HIV-1 genetic diversity and demographic characteristics in Bulgaria.

HIV-1 strain diversity in Bulgaria is extensive and includes contributions from nearly all major subtypes and the Circulating Recombinant Forms (CRF): 01_AE, 02_AG, and 05_DF. Prior to this study, HIV-1 sequence information from Bulgaria has been based solely on the pro-RT gene, which represent less than 15% of the viral genome. To further characterize HIV-1 in Bulgaria, assess participant risk behaviors, and strengthen knowledge of circulating strains in the region, the study "Genetic Subtypes of HIV-1 in Bulgaria (RV240)" was conducted. This study employed the real time-PCR based Multi-region Hybridization Assay (MHA) B/non-B and HIV-1 sequencing to survey 215 of the approximately 1100 known HIV-1 infected Bulgarian adults (2008-2009) and determine if they were infected with subtype B HIV-1. The results indicated a subtype B prevalence of 40% and demonstrate the application of the MHA B/non-B in an area containing broad HIV-1 strain diversity. Within the assessed risk behaviors, the proportion of subtype B infection was greatest in men who have sex with men and lowest among those with drug use risk factors. During this study, 15 near full-length genomes and 22 envelope sequences were isolated from study participants. Phylogenetic analysis shows the presence of subtypes A1, B, C, F1, and G, CRF01_AE, CRF02_AG, CRF05_DF, and one unique recombinant form (URF). These sequences also show the presence of two strain groups containing participants with similar risk factors. Previous studies in African and Asian cohorts have shown that co-circulation of multiple subtypes can lead to viral recombination within super-infected individuals and the emergence of new URFs. The low prevalence of URFs in the presence of high subtype diversity in this study, may be the result of successful infection prevention and control programs. Continued epidemiological monitoring and support of infection prevention programs will help maintain control of the HIV-1 epidemic in Bulgaria.

An Evaluation of Selected Populations for HIV-1 Vaccine Cohort Development in Nigeria.

Development of a globally effective HIV-1 vaccine will need to encompass Nigeria, one of the hardest hit areas, with an estimated 3.2 million people living with HIV. This cross-sectional Institutional Review Board (IRB) approved study was conducted in 2009-12 at four market sites and two highway settlements sites in Nigeria to identify and characterize populations at high risk for HIV; engage support of local stakeholders; and assess the level of interest in future vaccine studies. Demographic, HIV risk data were collected by structured interviewer-administered questionnaires. Blood samples were tested on site by HIV rapid diagnostic tests, followed by rigorous confirmatory testing, subtype evaluation and testing for HBV and HCV markers in a clinical reference laboratory. Of 3229 study participants, 326 were HIV infected as confirmed by Western Blot or RNA, with a HIV prevalence of 15.4%-23.9% at highway settlements and 3.1%-9.1% at market sites. There was no observable correlation of prevalence of HIV-1 (10.1%) with HBV (10.9%) or HCV (2.9%). Major HIV-1 subtypes included CRF02_AG (37.5%); G (27.5%); G/CRF02_AG (25.9%); and non-typeable (8.9%), with 0.3% HIV-2. Univariate analysis found age, gender, marital status, level of education, and sex under substance influence as significant risk factors for HIV (p<0.001). Educating and winning the trust of local community leadership ensured high level of participation (53.3-77.9%) and willingness to participate in future studies (95%). The high HIV prevalence and high risk of HIV infection at highway settlement and mammy markets make them well suited for targeting future vaccine trials in Nigeria.

Deployment of the 1st Area Medical Laboratory in a Split-Based Configuration During the Largest Ebola Outbreak in History.

BACKGROUND: The U.S. Army 1st Area Medical Laboratory (1st AML) is currently the only deployable medical CBRNE (Chemical, Biological, Radiological, Nuclear, and Explosives) laboratory in the Army's Forces Command. In support of the United States Agency for International Development Ebola response, the U.S. military initiated Operation United Assistance (OUA), and deployed approximately 2,500 service members to support the Government of Liberia's Ebola control efforts. Due to its unique molecular diagnostic and expeditionary capabilities, the 1st AML was ordered to deploy in October of 2014 in support of OUA via establishment of Ebola testing laboratories. To meet the unique mission requirements of OUA, the unit was re-organized to operate in a split-based configuration and sustain four separate Ebola testing laboratories. METHODS: This article is a review of the 1st AML's OUA participation in a split-based configuration. Topics highlighted include pre-deployment planning/training, operational/logistical considerations in fielding/withdrawing laboratories, laboratory testing results, disease and non-battle injuries, and lessons learned. FINDINGS: Fielding the 1st AML in a split-based configuration required careful pre-deployment planning, additional training, optimal use of personnel, and the acquisition of additional laboratory equipment. Challenges in establishing and sustaining remote laboratories in Liberia included: difficulties in transportation of equipment due to poor road infrastructure, heavy equipment unloading, and equipment damage during transit. Between November 26, 2014 and February 18, 2015 the four 1st AML labs successfully tested blood samples from patients and oral swabs collected by burial teams in rural Liberia. The most significant equipment malfunction during laboratory operations was generators powering the labs, with the same problem impacting headquarters. Generator failures delayed laboratory operations/result reporting, and put temperature sensitive reagents at risk. None of the 22 1st AML soldiers (at remote labs or headquarters) had an Ebola exposure, none were infected with malaria or other tropical diseases, and none required evacuation from the time deployed to remote sites. The primary medical condition encountered was acute gastroenteritis, and within the first week of arrival to Liberia, 19 (86%) soldiers were affected. DISCUSSION/IMPACT/RECOMMENDATIONS: With proper planning and training, the 1st AML can successfully conduct split-based operations in an outbreak setting, and this capability can be utilized in future operations. The performance of the 1st AML during the current Ebola outbreak highlights the value of this asset, and the need to continue its evolution to support U.S. military operations.

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG.

While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development.The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants.The sensitivity and specificity of MHA varied between 73-100% and 90-100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants.CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development.

Molecular epidemiology of early and acute HIV type 1 infections in the United States Navy and Marine Corps, 2005-2010.

The U.S. military represents a unique population within the human immunodeficiency virus 1 (HIV-1) pandemic. The last comprehensive study of HIV-1 in members of the U.S. Navy and Marine Corps (Sea Services) was completed in 2000, before large-scale combat operations were taking place. Here, we present molecular characterization of HIV-1 from 40 Sea Services personnel who were identified during their seroconversion window and initially classified as HIV-1 negative during screening. Protease/reverse transcriptase (pro/rt) and envelope (env) sequences were obtained from each member of the cohort. Phylogenetic analyses were carried out on these regions to determine relatedness within the cohort and calculate the most recent common ancestor for the related sequences. We identified 39 individuals infected with subtype B and one infected with CRF01_AE. Comparison of the pairwise genetic distance of Sea Service sequences and reference sequences in the env and pro/rt regions showed that five samples were part of molecular clusters, a group of two and a group of three, confirmed by single genome amplification. Real-time molecular monitoring of new HIV-1 acquisitions in the Sea Services may have a role in facilitating public health interventions at sites where related HIV-1 infections are identified.

trans-Complementation of HBV rtM204I mutant replication by HBV wild-type polymerase.

The function of the hepatitis B virus (HBV) wild-type (WT) polymerase (pol) expressed alone or in the context of the intact genome when interacting with HBV rtM204I in HepG2 cells was compared. We show that WT pol expression from a packaging-defective RNA can complement defective rtM204I pol activity resulting in increased levels of HBV replicative intermediates (RI). Analysis of the genetically marked genomes showed that this restoration resulted from trans-complementation, rather than recombination. In contrast, we demonstrate that enhanced levels of total HBV RI observed when cells were cotransduced with both WT and rtM204I baculoviruses were predominantly WT RI. In this case, WT pol was produced from a full-length pregenomic RNA (pgRNA). We conclude that the WT pol has the capacity to trans-complement the replication defect of rtM204I; however, when expressed from an authentic pgRNA, as in a mixed infection, pol may not trans-complement efficiently.

In vitro study of the effects of precore and lamivudine-resistant mutations on hepatitis B virus replication.

Understanding the consequences of mutation in the hepatitis B virus (HBV) genome on HBV replication is critical for treating chronic HBV infection. In this study, HBV replication in HepG2 cells initiated by transduction with precore (PC), rtM204I, and wild-type (wt) HBV recombinant baculoviruses was compared. The pattern and magnitude of HBV replication initiated by the PC HBV recombinant baculovirus were similar to those observed for wt HBV throughout the time course examined. In contrast, when the rtM204I mutation was introduced into wt HBV, by day 10 postinfection the levels of intra- and extracellular HBV DNA were markedly reduced compared to those for wt HBV. Although the rtM204I mutation reduced the production of HBV replicative intermediates, no effect on the level of covalently closed circular DNA or HBV transcripts was observed at late time points. Coinfection studies with different ratios of wt and rtM204I baculoviruses showed that the rtM204I variant did not produce a product that inhibited HBV replication. However, the combination of the wt and rtM204I baculoviruses yielded HBV DNA levels at late time points that were greater than those for the wt alone, suggesting that wt polymerase may function in trans to boost rtM204I replication. We concluded that the rtM204I mutation generates a polymerase that is not only resistant to lamivudine but also replicates nucleic acids to lower levels in vitro.