using multiple in silico tools to characterize the genetic mosaicism, and phylogenetic approaches to infer their relationships. The evolutionary history of BF recombinants and subtype B sequences was reconstructed by a Bayesian coalescent-based method. By phylogenetic inference, 81/312 pol sequences clustered within BF clade. Of them, 46 sequences showed a genetic mosaic with CRF12_BF-like patterns, including plausible second-generation recombinants. Other CRFs_BF like (CRF17, 28, 29, 39, 42, 44, 47) and probable URFs_BF were less frequently found. Phylogenetic and recombination analyses on NFL sequences allowed a meticulous definition of new BF mosaics of genomic patterns. The Bayesian analyses pointed out quite consistent onset dates for the CRFs_BF clade based on B and F gene datasets (~1986 and ~1991 respectively). These results indicate that the CRFs_BF variants have been circulating among Argentinean MSM for about 30 years. This study reveals, through growing evidence showing the importance of MSM in the dynamics of the HIV-1 epidemic in Argentina, the coexistence of CRF12_BF-like and high diversity of strains exhibiting several BF mosaic patterns, including non-reported URFs that may reflect active clusters as potential intervention targets to hinder HIV-1 transmission.

First report of an HIV-1 triple recombinant of subtypes B, C and F in Buenos Aires, Argentina.

We describe the genetic diversity of currently transmitted strains of HIV-1 in men who have sex with men (MSM) in Buenos Aires, Argentina between 2000 and 2004. Nearly full-length sequence analysis of 10 samples showed that 6 were subtype B, 3 were BF recombinant and 1 was a triple recombinant of subtypes B, C and F. The 3 BF recombinants were 3 different unique recombinant forms. Full genome analysis of one strain that was subtype F when sequenced in pol was found to be a triple recombinant. Gag and pol were predominantly subtype F, while gp120 was subtype B; there were regions of subtype C interspersed throughout. The young man infected with this strain reported multiple sexual partners and sero-converted between May and November of 2004. This study reported for the first time the full genome analysis of a triple recombinant between subtypes B, C and F, that combines in one virus the three most common subtypes in South America.

HIV-1 genetic diversity and genotypic drug susceptibility in the Republic of Georgia.

The genetic diversity and genotypic drug susceptibility of HIV-1 strains circulating in the Republic of Georgia, formerly part of the Soviet Union, were investigated for first time. Forty-eight HIV-positive drug-naive Georgian individuals contributed PBMC DNA between 1998 and 2003. On the basis of phylogenetic analyses of partial pol sequences, the predominant HIV-1 genetic forms were subtype A (70%), followed by subtype B (26%); both genetic forms were carried by injecting drug users and heterosexuals. There was also one subtype C (2%) and one CRF18_cpx (2%). The Georgian subtype A strains clustered with subtype A from Russia, designated A(FSU). Twelve of the subtype A strains (25%) contained the secondary protease inhibitor mutation V77I and 9 also had two other silent mutations. This "V77I haplotype" marks one particular genetic lineage of the epidemic in the former Soviet Union. Two strains (4%) carried antiretroviral (ARV) drug resistance mutations. Nearly full-length genome sequences of five Georgian strains were also completed. Two, 98GEMZ011 (subtype A) and 98GEMZ003 (subtype B), closely resembled the parental strains that recombined to create CRF03_AB. The use of these parental strains in the analysis revealed an additional segment of subtype A in CRF03_AB. Thus, the HIV-1 epidemic in Georgia was composed of a mixture of subtype A(FSU) and subtype B.

First case of HIV Type 1 subtype F among men who have sex with men in Colombia.

To study the molecular epidemiology of human immunodeficiency virus (HIV) strains among men who have sex with men (MSM), the main high-risk group for HIV infection in Colombia, 113 HIV-positive MSM subjects recruited in Bogotá during the year 2002 were genotyped. By heteroduplex mobility assay (env HMA) all samples were classified as subtype B. Partial sequencing of the protease and the reverse transcriptase (Pro/RT) regions performed on a random subset of 10 samples revealed that nine were classified as subtype B, and one sample was subtype F. The specimen that is subtype F in pol and subtype B in env is likely to be is either a recombinant or a dual infection. In this study, we identify the HIV F subtype for the first time in Colombia.

Genetic forms of HIV Type 1 in the former Soviet Union dominate the epidemic in Azerbaijan.

A total of 125 strains collected in Azerbaijan between 1999 and 2002 from HIV seropositives were genetically classified. Of 84 strains classified using HMA, 91.6% were subtype A, 1.2% subtype B, and 7.1% untypeable. Of 41 strains analyzed using partial pol gene sequences, 90.2% were subtype A, 7.3% subtype B, and 2.4% CRF03_AB. Most sequenced A strains clustered with those circulating in countries of the former Soviet Union (FSU). Two of three sequenced B strains were from individuals who traveled to FSU clustering tightly with B strains from Nikolayev, Ukraine. CRF03_AB, characteristic of the 1996 epidemic in Kaliningrad, Russia, was sequenced from an individual who lived in Russia from 1995 until 2001. The HIV epidemic in Azerbaijan is concentrated in IDU and is closely connected to other such epidemics to the east based on genetics. Of the 41 sequenced strains, 95% were close genetic relatives of HIV strains in IDU networks in the FSU.

Molecular epidemiology of HIV Type 1 in preparation for a Phase III prime-boost vaccine trial in Thailand and a new approach to HIV Type 1 genotyping.

To characterize HIV-1 genotypes in candidate populations for a prime-boost phase III vaccine trial in Thailand, specimens from prevalent and incident HIV-1 infections from a family planning clinic population in Rayong Province and a community cohort in Chon Buri Province, collected from 1998 to 2001, were genotyped. A new multiregion hybridization assay, MHAbce, capable of distinguishing HIV-1 CRF01_AE, subtype B, and subtype C and their recombinants, was developed and applied to prevalent infections. Most incident and selected prevalent infections were studied by complete genome sequencing. By MHAbce, 168 of 194 prevalent infections were genotyped. Of these, 90.5% were CRF01_AE, 2.4% were subtype B, and 7.2% showed discordant or dual probe reactivity, indicative of recombination or dual infection, respectively. Among 23 incident infections, 20 were sequenced. Eighteen CRF01_AE, one subtype B, and one CRF01/B recombinant strains were seen. Two CRF01/B and one CRF01/C recombinant were identified among selected prevalent infections. These results indicate that incident and prevalent HIV-1 infections in Rayong and Chon Buri during 1998-2001 were 90% CRF01_AE, 3% subtype B, and 7% either recombinant or dual. This study frames the genetic diversity of HIV-1 in these cohorts in their preparatory phase for the ongoing ALVACHIV (vCP1521) prime, AIDSVAX B/E boost, phase III vaccine trial and will provide a benchmark for interpretation and analysis.

Short report: HIV infection among commercial sex workers and injecting drug users in the Czech Republic.

Since the first HIV case was diagnosed in the Czech Republic in 1985, there is a lack of information regarding the epidemiology of HIV infection in most high-risk groups. To determine the prevalence of, and risk factors for, HIV among female and male commercial sex workers (FCSW and MCSW, respectively) and injecting drug users (IDUs), cross-sectional studies were conducted in the cities of Cheb, Usti nad Labem, Ostrava, and Prague of the Czech Republic. A total of 1,277 subjects were enrolled, which included 585 FCSWs, 230 MCSWs, and 462 IDUs. The HIV prevalences were 0.7% (95% CI: 0.2-1.7%), 0.9% (95% CI: 0.1-3.1%), and 0.2% (95% CI: 0.005-1.2%) among FCSWs, MCSWs, and IDUs, respectively. Although low HIV prevalences were found, ongoing sentinel surveillance studies, which address modifiable behavioral and biologic risk factors among high-risk groups, are necessary to guide strategies to stem the tide of the epidemic in this country.